Kits and methods for diagnosis of neurological diseases

Abstract

The present invention relates to kits and diagnostic methods for multiple biomarkers or their isoforms as well as to methods for determining and selecting treatment methods based on the results obtained from said kits and diagnostic methods. In particular, the invention relates to improved lateral flow (LFA) strips, cassettes containing the LFA assay strips as well as to kits containing the strips and cassettes. The invention also relates to methods for subclassing patients into various subtypes based on the simultaneous detection of three or more different biomarkers or isoforms of biomarkers.

Description

Kits and Methods for Diagnosis of Neurological DiseasesBACKGROUN D OF THE INVENTION

[0001] Age-related neurodegenerative diseases (ND) are increasing exponentially as life expectancy is prolonged. This phenomenon is due to effective management of cardiovascular disease and stroke. The increased frequency of ND with aging is a growing social problem that impacts single families in the overall global economy. Currently, there are no cures for any age- related ND.

[0002] Most ND are considered to have multiple factors associated with development and progression of disease, though one or two molecules are recognized as the pathological factors. It is now accepted that one disease may be associated with more than one pathological process or factor.

[0003] Unfortunately, most diagnosis and treatment methods focus on a single factor and do not address other pathological factors. Therefore, the outcome of single target treatment has rarely reached the therapeutic threshold in clinical trials.

[0004] There is accordingly an urgent need to develop more effective diagnosis and treatment methods for these diseases.SU MMARY OF TH E I NVENTION

[0005] The present invention relates to apparatuses which include a lateral flow assay (LFA) strip, a cassette containing the LFA strip and kits which include the LFA assay strip and / or cassette. The LFA strip and the cassette includes an improved design which allows for detection of three or more biomarkers and or biomarker isoform(s) simultaneously.

[0006] In one embodiment, the cassette includes a longer open slot which can thereby accommodate a test strip having four or more detection lines. In such an embodiment, the four or more detection lines can be three separate biomarker test lines and a control test line. The apparatus can accordingly detect three or more biomarkers or biomarker isoforms simultaneously in a single test strip.

[0007] In a separate embodiment, the LFA assay strip includes a larger absorbent pad made possible by a cassette which is configured to allow for the larger absorbent pad and unrestricted sample flow to the absorbent pad. This configuration allows for increased sample flow and increased sensitivity for detection of the biomarkers.

[0008] In a separate aspect, the invention relates to determining and selecting treatment methods based on the detection results obtained from LFA assay / cassette / kits.

[0009] In a separate aspect, the invention relates to differentiation of neurological diseases into different subtypes for precision and personalized medicine.

[0010] In preferred embodiments, the invention relates to lateral flow assays (LFA) used in methods of detecting biomarkers for neurological diseases where multiple biomarkers are detected simultaneously. Accordingly, said LFA kits are part of multiplex lateral flow assays (LFA) kits.

[0011] In other preferred embodiments, the biomarkers for neurological diseases are selected from at least three biomarkers for different neurological diseases. Said biomarkers and neurological diseases are selected among the group consisting of Amyloid beta (AP) and Tau for Alzheimer's disease (AD), alpha synuclein for Parkinson's disease (PD), Huntington protein (htt) for Huntington disease (HD), and TDP43 or SOD for Amyotrophic lateral sclerosis (ALS). In other embodiments, the biomarkers are A oligomers, aggregated a-Syn, and p-tau217.

[0012] Some embodiments of the invention include said biomarkers which make them particularly representative of their respective disease when being used in the LFA kits and method of making said LFA kits. For example, in particular embodiment the invention a composition which includes an aggregated isoform of a-Syn is used in the described LFA methods and kits.

[0013] The invention includes antibodies which to the above biomarkers which exhibit extremely high affinity and use of said antibodies as part of the LFA kits for detection of said biomarkers. In particular embodiments, said antibodies can distinguish better more toxic forms of said biomarkers. For example, in particular embodiment the invention includes antibodies which are more specific for an aggregated isoform of a-Syn, oligomer AP, phosphorylated tau217.

[0014] In preferred embodiments, the antibodies are conjugated to gold particles as part of the LFA kit.

[0015] The invention also relates to the use of said kits and diagnostic methods for determining the presence of said neurological diseases as well as to methods of making individual treatment determinations based on the results form said kits and methods.BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1. Western blotting results for anti-human a-Syn antibody screening. FigurelA demonstrates the antibody mapping results of different antibodies against different lengths of recombinant human a-Syn. Lane 1, 2, 3, 4, 5, 6 in FigurelA represent human a-Syn 1-65, 15-65, 1-95, 15-95, 1-140, 15-140 respectively. Each blot was detected with different antibody. Poly G-16- 33 is a polyclonal antibody generated from goat and Poly R251 is a polyclonal antibody generated from rabbit against a-Syn. Clone 3H10, 2B5, 1H5 and 2E11 are monoclonal antibody against human a-Syn. FigurelA indicated that 3H10 and 1H5 are specific for N-terminal, and 2B5 and 2E11 are specific for C-terminal; Figure IB panel is the results of anti-human a-Syn monoclonal antibodies against different samples. Spl and 2 are mouse brain tissue from human a-Syn with mutation as A53T transgenic mouse (Tg), No-Tg mouse, Sp3, 4 are two different human PD brain tissue. 1F5, 2A4, 2B5, 2C6, 2G3, 2H1, 3G7 and 97A are monoclonal antibodies against human a-Syn. 2A4, 2G3, 2H1 and 3G7 are specific for human a-Syn. Upon those results, four a-Syn antibodies, 2A4, 1H5, 2C6, and 2G3 are specific for human a-Syn protein and selected for further characterization.

[0017] Figure 2. Thermodynamic studies on the interaction between recombinant human a-syn with different antibodies. Figure 2A-2D are the titration data of different antibody affinity to a-Syn protein, and 1, 2 represent response time and mole ratio of two molecules. The results were analyzed using one-site binding model by Launch NanoAnalyzer software to give the Number of binding Site(n), binding constant (Ka), binding enthalpy (AH), Entropy contribution(-TAS), and the Gibbs energy(AG). AG=AH-TAS=-RTInKa, where AG, AH, and-TAS are the changes in free energy, enthalpy, and entropy of binding. Figure 2E is the bar graph of interaction Ka value. The study's findings demonstrated that 2A4 exhibits a higher number of binding sites for a-Syn, indicating greater affinity compared to the other antibodies (Figure IB). This suggests that 2A4 displays a higher specificity in binding with a-syn FL.

[0052]

[0018] Figure 3. Monoclonal antibody 2A4 has more specific binding to oligomer isoform of a- Syn. Figure 3A-3C shows the ITC binding of 2A4 to monomer a-Syn (A) and oligomer (B). The findings shown in Figure 3C showed that 2A4's affinity for oligomeric a-syn was twice as strong as its affinity for monomeric a-syn. This suggests that 2A4 displays greater specificity for binding to oligomeric a-syn. DI and D2 are results of western blot. D2 proved that 2A4 can recognize oligomer a-Syn.

[0019] Figure 1: The bioactivity of 2A4 before and after conjugation to a-Syn. To validate the ability of 2A4 to maintain its bioactivity and specificity to bind to a-Syn, sandwich ELISA was performed to test the binding of 2A4 with standard aggregated a-Syn (1-140). As shown in Figure 4A, the conjugation enhanced the activity of 2A4 by increasing the amount of antibody attached to the AuNPs, increasing the capacity to bind to a-Syn (**P<0.01) with the standard curve (4B). Itshowed that 2A4 antibody maintained its bioactivity after conjugation, indicating that the conjugation didn't hinder its binding site.

[0020] Figure 2. The sensitivity test of LFA for the detection of aggregated a-syn protein. Figure 5A represent testing results of a-Syn LFA kit to the standard of aggregated a-Syn (1-140), from left to right is the lowest concentration (0.97 ng / ml) to the highest concentration (500 ng / ml). The lowest detected concentration was 1.95 ng / ml (Figure 5A). The figure 5B is the quantification result of 5A by scanning all strips to obtain density using image J software, and the quantification measure (T / C) were graphed for the test line (a-Syn) to control line (C) with the increase in cone of the analyte. This standard curve can be used for the quantification of other testing sample in the future.

