Biomarkers for motor neuron disease

Abstract

Disclosed herein are methods and related compositions for determining a likelihood that a subject has a motor neuron disease (e.g., amyotrophic lateral sclerosis) based on levels of a combination of lipid biomarkers in a biological sample from the subject. Also disclosed are methods for determining a rate of progression of a motor neuron disease based on changes in lipid biomarker levels in a subject over time. Also disclosed are methods for detecting the presence and / or determining the level of at least three lipid biomarkers disclosed herein in a subject suspected of suffering from a motor neuron disease. Also disclosed are related kits useful for carrying out the disclosed diagnostic or prognostic methods.

Description

[0001] Biomarkers for Motor Neuron Disease

[0002] All documents cited or referenced herein, and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference in their entirety.

[0003] FIELD

[0004] The present invention relates to the identification of biomarkers which are associated with a higher risk for neurodegenerative disease and particularly motor neuron disease.

[0005] BACKGROUND

[0006] Motor neuron disease (MND) is a progressive neurodegenerative disorder which leads to death 2-5 years after diagnosis. Currently, it can take up to 2 years from presentation of symptoms to definite diagnosis of MND. The complexity of the disease is one of the main reasons that diagnosis is so lengthy. There is no single test that can diagnose MND, and patients must endure numerous neurophysiology examinations, blood tests, and cerebrospinal fluid examinations, resulting in high diagnostic burden and, for those afflicted by the condition, progression of symptoms. Single, easy, accessible biomarkers for MND are scarce and, to date, not sufficiently specific or reliable.

[0007] Thus, there is an urgent and ongoing need to identify specific and reliable diagnostic and prognostic tests for MND.

[0008] SUMMARY

[0009] The inventors of the present disclosure have identified that despite the heterogeneity of MND, metabolic disturbances before onset of symptoms are consistently present in all patients. In particular, increased fat free mass ( / .e., less fat) is correlated with shorter survival, whereas greater fat mass seems to be protective in MND. Further, while patients with MND rapidly lose weight and fat mass, the fat content of muscle is increased, indicating a major dysregulation in lipid homeostasis.

[0010] The present inventors have performed targeted lipidomic analysis to identify specific biomarkers for detection and monitoring of MND in a subject.

[0011] The invention relates to specific combinations of biomarkers as well as methods and kits for detecting, diagnosing and treating MND, methods and kits for monitoring the rate of progression of MND, and methods and kits for determining the likelihood that a subject has a MND.

[0012] Accordingly, in one aspect, the present disclosure provides a method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid. In a related aspect, the present disclosure provides a method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least one lipid wherein the at least one lipid is selected from the group consisting of glycerolipids, sphingolipids, and sterol lipids. For example, the method comprises measuring a biological sample. For example, the method comprises having measured a biological sample.

[0013] In a further aspect, the present disclosure provides a method for determining a rate of progression of motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid. For example, the method comprises measuring a biological sample. For example, the method comprises having measured a biological sample.

[0014] In another aspect, the present disclosure provides a method detecting the presence and / or level of at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: (i) providing a biological sample obtained previously from the subject; (ii) contacting the biological sample with at least one lipid-binding agent that binds specifically to a lipid selected from the group consisting of: sphingolipids, glycerolipids, and sterol lipids; and (iii) determining the level of at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid, based on the amount of specifically bound lipid-binding agents.

[0015] In some examples the at least one lipid-binding agent is an antibody.

[0016] In some examples of the disclosed methods: (i) a sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4- enine, N-(tetracosanoyl)-4E, 14Z-sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1 -p-lactosyl-sphing-4-enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N- (2-hydroxydocosanoyl)-eicosasphinganine; (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1 -hexadecyl-sn-glycero-3-phosphocholine, 1 -

[0017] (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)- sn-glycero-3-phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z- hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3- phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phosphocholine, 1 -octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1 -hexadecyl-2- (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2- octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero- 3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1-(11Z,14Z-eicosadienoyl)- glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1- octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1-(9Z- tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0018] For example, the sphingolipid is N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3- phosphocholine, 1 -hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,1 1Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3- phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z-hexadecenyl)-sn- glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1- octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,1 1Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3- phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z, 12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1 - eicosanoyl-sn-glycero-3-phosphocholine, 1 -(11 Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1 - (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3- phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '- myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2- octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0019] For example, the sphingolipid is N (tetracosanoyl)-sphing-4-enine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn- glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1- (5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1-(10Z-heptadecenoyl)-sn-glycero-3- phosphocholine, 1 -(1 Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z, 12Z,15Z- octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3- phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z- octadecadienoyl)-glycero-3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1- (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn- glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0020] For example, the sphingolipid is N-(tetracosanoyl)-4E, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3- phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-

[0021] (5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1-(10Z-heptadecenoyl)-sn-glycero-3- phosphocholine, 1 -(1 Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z, 12Z,15Z- octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3- phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z- octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z, 12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1 -eicosanoyl-sn-glycero-3-phosphocholine, 1 - (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1 -(4Z,7Z, 10Z, 13Z, 16Z-docosapentaenoy l)-sn- glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1 -hexadecanoyl-2-(5Z,8Z, 11 Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1 -(9Z-octadecenoyl)-2-(8Z,11 Z, 14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0022] For example, the sphingolipid is 14Z-sphingadienine-1 -phosphocholine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn- glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1- (5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1-(10Z-heptadecenoyl)-sn-glycero-3- phosphocholine, 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z- octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3- phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z- octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1-

[0023] (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1 -(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn- glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2-

[0024] (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1 -hexadecanoyl-2-(5Z,8Z, 11 Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1 -(9Z-octadecenoyl)-2-(8Z,11 Z, 14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0025] For example, the sphingolipid is N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)- glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3- phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z-hexadecenyl)-sn- glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1- octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,1 1Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3- phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1 - eicosanoyl-sn-glycero-3-phosphocholine, 1 -(11 Z, 14Z-eicosadienoyl)-glycero-3-phosphocholine, 1 - (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3- phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11 Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '- myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2- octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0026] For example, the sphingolipid is N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)- glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3- phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z-hexadecenyl)-sn- glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1- octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3- phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1 - eicosanoyl-sn-glycero-3-phosphocholine, 1 -(11 Z, 14Z-eicosadienoyl)-glycero-3-phosphocholine, 1 - (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3- phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine, 1 -hexadecanoyl-2-(5Z,8Z,11 Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '- myo- inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2- octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0027] For example, the sphingolipid is N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1 -(5Z,8Z, 11 Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1 -(1 OZ-heptadecenoyl)- sn-glycero-3-phosphocholine, 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di- (9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn- glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1 -hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1 -(1 Z-octadecenyl)-2- (9Z,12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1- (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn- glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,1 1Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0028] For example, the sphingolipid is N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1 -(5Z,8Z, 11 Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1 -(1 OZ-heptadecenoyl)- sn-glycero-3-phosphocholine, 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di- (9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn- glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1 -hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1 -(1 Z-octadecenyl)-2- (9Z,12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1- (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn- glycero-3-phosphocholine, 1 -octadecanoyl-sn-glycero-3-phosphoethanolamine, 1 -octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0029] For example, the sphingolipid is N-(2-hydroxydocosanoyl)-eicosasphinganine, and (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1 -(5Z,8Z, 11 Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1 -(1 OZ-heptadecenoyl)- sn-glycero-3-phosphocholine, 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di- (9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn- glycero-3-phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine, 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2- (9Z, 12Z-octadecadienoyl)-glycero-3-phosphoethanolamine, and 1 -(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1- (11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn- glycero-3-phosphocholine, 1-octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z- eicosatrienoyl)-sn-glycerol, 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn- glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0030] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0031] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-hexadecyl-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0032] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3- phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0033] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol. For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0034] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine; and (iii) the sterol lipid comprises free cholesterol.

[0035] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3- phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0036] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0037] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0038] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn- glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0039] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0040] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero-3- phosphoethanolamine; and (iii) the sterol lipid comprises free cholesterol.

[0041] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(9Z-octadecenoyl)-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine; and (iii) the sterol lipid comprises free cholesterol.

[0042] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-eicosanoyl-sn-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0043] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0044] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; and (iii) the sterol lipid comprises free cholesterol.

[0045] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; and (iii) the sterol lipid comprises free cholesterol.

[0046] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and (iii) the sterol lipid comprises free cholesterol.

[0047] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and (iii) the sterol lipid comprises free cholesterol.

[0048] For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol. For example, the sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)- sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, 14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4- enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N-(2-hydroxydocosanoyl)- eicosasphinganine; the glycerolipid is 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z- octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0049] In some examples, the at least one sphingolipid comprises N (17Z hexacosenoyl)-sphing-4- enine-1 -phosphocholine. In some examples, the at least one sphingolipid comprises N-(dodecanoyl)- 1-p-lactosyl-sphing-4-enine. In some examples, the at least one sphingolipid comprises N-(9Z- octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine. In some examples, the tat least one sphingolipid comprises N-(2-hydroxydocosanoyl)-eicosasphinganine.ln some examples, he at least one sphingolipid comprises N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine. In some examples, the at least one sphingolipid comprises N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide.

[0050] In some examples, at least one glycerolipid is selected from 1-(9Z-nonadecenoyl)-glycero- 3-phosphocholine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine, 1 -(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1 - octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, and 1-(9Z- tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol.

[0051] In some examples, the at least one sterol lipid is free cholesterol.

[0052] In some examples, at least three lipids comprise: a) N (17Z hexacosenoyl)-sphing-4-enine-1- phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4- (trimethylazaniumyl)butanoate; b) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)- glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; d) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine;Free cholesterol; and 1-octadecanoyl-sn-glycero-3- phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,1 1 Z, 14Z-eicosatrienoyl)-sn-glycerol; f) N-(dodecanoyl)-1 -p-lactosyl-sphing-4- enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; g)) N-(dodecanoyl)- 1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn-glycero-3-phosphocholine; h) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine; Free cholesterol; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; I) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; m) Free cholesterol; 1- hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo- inositol); and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing- 4-enine-1-phosphocholine; o) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; r) Free cholesterol; N-[(2S,3R)-1 ,3- dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3- phospho-(1 '-myo- inositol); or s) free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

[0053] In some examples, the method comprises measuring the level of up to 24 lipids.

[0054] In some examples, the levels of the at least three lipids to be measured consist of the levels of a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3- phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; b) free cholesterol; 1- eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; d) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and

[0055] 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; f) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine; g)) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl- sn-glycero-3-phosphocholine; h) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4- enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; Free cholesterol; N-[(2S,3R)- 1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-

[0056] 2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; I) free cholesterol; N-(2- hydroxydocosanoyl)-eicosasphinganine; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; m) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); and N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; o) free cholesterol; N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3- (9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2- (13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) free cholesterol; 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4- enine-1 -phosphocholine; r) free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or s) free cholesterol;

[0057] 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

[0058] In some examples, the method further comprises determining or having determined a higher likelihood of motor neuron disease in the subject by comparing the measured levels of each of: a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)-glycero-3- phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; b) free cholesterol; 1- eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; c) free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; d) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1 -octadecanoyl-sn-glycero-3-phosphoethanolamine; e) N-(dodecanoyl)-1 -p-lactosyl-sphing-4-enine; free cholesterol; and 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; f) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine; g)) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Fr free ee cholesterol; and 1- eicosanoyl-sn-glycero-3-phosphocholine; h) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4- enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; free cholesterol; N-[(2S,3R)- 1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1-octadecanoyl-

[0059] 2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; I) free cholesterol; N-(2- hydroxydocosanoyl)-eicosasphinganine; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; m) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); and N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; o) free cholesterol; N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3- (9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2- (13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) free cholesterol; 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4- enine-1 -phosphocholine; r) free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); or s) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine to established threshold levels for each of these lipids, where levels falling above the threshold indicate a higher likelihood of a motor neuron disease.

