Bacterial vaginosis diagnosis

Abstract

The present invention provides a sialidase activity detection kit or device, comprising: (i) an indicator molecule, the indicator molecule comprising a sialylated peptide and a capture site; (ii) a capture zone, the capture zone comprising a capture molecule; and (iii) a binding molecule, the binding molecule being capable of binding to a desialylated derivative of the indicator molecule. Also provided are a method for using the kit or device, a specific indicator molecule, and a specific binding molecule.

Description

Technical Field

[0001] The present invention relates to detecting the cleavage activity of sialidase and its use in detecting bacterial vaginosis. Specifically, the present invention provides specially designed peptides and indicator molecules that can be used to detect the cleavage activity of sialidase. Various other aspects of the present invention include enzyme detection devices, kits, methods and uses for detecting or measuring the presence of the activity of sialidase capable of cleaving the indicator molecule of the present invention in a test sample. Background Art

[0002] Sialidases (otherwise known as neuraminidase) are glycoside hydrolases that split into two major classes (EC 3.2.1.18 and EC 3.2.1.129, respectively) that cleave exo- or endo(poly)sialic acid. Sialic acid is an N- or O-substituted derivative of neuraminic acid (shown below):

[0003]

[0004] Sialic acid is usually found in glycoproteins and glycolipids (especially gangliosides) in nature. Viruses, bacteria and mammalian sialidases are all known in nature. It is known that elevated levels of bacterial sialidase activity serve as diagnostic markers for bacterial vaginosis (BV; Briselden et al., "Clinical Microbiology (J.Clin.Microbiol.)", 1992, Vol. 30, pp. 663-666). The theory behind this is based on the bacterial imbalance that may occur in the vagina and the transition from a typical healthy environment with high lactobacilli to an anaerobic driven microbiome (Petrova et al., "Front.Physiol., 2015, 6:81). Some anaerobic bacteria secrete sialidases and destroy the natural mucus barrier in the vagina. Nevertheless, the exact mechanism for BV remains to be fully established. Patients with symptomatic BV may experience significant discomfort, vaginal discharge, unpleasant odor, and a more general sense of lack of control over their vaginal health (Bilardi et al., PLoS One, 2013, Vol. 8, e74378). Treatment options typically involve topical or oral antibiotics. Topical prebiotic treatments are also available over-the-counter (e.g., VH ) and pH gel (e.g., balancing active BV). BV may be refractory, and patients may relapse after antibiotic treatment (Bilardi et al., Public Library of Science Comprehensive, 2013, Vol. 8, e74378). Some refractory cases are thought to be associated with the formation of biofilms in the vagina (Verstraelen and Swidsinski, Curr. Opin. Infect. Dis., 2013, 26: 86-89). Risks should not be taken. The presence of BV is more sensitive to sexually transmitted infections (including HIV) and the associated risk of premature birth. The risk of premature birth is thought to be associated with premature weakening of the cervical mucus barrier (Lewis et al., J. Biol. Chem., 2013, Vol. 288, pp. 12067-12079). Currently in the clinic, BV testing is based on vaginal sample smears, which are sent for analysis in the laboratory. Analytical methods include searching for clue cells, potassium hydroxide (KOH) odor testing, or applying set criteria such as Nugent scoring (Nash, Jungmann and Gubert, BMJ Learning, 2015, 1-29). The use of rapid tests at the point of care will benefit patients by delivering quick answers, allowing patients to immediately follow up on treatment plans, and speeding up the entire process for delivering results. Likewise, there will be potential benefits for healthcare providers by avoiding outsourcing to laboratories and avoiding the resource and time costs involved and reducing patient time to a single session in the first place.

[0005] Two main types of products for diagnosing BV are currently available in the United States and Europe. One group of products is based on a color-changing swab that measures pH. Another group of products is also colorimetric and is based on sialidase activity measurement. pH-based products are available over-the-counter at point-of-care locations and include (sold by Common Sense) and (Sold by Canesten). The main product currently sold based on sialidase detection is BV Blue (sold by Sekisui Diagnostics and Gryphus Diagnostics). BV Blue is a swab-based test; a sample swab is placed in an indicator solution, which will change color to indicate the presence of sialidase activity in the sample. The test takes about 15 minutes to run.