[0021] Figure 6. The results of a-Syn LFA for PD patient. Figure 6A (al-a5) are detection results using a-Syn specific LFA kit to human PD patient. The al, a2, a3, a4 and a5 represent normal, stage 1, 2, 3 and 4 respectively. Human a-Syn LFA kit can successfully detect stage 1 and 2 at 2 / 7, 4 / 7 and increase the detection rate with disease dependent manner; Figure 6B is the bar graph of the scanned and quantified result of FigureSA. The aggregated a-Syn specific LFA kit show high specificity to PD patient and has showed disease dependent detection rate.

[0022] Figure 7. The detection results of using triplex LFA kit for human PD patient. The figure is the conjugated gold particle mixture of conjugated A(3 specific antibody, anti-pTau specific antibody and anti-a-Syn conjugated gold particle. The line is an oligomer specific antibody, p- Tau217 and agg a-Syn (2A4) specific antibody. PD patients can be divided into subtype 1-4 respectively. The co-existence of other disoriented proteins in PD was detected by Lateral Flow Assay.

[0023] Figure 8: Sandwich ELISA results to validate the LFA results in detecting a-syn in plasma samples with different PD's stages (S1-S4). The results shown in Figure 8A and 8B indicate that aggregated a-syn is increased in SI, but it wasn't statistically significant. There was a higher increase in aggregated a-syn in S2 samples compared to SI and normal samples (Control), confirming the stronger signal that LFA detected with S2 samples. Figure 8C shows the strong positive correlation between LFA and ELISA results that indicates the accuracy and reliability of both diagnostic methods, with the advantage of LFA as a more rapid and cost-effective detection method (****P<0.0001), and the correlation between ELISA (X-axis) and LFA results (Y-Axis). Pearson correlation coefficient r = 0.9662. P < 0.001, n=33 Calculated by https: / / www.socscistatistics.com / tests / pearson / default2.aspx

[0024] Figure 9: Triplex Lateral Flow kit development plan. A is the single lateral flow for Oligomer AP, aggregated alpha synuclein and phosphorated tau217 carton, and the B is the carton for triplex lateral flow kit.

[0025] Figure 10: Optimization and characterization of protein aggregation (AP and Alpha Synuclein): Growth and Characterization of distinct A 42 isoforms by ThT and AFM at different time points. A[342 was dissolved in lOOmM NaOH, purified by FPLC using 35 mM Na2HPO4 buffer at pH 11, and incubated in 35 mM Na2HPO4 buffer at pH 7.4 / 27 °C at the indicated concentrations. (A) ThT-monitored (15 pM) growth kinetics (left panel) and (B) AFM images of the dominant isoform at 6h (top panels) and 120 h (bottom panels) of incubation. The 1 pM sample serves as monomer control, the 5 pM after 5 days is predominately fibrillar, and the 20 pM sample at 6 h provides large numbers of uniform oligomers.

[0026] Figure 11: The results of Isothermal Titration Calorimetry (ITC) of antibody affinities. (A- B) Thermodynamic analysis of the binding of a-synuclein to anti-a-synuclein antibodies. (A) Thermodynamic binding parameters of monomeric a-synuclein(40 or 80pM) to mAb 2A4 (0.5pM), 1H5 (lpM), 2C6 (0.25pM) and 2G3 (lpM) at pH 7.4 and 37 °C. This shows an exothermic interaction between a-synuclein FL and 4 antibodies.(B) Thermodynamic binding parameters of monomeric or oligomeric a-synuclein(40) to mAb 2A4 (0.5pM) at pH 7.4 and 37 °C. (C-D) Thermodynamic data of the binding of A to anti- A antibodies. (C-D). Thermodynamic analysis of the binding of ap to anti-A|3 antibodies. C is the ITC binding parameters of A 42 E22W(100pM or 50pM) to mAb 9A9 (0.5pM), 5D12 (0.5pM), MoA 2 (O.lpM) and M4009C (O.lpM) at pH 7.4 and 37 °C. (D) The ITC binding parameters of monomeric and oligomeric A 42 wildtype to mAb 2A4. The parameters included n, Ka, AH, -TAS and AG value determined by One-site model.

[0027] Figure 12. Dot blot result using different antibody to plasma from different neurological diseases. Human plasma was diluted and applied to the dot blot, all three memberane are loaded at the same pattern and then detected with specific antibody respectively . Figure 3A is detected with 9A9 (oligomer A specific antibody), Figure 3B is detected with 2A4 (specific antibody against aggregated alpha synuclein), and Figure 3C is detected with anti-pTau217 (pathological tau isoform). Red arrow indicated that those protein are highly expressed at each individual with different health condition.

[0028] Figure 13. Lateral Flow result for PD patient. Gold nanopaticle was conjugated with 2A4 antibody (specific for aggregated alpha synuclein), and the detection antibody is goat-anti-alpha synuclein aa93-115 (generated by MND). One of each age-gender matched control sample wasselected for the initial testing. Our result demonstrated that the aggregated alpha synuclein shows disease specific trends. In our initial test. It strongly indicates that it is highly possible to use our uniqe antibody to assembly quick home use assay treatment benefit. The right figure is the percentage of each band compared to the positive control. From stage 2-4 are clearly positive.

[0029] Figure 14: Triplex lateral flow for disease differentiation: We will use the established assays to test different plasma from different neurological diseases: Though each disease has its own disease specific pathological protein, most of the protein abundantly existed in our body, and will interact with each other and will contribute to the disease development and / or interfere to treatment benefit. It is pivotal to know the nature of protein level in each neurological disease. We will use our triplex lateral flow kit to test 30 patients of each neurological disease and make informed treatment decisions based on the results as shown in the figure.

[0030] Figure 15: Diagram of lateral flow cassette with preferred size of slots of the cassettes and line distance on the membrane according to a preferred embodiment of the invention. Top part is the membrane with preferred line distance and lower part is the cassette.

[0031] Figure 16: Diagram of a lateral flow assay strip according to an embodiment of the invention. The Lateral Flow Assay includes the following parts: sample pad 150, conjugate release pad containing antibody conjugated gold particles 134, membrane 110 (e.g., nitrocellulose membrane) that contains different antibodies on 112, 114, 116 and an absorbent pad 160.

[0032] Figure 17: Illustrates sample application to the lateral flow assay strip, flow direction of the sample and binding of antigen to conjugated antibody to form an antien-antibody conjugated complex. When the sample is applied to the device, it will flow to the right and then meet gold particles which have conjugated antibody (130) and then antigen (154) will bind to the particles.

[0033] Figure 18: Illustrates antigen-antibody conjugated complex meeting detection antibody and aggregated into detect line. When the antibody binds the target antigen / protein, it will aggregate together to form the detection line (positive 112, 114, 116) and the control line (118).

[0034] Figure 19: Diagram of a modified lateral flow assay with an enlarged and thicker absorbent pad 161, according to a preferred embodiment of the invention. The LF assay 100(a) can detect three or more proteins on the same strip. The enlarged absorbent pad allows more sample loading to increase detection sensitivity for different body fluids.

[0035] Figure 20: Diagram of lateral flow cassette with preferred size slots of the cassettes and line distance on the membrane according to a preferred embodiment of the invention (top portion). Bottom part of the figure shows a comparison between tradeitional LFA (A) versus ourmodified LFA (B). The traditional LFA only detects one target protein and one control (two lines). Our modified LFA can detect four or more lines (three targeted proteins or more and a control line)

[0036] Figure 21: Diagram of modified lateral flow cassette according to a preferred embodiment. Unlike with conventional cassette designs which can only accommodate two test lines (one target protein and a control line), the modifed cassette can accommodate and detect three or more test lines. In addition, the cassette according to a preferred embodiment includes a more open structure at its distal end by eliminating ridges contained in most traditional LFA cassette designs which restrict sample flow and by doing so allows loading of over 300 pl, significantly enhancing sensitivity.