[0060] In some examples, the method further comprises determining or having determined a rate of progression of motor neuron disease in the subject by comparing the measured levels of each of: a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)-glycero-3- phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; b) free cholesterol; 1- eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; c) free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; d) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and

[0061] 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; f) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine; g)) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl- sn-glycero-3-phosphocholine; h) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4- enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; free cholesterol; N-[(2S,3R)- 1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; free cholesterol; and 1-octadecanoyl-

[0062] 2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; I) free cholesterol; N-(2- hydroxydocosanoyl)-eicosasphinganine; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; m) free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); and N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; o) free cholesterol; N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3- (9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2- (13Z,16Z-docosadienoyl)-glycero-3-phospho-(1'-myo-inositol); q) free cholesterol; 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4- enine-1 -phosphocholine; r) free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); or s) free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine to established threshold levels for each of these lipids. In one example, the measured levels from biological samples that were obtained from at least two different time points are compared to the threshold levels.

[0063] In some examples, the biological sample is a whole blood sample, a plasma sample, a serum sample, or a cerebrospinal fluid (CSF) sample. For example, the biological sample is a blood sample. For example the biological sample is a serum sample. For example, the biological sample is a cerebrospinal fluid (CSF) sample.

[0064] In some examples, the levels of the at least three lipids wherein are measured by one or more immunoassays. In one example, the one or more immunoassays comprise an ELISA. In one example, the ELISA is a competitive ELISA. In some examples, the ELISA is a multiplex ELISA.

[0065] In some examples, the levels of the at least three lipids are measured by high performance liquid chromatography (HPLC) or mass spectrometry. For example, the levels of the at least three lipids are measured by high performance liquid chromatography (HPLC). For example, the levels of the at least three lipids are measured by mass spectrometry. In one example, the levels of the at least three lipids are measured by HPLC and mass spectrometry.

[0066] In some examples, a higher abundance of one or more lipids is indicative of a higher risk of MND. For example, a higher risk compared to a reference sample. For example, a higher abundance of lipids correlates to a higher level of the lipid. For example, higher than a reference sample.

[0067] In some examples, the motor neuron disease is a familial form of motor neuron disease. In some examples, the motor neuron disease is a sporadic form of motor neuron disease. In some examples, the motor neuron disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy, and spinal and bulbar muscular atrophy (SBMA). For example, the motor neuron disease is amyotrophic lateral sclerosis (ALS). For example, the motor neuron disease is primary lateral sclerosis. For example, the motor neuron disease is progressive muscular atrophy (PMA). For example, the motor neuron disease is progressive bulbar palsy. For example, the motor neuron disease is spinal and bulbar muscular atrophy (SBMA).

[0068] In another aspect, the present disclosure provides a method for treating a subject identified as suffering from a motor neuron disease, the method comprising administering or having administered to the subject a therapeutically effective amount of an agent selected from the list consisting of: agents that inhibit protein misfolding, protein aggregation, and / or reduce endoplasmic reticulum stress; agents that reduce oxidative stress agents that reduce inflammation; agents that modulate lipid metabolism, agents that block excitotoxicity; wherein the subject was identified as suffering from a motor neuron disease based on the levels of at least three lipids in a biological sample from the subject, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.

[0069] In one example, the at least three lipids comprise: a) N (17Z hexacosenoyl)-sphing-4-enine-1- phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4- (trimethylazaniumyl)butanoate; b) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)- glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; d) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine;Free cholesterol; and 1-octadecanoyl-sn-glycero-3- phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,1 1 Z, 14Z-eicosatrienoyl)-sn-glycerol; f) N-(dodecanoyl)-1 -p-lactosyl-sphing-4- enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; g)) N-(dodecanoyl)- 1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn-glycero-3-phosphocholine; h) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine; Free cholesterol; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; I) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; m) Free cholesterol; 1- hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo- inositol); and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing- 4-enine-1-phosphocholine; o) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; r) Free cholesterol; N-[(2S,3R)-1 ,3- dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3- phospho-(1 '-myo-inositol); or s) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

[0070] In a further aspect, the present disclosure provides a method for treating a subject identified as suffering from a motor neuron disease, the method comprising determining, in a biological sample previously obtained from the subject, the level of: a) N (17Z hexacosenoyl)-sphing-4-enine-1- phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4- (trimethylazaniumyl)butanoate; b) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)- glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; d) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine;Free cholesterol; and 1-octadecanoyl-sn-glycero-3- phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,1 1Z,14Z-eicosatrienoyl)-sn-glycerol; f) N-(dodecanoyl)-1 -p-lactosyl-sphing-4- enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; g)) N-(dodecanoyl)- 1 -p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn-glycero-3-phosphocholine; h) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; i) N-(dodecanoyl)-1 -p-lactosyl- sphing-4-enine; Free cholesterol; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; I) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; m) Free cholesterol; 1- hexadecanoyl-2-(5Z,8Z,11 Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo- inositol); and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing- 4-enine-1-phosphocholine; o) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; r) Free cholesterol; N-[(2S,3R)-1 ,3- dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3- phospho-(1 '-myo- inositol); or s) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

[0071] In another aspect, the present disclosure provides a kit comprising at least one, two, or three distinctly labelled binding agents, wherein at least one binding agent binds to a glycerolipid, and wherein at least one binding agent binds to at least one of either a sphingolipid or a sterol lipid, together with instructions for using the kit according to a method described herein.

[0072] In one example, the at least three distinctly labelled binding agents bind to a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; b) Free cholesterol; 1-eicosanoyl-sn- glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3- phosphoethanolamine; d) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine;Free cholesterol; and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; e) N-(dodecanoyl)-1 -p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; f) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine; g)) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl- sn-glycero-3-phosphocholine; h) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4- enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; Free cholesterol; N-[(2S,3R)- 1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- 2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; I) Free cholesterol; N-(2- hydroxydocosanoyl)-eicosasphinganine; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; m) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); and N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; o) Free cholesterol; N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3- (9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2- (13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) Free cholesterol; 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4- enine-1 -phosphocholine; r) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or s) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.,

[0073] In one example, the kit further comprises one or more lipid control standards for each of the at least three lipids. In one example, the one or more lipid control standards are provided in separate containers.

[0074] In some examples, the labelled binding agents each comprise at least one immunoglobulin variable domain that binds specifically to one of the at least three lipids. In one example, the labelled binding agents are selected from the group consisting of: mAbs; a scFvs or Fab’s; and sdAbs. In one example, the kit comprises one or more agents for an ELISA. In one example, the ELISA is a competitive ELISA.

[0075] In another aspect, the present disclosure provides a method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the level of at least one lipid selected from the group consisting of: a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3- phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; b) Free cholesterol; 1- eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; c) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; d) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine;Free cholesterol; and 1-octadecanoyl-sn-glycero-3-phosphoethanolamine; e) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; f) N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z-eicosadienoyl)-glycero-3- phosphocholine; g)) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl- sn-glycero-3-phosphocholine; h) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4- enine-1 -phosphocholine; i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; Free cholesterol; N-[(2S,3R)- 1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; k) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- 2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; I) Free cholesterol; N-(2- hydroxydocosanoyl)-eicosasphinganine; and 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3- phosphocholine; m) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; n) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'-myo-inositol); and N- (9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; o) Free cholesterol; N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3- (9Z,12Z-octadecadienoyl)-sn-glycerol; p) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2- (13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); q) Free cholesterol; 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4- enine-1 -phosphocholine; r) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and (iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or s) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; and N-(9Z- octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

[0076] In some examples the methods comprises measuring or having measured the level of at least two lipids selected from any one of a) to s). In some examples the method comprises measuring the level of 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate. In some examples the method comprises measuring the level of free cholesterol. In some examples the method comprises measuring the level of N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine. In some examples the method comprises measuring the level of a glycerolipid. In some examples, the glycerolipid is a glycerophospholipid.

[0077] FIGURES

[0078] Figure 1 : Random forest plots of 4 potential lipid sets. (A) Lipid set 1 . (B) Lipid set 2. (C) Lipid set 3. (D) Lipid set 4, highly MND specific.

[0079] Figure 2: Lipid peak areas for the human dataset. The figure shows high performance liquid chromatography-Mass spectrometry peak areas for lipid classes of lipid set 4 when tested on the human dataset. Ctr = control, ALS = MND and PD = Parkinsons disease.

[0080] Figure 3: Forest plot of top 18 lipid biomarker sets. The figure shows shows top 18 sets of 3 lipid biomarker sets based on analysis of mouse models of PD and MND. Thresholds were set to AUC >0.7, <0.95; p<0.05. OddsRatio 1. Only positive relationships were included ( / .e., higher abundance correlates with a greater likelihood of MND).

[0081] DETAILED DESCRIPTION

[0082] General techniques and definitions

[0083] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, lipidomics, proteomics, neuroscience, molecular genetics, immunology, immunohistochemistry, lipid chemistry, protein chemistry and biochemistry).

[0084] Unless otherwise indicated, the naming convention for lipids used herein follows the guidelines established by the Lipid Maps Consortium and the shorthand notation of Liebisch et al. (see for example Fahy et al. 2005. J. Lipid Res. 46: 839-861 ; Fahy et al 2009. J. Lipid Res. 50 (Suppl.): S9-S14; Liebisch et al. 2013. J. Lipid Res. 54: 1523-1530; Liebisch et al. 2020. J Lipid Res. 2020 Dec; 61 (12): 1539- 1555). Lipids can be divided into primary categories including fatty acyls (FA), glycerolipids (GL), glycerophospholipids (GP), sphingolipids (SP), sterol lipids (ST), prenol lipids (PR), saccharolipids (SL), and polyketides (PK), which can be divided into molecule classes, subclasses and species representing chemical structures. It will be understood that glycerolipids comprise glycerophospholipids, for exmasple, glycerophospholipids are a subset of a broader category of glycerolipids.

[0085] Fatty acyls (FA) are a diverse group of molecules synthesised by chain elongation of an acetyl- CoA primer with malonyl-CoA (or methylmalonyl-CoA) groups that may contain a cyclic functionality and / or are substituted with heteroatoms. Structures with a glycerol group are represented by two distinct categories: the glycerolipids (GL), which include acylglycerols but also encompass alkyl and 1Z-alkenyl variants, and the glycerophospholipids (GP), which are defined by the presence of a phosphate (or phosphonate) group esterified to one of the glycerol hydroxyl groups. Sterol lipids (ST) and prenol lipids (PR) share a common biosynthetic pathway via the polymerization of dimethylallyl pyrophosphate / isopentenyl pyrophosphate but otherwise differ in structure and function. Sphingolipids (SP) contain a long-chain base as their core structure. Saccharolipids” (SL) contain fatty acyl groups linked directly to a sugar backbone.

[0086] Glycerolipids (GL) are a structurally heterogeneous group of lipids that all have at least one hydrophobic chain linked to a glycerol backbone in an ester or ether linkage. Glycerolipids comprise neutral (uncharged) glycerolipids (mono-, di-, and triacylglycerols), neutral glyceroglycolipids (mono- and digalactosylglycerides; sulfoglucolipids), and polar glycerolipids (phospholipids and betaine glycerolipids).

[0087] Glycerophospholipids (GP) typically contain two fatty acid chains and in the absence of detailed characterisation are expressed as the sum composition of carbon atoms and double bonds ( / .e. PC(38:6)). However, where an acyl chain composition has been determined the naming convention indicates this ( / .e. PC(38:6) is changed to PC(16:0_22:6)). This is also extended into other lipid classes or subclasses. Sterol lipids include all lipids based on the cyclopentanoperhydrophenanthrene skeleton. In general, sterols are characterised by a complex ring structure derived from squalene, consisting in a tetracyclic cyclopenta[a]-phenanthrene structure with a hydroxyl group at C-3 and a flexible side chain with 8-10 carbons at C-17

[0088] Unless otherwise indicated, the present disclosure refers to lipid molecules using the numbering system X:Y. The number X represents the number of carbon atoms present in the chain.

[0089] It will also be recognized that the compounds described herein may possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. The disclosure thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres e.g., greater than 90% ee, such as 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be naturally occurring or may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.