[0006] pH-based tests lack accuracy because various conditions may cause changes in the local pH of the vagina (e.g., candidiasis and trichomoniasis and BV). Known tests that detect sialidase activity, such as BV Blue, lack sensitivity. Summary of the invention

[0007] The present invention arises from an attempt to improve the sensitivity and / or specificity of sialidase activity detection. The inventors have now specifically designed peptides and corresponding indicator molecules for detecting the cleavage activity of sialidase. As further described herein, each peptide is conjugated to a sialylated group via a galactosyl group (and therefore acts as a substrate for sialidase), wherein the peptide architecture is specifically designed so that once the sialylated group is cleaved from the peptide by one or more sialidases present in the sample, the desialylated derivative of the peptide is recognized and bound by a specific binding molecule (e.g., an antibody). In a preferred embodiment, the peptide incorporates one or more amino acids that enhance the resistance of the peptide to protease cleavage.

[0008] Thus, in one aspect, the invention provides a peptide comprising a sequence according to the formula:

[0009] X 1 -X 2 -X 3 [Gal-Sial]-X 4 -X 5

[0010] (Formula I) (SEQ ID NO: 9)

[0011] in:

[0012] (i) Sial is a sialylation group;

[0013] (ii)X 3 is a natural or unnatural amino acid that includes a glycosyl acceptor group; and

[0014] (iii)X 1 , X 2 , X 4 and X 5 are independently selected from any amino acid, provided that X 1 , X 2 , X 4 and X 5 At least one of D - amino acids and / or non-standard amino acids or unnatural amino acids.

[0015] The peptide may consist essentially of, or consist of, the sequence of Formula I.

[0016] According to all aspects of the invention, a "sialylated group" means a sialic acid substituent, wherein the sialic acid is an N- or O-substituted derivative of neuraminic acid (eg, N-acetylneuraminic acid; referred to as "Neu5Ac" or "NANA").

[0017] According to all aspects of the present invention, "Gal" means a galactosyl substituent. In a preferred embodiment, the galactosyl substituent is a group having the following structure:

[0018]

[0019] In a particular embodiment, the galactosyl substituent is a group of beta-galactose. In such embodiments, the galactosyl substituent is O-linked to the sialylated group. The present invention encompasses all possible regioisomers. The skilled artisan will also appreciate that other sugars may be used in place of the galactosyl group, such as, for example, groups of glucose, fructose, lactose, maltose, and sucrose. Therefore, references herein to "galactosyl substituents" should be interpreted accordingly.

[0020] As shown above, the -Gal-Sial group is 3 Conjugation, where X 3 is a natural or unnatural amino acid including a glycosyl acceptor group. 3 The side chain of the amino acid represented by includes a nucleophilic substituent (i.e., a glycosyl donor) that forms a bond with the Gal substituent. For example, the nucleophilic substituent can be a hydroxyl or amine substituent. Thus, in certain embodiments, X 3 X may be selected from serine (Ser), threonine (Thr), tyrosine (Tyr), hydroxylysine (Hyl), hydroxyproline (Hyp), asparagine (Asn), arginine (Arg) and phosphoserine (SEP). 3 It's Ser.

[0021] It will be understood from Formula I that the -Gal-Sial group is only bonded to X 3 (as indicated by the square brackets in Formula I). 3 Bridge to X 4 For the avoidance of doubt, an alternative and equivalent formula (Ia) is shown below:

[0022]

[0023] Thus, the -Gal-Sial group branches from the peptide backbone. Thus, the -Gal substituent is centrally positioned in the structure such that it is flanked by amino acids X 1 , X 2 , X 4 and X 5Thus, as further described herein, binding molecules (e.g., antibodies) can be generated that have very high affinity and specificity for the desialylated derivatives of each peptide, while minimizing the generation of peripheral binding molecules (e.g., antibodies) that lack interaction with the Gal substituent.