[0037] Fig. 22 illustrates results obained with our LFA assay strip having an enlarged sample pad. The top portion is with a sample load of 40 microliters (40 ml) and the bottom portion is with a sample load of 80 microliters (80 ml). We found that even with the load of 80 ml, the absorbent pad had not become fully saturated.DETAI LED DESCRI PTION OF TH E I NVENTION

[0038] Age-related neurodegenerative diseases (ND) are increasing exponentially as life expectancy is prolonged. This phenomenon is due to effective management of cardiovascular disease and stroke. The increased frequency of ND with aging is a growing social problem that impacts single families in the overall global economy. Currently, there are no cures for any age- related ND.

[0039] Almost all NDs are diagnosed at a late stage when the neurological symptoms are first observed, since there are no early and reliable diagnostic tools available. Fortunately, scientists have made breakthroughs on biomarker discovery for many NDs, including genetic, proteomic and biochemical markers. Some biomarkers are disease-specific or ND-specific feature and will be used clinically in the future. Most NDs are associated with accumulation of misfolded proteins (amyloid) that form different isoforms (toxic molecules), such as Amyloid beta (A ) and Tau for Alzheimer's disease (AD), alpha synuclein for Parkinson's disease (PD), Huntington protein (htt) for Huntington disease (HD), and TDP43 or SOD for Amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease):

[0040] In fact, most ND are considered to have multiple factors associated with development and progression of disease, though one or two molecules are recognized as the pathological factors. It is now accepted that one disease may be associated with more than one pathological process or factor. Unfortunately, most treatments focus on a single factor and do not addressother pathological factors. Therefore, the outcome of single target treatment has rarely reached the therapeutic threshold in clinical trials. This is because of a lack of practical biomarkers to predict the disease course or monitor the treatment benefit. Undoubtedly, reliable and sensitive biomarkers provide the best solution, since the biomarkers can guide treatment and predict the disease status. There is a strong need to develop such tools for clinical use.

[0041] We have surprisingly discovered that there is more than abnormally folded protein in a single patient: an HD patient has in addition to htt protein, oligomers of AP or phosphorylated Tau (p-tau). PD patients also have p-tau and oligomer A in addition to misfolded alpha-synuclein. Further, in AD subjects, some of the blood has more AP or oligomer AP than Tau or p-tau, but some of them have more Tau than Ap.

[0042] We have used this surprising discovery to develop kits for toxic molecule detection, such as oligomer Ap, aggregated alpha synuclein and p-tau217. We further conducted a cross sectional study among AD, PD HD and ALS patients for these biomarkers using our established method to identify a method to differentiate ND and grouped patients into different subtypes for each neurological disease to lay a foundation for precision medicine and personalized medicine.

[0043] Parkinson's disease (PD) is the second most prevalent and rapid growing neurodegenerative disease worldwide. [Palmqvist, S., et al., Cerebrospinal fluid and plasma biomarker trajectories with increasing amyloid deposition in Alzheimer's disease. EMBO Mol Med, 2019. 11(12): p. elll70.] Currently, PD affects approximately 3% of individuals aged 65 to 85 years and up to 5% of people over 85 years. [Akiyama, H., et aL, Inflammation and Alzheimer's disease. Neurobiol Aging, 2000. 21(3): p. 383-421.] PD has been associated with various molecules and different pathological pathways. Consequently, several biomarkers have been proposed as potential indicators for the diagnosis and prognosis of PD. These markers include vesicles that transport different proteins such as alpha synuclein, parkin, as well as extracellular and nucleic acids which contribute to the progress of pathogenic processes in the central nerve system (CNS). [PMID: 30685501] Additionally, MicroRNAs (miRNAs) are also involved in regulating PD-related genes, and any change to certain miRNAs is related to the disease's onset or progress [PMID: 29236052], Among them, Lewy bodies are well accepted as the major pathological hallmark of PD. They are abnormal intracellular inclusions that contain a presynaptic neuronal protein called a- Synuclein (a-Syn).

[0044] Therefore, a-Syn has become the major disease marker for PD. Various studies have shown that the aggregation and misfolding of a-Syn are major events in Parkinson's diseasedevelopment. [Hodges, E., et al., Diagnostic role of tests for T cell receptor (TCR) genes. J Clin Pathol, 2003. 56(1): p. 1-11; Sperling, R.A., et al., Toward defining the preclinical stages of Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimer's and Dementia, 2011. 7(3): p. 280-92.] Monomer a-syn can aggregate into small species to form oligomers that are stabilized by -sheet-like interactions. The aggregation process continues to develop larger sizes of insoluble fibrils. Then, they can form annular, pore-like structures to interact with membranes. Eventually, a-syn is deposited into Lewy bodies. [Gaskin, F., et al., Human antibodies reactive with beta-amyloid protein in Alzheimer's disease. J Exp Med, 1993. 177(4): p. 1181-6.] Among all isoforms, the oligomer a-Syn is considered as the most toxic molecule and associated with disease development. Thus, the method that is specific for oligomer a-Syn will have potential for disease diagnostic and monitor the treatment benefit.

[0045] Unfortunately, there is no such method available, the current diagnostic methods are still based on the clinical features of the disease, which don't allow for a definitive diagnosis of the disease at early stages, nor do they provide an accurate diagnosis as many clinical features could overlap with other neurodegenerative diseases. [Tai, Y., et al., Molecular Mechanisms of T Cells Activation by Dendritic Cells in Autoimmune Diseases. Front Pharmacol, 2018. 9: p. 642; Lin, J. and A. Weiss, T cell receptor signaling. J Cell Sci, 2001. 114(Pt 2): p. 243-4.] Thus, a biomarker that can be accurately detected with high sensitivity and specificity is urgently needed.

[0046] Many studies have focused on detecting a-syn in peripheral tissues, such as blood and saliva, because the detection of phosphorylated and aggregated a-syn in blood and saliva can distinguish PD from healthy control and, more importantly, serve as a biomarker for patients with prodromal stage. [Okkenhaug, K., et al., Phosphoinositide 3-kinase in T cell activation and survival. Biochem Soc Trans, 2004. 32(Pt 2): p. 332-5.] In addition, studies have shown that 31.3% of PD patients will develop cognitive impairment and dementia. [PMID: 20522088] This indicates that a- syn, phosphorylated tau, and aggregated amyloid-beta (A[3) pathologies signal PD. [PMID: 35833836]

[0047] Peripheral blood is extremely appealing for biomarker discovery due to the easy access and less invasive. However, there is currently no effective, early, and convenient detection tool for PD and not to mention the way to differentiate disease subtypes other than the clinical symptoms. PD is associated with multiple factors and has cross interaction with other factors that may cause other neurological diseases. This situation well defined the reason why there is no cure for PD. Thefuture successful treatment of PD will require the development of precision and personalized medicine based on the patient's specific biomarkers.

[0048] We have surprisingly found that of the coexisting of a-Syn, AfJ and p-Tau are very important for the treatment of PD and other neurological diseases, because neurological diseases such as PD can be further differentiated into different subtype based upon the presence of these proteins in the blood. This surprising fact can be used to create detection and diagnostic methods for precision medicine and personalized medicine to increase the treatment efficacy of neurological diseases such as PD. For example, embodiments of the methods described herein can differentiate neurological diseases such as PD into different subtypes, and based upon this differentiation, individual patients can be classified into subgroups. Appropriate treatment methods can then be made based upon this classification.

[0049] Accordingly and in some embodiments, the present invention includes the major isoforms of a-Syn that are associated with neuronal loss and methods of detection of aggregated a-Syn using a lateral flow immunoassay (LFA). Importantly and according to preferred embodiments of the invention, the LFA that can simultaneously test the presence of AfJ oligomers, aggregated a- Syn, and p-tau217 in blood samples. The LFA is a convenient diagnostic, cost-effective and rapid diagnostic approach. The assays and kits of the current invention can be used for single sample tests, and it do not require expertise to use or read the results compared to the other lab techniques.