[0090] The present disclosure relates to derivatives of glycerol. Whilst glycerol is achiral, derivatives are typically chiral. Typically the glycerol utilised will have a stereochemical configuration corresponding to that found in nature. In some embodiments, the glycerol derivatives utilised have the following stereochemical configuration:

[0091] Unless otherwise indicated, any recombinant protein, cell culture, and immunological techniques utilised in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rdedn, Cold Spring Harbour Laboratory Press (2001), R. Scopes, Protein Purification - Principals and Practice, 3rdedn, Springer (1994), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0092] The term “measurement” as used herein refers to assessing the presence, absence, quantity or amount of a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances. The term “measure” and its conjugations means quantifying the amount of biomarker(s) detected in, for example, a sample, and / or qualifying the type of biomarker detected by some quantifiable means. Measuring can be accomplished by methods known in the art and those further described herein, including but not limited to HPLC, mass spectrometry approaches, magnetic resonance spectroscopy and immunoassay approaches (e.g., ELISA), or combinations thereof, or any suitable methods can be used to detect and measure one or more of the markers described herein.

[0093] The term “detect” refers to identifying the presence, absence, or amount of the biomarker to be detected. Non-limiting examples include lipids, proteins, peptides, or nucleic acids.

[0094] The term “report” refers to a printed result provided from the methods of the present invention to the physician. The report can indicate the presence of, nature of, or risk forthe pathological condition. The report can also indicate what treatment is most appropriate e.g. no action, surgery, further tests, or administering a therapeutic agent.

[0095] The term “control” as used herein refers to a known steady state molecule or a non-diseased, healthy condition that is used as a relative marker in which to study fluctuations or compare the nonsteady state molecules or normal non-diseased healthy condition, or it can also be used to calibrate or normalise values. In some examples, a control reference value is a calculated value such as a combination of biomarker concentrations or a combination of ranges of concentrations.

[0096] The term “immunoassay” is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterised by the use of specific binding properties of a particular antibody to isolate, target, and / or quantify the antigen.

[0097] The term “antibody” refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope. Antibodies exist, e.g., as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab" and F(ab)"2 fragments. As used herein, the term “antibody” also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. “Fc” portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, but does not include the heavy chain variable region.

[0098] As used herein, the term “subject” refers to any animal that may develop MND and includes animals such as mammals, e.g. humans, or non-human mammals such as cats and dogs, laboratory animals such as mice, rats, rabbits or guinea pigs, and livestock animals. In a preferred embodiment, the subject is a human.

[0099] The term “sample” or “biological sample” as used herein refers to a sample of biological fluid, tissue, or cells in a healthy and / or pathological state obtained from a subject. Preferably, the term “sample” is a blood sample, a plasma sample, a serum sample, or a CSF sample. A reference sample refers to any reference sample, and includes a sample taken from the same subject at a different time point, as well as a reference sample taken from a different subject or a population of different subjects, or a sample represented by a reference level, for example, a reference level obtained from a reference population sample.

[0100] The terms “treating” or “treatment” as used herein, refer to both direct treatment of a subject by a medical professional (e.g., by administering a therapeutic agent to the subject), or indirect treatment, effected, by at least one party, (e.g., a medical doctor, a nurse, a pharmacist, or a pharmaceutical sales representative) by providing instructions, in any form, that (i) instruct a subject to self-treat according to a claimed method (e.g., self-administer a drug) or (ii) instruct a third party to treat a subject according to a claimed method. Also encompassed within the meaning of the term “treating” or “treatment” are prevention or reduction of the disease to be treated, e.g., by administering a therapeutic at a sufficiently early phase of disease to prevent or slow its progression.

[0101] The term “at risk” as used herein, refers to a probability of developing a health condition that is higher than in the general population. Accordingly, treatment of an individual considered to be “at risk” of a particular condition is designed to prevent a subject from the developing the condition or at least to reduce the risk of developing the condition to a level no higher than that found in general population as a whole.

[0102] The terms "effective amount" or "therapeutically effective amount” as used herein, refer to a sufficient amount of a MND treatment being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. It is known to those of skill in the art that therapeutically-effective dosages can vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

[0103] Motor neuron disease

[0104] Motor neuron disease (MND) is the name for a group of rare progressive neurological disorders that result in degeneration of motor neurons over time and leads to disability or death. This causes skeletal muscles to become increasingly weaker, impacting activity such as walking, breathing, speaking and swallowing, and eventually leads to paralysis. The group of diseases includes amyotrophic lateral sclerosis (ALS), progressive bulbar palsy, primary lateral sclerosis, progressive muscular atrophy, spinal muscular atrophy, bulbar muscular atrophy, Kennedy's disease (spinal and bulbar muscular atrophy), and post-polio syndrome. Although ALS is referred to as classical motor neuron disease and ALS and MND are sometimes used interchangeably, the term “motor neuron disease” or “MND” as used herein means any motor neuron disease generally and is not limited or confined to amyotrophic lateral sclerosis (ALS). For the avoidance of doubt, as used herein, amyotrophic lateral sclerosis (ALS), also known in the field as motor neuron disease or Lou Gehrig's disease (LGD), for the purposes of this specification is only one type of MND, for example, a subset of MND.

[0105] MND may affect the upper and / or lower motor neurons. When the muscles cannot receive signals from the lower motor neurons, they begin to weaken and shrink in size (muscle atrophy or wasting). The muscles may also start to spontaneously twitch. These twitches (fasciculations) can be seen and felt below the surface of the skin. When the lower motor neurons cannot receive signals from the upper motor neurons, it can cause muscle stiffness (spasticity) and overactive reflexes. This can make voluntary movements slow and difficult, and can result, with time, in loss of the ability to walk or control other movements.

[0106] The different motor neuron diseases caught by the general term MND are classified according to whether the loss of function (degeneration) is inherited or sporadic; and whether the upper, lower, or both motor neurons are affected. Inherited (also known as familial) MND is usually caused by mutations in a single gene, which can be autosomal dominant, autosomal recessive or X-linked.

[0107] General overview

[0108] The present disclosure provides methods for the analysis of an easily accessible biological sample, for example, blood, from a subject. The analysis includes a lipid biomarker assay which can be with an algorithm executable by a computer for analysis, or which may be analysed by any other suitable means. Generally, the methods use proteins present in the biological sample of the subject to identify biomarkers or a biomarker profile and thus identify subjects who have MND or are at a higher risk for MND, and also to monitor the progression of MND in a subject diagnosed with MND.

[0109] The present disclosure also provides a kit that in general will include compositions used for the detection of biomarkers provided herein.

[0110] Biomarkers

[0111] The present disclosure utilises a set or panel of biomarkers measured in a biological sample obtained from a subject to identify subjects that have, or are suspected of having, MND. For example, by comparing measured levels of biomarkers to pre-determined or established threshold levels. The panel of biomarkers is also utilised to determine the rate of progression of MND in a subject. For example by comparing measured levels of biomarkers taken on two or more separate occasions, or by comparing measured levels of biomarkers to pre-determined or established threshold levels.

[0112] As used herein, the term ‘biomarker” refers to any biological compound or aspect that can be measured as an indicator of the physiological status of a biological system. In some examples, the biomarker is a lipid biomarker.

[0113] The present studies have demonstrated a particular role for a lipid panel comprising a combination of at least two different lipids taken from the group consisting of lipid classes sphingolipids, glycerolipids (including glycerophospholipids), sterol lipids, and fatty acyls. Preferably, the combination consists of at least three different lipids, wherein at least one lipid is a glycerolipid, and at least one lipid is at least one of either a sterol lipid or a sphingolipid. In some examples, the combination consists of at least three different lipids, selected from the lipid sets described in Table 7. In some examples, the combination consists of at least three different lipids, at least one selected from each of a sphingolipid, a glycerophospholipid, and a fatty acyl. When the lipid panel comprises at least AcylCarnitine 16:0 (3- (hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate), LPC 19:1 (1-(9Z-nonadecenoyl)-glycero-3- phosphocholine) and SM 44:2 (N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine), 97-100% of subjects will be correctly diagnosed with MND specifically (average confidence 0.87-0.95). Other exemplified lipid combinations are shown in Table 7.

[0114] In some examples provides a method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one lipid from each of sphingolipids, glycerophospholipids, and fatty acyls.

[0115] In some examples, the present disclosure provides a method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least one lipid selected from the group consisting of sphingolipids, glycerophospholipids, and fatty acyls.

[0116] In some examples, the present disclosure provides a method for determining a rate of progression of a motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.

[0117] In some examples, the present disclosure provides a method for determining a rate of progression of motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one lipid from each of sphingolipids, glycerophospholipids, and fatty acyls.

[0118] In some examples, the present disclosure provides a method detecting the presence and / or level of at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: (i) providing a biological sample obtained previously from the subject; (ii) contacting the biological sample with at least one lipid-binding agent that binds specifically to a lipid selected from the group consisting of: sphingolipids, glycerolipids, and sterol lipids; and (iii) determining the level of at least glycerolipid, and at least one of either a sphingolipid or a sterol lipid, based on the amount of specifically bound lipid-binding agents.

[0119] In some examples, the present disclosure provides a method detecting the presence and / or level of at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: (i) providing a biological sample obtained previously from the subject; (ii) contacting the biological sample with at least one lipid-binding agent that binds specifically to a lipid selected from the group consisting of: sphingolipids, glycerophospholipids, and fatty acyls; and (iii) determining the level of at least one lipid from each of sphingolipids, glycerophospholipids, and fatty acyls based on the amount of specifically bound lipid-binding agents.

[0120] In some examples of the disclosed methods: (i) a sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4- enine, N-(tetracosanoyl)-4E, and14Z-sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine, N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide, N- (2-hydroxydocosanoyl)-eicosasphinganine; (ii) a glycerolipid is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1- (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)- sn-glycero-3-phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z- hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3- phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phosphocholine, 1 -octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1 -hexadecyl-2- (5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2- octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z-octadecadienoyl)-glycero- 3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3- phosphoethanolamine, 1-eicosanoyl-sn-glycero-3-phosphocholine, 1-(11Z,14Z-eicosadienoyl)- glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1- octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1-(9Z- tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and (iii) the sterol lipid comprises free cholesterol.

[0121] In some examples of the disclosed methods: (i) a sphingolipid is selected from the group consisting of: N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, N (tetracosanoyl)-sphing-4- enine, N-(tetracosanoyl)-4E, and14Z-sphingadienine-1 -phosphocholine; (ii) a glycerophospholipids is selected from the group consisting of: 1 (9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-hexadecyl- sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3-phosphocholine, 1-(10Z-heptadecenoyl)-sn-glycero-3- phosphocholine, 1 -(1 Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z, 12Z,15Z- octadecatrienoyl)-sn-glycero-3-phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-glycero-3-phosphocholine, 1-octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3- phosphocholine, 1-hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1 -hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1 -(1 Z-octadecenyl)-2-(9Z,12Z- octadecadienoyl)-glycero-3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine; and (iii) the fatty acyl comprises 3 (hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate. In some examples, at least one sphingolipid comprises N (17Z hexacosenoyl)-sphing-4-enine- 1 -phosphocholine. In some examples, at least one sphingolipid comprises N-(dodecanoyl)-1-p-lactosyl- sphing-4-enine. In some examples, at least one sphingolipid comprises N-(9Z-octadecenoyl)- hexadecasphing-4-enine-1 -phosphocholine. In some examples, at least one sphingolipid comprises N- (2-hydroxydocosanoyl)-eicosasphinganine. In some examples, at least one sphingolipid comprises N- (9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine. In some examples, at least one sphingolipid comprises N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide.

[0122] In some examples, at least one glycerophospholipid comprises 1-(9Z-nonadecenoyl)-glycero-3 phosphocholine. In some examples, at least one fatty acyl comprises 3-(hexadecanoyloxy)-4- (trimethylazaniumyl)butanoate.

[0123] In some examples, at least one glycerolipid is selected from 1-(9Z-nonadecenoyl)-glycero-

[0124] 3-phosphocholine, 1 -eicosanoyl-sn-glycero-3-phosphocholine, 1 -(11 Z, 14Z-eicosadienoyl)-glycero-3- phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1- octadecanoyl-sn-glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)- glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, and 1-(9Z- tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol.

[0125] In some examples, the at least one sterol lipid is free cholesterol.

[0126] In some examples, at least three lipids comprise at least one of N (17Z hexacosenoyl)-sphing-

[0127] 4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)- 4-(trimethylazaniumyl)butanoate.

[0128] In some examples, at least three lipids comprise: a) N (17Z hexacosenoyl)-sphing-4-enine-1- phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4- (trimethylazaniumyl)butanoate.