[0024] X 1 , X 2 , X 4 and X 5 are independently selected from any amino acids (natural and unnatural), provided that X 1 , X 2 , X 4 and X 5 At least one of D -amino acids and / or (i) non-standard amino acids or (ii) unnatural amino acids. When generating binding molecules as antibodies with high affinity for desialylated derivatives of peptides, non-standard and unnatural amino acids increase sequence diversity and help promote an immune response in a host organism. D -amino acids help reduce sensitivity to proteases. In certain embodiments, X 1 , X 2 , X 4 and X 5 At least two, three or all four of D - amino acids and / or non-standard amino acids or unnatural amino acids.

[0025] The skilled person will be very familiar with the terms "natural" and "non-natural" amino acids. For the avoidance of doubt, "natural" amino acids are amino acids found in nature, and include both standard and non-standard amino acids. As is known in the art, standard amino acids are amino acids that are directly encoded by triplet codons in the universal genetic code. In contrast, non-standard amino acids are amino acids that are found in nature but are not directly encoded by triplet codons in the universal genetic code. "Non-natural" amino acids are amino acids that are not found in nature but exist only through artificial synthesis. As used herein, amino acids without the prefix " D "or" L " is used to refer to the D and L stereoisomers. D "or" L The " prefix indicates the specific stereoisomer being described.

[0026] A "desialylated derivative" of a peptide or indicator molecule is a product resulting from the cleavage of a sialylated group from the peptide or indicator molecule, typically by a sialidase. Thus, a "desialylated derivative" of a peptide or indicator molecule typically differs only or substantially only from a sialylated peptide or indicator molecule insofar as the sialylated group is not present. Thus, any reference herein to a "desialylated derivative" of a peptide or indicator molecule should be understood to mean a desialylated form of the peptide or indicator molecule.

[0027] Further increasing the diversity of peptide sequence topology is beneficial, as it has been shown that this further facilitates the generation of binding molecules with very high affinity and specificity for the desialylated derivatives of each peptide. This is particularly relevant for binding molecules that are antibodies. Thus, in a preferred embodiment, X 1 , X 2 , X 4 and X 5 In some embodiments, X 1 , X 2 , X 4 and X 5 At least one of X is a hydrophobic amino acid. 1 , X 2 , X 4 and X 5 At least one of the amino acids may be selected from alanine (Ala), valine (Val), isoleucine (Ile), leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), and proline (Pro), D -alanine (DAla), 1-aminocyclohexane-carboxylic acid (Cyc), β-alanine (βAla), norleucine (Nle), norvaline (Nva), 2'-(aminomethyl)biphenyl-2-carboxylic acid (Bip) and cyclohexylalanine (Cha). Preferably, the L-stereoisomers of Nle, Nva and Cha are used. In another embodiment, X 1 , X 2 , X 4 and X 5 At least two or three of the amino acids are hydrophobic amino acids. 1 , X 2 , X 4 and X 5 All four of can be hydrophobic amino acids, provided that X 1 , X 2 , X 4 and X 5 At least one of D-amino acids and / or non-standard amino acids or non-natural amino acids. In certain embodiments, at least one hydrophobic amino acid is Ala. In other embodiments, X 1 and X 2 In a preferred embodiment, Ala is D Ala or βAla. In another embodiment, each hydrophobic amino acid is selected from D Ala, βAla, Ile, Val, Pro, Nle, Nva, Cyc, Cha and Bip. Additionally or alternatively, in some embodiments, X 1 , X 2 , X 4 and X 5 At least one of X is a charged amino acid. 1 , X 2 , X 4 and X 5 At least one of X can be selected from arginine (Arg), histidine (His), lysine (Lys), aspartic acid (Asp), glutamic acid (Glu), ornithine (Orn) and phosphoserine (SEP). Preferably, the L-stereoisomer of Orn and the D-stereoisomer of Asp are used. In another embodiment, X 1 , X 2 , X 4 and X 5 At least two or three of them are charged amino acids. 1 , X 2 , X 4 and X 5 All four of them can be charged amino acids, provided that X 1 , X 2 , X 4 and X 5 At least one of D -amino acids and / or non-standard amino acids or non-natural amino acids. In a specific embodiment, each charged amino acid is selected from Arg, Glu, D Asp, L Additionally or alternatively, in some embodiments, X 1 , X 2 , X 4 and X 5 At least one of X is a polar amino acid. 1 , X 2 , X 4 and X 5At least one of X can be selected from serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn), glutamine (Gln) and cysteine ​​(Cys). Preferably, the D-stereoisomer of Ser is used. In another embodiment, X 1 , X 2 , X 4 and X 5 At least two or three of them are polar amino acids. 1 , X 2 , X 4 and X 5 All four of can be polar amino acids, provided that X 1 , X 2 , X 4 and X 5 At least one of D -amino acids and / or non-standard amino acids or non-natural amino acids. In a specific embodiment, each polar amino acid is selected from L Ser, D Ser and Thr. In a preferred embodiment, in order to maximize the diversity of peptide sequence topology, X 1 , X 2 , X 4 and X 5 It is a combination of hydrophobic, charged and polar amino acids. Pro and βAla are particularly useful in the peptides of the present invention because they act as "hinge groups", allowing the overall structure to fold with additional degrees of freedom, thereby further increasing the diversity of peptide sequence topology.