[0050] LFAs are attractive due to their simplicity, rapid results, and minimal equipment requirements. These assays employ a user-friendly, paper-based platform that allows for the detection of a-syn and other relevant biomarkers directly from blood or saliva samples with ease and speed. [Schroeder, A., et al., The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol, 2006. 7: p. 3.] The convenience of LFAs makes them suitable for point-of-care applications, enabling healthcare providers to perform quick and cost-effective screenings in diverse clinical settings. This accessibility can greatly enhance the early diagnosis of Parkinson's disease by bringing testing closer to patients, reducing the burden of invasive procedures like CSF collection, and offering a more efficient means of monitoring disease progression and response to treatment.

[0051] The kits according to embodiments of the present invention importantly identify dominant subtypes of PD, enabling their categorization into different groups as precision medicine to identify optimal therapeutic strategies for each subtype (personalized medicine).

[0052] Accordingly to some embodiments the present invention is directed to kits and employing proprietary antibodies highly specific for pathological species of alpha synuclein and specifically a multiplex lateral flow assay to differentiate PD into different subtypes to guide clinical treatment.

[0053] Unlike other diseases, progress in treating ND is slow and has been frustrating to patients as well as to the pharmaceutical companies. Billions of dollars of investment have yielded very small returns. This is largely attributed to the misunderstanding of the natural interaction among various amyloid proteins. The treatment approach has traditionally been to focus on one target without considering targeting the various amyloid proteins found in a specific ND. At present, there is no effective, early, and convenient tool to differentiate subtypes of each ND based on analysis of the various amyloid proteins present and which would allow targeting the other pathological factors responsible for the ND. The future for successful treatment of specific NDs will require development of precision, personalized medicine.

[0054] Unlike other disease, treatment progress for neurological disease (ND) is the most frustrated and sentimental experience to most pharmaceutical companies. Since billions of dollars investment turn to be fruitless, due to the zero rewards from it. This is largely attributed to the misunderstanding to the natural interaction among amyloid protein, and the treatment approach is single target approach. One method designed for one subtype of disease applied to all subtypes. The critical issue is due to lack of effective, handy, early, convenient tool to differentiate subtype of each ND and use the proper approach to harness the pathological factor effectively. Thus, precision medicine and personalize medicine is the key factor to treat ND. We have developed several unique methods to significantly increase the chance to benefit patients.

[0055] First, we developed unique antibodies supported by MEGANANO DIAGNOSTIC INC. (MND) that only target toxic molecules (antibody against oligomer Ap, aggregated alpha synuclein, p-tau217) and a developed lateral flow kit as in-home quick screening method for neurological disease.

[0056] Second, we developed a triplex lateral flow kit and use it to identify dominant subtypes of each type of ND and group them into different groups as precision medicine, and then identified the best treatment approaches for each subtype (personalized medicine). Specifically, we used these unique antibodies supported by MEGANANO DIAGNOSTIC INC. (MND) that only target toxic molecules (antibody against oligomer A , aggregated alpha synuclein, p-tau217) to develop a lateral flow kit as an in-home quick screening method for neurological disease. In other words, we used these antibodies to develop a triplex lateral flow kit and use it to identify dominant subtypesof each type of ND and grouped them into different groups as precision medicine. We then identified the best treatment approaches for each subtype (personalized medicine).

[0057] Third, we applied our kits to clinical application for diagnosis and prognosis.

[0058] We have surprisingly discovered that it is possible to detect more than three proteins or isoforms of one protein in a single test strip for different samples obtained from samples such as whole blood, serum, plasma, saliva, or urine. This has been possible due to the unique design of our test kits. In particular, the test strip is placed into a cassette with a longer open slot to accommodate four or more detection lines. This unique configuration means that three or more test antibodies including a control line can be accommodated for testing in a single test strip. To our knowledge, this is the first time that four or more detection lines have been included in a single test strip as part of a diagnostic strip.

[0059] Now turning to the figures, accordingly to the various embodiments of the lateral flow assay and its cassette, Fig. 16 shows a triplex lateral flow assay strip 100 according to an embodiment of the invention. From the proximal to distal end on membrane 100, LF assay 100 includes sample pad 150, conjugate release pad 134 with antibody conjugate(s) 130, followed by three separate test lines which contain different antibodies 112, 114, and 116 and control line 116 with control antibody 120. An absorbent pad 160 is at the distal end of the LF assay strip. The LF assay can simultaneously detect three different proteins (three lines) and control line on the same membrane that is normally designed for one protein detect and a control protein

[0060] Figs. 17-18 illustration operation of the LFA assay strip. Fig. 17 shows sample 152 which contains antigen in sample 154 being loaded onto sample pad 150 of the LF assay strip 100. Antigen 154 binds to antibody conjugate 130 to form an antigen-antibody conjugated complex. Fig. 18 shows the antigen-antibody conjugated complex meeting detection antibody 113 on detection line (detected results of the device). When the antibody binds the target antigen / protein, it will aggregate together to form the detection line (positive 112, 114, 116) and the control line (118).

[0061] Fig. 12 shows a modified lateral flow assay 100(a) according to a preferred embodiment, which includes an enlarged and thicker absorbent pad 161. The LF assay 100(a) as disclosed above can detect three or more proteins on the same strip. Importatnly, the enlarged absorbent pad in this embodiment allows more sample loading to increase detection sensitivity for different body fluids. We surprisingly found that ridges in traditional commercial cassettes restrict the size of sample flow to the adsorbent pad, so that the total volume of the sample application is limited by the size of the pad. We surprisingly found that by removing these ridges, the absorbent pad can beenlarged. By enlarging the absorbent pad, we discovered that an increased volume of sample can be loaded onto the LFA assay strip. With the allowance of an increased sample flow, we were able to obtained increased sensitivity for biomarker detection.

[0062] Fig. 21 shows a novel LFA cassette design 100(a) according to a preferred embodiment of the invention. Unlike with the conventional cassette design slot 139 which can only accommodate two test lines, the modifed cassette can accommodate and detect three test lines. This is made possible by the design of the cassette which includes an enlarged slot 140 which can accomodate four or more lines (3 biomarker test lines and a control test line in the embodiment shown).

[0063] In addition, most LFA cassettes include three ridges in the cassette as the distal end (not shown). We surprisingly found that these ridges limits the size of the aborbent pad which can be used. We surprisingly found that by removing these ridges, sample flow was unrestricted and a larger absorbent pad could be included.

[0064] A conventional cassette design which restricts flow and limits sample loading to 120 .L The novel cassette design according to a preferred embodiment as described herein includes a more open structure by eliminating these ridges which restrict sample flow; this allows loading over 300 .1, significantly enhancing sensitivity.

[0065] One of the problems we also encountered with using conventional cassettes was that due to ridges at the distal end of the cassette which would restrict sample flow, the smaller absorbent pad which could be accomodated by the cassette would quickly become saturated and the sample would start to reverse back into the test strip of the LFA assay. By including a larger open slot to accommodate an enlarged absorbent pad, we could load a larger volume of sample before we would encounter this problem Again, loading a larger volume also increased sensitivity of the assay.

[0066] Fig. 22 illustrates results obained with our LFA assay strip having an enlarged sample pad. The top portion is with a sample load of 40 microliters ("40 ml") and the bottom portion is with a sample load of 80 microliters ml ("80 ml). We found that even with the load of 80 ml, the absorbent pad had not become fully saturated. With accomodation of a larger sample load, we surprisingly found that the sensitivity of the assay was greatly improved.

[0067] Experiments:

[0068] Experiment 1:

[0069] Screening for anti-human a-Syn specific antibodies: Antibody is the most specific diagnostic reagent to protein related disease; thus, it is pivotal to have highly specific antibodyagainst human alpha synuclein (a-Syn). We generated different anti-human alpha synuclein polyclonal and monoclonal antibodies and tested them against a-Syn proteins through ELISA and western blotting assay (Figurel).

[0070] Clone 2A4 has the highest affinity to recombinant a-Syn. To generate a more sensitive and specific detection assay, we choose the antibody with the highest binding property to the targeted protein. Isothermal Titration Calorimetry (ITC) is considered as the most sensitive method to lest the antibody antigen interaction; We accordingly tested those selected four clones and conducted the antibody affinity to recombinant human a-Syn. We found that 2A4 showed the highest affinity to full length a-Syn protein (see Figure 2).