[0129] In some examples, at least three lipids comprise those described in Table 7.

[0130] In some examples, the levels of the at least three lipids to be measured consist of the levels of a) N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1-(9Z-nonadecenoyl)-glycero-3- phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate.

[0131] In some examples, the levels of the at least three lipids to be measured consist of the levels of the sets of lipids described in Table 7.

[0132] In some examples, the biomarker panel may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14,15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or more biomarkers selected from the group consisting of lipids described in Table 1 . In some examples, the biomarker panel contains no more than 24 lipids. In some examples, the no more than 24 lipids are selected from the lipids described in Table 1 .

[0133] Reference to any of these lipids includes reference to all relevant variants and functional equivalents as would be known or understood by the person skilled in the art.

[0134] It will be understood by the skilled person that demographic or morphometric terms may also be factored into the analysis, for example, logistic regression algorithm. Demographic or morphometric terms, include but are not limited to, age, biological sex, body mass index (BMI), levels of exercise, exercise history, exposure to environmental factors such as agricultural or industrial chemicals or heavy metals, and physical factors such as exposure to mechanical and / or electrical trauma. In some examples, a biomarker panel will include demographic or morphometric terms and biomarkers, or biomarkers associated with demographic or morphometric factors. These types of markers can be assigned arbitrary values to allow statistical analysis, for example logistic regression and other algorithmic analyses known in the art.

[0135] In some examples, a subject is identified for analysis based on one or more symptoms related to motor neuron disease, particularly early motor neuron disease, or based on known genetic associations or mutations. Early symptoms of motor neuron disease include, but are not limited to, weakness in hands or grip, slurred speech, weakness legs, a tendency to trip, difficulties in lifting, cramps, and muscle twitching. Once a subject is identified as a potential candidate for diagnosis, a biological sample is obtained for use in the methods disclosed herein. In some preferred examples, the biological sample is whole blood, plasma, serum, or CSF. Before analysing the biological sample, it may be desirable to perform one or more sample preparation operations upon the sample. Generally, these sample preparation operations may include such manipulations as extraction, from whole blood, plasma serum, or a fraction thereof, such as, the extraction of lipids, or other macromolecules from the samples.

[0136] Sample preparation which can be used with the methods of disclosure include but are not limited to, centrifugation, affinity chromatography, magnetic separation, fractionation, precipitation, and combinations thereof. Sample preparation can further include dilution by an appropriate solvent and amount to ensure the appropriate range of concentration level is detected by a given assay.

[0137] Samples may be prepared according to standard biological sample preparation depending on the desired detection method. For example, for mass spectrometry detection, biological samples obtained from a patient may be centrifuged, filtered, processed by immunoaffinity column, separated into fractions, partially digested, and combinations thereof. Various fractions may be resuspended in appropriate carrier such as buffer or other type of loading solution for detection and analysis, including LCMS loading buffer.

[0138] The skilled person will be aware of the relevant sample preparation techniques and it is not the intention of the inventors to limit the present disclosure to any particular means of sample preparation.

[0139] Biomarker measurement

[0140] Measurement of a biomarker panel relates to a quantitative measurement of a plurality of biomarkers. The present disclosure provides for methods for detecting biomarkers in biological samples. Biomarkers include but are not necessarily limited to lipids. More specifically the present disclosure is based on the discovery of lipid biomarkers that are differentially expressed in subjects that have an increased risk of acquiring MND or have MND. Therefore, the measurement of one or more of these differentially expressed biomarkers in a biological sample provides useful information as to the relative likelihood that a subject is at risk of, or suffering from MND. In some examples, and the rate of progression of their MND once diagnosed or from one time point to another. Any suitable method known to the skilled person can be used to detect one or more of the biomarkers described herein.

[0141] Useful analyte capture agents that can be used with the present disclosure include but are not limited to antibodies, such as crude serum containing antibodies, purified antibodies, monoclonal antibodies (mAbs), polyclonal antibodies, single-domain antibodies (sdAbs), synthetic antibodies, antibody fragments (for example, fragment variables (Fvs), single chain fragment variables (scFv) or fragment antigen binding regions (Fab fragments)); antibody interacting agents, such as protein A, carbohydrate binding proteins, and other interactants; protein interactants (for example avidin and its derivatives); peptides; and small chemical entities, such as enzyme substrates, cofactors, metal ions / chelates, and haptens. Antibodies may be modified or chemically treated to optimise binding to targets or solid surfaces (e.g. biochips and columns).

[0142] As used herein, the term “lipid-binding agent” means any agent that binds to a specific lipid of interest. In one example, the lipid-binding agent is a protein. In one example, the lipid-binding agent is an antibody. Binding between the lipid-binding agent and the lipid may be facilitated through means known in the art, including but not limited to unspecific hydrophobic association, electrostatic interaction, binding pockets and lipid anchors.

[0143] As used herein, the term “binds” or “binding” in reference to the interaction of a lipid-binding agent means that the interaction is dependent upon the presence of a particular structure. For example, an antibody recognises and binds to a specific lipid structure rather than to lipids generally. If an antibody binds to epitope "A", the presence of a molecule containing epitope “A” (or free, unlabelled “A”), in a reaction containing labelled “A” and the protein, will reduce the amount of labelled “A” bound to the antibody. Binding may be reversible or irreversible.

[0144] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that a binding agent of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a binding agent with an immunoglobulin domain that binds N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine specifically, will bind with greater affinity to N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine than to any other lipid. Generally, but not necessarily, reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term. In some examples, a binding agent comprises at least one immunoglobulin variable domain that binds specifically to at least one lipid. In some examples, several binding agents each comprise at least one immunoglobulin variable domain that binds specifically to one of the at least one of several different lipids, for example to one of at least three different lipids. In some examples, at least three distinctly labelled binding agents each specifically bind to a different type of lipid selected from the group consisting of: sphingolipids, glycerophospholipids, and fatty acyls. In some examples, the sphingolipids comprise N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine; the glycerophospholipids comprise 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine; and the fatty acyls comprise 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate. In some examples, at least three distinctly labelled binding agents each specifically bind to a different type of lipid selected from at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid. In some examples, the lipids comprise the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid as shown in Table 7.

[0145] In some examples, the lipid-binding agent is labelled. The type of label used will depend on the specific lipid-binding agent, and suitable labels are well-known in the art. As used herein, “labelled” means having a detectable molecular or atomic tag or marker that generates or can be induced to generate an optical or other signal or product that can be detected visually or by using a suitable detector. Detectable labels are well-known in the art and include, for example, a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, a prosthetic group, a contrast agent and an ultrasound agent.

[0146] Fluorescent labels commonly used include Alexa, cyanine such as Cy5 and Cy5.5, and indocyanine, and fluorescein isothiocyanate (FITC), but they are not so limited. Fluorescent labels useful in the practice of the present disclosure can include, also without limitation, 1 ,5 IAEDANS; 1 ,8- ANS; 4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5- Carboxynapthofluorescein (pH 10); 5-Carboxytetramethylrhodamine (5-TAMRA); 5-FAM (5- Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-Hydroxy Tryptamine (HAT); 5-ROX (carboxy-X- rhodamine); 5-TAMRA (5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6C; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-methylcoumarin; 9-Amino-6- chloro-2-methoxyacridine; ABQ; Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); Acridine Orange+DNA; Acridine Orange+RNA; Acridine Orange, both DNA & RNA; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); Alexa Fluor 350; Alexa Fluor 430; Alexa Fluor 488; Alexa Fluor 532; Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 633; Alexa Fluor 647; Alexa Fluor 660; Alexa Fluor 680; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin; Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC (Allophycocyanin); APC-Cy7; APTRA-BTC=Ratio Dye, Zn2+; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAG CBQCA; ATTOTAG FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisamninophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET Bimane; Bisbenzamnide; Bisbenzimide (Hoechst); bis-BTC=Ratio Dye, Zn2+; Blancophor FFG; Blancophor SV; BOBO-1 ; BOBO-3; Bodipy 492 / 515; Bodipy 493 / 503; Bodipy 500 / 510; Bodipy 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591 ; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO-1 ; BO-PRO-3; Brilliant Sulphoflavin FF; BTC-Ratio Dye Ca2+; BTC-5N-atio Dye, Zn2+; Calcein; Calcein Blue; Calcium Crimson; Calcium Green; Calcium Green-1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green- Ci 8 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue; Cascade Yellow 399; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP--Cyan Fluorescent Protein; CFP / YFP; FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF (Ratio Dye, pH); CMFDA; Coelenterazine; Coelenterazine cp (Ca2+ Dye); Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM Methylcoumarin; CTC; CTC Formazan; Cy2; Cy3.1 8; Cy3.5; Cy3; Cy5.1 8; Cy5.5; Cy5; Cy7; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); CyQuant Cell Proliferation Assay; Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3; DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4- Di- 16- ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; Red fluorescent protein; DTAF; DY-630-NHS; DY-635- NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; FITC Antibody; Flazo Orange; Fluo- 3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43; FM 4-46; Fura Red (high pH); Fura Red / Fluo-3; Fura-2, high calcium; Fura-2, low calcium; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); GFP (S65T); GFP red shifted (rsGFP), GFP wild type, non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular Blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; lndo-1 , high calcium; lndo-1 , low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1 ; JO-JO-1 ; JO-PRO-1 ; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; LIVE / DEAD Kit Animal Cells, Calcein / Ethidium homodimer; LOLO-1 ; LO-PRO-1 ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue, LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1 ; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X; Oregon Green; Oregon Green 488; Oregon Green 500; Oregon Greene 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1 ; POPO-3; PO-PRO-1 ; PO-PRO-3; Primuline; Procion Yellow; Propidium Iodide (PI); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613 [PE- TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T ; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP; sgBFP (super glow BFP); sgGFP; sgGFP (super glow GFP); SITS; SITS (Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1 ; SNAFL-2; SNARF calcein; SNARF1 ; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine B can C; Sulphorhodamine G Extra; SYTO 11 ; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYT; SYTO 17; SYTO 18; SYTO 20; SYTO 21 ; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41 ; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61 ; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81 ; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red; Texas Red- X conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1 ; TO- PRO-3; TO-PRO-5; TOTO-1 ; TOTO-3; TriColor (PE-Cy5); TRITC (TetramethylRodamine- IsoThioCyanate); True Blue; TruRed; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW781 ; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1 ; YO-PRO-3; YOYO-1 ; and YOYO-3.

[0147] In one example, a detectable label is an enzyme. Examples of enzymes useful in the disclosure include, without limitation, alkaline phosphatase and horseradish peroxidase. Alternatively or in addition, the enzyme can be, for example, luciferase. The enzyme can be linked to the antibody by conventional chemical methods, or it can be expressed together with the antibody as a fusion protein. In one example, the enzyme is horseradish peroxidase.

[0148] Radioisotopes useful as detectable labels in the disclosure are well known in the art and can include 3H, 11C, 18F, 35S, 64Cu, 67Ga, 68Ga, 99mTc, 1111n, 1231, 1241, 1251, and 1311.

[0149] In one particular example of the disclosure, the biomarker can be detected in a biological sample using an immunoassay. Immunoassays are assay that use an antibody that specifically bind to or recognises an antigen (e.g. site on a protein or peptide, biomarker target). The method includes the steps of contacting the biological sample with the antibody and allowing the antibody to form a complex of with the antigen in the sample, washing the sample and detecting the antibody-antigen complex with a detection reagent. In one example, antibodies that recognise the biomarkers may be commercially available. In another examples, an antibody that recognises the biomarkers may be generated by known methods of antibody production.

[0150] Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labelled antibody is used to detect bound marker-specific antibody. Exemplary detectable labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes, radiolabels, enzymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used), and colorimetric labels such as colloidal gold or coloured glass or plastic beads. The marker in the sample can be detected using and / or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker is incubated simultaneously with the mixture.

[0151] The conditions to detect an antigen using an immunoassay will be dependent on the particular antibody used. Also, the incubation time will depend upon the assay format, marker, volume of solution, concentrations and the like. In general, the immunoassays will be carried out at room temperature, although they can be conducted over a range of temperatures, such as 10 degrees to 40 degrees Celsius depending on the antibody used.