[0028] In a preferred embodiment, the molecular weight of the peptide is less than 5000 Daltons. This helps stimulate an immune response in the host organism to produce antibodies that bind to the desialylated derivative of the peptide. Therefore, in a specific embodiment, the length of the peptide can be 5-20 amino acids, more preferably 9-20 amino acids in length.

[0029] Thus, in a related aspect, the invention provides a peptide comprising a sequence according to the formula:

[0030] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 [Gal-Sial]-X 13 -X 14-X 15 -X 16 -X 17 -X 18 -X 19 -X 20

[0031] (Formula II)(SEQ ID NO:10)

[0032] in:

[0033] Sial is the sialylation group;

[0034] X 1 does not exist or is Thr;

[0035] X 2 Does not exist or D Ala;

[0036] X 3 does not exist or is Nle;

[0037] X 4 Not present or Glu;

[0038] X 5 Does not exist or D Ala;

[0039] X 6 does not exist or is Arg;

[0040] X 7 Not present or selected from Glu, Arg, Ser, Nva, βAla;

[0041] X 8 Does not exist or is selected from D Ser, D Ala, SEP, Cyc;

[0042] X 9 Not present or selected from Nva, BIP, D Ala, βAla, Orn;

[0043] X 10 Selected from Cyc, Ser, Ile, D Ala, D Ser;

[0044] X 11 Selected from D Ala, Pro, Orn, Nle;

[0045] X 12 Selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or SEP;

[0046] X 13 Selected from D Ala, BIP, βAla;

[0047] X 14 Selected from Arg, D Asp, Nle, Orn, Nva;

[0048] X 15 Not present or selected from Phe, BIP, Ser, Glu, D Ala, D Ser;

[0049] X 16 Does not exist or is selected from D Ser, Glu;

[0050] X 17 Not present or selected from Val, Ser, Thr;

[0051] X 18 Does not exist or is Cha;

[0052] X 19 Does not exist or D Ser;

[0053] X 20 Does not exist or is Val.

[0054] It will be understood from Formula II that the -Gal-Sial group is bonded only to X 12 (As indicated by the square brackets in Formula II). 12 Bridge to X 13 For the avoidance of doubt, an alternative and equivalent formula (IIa) is shown below:

[0055]

[0056] Thus, the -Gal-Sial group branches from the peptide backbone. Thus, the -Gal substituent is positioned so that it is flanked by amino acids X 10-11 and X 13-14 and amino acid X 1-9 and X 15-20 (if present). Thus, as further described herein, binding molecules (e.g., antibodies) with very high affinity and specificity for each desialylated derivative of the peptide can be generated, while minimizing the generation of peripheral binding molecules (e.g., antibodies) that lack interaction with the Gal substituent.