[0071] Clone 2A4 antibody is more specific to the aggregated isoform of a-Syn. We determined the affinity of 2A4 to the oligomeric or the monomeric a-Syn. To verify this property, we have reaggregated recombinant human a-Syn protein, and the conducted different assays to mapping the binding property to different isoforms (Figure 3).

[0072] Experiment 2:

[0073] Aim: Develop a highly sensitive and specific a-Syn specific LFA for aggregated alpha synuclein and use it for early detection of PD. Use the results to determine appropriate courses of treatment and further use the assay to monitor the treatment benefit throughout treatment. This goal is conducted with the University of South Florida (USF) Parkinson’s Disease (PD) center.

[0074] Experimental design: 1. Create standard curve with aggregated recombinant alpha synuclein (ra-Syn) and test them on Later Flow Assay (LFA); 2. Select 10 samples from different stages of PD patient (10 samples of stage 1, 2, 3, and 4) and 20 age and gender match controls. Test those samples at different dilutions (1:2, 1:4 and 1:8) to test them using LFA and calculate the different concentration.

[0075] We may fail to have a clear cutoff value between normal and PD patient because a-Syn protein is an endogenous protein with high level in the blood. The variability among individuals may prevent this to directly transfer into a sectional diagnostic tool. To overcome this potential problem, we will test it on clinical samples with multiple blood draw to see the changes with time dependent to prove the value of this kit for clinical diagnosis and prognosis.

[0076] Validation of the 2A4 conjugated gold particles. To use the antibody for LFA assay, we need to conjugate the 2A4 antibody to the gold particle. The antibody should retain its affinity for downstream application. Our result demonstrated the conjugation has no impact the antibody function (Figure4).

[0077] Sensitivity of the a-Svn specific LFA kit. The most important issue is the sensitivity of the LFA kit. Thus, we generated a standard curve with the aggregated recombinant a-Syn protein for future application (Figures).

[0078] Performance of the a-Svn specific LFA in detecting aggregated a-Svn in PD patients. The purpose of developing this pathological a-Syn isoform LFA is to use for clinical diagnosis, thus we have selected age and gender matched some clinical samples at different stages of PD as well as normal controls (Table 1 and Figure 6).

[0079] Table 1: Gender and age characteristics of PD and healthy subjects' plasma samples used to detect a-syn with LFA.Total 19 14

[0080] The above table illustrated human samples that we used for LFA kit. Only normal, stage1, 2, 3 and 4 were selected for illustration purposes.

[0081] Human aggregated a-Syn specific LFA kit can effectively detected PD samples (Figure 6).

[0082] Development multiplex LFA for Precision and personalized medicine for PD. PD treatment is a very challenged health issue, and there no cure now. The major hurdle is due to the multiple pathological factors related to the disease. In addition, about 30% of PD has AD at the late stage. Since AD is related to highly phosphorylated Tau (p-Tau) and oligomer AP, and those proteins have been tested in PD patients. Therefore, we want to use p-Tau217, oligomer A and aggregated a-Syn to develop a tool to serve as precision and personalized medicine for PD treatment. We have successfully developed the triplex LFA kit and tested our PD samples (See Figure 7).

[0083] Validating human a-Syn specific LFA results with sandwich ELISA. To validate the accuracy of the LFA in detecting aggregated a-syn in plasma samples, sandwich ELISA was performed to detect and quantify the aggregated a-syn in human plasma samples (see Figures ).

[0084] Our results strongly indicated that the aggregated a-Syn LFA assay can be an early, easy, and specific detection assay for PD. Also, the triplex LFA kit can be used to differentiate different subtypes of PD for precision medicine and personalized medicine.

[0085] Experiment 3:

[0086] Lateral Flow Assay Protocol: We developed a LFA protocol which included a Gold Nanoparticles (AuNP) Preparation, antibody conjugation to AuNP, and the Preparation of LFA.

[0087] 1. Gold Nanoparticles (AuNP) Preparation: 21-40 nm gold particles are formulated by reduction reaction of AuCI3 with sodium citrate. The particles will be stored at 4 °C and protected from light.

[0088] 2. Conjugate antibody to AuNP: To prepare 5 ml of the conjugated mixture, add 200 pL of0.1 mg / mL Borate buffer (pH 9.5) to a 15 mL tube. Add 4800 pL of AuNP solution to the buffer and mix by pipetting. Add 26 pL of 2A4 antibody (stock conc= 6.7 mg / mL) to the solution for a final concentration of the antibody 35 pg / mL. Vortex, then incubate for 1 hr at RT while gently shaking (150 rpm). Add 5 mL blocking buffer (10% BSA), then incubate for 30 mins at RT with gentle shaking. Centrifuge at lOOOOxg for 1 hr at RT. Remove the supernatant and resuspend the pellet again with a 5 mL storage buffer. Cover with Aluminum foil and store at 4°C.

[0089] The aggregation method of a-Synuclein:

[0090] The monomeric preparation: recombinant full length a-syn (rFL) and a-syn 15-140 fragments were treated with HFIP lmg / mL for 7 days at 37 °C, then removed HFIP by evaporation in Eppendorf Concentrator Plus with the integrated vacuum pump.

[0091] To prepare oligomer a-syn: HFIP treated a-syn FL incubated with a-syn 15-140 fragments, at final concentrations 40 pM, in an orbital culture shaker and agitated at 250 rpm and 37 °C for 7 days. The aggregated oligomer alpha synuclein was aliquoted into 1 uM and stored at -80C for future applications.

[0092] 3. Preparation of LFA: Pretreat the sample pad (Millipore Sigma. Cat: 32031602) in a buffer solution (1% BSA, 0.1% PEG 5000, 0.05% Tween in PBS), incubate for 30 minutes, then leave overnight to dry at RT. Dip the conjugate pad (Millipore Sigma, Cat: GFCP103000) in the conjugate solution and incubate for 15 minutes, then leave it to dry at 37 °C for 45 minutes. Dilute the antimouse IgG whole molecule (Millipore Sigma, Cat: M7023-2ML) 1:10, then add it to the membrane (Millipore Sigma, Cat: HF075MC100) as a control line (1.4 cm away from the edge and 7 mm from the test line). Dilute detection antibody (MEGANANO BIOTECH INC., stock cone: 12.7 mg / mL) to afinal 1 mg / mL concentration, then add it to the membrane as a test line (7 mm away from the edge). Dry the membrane at 37°C for 1 hr.

[0093] 4. Instructions for building the kit: 1) Place the NC membrane on the adhesive pad. 2)Place the conjugate pad on the adhesive pad so that around 1-2 mm overlaps the NC membrane. 3) Ensure the test line is at least 5 mm from the conjugate pad. 4) Place the sample pad at the beginning of the kit with a 2 mm overlap on the conjugate pad. 5) Place the absorbent pad at the end of the pad with a 2 mm overlap on the NC membrane.

[0094] 5. Statistical methods: All data is collected and analyzed with one-way ANOVA and followed by using Tukey t-test between two groups. The significant level is set at a=0.05.

[0095] Experiment 3:

[0096] Aim: Optimize the conditions for detecting Oligomer A0, p-Tau 217, and aggregated a- synuclein_to use them as tools for precision medicine and personalized medicine for PD. Since 30% of PD patients will develop AD, the oligomer A|3 and p-Tau217 levels will help differentiate PD into different subtypes. Such results will help clinicians to differentiate PD into different subtypes and use that information to develop proper treatment options to individuals to reach the maximum treatment benefit. We will use oligomer AfJ, aggregated a-synuclein, and p-Tau217 protein to create a standard and then check those clinical samples to see the ability to differentiate PD samples into different subtypes. The highly sensitive and specific Triplex LFA for the differentiating of PD can be used for the precision and personalized medicine for PD.

[0097] The ideal situation is to detect all three proteins in the blood samples. There is the possibility that results from duplex and triplex kit do not agree with single plex assay, because those proteins have the nature to interact with each other. Thus, the potential issue will be no correlation among the three protein levels to the disease stages. The triplex is used for directing clinical treatment to avoid the non-responder to single target treatment approach. However, it may not work for all three proteins when we put them together, or the concentration is too low to be detected due to the interaction among them. In this unlikely scenario, we have developed dual assay kits.