[0152] There are various types of immunoassay known in the art that, as a starting basis, can be used to tailor the assay for the detection of the biomarkers of the present disclosure. Useful assays can include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), including the sandwich ELISA, competitive ELISA (inhibition assay), and multiplex ELISA. There are many variants of these approaches, but those are based on a similar idea. For example, if an antigen can be bound to a solid support or surface, it can be detected by reacting it with a specific antibody and the antibody can be quantitated by reacting it with either a secondary antibody or by incorporating a label directly into the primary antibody. Alternatively, an antibody can be bound to a solid surface and the antigen added. A second antibody that recognises a distinct epitope on the antigen can then be added and detected. This is frequently called a ‘sandwich assay’ and can frequently be used to avoid problems of high background or non-specific reactions. These types of assays are sensitive and reproducible enough to measure low concentrations of antigens in a biological sample.

[0153] A Competitive ELISA, also known as an inhibition assay, is an ELISA that can measure the concentration of an analyte through its interference in the ELISA assay signal.

[0154] A multiplex assay is a type of immunoassay that uses magnetic beads to simultaneously measure multiple analytes in a single experiment. A multiplex assay is a derivative of an ELISA using beads for binding the capture antibody.

[0155] Immunoassays can be used to determine presence or absence of a marker in a sample as well as the quantity of a marker in a sample. Methods for measuring the amount of, or presence of, antibodymarker complex include but are not limited to, fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). In general these regents are used with optical detection methods, such as various forms of microscopy, imaging methods and non-imaging methods. Electrochemical methods include voltammetry, amperometry and electrochemiluminescence methods. Radio frequency methods include multipolar resonance spectroscopy.

[0156] In one example, the disclosure can use antibodies for the detection of the biomarkers. Antibodies can be made that specifically bind to the biomarkers of the present assay can be prepared using standard methods known in the art. For example, polyclonal antibodies can be produced by injecting an antigen into a mammal, such as a mouse, rat, rabbit, goat, sheep, alpaca, llama, camel, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodies — multiple antibodies that bind to the same antigen. Alternatively polyclonal antibodies can be produced by injecting the antigen into chickens for generation of polyclonal antibodies in egg yolk. In addition, antibodies can be made that specifically recognise modified forms for the biomarkers such as a phosphorylated form of the biomarker, that is to say, they will recognise a tyrosine or a serine after phosphorylation, but not in the absence of phosphate. In this way antibodies can be used to determine the phosphorylation state of a particular biomarker.

[0157] Antibodies can be obtained commercially or produced using well-established methods. To obtain antibody that is specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas, and will continually grow and secrete antibody in culture. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies.

[0158] Monoclonal antibodies are exemplary antibodies contemplated by the present disclosure. The term “monoclonal antibody" or “mAb” or “MAb” refers to a homogeneous antibody population capable of binding to the same antigen(s) and, for example, to the same epitope within the antigen. This term is not intended to be limited with respect to the source of the antibody or the manner in which it is made.

[0159] The skilled person will be aware that scFvs comprise VH and VL regions in a single polypeptide chain. The polypeptide chain further comprises a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding ( / .e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly4Ser)3 being one of the more favoured linkers for a scFv. For a review of scFv, see Pluckthun (1994), “Recombinant antibodies” in: Van Oss CJ, Van Regenmortel MHV (eds), Immunochemistry. Marcel Dekker, New York, pp 201-236.

[0160] A "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain or can be produced using recombinant means. A "Fab1fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain comprising a VH and a single constant domain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab’ fragment can also be produced by recombinant means. A "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A “Fab2” fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

[0161] Polyclonal and monoclonal antibodies can be purified in several ways. For example, one can isolate an antibody using antigen-affinity chromatography which is couple to bacterial proteins such as Protein A, Protein G, Protein L or the recombinant fusion protein, Protein A / G followed by detection of via UV light at 280 nm absorbance of the eluate fractions to determine which fractions contain the antibody. Protein A / G binds to all subclasses of human IgG, making it useful for purifying polyclonal or monoclonal IgG antibodies whose subclasses have not been determined. In addition, it binds to IgA, IgE, IgM and (to a lesser extent) IgD. Protein A / G also binds to all subclasses of mouse IgG but does not bind mouse IgA, IgM or serum albumin. This feature, allows Protein A / G to be used for purification and detection of mouse monoclonal IgG antibodies, without interference from IgA, IgM and serum albumin.

[0162] Antibodies can be derived from different classes or isotypes of molecules such as, for example, IgA, IgA IgD, IgE, IgM and IgG. The antibody that is most useful in biological studies is the IgG class, a protein molecule that is made and secreted and can recognize specific antigens. The IgG is composed of two subunits including two “heavy” chains and two “light” chains. These are assembled in a symmetrical structure and each IgG has two identical antigen recognition domains. The antigen recognition domain is a combination of amino acids from both the heavy and light chains. The molecule is roughly shaped like a “Y” and the arms / tips of the molecule comprise the antigen-recognizing regions or Fab (fragment, antigen binding) region, while the stem of Fc (Fragment, crystallizable) region is not involved in recognition and is fairly constant. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes.

[0163] In one example, the method of the disclosure can use immunohistochemistry for detecting the expression levels of the biomarkers of the present disclosure. Thus, antibodies specific for each marker are used to detect expression of the claimed biomarkers in a biological sample. The antibodies can be detected by direct labelling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody is used in conjunction with a labelled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols are well known in the art and protocols and antibodies are commercially available. Alternatively, one could make an antibody to the biomarkers or modified versions of the biomarker or binding partners as disclosure herein that would be useful for determining the expression levels of in a biological sample. In some embodiments one or more CDRs VH domain from can be used to generate a single domain antibody AKA a “nanobody.” Methods for generating a nanobody by CDR grafting from a mammalian antibody onto frameworks derived from variable domains of heavy-chain-only antibodies (VHH) to generate nanobodies are known in the art as exemplified in, e.g., Wagner et al., (2018), International Journal of Molecular Sciences, 19(11), 3444. See also, Vincke et al., (2009), Journal of Biological Chemistry, 284(5):3273-3284.

[0164] In some examples, the present disclosure provides for the detection and / or quantitative analysis of biomarkers disclosed herein (e.g., combinations of lipids disclosed herein) by mass spectrometry. Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is primarily used for determining the elemental composition of a sample or molecules, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionising chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. MS instruments typically consist of three modules (1) an ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase) (2) a mass analyser, which sorts the ions by their masses by applying electromagnetic fields and (3) detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present.

[0165] In some examples, a higher abundance of one or two or three lipids indicates a higher risk of MND. For example, a higher abundance of one lipid compared to a reference abundance indicates a higher risk of MND. For example, a higher abundance of two lipids compared to a reference sample or compared to a sample with higher abundance in one lipid indicates a higher risk of MND than for either compared sample. In some examples, a higher abundance of three lipids compared to the abundance in a reference sample indicates increased risk of MND. In some examples, a higher abundance of three lipids indicates a greater risk of MND compared to a sample with higher abundance of one or two lipids.

[0166] Suitable mass spectrometry methods to be used with the present disclosure include but are not limited to, one or more of electrospray ionisation mass spectrometry (ESI-MS), ESI-MS / MS, ESI- MS / (MS)n, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF- MS), surface-enhanced laser desorption / ionisation time-of-flight mass spectrometry (SELDI-TOF-MS), tandem liquid chromatography-mass spectrometry (LC-MS / MS) mass spectrometry, desorption / ionisation on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of- flight (Q-TOF), atmospheric pressure chemical ionisation mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS), atmospheric pressure photoionisation mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n, quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), and ion trap mass spectrometry, where n is an integer greater than zero.

[0167] One of the challenges related to MS-based lipidomics is the complexity of the sample with many diverse species in a small mass range between 300 and 900 Da, as conventional mass spectrometry has a limited mass resolution (ability to separate molecules with different masses), usually multiple lipid species with similar masses (referred to as isobaric species) are co-detected, hampering the exact assignment of a mass to a specific individual lipid species. This can in part be solved by using mass spectrometers with higher resolution mass analysers such as the time-of-flight (TOF) analyser, which separates ions based on their m / z-dependent acceleration or an “Orbitrap” in which ions oscillate around an inner rod. By applying a Fourier transformation to the measured ion oscillations, the use of an Orbitraps can resolve very small differences in mass. Alternatively, or additionally, lipids can be preseparated by liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), or liquid chromatography with tandem mass spectrometry (LC-MS / MS).

[0168] Prior to LC, a crude lipid extract is prepared from a biological sample, e.g., plasma. Most commonly, chloroform-containing liquid-liquid extraction methods, such as the Bligh-Dyer and Folch extraction protocols, are used (see, e.g., Patterson et al., 2015, J Chromatogr B Analyt Technol Biomed Life Sci, 1002:260-262). Typically, a mixture of chloroform and methanol is added to the biological sample as well as an antioxidant to prevent oxidation of double bonds and a lipid standard mix for later correction of eventual differences in lipid extraction efficiency and ionization. Lipids are recovered in the organic phase after centrifugation and evaporation. Alternatively, the raw extract is applied to a resin in a column for solid phase extraction and elution. Lipid extracts are then subjected to MS. Preferably, lipids are separated by LC. LC separation of lipids can be based on the hydrophobicity of the fatty acyl chains (reversed phase chromatography, e.g., using a C18 column) or based on the hydrophilicity of the headgroups (HILIC).

[0169] In some preferred examples, MS uses a targeted approach to identify the specific and predefined biomarker lipid species disclosed herein. Quadrupole instruments are well suited for a targeted analysis, as specific voltages can be applied on the mass filters in a sequential manner corresponding to only the lipid biomarkers of interest. This targeted analysis allows a longer time for data integration of each individual species, which facilitates quantification of each lipid species.

[0170] One particularly useful MS technique for targeted analysis that can be used for quantifying biomarker lipids in the present disclosure is Multiple Reaction Monitoring Mass Spectrometry (MRM- MS), or alternatively referred to as Selected Reaction Monitoring Mass Spectrometry (SRM-MS).

[0171] The MRM-MS technique uses a triple quadrupole (QQQ) mass spectrometer to select a positively charged ion from the lipid of interest, fragment the positively charged ion and then measure the abundance of a selected positively charged fragment ion. This measurement is commonly referred to as a transition.

[0172] In some applications the MRM-MS is coupled with High-Pressure Liquid Chromatography (HPLC) and more recently Ultra High-Pressure Liquid Chromatography (UHPLC). In other applications MRM-MS is coupled with UHPLC with a QQQ mass spectrometer to make the desired LC-MS transition measurements for all of the peptides and proteins of interest.

[0173] In some examples, methods capable of analysing lipid species include classical lipid extraction methods, mass spectrometry together with electrospray ionization and matrix-assisted laser desorption ionisation, with mass analysis such as quadruple and / or TOF (e.g.,. Quadrapole / TOF) or orbitrap mass analysers. Chromatographic methods are used for the separation of lipid mixtures such as gas chromatography, high pressure liquid chromatography (HPLC), ultra-high pressure liquid chromatography (UHPLC), capillary electrophoresis (CE). These may be used with mass spectrometry based detection systems or other detectors including optical detectors. Clinical mass spectrometry systems are used by clinical laboratories to provide lipid profiles and ratios upon request. Another suitable technique for quantitative lipid analysis is one or two dimensional nuclear magnetic resonance (NMR). Two dimensional techniques such as heteronuclear single quantum coherence (HSQC) are suitable for lipid profiling through the ability to elucidate C-H bonds within a structure. Any technique capable of identifying individual lipid species in the sample can be used for collecting information on the lipid species. Typically MS is used coupled to a separation method such as various forms of chromatography.

[0174] In some examples, the methods described herein include separating sample analytes by liquid chromatographic separation, for example, in a sample obtained from a subject, for example a human subject, for example a plasma sample. In some examples, the analytes are analysed using a mass spectrometer. In some examples, the methods described herein further comprise using HPLC, LC, LC- MS and / or LC-MS / MS to measure, detect, and / or determine the level of lipids in a sample. In some examples, the analysis is high throughput. In some examples, the analysis is automated. In some examples, the analysis is partially automated. In some examples, the analysis is manual.