[0057] In a preferred embodiment, X 12 It's Ser.

[0058] In particular embodiments, according to all aspects of the invention, the peptide comprises, consists essentially of, or consists of the following sequence:

[0059] (i) Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg (SEQ ID NO: 11);

[0060] (ii)Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-Phe- D Ser-Val (SEQ ID NO: 12);

[0061] (iii) Arg- D Ala-Bip-Ser-Pro-Ser[Gal-Sial]- D Ala- D Asp-Ser (SEQ ID NO: 13);

[0062] (iv)Ser-Ser(PO 3 )- D Ala-Ile-Orn-Ser[Gal-Sial]- D Ala-Nle-Glu (SEQ ID NO: 14);

[0063] (v) D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal-Sial]-Bip-Orn- D Ala-Glu-Ser (SEQ ID NO: 15); or

[0064] (vi)Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal-Sial]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Val (SEQ ID NO: 16);

[0065] Among them, Cyc- D Ala-Ser[Gal-Sial]-D Ala-Arg is particularly preferred.

[0066] These specific peptides have been found by the inventors to be particularly useful for detecting sialidase activity and are further described in the Examples section.

[0067] Preferably, Nle, Nva, Orn and / or Cha (when present) are respectively L Nle, L Nva, L Orn and L Cha.

[0068] In some embodiments, the peptide is biased towards cleavage by one or more specific sialidases by constructing the peptide accordingly, so that the amino acid located at the flanking of the -Gal Sial group ensures the specificity and sensitivity of the cleavage. Therefore, the specific sialidase can have different affinities for the peptide. This allows the present invention to be used to detect specific sialidase activity in a test sample. As described herein, the peptide of the present invention is particularly useful for detecting bacterial vaginosis. Therefore, in a preferred embodiment, one or more specific sialidases are of bacterial origin. Specifically, one or more specific sialidases can be from Prevotella species, Bacteroides species and / or Mobiluncus species and / or Gardnerella vaginalis.

[0069] In a related aspect, the peptide of the present invention is incorporated into an indicator molecule for detecting the presence of the cleavage activity of sialidase in a test sample. The indicator molecule is a core component of an enzyme detection device, an enzyme detection kit, an enzyme detection material composition, and a method for detecting the presence of the cleavage activity of sialidase in a test sample as further described herein. Based on the inventor's previous disclosure of the general concept of indicator molecules for detecting the cleavage activity of an enzyme and binding molecules specifically bound to cleavage products, in the context of detecting sialidase activity to diagnose BV, the inventor has specially developed an indicator molecule described herein (see PCT / GB2014 / 053171, which is incorporated herein by reference). Therefore, the present invention provides an indicator molecule for detecting the presence of the cleavage activity of sialidase in a test sample, the indicator molecule comprising:

[0070] a) a peptide of the invention as described herein; and

[0071] b) A capture site which remains intact after cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample.

[0072] Capture site is the discrete region (as described in more detail below) of the combination of the mediation indicator molecule of indicator molecule and the capture molecule present in the capture zone.Therefore, capture site is the part in the indicator molecule responsible for retaining or positioning the indicator molecule in the capture zone.After the cracking of the indicator molecule, the capture site can remain intact or substantially intact so that the site is still recognized and combined by the capture molecule present in the capture zone of the device.In these cases, both the complete indicator molecule and the indicator molecule include the part of the capture site after the cracking will be combined with the capture molecule in the capture zone.The capture site can include any suitable molecule, for example, biotin molecule or oxime part.The capture site can be located at the N-terminal or C-terminal of the peptide.The key to the effectiveness of the indicator molecule is to be fixed by the interaction between the capture site and the capture molecule at the capture zone, and to be combined simultaneously by binding molecules after the cracking occurs.