[0098] Experimental design: 1. Optimize each individual marker with LFA (oligomer A , p-Tau217 and aggregated a-Syn); 2. Develop duplex LFA kit. 3. Develop Triplex LFA kit (See Fig. 9), and 4. Select 30 samples from different stages of PD patient (10 samples of stage 1, 2, 3, and 4) and 30 age and gender match controls. Test those samples at different dilutions (1:2, 1:4 and 1:8) to test them using LFA and calculate the different concentration.

[0099] Validation with Sandwich ELISA:

[0100] 1. Oligomer AB Sandwich ELISA Assay: Coat a plate 9A9 antibody with 50 pil / well at 5 pg / ml as capture antibody and incubate at 4°C overnight. Then, block with 1.5% BSA (200 pL / well, for 2 hr at room temperature). Add AB oligomers standard or sample to the designated well at 100 pL / well (concentration from 20 ng / mL to 0 ng / mL, followed by incubation at 4°C overnight. Washed twice with IX wash buffer (PBS with 0.05% Tween 20) and added 100 pL / well of biotin- conjugated 9A9 at 1:4000 (1 mg / mL stock solution), incubated for 2 hr at room temperature. Perform two washes, add 100 pL per well of streptavidin-HRP diluted at 1:5000, and incubate for 45 min at room temperature. Wash four times and add 100 ptL / well of TMB substrate; develop for 5 min and then stop the reaction with 100 pL / well of 0.4 N H2SO4. Finally, read at 450 nm.

[0101] 2. P-tau217 ELISA Description: Coat a plate with 50 pL / well at 5 pg / mL as a capture antibody, rabbit antibody- human phospho-Tau217 polyclonal antibody (p-tau217), and the detection antibody is Biotin conjugated p-tau217. All other steps and buffers are the same as the oligomer AB assay.

[0102] 3. Aggregated a-Synuclein ELISA Assay: The method is the same as oligomer AB and p- tau217 assay except the capture antibody is 2A4, the detection antibody is G93-115, and the detection antibody is anti-Goat HRP

[0103] Experiment 4:

[0104] 1. Isoform preparation of different proteins: Most neurological disease is caused by the abnormal accumulation and aggregation of disoriented protein in the body. The major challenge is how to detect these aggregated protein since they are closely associated to the pathological changes. We have produced an aggregated condition for AB and alpha synuclein. We further developed an assay to characterize them (see figure 10)

[0105] Conclusion: Incubation of A 42 at different concentrations and physiological pHs generates distinct amyloid isoforms. These can be harvested and preserved via freezing for analysis of subsequent vaccine selectivity.

[0106] 2. Antibody characterization to amyloid beta, alpha synuclein: To develop diagnostic kits, antibodies are a very useful tool. MEGANANO DIAGNOSTICS INC. (MND) has collaborated with Dr. Cao of the Pathology Department at the University of South Florida (USF) in the past and developed extremely effective antibodies against oligomer AB, aggregated alpha synuclein and p-tau217. The specificity and affinity of these antibodies have been fully characterized using ITC assay.

[0107] Conclusion: The mAb 2A4 has higher association constant (Ka=13.49 M-lxl03) to monomeric than other a-synuclein antibodies. Furthermore, the affinity of 2A4 towards oligomers is demonstrated to be higher compared to its binding affinity towards monomers. Monoclonal antibody 9A9 demonstrates robust binding affinity towards both wildtype and E22W mutant variant, with its binding constants reaching a remarkable 432 M-lxl03 for the oligomeric A 42 PWT. This distinctive capability positions 2A4 and 9A9 as highly promising candidates for the sensitive detection of oligomeric forms of a-synuclein and A .

[0108] 3. Detection and Quantification of each toxic molecules in different neurological disease:It is very important to develop a method or markers that can be used to differentiate or diagnose disease with specificity and sensitivity. We have used accepted pathological markers for AD and PD (oligomer AB, aggregated alpha synuclein and phosphorylated Tau217) to test plasma from different neurological patient and control subjects. (See Fig. 12).

[0109] Conclusion: As can be seen from Figure 12, it is very different to use any marker as a sectional test result to predict the disease status, so a multiple test to monitor the longitutinal change is required for disease diagnosis and treatment prognosis.

[0110] 4. Quantification assay to validate the result of dot blot assay: We developed an anELISA asay for Oligomer AB, aggregated alpha synuclein and p-tau 217.

[0111] Lateral Flow result for PD patient. Gold nanopaticle was conjugated with 2A4 antibody (specific for aggregated alpha synuclein), and the detection antibody is goat-anti-alpha synuclein aa93-115 (generated by MND). One of each age-gender matched control sample was selected for the initial testing. Our results (See Fig. 13) demonstrated that the aggregated alpha synuclein shows disease specific trends. The results strongly indicate that it is highly possible to use our uniqe antibody to assembly quick home use assay treatment benefit. The right figure in figure 13 is the percentage of each band compared to the positive control. From stage 2-4 are clearly positive.

[0112] Experiment 5:

[0113] Antibody screening for lateral flow assay development for oligomer AB, aggregated Alpha Synuclein and p-tau217: The key factor for lateral flow assay are antibody pairs. MND has developed antibodies that are specific for major neurological diseases such as AD, PD. We characterized and found highly effect sets of antibodies for aggregated alpha synuclein, oligomer A and p-tau 217. Goat anti-A , anti-alpha synuclein, anti-ptau217 have been developed and purified with peptide. We first test them by using sandwich ELISA assay to get the sensitivity, thentested them individually on lateral flow assay. Once we determined that the assay works for each specific disease, we will applied all of them into triplex assay by using the mixture protein as the standard to test the ability and sensitivity of the kits.

[0114] Material and Methods:

[0115] la. Oligomer AB preparation and aggregared alpha synuclein preparation: Preparation and incubation of A|342 for comparison of monomers and aggregates: Lyophilized WT A|342 peptide (Genescript., NJ) is dissolved in 100 mM NaOH and injected into a Superdex 75 10 / 300 GL column on an FPLC (Akta Pure, GE) using 35 mM Na2HPO4 running buffer at pH 11. The monomer fraction (typ. at 50-80 pM) is collected and kept on ice. Resulting AB monomer concentrations are measured using optical absorption at 280 nm with 8280 = (1,470 ± 20) M-l cm-1 [74,75]. This AB stock is diluted into ice-cold 35 mM Na2HPO4 at pH 11 at the highest A concentration, and the pH is adjusted to pH 7.4 by the addition of 1.5% (by vol) of IM NaH2PO4. Based on ThT kinetics and AFM imaging, we will incubate AB42 samples at 1, 10, and 20 pM at 1 C to produce different aggregated forms of AB- The preparation and isolation of different isoforms of AB will be conducted using the reported method with minor modifications [Larbi, A., et aL, Dramatic shifts in circulating CD4 but not CD8 T cell subsets in mild Alzheimer's disease. J Alzheimers Dis, 2009. 17(1): p. 91- 103],

[0116] ThT Response and Aggregate Morphology: Measuring Amyloid Growth Kinetics with Thioflavin T. Aliquots are withdrawn at 3, 10, and 20 hours for off-line ThT and aggregate morphology determination. ThT fluorescence spectra are measured offline in a microvolume cuvette using a Fluoromax-4 spectrofluorimeter. ThT stock (5 mM) is mixed with the AB aliquots for a final concentration of 15 pM ThT. ThT fluorescence will be excited at 450 nM and the emission measured between 460 and 550 nm. Amyloid aggregates are imaged in air with an MFP-3D atomic- force microscope (Asylum Research, Santa Barbara, CA) using NSC36 / NoAI (Mikromasch, San Jose, CA) or PFP-FMR-50 (Nanosensor, Neuchatel, Switzerland) silicon tips with nominal tip radii of 10 nm and 7 nm, respectively (typical spring constant and resonance frequency are 2 nN / nm and 70 kHz, respectively, driven at 60-70 kHz in alternating current mode and at a scan rate of 0.5 Hz, acquiring images at 512 x 512-pixel resolution). Raw image data are corrected for image bow and slope. For imaging, 50 pL of the sample solution is diluted up to 100-fold into the same salt / buffer solution used during growth, deposited onto freshly cleaved mica for 3-5 minutes, rinsed with deionized water, and dried with dry nitrogen. Amplitude, phase, and height images are collected for the same sample area.