[0175] In some applications the utilisation of a quadrupole time-of-flight (qTOF) mass spectrometer, time-of-flight (TOF-TOF) mass spectrometer, Orbitrap mass spectrometer, quadrupole Orbitrap mass spectrometer or any Quadrupolar Ion Trap mass spectrometer can be used to select for a positively charged ion from one or more lipids of interest. The fragmented, positively charged ions can then be measured to determine the abundance of a positively charged ion for the quantitation of the lipid of interest.

[0176] In some applications the utilisation of a time-of-flight (TOF), quadrupole time-of-flight (qTOF) mass spectrometer, time-of-flight (TOF-TOF) mass spectrometer, Orbitrap mass spectrometer or quadrupole Orbitrap mass spectrometer can be used to measure the mass and abundance of a lipid ion for quantitation. In this application, the accuracy of the analyte mass measurement can be used as selection criteria of the assay. An isotopically labelled internal standard of a known composition and concentration can be used as part of the mass spectrometric quantitation methodology.

[0177] In some applications, time-of-flight (TOF), quadrupole time-of-flight (qTOF) mass spectrometer, time-of-flight (TOF-TOF) mass spectrometer, Orbitrap mass spectrometer or quadrupole Orbitrap mass spectrometer can be used to measure the mass and abundance of a lipid of interest for quantitation. In this application, the accuracy of the analyte mass measurement can be used as selection criteria of the assay. An isotopically labelled internal standard of a known composition and concentration can be used as part of the mass spectrometric quantitation methodology.

[0178] In some applications, various ionisation techniques can be coupled to the mass spectrometers provided herein to generate the desired information. Non-limiting exemplary ionization techniques that can be used with the present disclosure include but are not limited to Matrix Assisted Laser Desorption Ionization (MALDI), Desorption Electrospray Ionization (DESI), Direct Assisted Real Time (DART), Surface Assisted Laser Desorption Ionization (SALDI), or Electrospray Ionization (ESI).

[0179] Typically, quantification of biomarkers as performed in the present disclosure will include referenced control samples. In some examples, the control reference is determined from measurements of the biomarkers in corresponding panel of biomarkers from a population of healthy individuals. The term “healthy individual” as used herein refers to a person or populations of persons who are known not to have MND, such knowledge being derived from clinical data on the individual, or lack of indications for MND. In some examples, the control reference is determined from measurements of the corresponding biomarkers in a “typical population”. Preferably, a "typical population" will exhibit a spectrum of MND at different stages of disease progression. It is particularly preferred that a “typical population” exhibits the expression characteristics of a cohort of subjects as described herein. In one example, the control reference is taken from the subject to be tested at an earlier time point, for example to monitor MND progression.

[0180] In some examples, provided herein is method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least one or at least two or at least three lipids, wherein the at least three lipids include at least one lipid from each of sphingolipids, glycerophospholipids, and fatty acyls.

[0181] In some examples, provided herein is method for determining a rate of progression of motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least one or at least two or at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.

[0182] In some examples, provided herein is method for determining a rate of progression of motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least one or at least two or at least three lipids, wherein the at least three lipids include at least one lipid from each of sphingolipids, glycerophospholipids, and fatty acyls.

[0183] In some examples, provided herein is a method for detecting the presence and / or level of at least one or at least two or at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: providing a biological sample obtained previously from the subject; contacting the biological sample with at least three lipid-binding agents that bind specifically to at least three different lipids, wherein each of the different lipids is a different type of lipid selected from the group consisting of: at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid; and determining the level of each of the at least three lipids based on the amount of specifically bound lipid-binding agents.

[0184] In some examples, provided herein is a method for detecting the presence and / or level of at least one or at least two or at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: providing a biological sample obtained previously from the subject; contacting the biological sample with at least three lipid-binding agents that bind specifically to at least three different lipids, wherein each of the different lipids is a different type of lipid selected from the group consisting of: sphingolipids, glycerophospholipids, and fatty acyls; and determining the level of each of the at least three lipids based on the amount of specifically bound lipid-binding agents.

[0185] In some examples, the at least three different lipids are the sets shown in Table 7.

[0186] Data

[0187] In some examples, methods of determining whether a subject has MND or is otherwise at an increased risk of developing MND, or of determining the rate of progression of MND are based upon the biomarker panel measurement compared to a reference profile that can be made in conjunction with statistical analysis.

[0188] A quantitative score may be determined by the application of a specific algorithm. The algorithm used to calculate the quantitative score in the methods disclosed herein may group the expression level values of a biomarker or groups of biomarkers. The formation of a particular group of biomarkers, in addition, can facilitate the mathematical weighting of the contribution of various expression levels of biomarker or biomarker subsets (e.g. classifier) to the quantitative score.

[0189] In some examples, SPSS software may be used for the statistical analysis. In some examples, binary logistic regression analysis may be used to predict the diagnostic efficiency of the selected biomarkers. In some examples, a statistical algorithm used with a computer to implement the statistical algorithm may be used. In some examples, the statistical algorithm is a learning statistical classifier system. Examples of such systems include Receiver operating characteristic (ROC), Random Forest, interactive tree, classification and regression tree classification or neural networks. In some examples, logistic regression is used.

[0190] A fair evaluation of a test requires its assessment using “out-of-sample” subjects, that is, subjects not included in the construction of the initial predictive model. This is achieved by assessing the test performance using n-fold cross validation.

[0191] Tests for statistical significance include linear and non-linear regression, including ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio, Bayesian probability algorithms. As the number of biomarkers measured increases however, it can be generally more convenient to use a more sophisticated technique such as Random Forests, simple logistic, or Bayes Net to name a few.

[0192] In some examples, Bayesian probability may be adopted. Other non-linear or linear logistic algorithms that would be equally applicable include Random Forest, Linear Models for MicroArray data (LIMMA) and / or Significance Analyses of Microarray Data (SAM), Best First, Greedy Stepwise, Naive Bayes, Linear Forward Selection, Scatter Search, Linear Discriminant Analysis (LDA), Stepwise Logistic Regression, Receiver Operating Characteristic, and Classification Trees (CT).

[0193] The skilled person will be familiar with determination of co-efficient values in regression algorithms. In some examples, an algorithm is used to derive a MND likelihood score. In some examples, a quantitative score is derived which may indicate an increased likelihood of poor clinical outcome, good clinical outcome, high risk of CRC, or low risk of CRC. The score may then inform treatment management.

[0194] Data Handling

[0195] It will be apparent from the discussion herein that knowledge-based computer software and hardware for implementing an algorithm or other forms of statistical analysis are used in implementing the methods of the present disclosure. Such computer software and / or hardware are useful for performing a method of detecting MND, assessing the likelihood a subject has MND, and for determining the rate of progression of MND in a subject according to the disclosure.

[0196] The values from the assays described herein can be calculated and stored manually. Alternatively, the statistical analysis steps can be completely or partially performed by a computer program product. The present disclosure thus provides a computer program product including a computer readable storage medium having a computer program stored on it. The_program can, when read by a computer, execute relevant calculations based on values obtained from analysis of one or more biological samples from a subject (e.g., lipid levels, normalisation, standardisation, thresholding, and conversion of values from assays to a clinical outcome score and / or text or graphical depiction of clinical status or stage and related information). The computer program product has stored therein a computer program for performing the calculation.

[0197] The present disclosure also provides systems for executing the data collection and handling or calculating software programs described above, which system generally includes: a) a central computing environment; b) an input device, operatively connected to the computing environment, to receive patient data, wherein the patient data can include, for example, gene or protein expression level or other value obtained from an assay using a biological sample from the subject, or mass spec data or data for any of the assays provided by the present disclosure; c) an output device, connected to the computing environment, to provide information to a user (e.g., medical personnel); and d) an algorithm executed by the central computing environment (e.g., a processor), where the algorithm is executed based on the data received by the input device, and wherein the algorithm calculates an expression score, thresholding, or other functions described herein. The methods provided by the present disclosure may also be automated in whole or in part. In some examples, the methods comprise a combination of laboratory-based methods and computer-based methods.

[0198] In one example, a method of the disclosure may be used in existing knowledge-based architecture or platforms associated with pathology services. For example, results from a method described herein are transmitted via a communications network (e.g. the internet) to a processing system in which an algorithm is stored and used to generate a predicted posterior probability value which translates to the score of disease probability which is then forwarded to an end user in the form of a diagnostic or predictive report. The method of the disclosure may, therefore, be in the form of a kit or computer-based system which comprises the reagents necessary to detect the concentration of the biomarkers and the computer hardware and / or software to facilitate determination and transmission of reports to a clinician.

[0199] The assays described herein can be integrated into existing or newly developed pathology architecture or platform systems. For example, the present disclosure contemplates a method of allowing a user to determine a subject’s risk with respect to MND, the presence of MND in a subject and / or the rate of progression of MND in a subject, the method including: receiving subject data obtained from determining a measurement of each biomarker in a biomarker panel described herein; processing the data via multivariate analysis (for example, regression analysis) to provide a disease score; determining the status of the subject in accordance with the results of the disease score in comparison with predetermined values; and transferring an indication of the status of the subject to the user via the communications network reference to the multivariate analysis which includes an algorithm which performs the multivariate analysis function.

[0200] In one example, provided herein is a system for determining the likelihood that a subject has a motor neuron disease and / or for determining a rate of progression of a motor neuron disease in a subject, the system comprising: a processor operable to execute programs; a memory associated with the processor; a database associated with said processor and said memory; and a program stored in the memory and executable by the processor, the program being operable for any of the methods for determining the likelihood that a subject has a motor neuron disease and / or for determining a rate of progression of a motor neuron disease described herein.

[0201] Treatment

[0202] In some examples, methods of treating a subject identified as suffering a MND based on the biomarkers of the present application are provided. Such methods include administering or having administered to the subject a therapeutically effective amount of an agent selected from the list consisting of: agents that inhibit protein misfolding, protein aggregation, and / or reduce endoplasmic reticulum stress; agents that reduce oxidative stress agents that reduce inflammation; agents that modulate lipid metabolism, and agents that block excitotoxicity. Examples of therapeutic agents for ALS include, but are not limited to, methylcobalamin (CAS 13422-55-4), masitinib (CAS 13422-55-4), lbudilast / MN-166 (CAS 1204192-90-4), verdiperstat (CAS 890655-80-8), memantine (CAS 41100-52- 1), AMX0035 (a combination of sodium phenylbutyrate and tauroursodeoxycholic acid), trazodone (CAS 25332-39-2), CNM-Au8 (oral suspension of gold nanoparticles), pridopidine (CAS 346688-38-8), SLS-005 (trehalose, a low molecular weight disaccharide), IONN363 (antisense oligonucleotide against mutated FUS), tofersen (CAS 2088232-70-4), riluzole (CAS 1744-22-5), edaravone (CAS 89-25-8), Ambroxol (23828-92-4), and reldesemtiv (CAS 89-25-8).

[0203] Kits The present invention provides kits for the measuring, detection, and determining the level of lipid(s) in a sample, for example lipid biomarkers.

[0204] Kits may include any agent capable of binding specifically to a lipid of the present disclosure. Other components of the kits will typically include binding agents, labels, labelled binding agents, mAbs, scFvs, Fabs, sdAbs, secondary antibodies, inhibitors, co-factors, reagents and control preparations to allow the user to quantitate expression levels and / or to assess whether the measurement has worked correctly. Liquid chromatography, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography and tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography (HPLC) separation analysis and / or enzyme-linked immunosorbent assay-based (ELISA) tests and competitive ELISA tests are particularly suitable assays that can be carried out easily by the skilled person to perform the methods described herein and using kit components.

[0205] In some examples, the kit comprises one or more agents for performing LC.

[0206] In some examples, the kit comprises one or more agents for performing LC-MS.

[0207] In some examples, the kit comprises one or more agents for performing LC-MS / MS.

[0208] In some examples, the kit comprises one or more agents for performing HPLC.

[0209] In some examples, the kit may comprise a microtitre plate on which is immobilised capture antibodies corresponding to the lipids being measured.

[0210] In some examples, the kit comprises beads on which is immobilised capture antibodies corresponding to the lipids being measured.

[0211] In some examples, the kit comprises labelled binding agents, for example, the labelled binding agents each comprise at least one immunoglobulin variable domain that binds specifically to one of the at least three lipids.

[0212] In some examples, the labelled binding agents comprise one or more of mAbs; scFvs or Fab’s; and sdAbs.

[0213] In some examples, the kit comprises one or more agents for an ELISA, for example, a competitive ELISA.

[0214] Optionally, the kit further comprises means for the detection of the binding of an antibody to a lipid. Such means include a reporter molecule such as, for example, an enzyme (such as horseradish peroxidase or alkaline phosphatase), a dye, a radionucleotide, a luminescent group, a chemiluminescent group, a fluorescent group, biotin or a colloidal particle, such as colloidal gold or selenium. Preferably such a reporter molecule is directly linked to the antibody.

[0215] In one example, a kit may additionally comprise a reference sample. In one embodiment, a reference sample comprises a lipid that is detected by an antibody. Preferably, the lipid is of known concentration. Such a lipid is of particular use as a standard. Accordingly, various known concentrations of such a lipid may be detected using a diagnostic assay described herein.

[0216] In one example, the present disclosure provides a kit comprising at least one, two, or three distinctly labelled binding agents, wherein at least one binding agent binds to a glycerolipid, and wherein at least one binding agent binds to at least one of either a sphingolipid or a sterol lipid. In one example, the present disclosure provides a kit comprising at least one, two, or three distinctly labelled binding agents, wherein at least each specifically binds to a different type of lipid selected from the group consisting of: sphingolipids, glycerophospholipids, and fatty acyls. In one example, the at least three distinctly labelled binding agents bind to N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1- (9Z-nonadecenoyl)-glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-

[0217] (trimethylazaniumyl)butanoate, together with instructions for using the kit according to a method described herein.

[0218] In one example, the kit further comprises one or more lipid control standards.

[0219] In one example, the kit further comprises one or more lipid control standards for each of the at least three lipids. In one example, the one or more lipid control standards are provided in separate containers.

[0220] In one example, the kit further comprises one or more reagents for purification.

[0221] In one example, the kit further comprises one or more reagents for calibration.

[0222] In another example, the kit comprises a substrate on which is immobilised one or more binding agents that specifically bind to a different type of lipid selected from the group consisting of: sphingolipids, glycerophospholipids, and fatty acyls. In one example, the at least three distinctly labelled binding agents bind to N (17Z hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1-(9Z-nonadecenoyl)- glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate and secondary reagents for detecting binding between the binding agent and lipid.

[0223] In one example, the kit comprises one or more of lipid control standards, panels, analytical columns, platform for storing and / or analysing the sample, for example a well plat, chip, array, or column.

[0224] In further examples, the kit may also comprise a container for collecting the biological sample from the subject. The container may comprise an anti-coagulant or similar agent to avoid red blood cell clotting. Optionally, the container may also comprise an antioxidant to avoid lipid oxidation in the biological sample.

[0225] In one example, the kit is for diagnosis of MND.

[0226] In one example, the kit is for identifying a subject with MND.

[0227] In one example, the kit is for identifying MND in a subject.

[0228] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. EXAMPLES

[0229] Example 1 : Material and Methods

[0230] Mouse models

[0231] Three well-validated mouse models of motor neuron disease (MND) were used: C90RF72500 (most common hereditary gene mutation and genetic cause of MND); SOD1G93A (earliest MND associated gene and most robust mouse model); and TDP-43Q331 K (most clinically relevant mouse model, as 97% of all, both sporadic and familial cases, have TDP-43 pathology).

[0232] Lipid biomarkers and biomarker analysis

[0233] To determine whether a MND signature was detectable in blood, plasma and serum were isolated at pre-symptomatic, symptomatic and end-stage time points of the three genetic variants, their wild-type litter mates, and from both sexes in mice.

[0234] Lipids were extracted from the samples and targeted lipidomic analysis was run at metabolomics Australia and raw data was analysed by the inventors. For diagnostic properties, the receiver operating characteristic (ROC) of lipids in each mouse model (heterozygous (het) vs wild type (WT)) was determined. Area under the curve (AUC) value cut-off was set at 0.83 with p value cut-off of 0.05. ROC analysis of SOD1G93A vs WT at postnatal day 60 (P60) resulted in 53 lipids that met cutoff criteria; TDP-43Q331 K vs WT at P60 resulted in 43 lipids that met cut-off criteria, and C90RF72500 vs WT at P120 resulted in 92 lipids that met cut-off criteria. Cross comparison of these lipids resulted in a list of 24 lipids that were represented in 2 or all 3 ROC analysis datasets (Table 1).

[0235] Table 1 : Lipids that met ROC cut-off criteria of AUC->0.83 and p value <0.05 that were present in two or more genotypes

[0236] Example 2: Candidate lipid biomarker sets and validation of biomarkers in humans

[0237] The clinical characteristics for the subjects analysed in this study are shown in Table 2. Table 2: Human datasets clinical characteristics for selecting and testing lipid panels

[0238] Lipid biomarker set determination

[0239] Three lipids of distinct classes were chosen from the lipids in Table 1 , each set of three lipids forming a lipid set for analysis. Three distinct lipid classes were chosen to represent different disease states, given the heterogeneity of MND. A total of approximately 243(13,824) different lipid set combinations were tested on a human dataset which consisted of 103 ALS patients, 39 Parkinson’s disease patients and 30 healthy controls. Lipidomic analysis was performed on this dataset. Statistical analysis, principal component analysis (PCA) plot revealed that the lipid sets returned a distinct grouping of patients based on disease state, confirming that the human datasets chosen (DSP and PD datasets) were a suitably representative dataset. Lipidomic analysis and PCA showed that the 24 lipids in Table 1 are all suitable for inclusion in a lipid set for detection of MND.

[0240] Several lipid sets were further tested, as well as sets that consisted of 3 lipids from the same lipid class (Table 3). ROC of human data was performed on MetaboAnalyst software (version 5.0) (Xia et al. 2009) and several random samples were blinded, so the program was asked to allocate these samples to disease state based on the ROC of the lipid set. Three 2-way analyses on the human data were conducted. That is; MND vs Control (29 blinded), MND vs PD (29 blinded) and PD vs control (20 blinded).

[0241] From these analyses, several randomised lipid sets were tested.

[0242] Lipid set 1 : DG 38:4 -(20:3), Cer(d18:1_24:0), LPC(O-18:1);

[0243] Lipid set 2: LPC 20:5, PC(O-36:5), TG 18: 1_14:0_16:0;

[0244] Lipid set 3: LPE(P-16:0), TG 18:1 18:1 18:1 , CE 17:1 ; and

[0245] Lipid set 4: SM 44:2, LPC 19:1 , AcylCarnitine 16:0.

[0246] AUC with 95% Cl of each of these tests is plotted in Figure 1. Lipid set 4 performed the best overall (Table 4), the remaining three lipid sets not meeting the specificity criteria (Table 5). Each of the lipids in this set represents a major MND related pathophysiology (Table 6). Lipid set 4 was able to distinguish MND vs control and MND from PD, but did not distinguish PD from control, making it MND specific.

[0247] Table 3: Lipid sets consisting of three lipid classes from the same lipid species

[0248] Table 4: Analysis of lipid set 4 Table 5: Analysis of lipid sets that did not meet the specificity criteria

[0249] Table 6: Biological relevance of lipids

[0250] Example 3: Determination of 18 sets of 3 candidate lipid biomarker sets

[0251] Extensive unbiased data analysis of the plasma lipidomics was performed.

[0252] From this analysis 160 top lipids were identified from the mouse based on ROC. These 160 lipids were then used to discover novel sets of biomarkers, which were novel MND lipid signature. Sets of four were contemplated but the testing was not feasible as 10 supercomputers would have been needed, running for >7 days. Accordingly, sets of three were tested. This led to a total of 438,480 combinations that were tested.

[0253] Lipid sets that had a high confidence in predicting PD from Control were excluded, to ensure that the biomarker sets were unique to MND and would provide a reliable and robust MND lipid signature. The following thresholds were used: AUC >0.7, <0.95, p<0.05 OddsRatio 1. Only positive relationships were included (i.e. higher abundance correlates with a greater likelihood of MND). A final total of 18 sets of 3 lipid biomarkers was identified (Figure 3; Table 7).

[0254] Table 7: Sets of 3 lipid biomarkers

[0255] Biological relevance of each lipid:

[0256] COH: free cholesterol; higher levels are associated with improved survival in ALS, but this is not conclusive. Some research shows ALS patients have higher levels of cholesterol than controls and total cholesterol changes are instead indicative of ALS risk.

[0257] Hex2Cer: Gala1-4Galp-Cer(d18:1 / 18:0); precursor of gangliosides, upregulated in diabetic kidneys, linked to renal cell proliferation. Unknown brain function, could induce insulin resistance. Important for the long-term stability of myelin.

[0258] LPC: 1-acyl-sn-glycero-3-phosphocholine; LPC accumulation has been observed in preclinical mouse models of MND as well as in patient spinal cord and plasma. Causes demyelination.

[0259] LPE: 1-acyl-sn-glycero-3-phosphoethanolamine; LPE increases intracellular calcium influx in PC-12 neuron cells and SH-SY5Y neuroblastoma cells. Could be associated with hyperexcitability.

[0260] PE: 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine (Phosphatidylethanolamine); second most abundant phospholipid, associated with protein biogenesis, autophagy, mitochondrial stability and is a precursor to many other lipids.

[0261] PI: 1 ,2-diacyl-sn-glycero-3-phospho-(1 '-myo-inositol) (Phosphatidylinositol); ion channel regulators and autophagy control. dhCer: dihydroceramide, N-acyl-sphinganine; precursor to major fatty acids and ceramide. It is thought to be important in promoting glial function and linked to reactive oxygen species production in mitochondria. Accumulation is linked to neuronal dysfunction.

[0262] LPI; has affinity to G protein coupled receptor 55, causing excitation. SM; Membrane stability and inflammatory response.

[0263] DG; DG is a second messenger with many functions in cells. Its hydrolysis by DG lipase results in 2- arachidonoylglycerol, a molecule involved in endocannabinoid signalling.

[0264] Table 8: Lipid classification

[0265] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the invention as shown in the specific embodiments without departing from the scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0266] This application claims priority from Australian Provisional Application No. 2024901108 entitled “Biomarkers for Motor Neuron Disease” filed on 19 April 2024, the entire contents of which are hereby incorporated by reference.

[0267] All publications discussed and / or referenced herein are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Claims

Claims1 . A method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.

2. A method for determining a rate of progression of a motor neuron disease in a subject identified as suffering from motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the levels of at least three lipids, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.

3. A method for detecting the presence and / or determining the level of at least three lipid biomarkers in a subject suspected of suffering from a motor neuron disease, the method comprising: i) providing a biological sample obtained previously from the subject; ii) contacting the biological sample with at least one lipid-binding agent that binds specifically to a lipid selected from the group consisting of: glycerolipids, sphingolipids, and sterol lipids; and iii) determining the level of at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid based on the amount of specifically bound lipid-binding agents.

4. The method according to claim 3, wherein the at least one lipid-binding agent is an antibody.

5. The method according to any one of claims 1 to 4, wherein: i) a sphingolipid is selected from the group consisting of: N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine, N-(tetracosanoyl)-sphing-4-enine, N-(tetracosanoyl)-4E, and14Z- sphingadienine-1 -phosphocholine, N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine, N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine, N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine, N-(2-hydroxydocosanoyl)-eicosasphinganine; ii) a glycerolipid is selected from the group consisting of: 1-(9Z-nonadecenoyl)-glycero- 3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z,14Z,17Z- eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1-(5Z,8Z,11Z-eicosatrienoyl)-sn-glycero-3- phosphocholine, 1 -(10Z-heptadecenoyl)-sn-glycero-3-phosphocholine, 1 -(1 Z-hexadecenyl)- sn-glycero-3-phosphoethanolamine, 1 ,2-di-(9Z,12Z,15Z-octadecatrienoyl)-sn-glycero-3- phosphocholine, 1-(9Z,12Z-heptadecadienoyl)-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phosphocholine, 1 -octadecyl-2-(9Z-hexadecenoyl)-sn-glycero-3-phosphocholine, 1 -hexadecyl-2-(5Z,8Z,11Z,14Z,17Z-eicosapentaenoyl)-sn-glycero-3-phosphocholine, 1- hexadecanoyl-2-octadecanoyl-sn-glycero-3-phosphocholine, 1-(1Z-octadecenyl)-2-(9Z,12Z- octadecadienoyl)-glycero-3-phosphoethanolamine, and 1-(9Z-octadecenoyl)-2-(9Z,12Z- octadecadienoyl)-sn-glycero-3-phosphoethanolamine, 1-eicosanoyl-sn-glycero-3- phosphocholine, 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1-octadecanoyl-sn- glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, 1- (9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; and iii) the sterol lipid comprises free cholesterol.

6. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine (SM 44:2).

7. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer).

8. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine (SM 34:3).

9. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-(2-hydroxydocosanoyl)-eicosasphinganine (DhCer (22.0)(24.1)).

10. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine (SM 36:3).

11. The method according to any one of claims 1 to 5, wherein the at least one sphingolipid comprises N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide.

12. The method according to any one of claims 1 to 11 , wherein the at least one glycerolipid is selected from 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine, 1-eicosanoyl-sn-glycero-3- phosphocholine, 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine, 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine, 1-octadecanoyl-sn- glycero-3-phosphoethanolamine, 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine, 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myo-inositol), 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol, and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol.

13. The method according to any one of claims 1 to 12, wherein the at least one sterol lipid is free cholesterol.

14. The method according to any one of claims 1 to 5, wherein the at least three lipids comprise: a) (i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine (SM 44:2);(ii) 1-(9Z-nonadecenoyl)-glycero-3-phosphocholine (LPC 19:1); and(iii) 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate (AcylCarnitine 16:0); b) (i) Free cholesterol (COH 16:1);(ii) 1-eicosanoyl-sn-glycero-3-phosphocholine (LPC 20:0); and(iii) 1-octadecanoyl-sn-glycero-3-phosphoethanolamine (LPE 18:0); c) (i) Free cholesterol (COH 16:1);(ii) 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine (LPC 20:2); and(iii) 1-octadecanoyl-sn-glycero-3-phosphoethanolamine (LPE 18:0); d) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-octadecanoyl-sn-glycero-3-phosphoethanolamine (LPE 18:0); e) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-(9Z-octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol (DG 36:1 (18:1)); f) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine (LPC 20:2); g) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-eicosanoyl-sn-glycero-3-phosphocholine (LPC 20:0); h) (i) Free cholesterol (COH 16:1); ;(ii) 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1'-myo- inositol)(PI 36:4) ; and(iii) N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine (SM 34:3); i) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine (LPC 22:5); j) (i) Free cholesterol (COH 16:1);(ii) N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and(iii) 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine (LPC22:5); k) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine(Hex2Cer);(ii) Free cholesterol (COH 16:1); and(iii) 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine (PE 38:3); l) (i) Free cholesterol (COH 16:1);(ii) N-(2-hydroxydocosanoyl)-eicosasphinganine (DhCer (22.0)(24.1)); and(iii) 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine (LPC 22:5); m) (i) Free cholesterol (COH 16:1);(ii) 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3-phospho-(1 '-myoinositol) (PI 36:4); and(iii) N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine (SM 36:3); n) (i) Free cholesterol (COH 16:1);(ii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol) (LPI 20:4); and(iii) N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine (SM 34:3); o) (i) Free cholesterol (COH 16:1);(ii) N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine (SM 36:3); and(iii) 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol (DG 36:1 (18:1)); p) (i) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine (Hex2Cer);(ii) 1-(4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine (LPC 22:5); and(iii) 1-hexadecanoyl-2-(13Z,16Z-docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol) (PI 36:4) (PI 38.2); q) (i) Free cholesterol (COH 16:1);(ii) 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine (PE 38:3); and(iii) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine (SM 44:2); r) Free cholesterol (COH 16:1);(ii) N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and(iii) 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol)( LPI 20:4); or s) (i) Free cholesterol (COH 16:1);(ii) 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine (PE 38:3); and(iii) N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine (SM 36:3).

15. The method according to any one of claims 1 to 14, wherein the method comprises measuring the level of up to 24 lipids.

16. The method according to claim 14, wherein the levels of the at least three lipids to be measured consist of the levels of i) N- (17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine;xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

17. The method according to any one of claims 1 to 6, further comprising determining or having determined a higher likelihood of motor neuron disease in the subject by comparing the measured levels of each of: i) N- (17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine;xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine to established threshold levels for each of these lipids.

18. The method according to any one of claims 1 to 6, further comprising determining or having determined a rate of progression of motor neuron disease in the subject by comparing the measured levels of each of: i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine;viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine, to established threshold levels for each of these lipids.

19. The method according to claim 18, wherein the measured levels from biological samples that were obtained from at least two different time points are compared to the threshold levels.

20. The method according to any one of claims 1 to 19, wherein the biological sample is a whole blood sample, a plasma sample, or a serum sample.21 . The method according to any one of claims 1 to 20, wherein the levels of the at least three lipids wherein are measured by one or more immunoassays.

22. The method according to claim 21 , wherein the one or more immunoassays comprise an ELISA.

23. The method according to claim 22, wherein the ELISA is a competitive ELISA.

24. The method according to claim 22 or claim 23, wherein the ELISA is a multiplex ELISA.

25. The method according to any one of claims 1 to 20, wherein the levels of the at least three lipids are measured by high performance liquid chromatography (HPLC) or mass spectrometry.

26. The method according to any one of claims 1 to 20, wherein the levels of the at least three lipids are measured by HPLC and mass spectrometry.

27. The method according to any one of claims 1 to 6, wherein the motor neuron disease is a familial form of motor neuron disease.

28. The method according any one of claims 1 to 6, wherein the motor neuron disease is a sporadic form of motor neuron disease.

29. The method according to claim 27 or claim 28, wherein the motor neuron disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, (PLS), progressive muscular atrophy (PMA), progressive bulbar palsy, and spinal and bulbar muscular atrophy (SBMA).

30. A method for treating a subject identified as suffering from a motor neuron disease, the method comprising administering or having administered to the subject a therapeutically effective amount of an agent selected from the list consisting of: i) agents that inhibit protein misfolding, protein aggregation, and / or reduce endoplasmic reticulum stress; ii) agents that reduce oxidative stress iii) agents that reduce inflammation; iv) agents that modulate lipid metabolism; and v) agents that block excitotoxicity; wherein the subject was identified as suffering from a motor neuron disease based on the levels of at least three lipids in a biological sample from the subject, wherein the at least three lipids include at least one glycerolipid, and at least one of either a sphingolipid or a sterol lipid.31 . The method according to claim 30, wherein the at least three lipids comprise:i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine;Hi) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine;xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

32. A method for treating a subject identified as suffering from a motor neuron disease, the method comprising determining, in a biological sample previously obtained from the subject, the level of: i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate; ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,11Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine;xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,1 1Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

33. A kit comprising at least three binding agents, wherein each specifically binds to a different type of lipid, and wherein at least one binding agent binds to a glycerolipid, and wherein at least one binding agent binds to at least one of either a sphingolipid or a sterol lipid.

34. The kit according to claim 33, wherein the at least three binding agents bind to: i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine, 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine, and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,1 1Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11 Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine;xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,1 1Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine.

35. The kit according to claim 33 or claim 34, further comprising one or more lipid control standards for each of the at least three lipids, wherein the one or more lipid control standards are provided in separate containers.

36. The kit according to any one of claims 33 to 35, wherein the labelled binding agents each comprise at least one immunoglobulin variable domain that binds specifically to one of the at least three lipids.

37. The kit according to claim 36, wherein the labelled binding agents are selected from the group consisting of: i) mAbs; ii) a scFvs or Fab’s; and iii) sdAbs.

38. The kit according to claim 37, wherein the kit comprises one or more agents for performing high performance liquid chromatography.

39. The kit according to claim 37, wherein the kit comprises one or more agents for performing liquid chromatography-mass spectrometry and / or liquid chromatography tandem mass spectrometry.

40. The kit according to claim 37, wherein the kit comprises one or more agents for performing mass spectrometry.41 . The kit according to claim 37, wherein the kit comprises one or more agents for an ELISA.

42. The kit according to claim 41 , wherein the ELISA is a competitive ELISA.

43. The kit according to any one of claims 33 to 42, for diagnosis of MND.

44. The kit according to any one of claims 33 to 43, for identifying a subject with MND.

45. A method for determining the likelihood that a subject has a motor neuron disease, the method comprising measuring or having measured in a biological sample from the subject the level of at least one lipid selected from the group consisting of: i) N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; 1 -(9Z-nonadecenoyl)- glycero-3-phosphocholine; and 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate ii) Free cholesterol; 1-eicosanoyl-sn-glycero-3-phosphocholine; and 1-octadecanoyl-sn- glycero-3-phosphoethanolamine; iii) Free cholesterol; 1-(11Z,14Z-eicosadienoyl)-glycero-3-phosphocholine ;and 1- octadecanoyl-sn-glycero-3-phosphoethanolamine; iv) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl- sn-glycero-3-phosphoethanolamine; v) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(9Z- octadecenoyl)-2-(8Z,1 1Z,14Z-eicosatrienoyl)-sn-glycerol; vi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-(11Z,14Z- eicosadienoyl)-glycero-3-phosphocholine; vii) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-eicosanoyl-sn- glycero-3-phosphocholine; viii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11 Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1- phosphocholine; ix) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; x) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine;xi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; Free cholesterol; and 1-octadecanoyl-2- (8Z,11Z,14Z-eicosatrienoyl)-glycero-3-phosphoethanolamine; xii) Free cholesterol; N-(2-hydroxydocosanoyl)-eicosasphinganine; and 1- (4Z,7Z,10Z,13Z,16Z-docosapentaenoyl)-sn-glycero-3-phosphocholine; xiii) Free cholesterol; 1-hexadecanoyl-2-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-glycero-3- phospho-(1 '-myo-inositol); and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1- phosphocholine; xiv) Free cholesterol; 1-(5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1'- myo- inositol); and N-(9Z-octadecenoyl)-hexadecasphing-4-enine-1 -phosphocholine; xv) Free cholesterol; N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1-phosphocholine; and 1-(9Z-tetradecenoyl)-2-octadecanoyl-3-(9Z,12Z-octadecadienoyl)-sn-glycerol; xvi) N-(dodecanoyl)-1-p-lactosyl-sphing-4-enine; 1-(4Z,7Z,10Z,13Z,16Z- docosapentaenoyl)-sn-glycero-3-phosphocholine; and 1-hexadecanoyl-2-(13Z,16Z- docosadienoyl)-glycero-3-phospho-(1 '-myo-inositol); xvii) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(17Z-hexacosenoyl)-sphing-4-enine-1 -phosphocholine; xviii) Free cholesterol; N-[(2S,3R)-1 ,3-dihydroxyoctadecan-2-yl]formamide; and 1- (5Z,8Z,11Z,14Z-eicosatetraenoyl)-sn-glycero-3-phospho-(1 '-myo-inositol); or xix) Free cholesterol; 1-octadecanoyl-2-(8Z,11Z,14Z-eicosatrienoyl)-glycero-3- phosphoethanolamine; and N-(9Z-octadecenoyl)-4E,14Z-sphingadienine-1 -phosphocholine46. The method according to claim 45, comprising measuring or having measured the level of at least two lipids selected from any one of (i) to (xii).

47. The method according to claim 45 or claim 46, comprising measuring the level of 3-(hexadecanoyloxy)-4-(trimethylazaniumyl)butanoate.

48. The method according to claim 45 or claim 46, comprising measuring the level of free cholesterol.

49. The method according to claim 45 or claim 46, comprising measuring the level of N- (dodecanoyl)-1-p-lactosyl-sphing-4-enine.

50. The method according to any one of claims 47 to 49, further comprising measuring the level of a glycerolipid.51 . The method according to claim 48, wherein the glycerolipid is a glycerophospholipid.

52. A system for determining the likelihood that a subject has a motor neuron disease and / or for determining a rate of progression of a motor neuron disease in a subject, the system comprising: i) a processor operable to execute programs; ii) a memory associated with the processor; iii) a database associated with said processor and said memory; and iv) a program stored in the memory and executable by the processor, the program being operable for the methods of any one of claims 1 to 29 or 45 to 51 .

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