[0117] lb. ITC Assay for antibody affenitv: Isothermal Titration Calorimetry (ITC): Isothermal Titration Calorimetry (ITC) was used to measure antibody affinities. All ITC measurements were carried out at 37°C with the Nano ITC calorimeter (TA Instruments, New Castle, DE), using 150-200 rpm stirring and 350 s delay between succeeding injections to the sample cell. All the reactants and buffer solutions were prepared with 25 mM HEPES buffer or lxPBS buffer (pH 7.4) and degassed by vacuum pump before each test. The samples cell was filled with antibody solution and the 250 pl syringe was filled with the specific antigen solution (oligomeric A , monomeric AP, oligomeric alpha-synuclein, monomeric alpha-synuclein, or p-Tau). The antigen solution was injected to sample cell by 25 injections of 8 pl. The concentration of reagents varied depending on their affinity. For each antibody at least two titration experiments were done with optimized injection volume. The titration data were processed with the standard NanoAnalyze software using the one-site binding model to provide the Number of binding Site(n), binding constant (Ka), binding enthalpy (AH), Entropy contribution(-TAS), and the Gibbs energy(AG). AG=AH-TAS=-RTInKa, where AG, AH, and-TAS are the changes in free energy, enthalpy, and entropy of binding, respectively.

[0118] Experiment 6:

[0119] Triplex lateral flow for disease differentiation: We will use the established assays to test different plasma from different neurological diseases: Though each disease has its own disease specific pathological protein, most of the protein abundantly existed in our body, and will interact with each other and will contribute to the disease development and / or interfere to treatment benefit. It is pivotal to know the nature of protein level in each neurological disease. We will use our triplex lateral flow kit to test 30 patients of each neurological disease (AD, PD, HD, ALS) and make informed treatment determination based upon the results. (See Fig. 14)

[0120] Materials and Methods: We will use all antibody and antibody pairs using dot blot and sandwich ELISA assay to test different plasma samples from different neurological diseases (N=30 per disease, and 30 normal control). Then we will assemble lateral flow kit with optimized antibody pairs and test plasma sample using single analyte kit, and then test triplex kit with samples that are positive for oligomer A0, aggregated alpha synuclein and p-tau217.

[0121] Dot blot assay: Dilutel.Spil of human plasma in 1ml TBS, then load 200 pl into each designated well with water soaked the 0.45pm nitrocellulose membrane to allow the entire sample to filter through the membrane by gravity flow and wash with 200pl / well TBST by vacuum. Dry the NC membrane under fume hood for lhr. Blocking the membrane with 0.2% l-Block / PBST (Thermo Fisher T2015) for lhr at RT. Dilute primary 1:5000 (Img / ml stock of anti-pTau217, or anti-Abeta 9A9, or anti-alpha-synuclein 2A4) in 0.2% l-block / PBST, incubate lhr at RT then wash 3times with PBST. Add 1:5000 HRP conjugated secondary antibody in 0.2% l-Block / PBST for 45min at RT then wash 4times with PBST. Add ECL substrate, and exposure the membrane by X-ray film.

[0122] Sandwich ELISA: Oligomer A(3 Sandwich ELISA Assay: Coat a plate 9A9 antibody at with 50pl / well at 5pg / ml as capture antibody and incubate at 4°C for overnight. Then block with 1.5% BSA (200pl / well, for 2h at room temperature). Add oligomer AfS standard or sample to the designated well at lOOpl / well (concentration from 20ng / ml to Ong / ml), followed by incubation at 4°C for overnight. Washed twice with IX wash buffer (PBS with 0.05% Tween 20) and add lOOul / well of biotin conjugated 9A9 at 1:4000 (lmg / ml stock solution), incubate for 2h at room temperature. Washed twice and add lOOul / well of 1:5000 diluted streptavidin-HRP, incubate for 45min at room temperature. Washed four times and add lOOul / well of TMB substrate; develop for 5min and then stop reaction with lOOul / well of 0.4N H2SO4. Finally, read at 450nm.

[0123] p-Tau217 ELISA Description: Coat a plate with 50pl / well at 5pg / ml as capture antibody, rabbit antibody- human phospho-Tau217 polyclonal Antibody (pTau217), and the detection antibody is Biotin conjugated pTau217. All other steps and buffers as the same as oligomer AfJ assay.

[0124] Aggregated Alpha-Synuclein ELISA Assay: Method is the same as oligomer A0 and ptau217 assay except the capture antibody is 2A4 and detection antibody is G93-115, the detection antibody is anti-Goat HRP.

[0125] Lateral Flow Assay Protocol: the pressures include a Gold Nanoparticles (AuNP) Preparation and antibody conjugation to AuNP and the Preparation of LFA.

[0126] Gold Nanoparticles (AuNP) Preparation: Using sodium citrate heating AuCI3 to generate 40-100 nm gold particles. The particle will be stored in 4C and protect from light.

[0127] Conjugate antibody to AuNP: To prepare 5 ml of the conjugated mixture, add 213 pl of 0.1 Borate buffer (PH 9.5) to a 15 ml tube.Add 4737 pl of AuNP solution to the buffer and mix by pipetting. Add 50 pl of antibody (stock conc=6.7 mg / ml) to the solution to have a final cone of the antibody 67 pg / ml. Vortex, then incubate for 1 hour at RT while gently shaking (150rpm). Add 5 ml blocking buffer, then incubate for 30 mins at RT+ gentle shaking (150rpm). Centrifuge at 4000xg for 1 hr at RT. Remove the supernatant and resuspend the pellet again with 5 ml storage buffer. Cover with Aluminum foil and store at 4C.

[0128] Preparation of LFA: Pretreat the sample pad (Millipore Sigma. Cat: 32031602) in a buffer solution (1% BSA, 0.1% PEG 5000, 0.05% Tween in PBS), incubate for 30 minutes, then leaveovernight to dry at RT. Dip the conjugate pad (Millipore Sigma, Cat: GFCP103000) in the conjugate solution and incubate for 15 minutes, then leave to dry at 37C for 45 minutes. Dilute the antimouse IgG whole molecule (Millipore Sigma, Cat: M7023-2ML) 1:10, then add it to the membrane (Millipore Sigma, Cat: HF075MC100) as a control line (1.4cm away from the edge and 7mm from the test line). Dilute detection antibody (MEGANANO BIOTECH INC., stock cone: 12.7mg / ml) to a final concentration of lmg / ml, then add it to the membrane as a test line (7mm away from the edge). Dry the membrane at 37C for 1 hour.

[0129] Construction of Kit: Put the conjugate pad on the adhesive pad so that around 1-2 mm overlap on the NC membrane. Ensure the test line is at least 0.4 mm from the conjugate pad. Put the sample pad at the beginning of the kit with a 2mm overlap on the conjugate pad. Put the absorbent pad at the end of the pad with a 2mm overlap on the NC membrane. (See Figure 9)

[0130] Experiment 7:

[0131] Use the triplex lateral flow assay to differentiate and monitor treatment benefit: In this experiment, we will select 20 patients of each ND (AD, PD, HD and ALS), then test their baseline of each protein by using ELISA, and also test with our triplex kit, and then test at monthly under their routine treatment with available clinical drugs or approaches.

[0132] Blood collection: We will draw 5 ml blood with EDTA tube and separate plasma, then aliquot into 0.5 ml screw cap tube and freeze at -80C.

[0133] Sandwich ELISA assay: same procedure as described above.

[0134] Lateral flow assay: the same method as described above.

[0135] Statistical analysis: All initial cross-sectional data will be analyzed using ANOVA followed by post-hoc comparisons between groups (HC, MCI, AD) and gender using Fisher's LSD test. For those measurements which show a significant difference between AD, MCI, and HC cases, we will correlate the values with cognitive performance scores (e.g., MMSE, as well as specific cognitive domain tasks) within the AD population to ascertain if the measurement predicts disease severity. For the longitudinal analysis, we will examine the transition from HC to MCI, as well as MCI to AD. For one-year conversions, we will compare mean-level values using ANOVA, controlling for appropriate covariates (e.g., age and gender).

[0136] The ideal situation ia to detect all three proteins in the blood samples, but it may not work for all of three protein when we put them together, or the concentration is too low to be detected.We will develop dual assay kits as a back up plan to in this scenario.

[0137] Experiment s

[0138] This example describes experiments leading up to the design of a LFA assay strip and cassette having a larger cassette slot to accommodate 4 test lines but also with an enlarged absorbent pad.

[0139] One of the problems we encountered using our newly improved cassette which could accommodate 4 test lines was the limited volume of sample which we could load. We wanted to load more volume to obtain a higher volume of sample to obtain greater sensitivity on the detection lines. However, when we loaded a larger sample volume, we would find that due to the small size of the absorbent pad, the sample liquid would saturate the absorbent pad and start to reverse back up into the test strip.

[0140] To solve this problem, we removed ridges which are contained in most traditional LFA cassettes towards the distal end to create a more open design so as to accommodate a larger absorbent pad. Surprisingly, we found that by including a larger absorbent pad, more sample could be loaded before we encountered the problem of sample reversing back into the test strip. With the ability to now load more sample, we could now also obtain greater sensitivity of the biomarkers on the test strip.

[0141] To illustrate, traditional cassette design having three ridges in the distal part of the cassette restricted sample flow, limiting sample loading to 120 pL. The new design features a more open structure, allowed loading of over 300 pL, significantly enhancing sensitivity.

Claims

Claims1. A method of preparing a detection or diagnostic lateral flow assay (LFA) strip comprising:(a) preparing a test line and placing said test line onto a membrane of a lateral flow assay (LFA) strip, said test line comprising at least three antibodies, each antibody having specificity for at least three separate biomarkers;(b) preparing a control line and adding said control line (control antibody) to the membrane;(c) preparing a gold-antibody conjugate pad by (i) synthesizing a gold nanoparticle (ii) preparing a conjugate solution; (iii) conjugating each of the antibodies of step (a), each antibody having specificity for the at least three separate biomarkers of step (a) to said gold particle, each antibody at a time to form at least three separate conjugate solutions, (iii) mixing said at least three antibody conjugate solutions to form a conjugate solution mixture; (iv) dipping a conjugate pad into said antibody conjugate solution mixture of step (iii) to form a gold-antibody conjugate pad, (vi) blocking the gold-antibody conjugate pad and (v) drying said gold conjugate pad;(d) placing said gold-antibody conjugate pad onto an adhesive part at a proximal or top end of said LFA strip of step (a) by overlaying onto said adhesive part at the proximal or top end;(e) preparing a sample pad and placing said sample pad onto an adhesive end at the proximal or top end of the LFA strip by overlaying onto a top portion of the gold-antibody conjugate pad of step (d) at the proximal or top end of the LFA strip, said sample pad overlapping with the gold-antibody conjugate pad of step (d); and(f) preparing an absorption pad onto the adhesive part at the distal or bottom end of the lateral flow assay (LFA) strip by overlaying said absorption pad onto the membrane of step (a) after the control line at the distal end of the LFA strip prepared in step (b).

2. The LFA strip according to claim 1, wherein said gold-antibody conjugate pad overlaps about 1-2 mm with said membrane at the proximal end of said LFA strip.

3. The LFA strip according to claim 1, wherein said test line is at least 4 mm from said gold- antibody conjugate pad.

4. The LFA strip according to claim 1, wherein said control line of step (b) is prepared using an antimouse IgG or human IgG or any other species to be tested.

5. The LFA strip according to claim 1, wherein said control line of step (b) is added to said membrane about 1.4 cm away from the edge of said membrane and about 7 mm from said test line of step (a).

6. The LFA strip according to claim 1, wherein the three biomarkers are for neurological diseases selected from the group of Amyloid beta (A ) or Tau for Alzheimer's disease (AD), alpha synuclein for Parkinson's disease (PD), Huntington protein (htt) for Huntington disease (HD), and TDP43 or SOD for Amyotrophic lateral sclerosis (ALS).

7. The LFA strip according to claim 1, wherein the three biomarkers are for neurological diseases selected from the group of Huntington protein (htt) for Huntington disease (HD), and TDP43 or SOD for Amyotrophic lateral sclerosis (ALS).

8. The LFA strip according to claim 1, wherein the membrane is a nitrocellulose membrane.

9. A lateral flow assay (LFA) strip prepared according to the method of claim 1.

10. A kit which includes the lateral flow assay (LFA) strip prepared according to claim 1, wherein the kit contains a cassette and buffers for carrying out the process.

11. A method of diagnosis of a neurological disease which includes the steps of placing a sample obtained from a patient onto a lateral flow assay (LFA) strip prepared according to the method of claim 1 and detecting the presence or absence of three separate biomarkers for three separate neurological diseases.

12. A method of diagnosis of a neurological disease accordingly to claim 11, wherein the sample is whole blood, serum, plasma, saliva, or urine.

13. A method of determining a treatment using the LFA strip of claim 1 which includes the steps of placing a sample obtained from a patient onto the lateral flow assay (LFA) strip prepared according to the method of claim 1 and detecting the presence or absence of the three separate biomarkers.

14. A method of determining a treatment accordingly to claim 13, wherein said sample is whole blood, serum, plasma, saliva, or urine.

15. The method of determining a treatment using the LFA strip of claim 14, wherein said lateral flow assay (LFA) strip is part of a multiplex lateral flow assay to differentiate three separate neurological diseases into different subtypes to guide clinical treatment.

16. A method of monitoring a treatment of a neurological disease which includes the steps of placing a sample obtained from a patient onto a lateral flow assay (LFA) strip prepared according to the method of claim 1, detecting the presence or absence of said biomarkers and classifying a patient into a particular subtype in order to monitor treatment.

17. A method of monitoring a treatment of a neurological disease accordingly to claim 16, wherein said sample is whole blood, serum, plasma, saliva, or urine.

18. A method of preparing a lateral flow assay (LFA) strip according to claim 1 which further includes a step (g) of placing said lateral flow assay (LFA) strip into a cassette.

19. A method of preparing a lateral flow assay (LFA) strip according to claim 18, wherein said cassette includes an open slot configured to accommodate four or more detection lines.

20. A method of differentiating a neurological disease into different subtypes for precision medicine purpose comprising; (a) obtaining a sample from a patient to be subtyped; (b) detecting at least three separate biomarkers for at least three separate neurological diseases or for at least three different isoforms of the disease related protein; and (c) subtyping said patient into different subtypes based on the presence or absence of said at least three separate biomarkers detected in step (b).

21. A method of differentiating a neurological disease into different isoform related types for personalized medicine purpose comprising: (a) obtaining a sample from a patient to be subtyped; (b) detecting at least three separate biomarkers for at least three separate neurological diseases or for at least three different isoforms of the disease related protein; and (c) differentiating said neurological disease into different isoform related types based on the presence or absence of said at least three separate biomarkers detected in step (b)22. A method of simultaneously detecting three or more biomarkers or three or more different biomarker isoforms on a single test strip for disease differentiation and personalized medicine.

23. A panel of antibodies against oligomer A , aggregated alpha synuclein and p-Tau.

24. A lateral flow assay (LFA) cassette comprising: (a) a lateral flow assay (LFA) strip comprising four or more detection lines and (b) a cassette which is configured to accommodate four or more detection lines of said LFA strip.

25. The diagnostic assay cassette according to claim 24, wherein the LFA strip of the lateral flow LFA cassette strip further includes an absorbent pad at the distal end which is configured to absorb more sample flow and wherein the cassette is configured to allow unrestricted sample flow to the absorbent pad.

Images