[0073] Therefore, the complete indicator molecule can include a capture site portion connected to the peptide of the present invention, and the peptide includes a first sialylated group. The cleavage of the indicator molecule by sialidase produces a fragment comprising a desialylated form of the capture site portion and the peptide. The fragment (i.e., the cracked indicator molecule) is structurally different from the complete indicator molecule because the fragment lacks the first sialylated group present in the complete indicator molecule. This lack of the first sialylated group reveals or exposes an epitope (novel binding site) that does not exist, is hidden or is difficult to access in the complete indicator molecule on the fragment. Therefore, it can be considered that the complete indicator molecule includes a concealed epitope. As discussed below, the binding molecule of the device, test kit, material composition and method of the present invention is specific to this epitope, and therefore only or preferentially combines with the indicator fragment compared with the complete indicator.

[0074] The peptide and capture site of indicator molecule can be associated by any means known to those skilled in the art. In a preferred embodiment, peptide and capture site can be associated by direct covalent connection. Peptide and capture site can be closely adjacent or can be separated by joint or spacer (for example, polyethylene glycol (PEG) part). In certain embodiments, peptide is connected with biotin group at its N-terminal or C-terminal by the joint including polyethylene glycol part, consisting essentially of it or consisting of it. One or more other amino acids can be present at the N-terminal or C-terminal of peptide, to connect joint group (such as PEG) to peptide. In certain embodiments, one or more other amino acids can include Asp. PEG part can further include functional group (for example, amine group, optionally alkylamine group) that can react with terminal residue and / or capture site (for example, biotin molecule) of peptide at one end or both ends of PEG part to connect peptide and capture site to each other during synthesis. Therefore, in a specific embodiment, indicator molecule includes following structure, consists essentially of it or consists of it:

[0075] (i) Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin (SEQ ID NO: 11-PEG-biotin);

[0076] (ii) Biotin-PEG-Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-Phe- D Ser-Val(Biotin-PEG-Asp-SEQ ID NO: 12);

[0077] (iii) Biotin-PEG-Asp-Arg- D Ala-BIP-Ser-Pro-Ser[Gal-Sial]- D Ala- D Asp-Ser (biotin-PEG-Asp-SEQ ID NO: 13);

[0078] (iv) Biotin-PEG-Asp-Ser-SEP- D Ala-Ile-Orn-Ser[Gal-Sial]- D Ala-Nle-Glu (biotin-PEG-Asp-SEQ ID NO: 14);

[0079] (v) Biotin-PEG-Asp- D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal-Sial]-BIP-Orn- D Ala-Glu-Ser(Biotin-PEG-Asp-SEQ ID NO: 15); or

[0080] (vi) Biotin-PEG-Asp-Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal-Sial]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Val(Biotin-PEG-Asp-SEQ ID NO: 16);

[0081] Among them, Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin (SEQ ID NO: 11-PEG-biotin) is particularly preferred.

[0082] In the context of the present invention, the indicator molecule can bind to the capture molecule in the capture zone (via the capture site) with relatively high affinity (as described in more detail below). In some embodiments, the dissociation constant (k d ) will be relatively low, and preferably between 0M and 1×10 -7 M (depending on the sensitivity required for the assay). In certain embodiments of the invention, the dissociation constant of the indicator molecule will be between 1×10 -15 M and 1×10 -9 Between M.

[0083] In certain embodiments of the invention, this binding interaction can be achieved due to direct binding of the capture site of the indicator molecule to the capture molecule present in the capture zone. In this context, direct binding means the binding of the indicator molecule (through the capture site) to the capture molecule without any intermediates.

[0084] In a preferred embodiment of the present invention, the capture molecule existing in the capture site of the indicator molecule and the capture zone is in conjunction with two right halves. In this context, in conjunction with two molecules or entities that can be combined with each other. In certain embodiments of the present invention, the binding interaction is specific so that each member in conjunction with right can only be in conjunction with its corresponding partner or a limited number of binding partners. In addition, as described in detail above, preferably in conjunction with showing relatively high affinity. In conjunction with can be found in nature in conjunction with or can be artificially produced interaction molecules or entity pairs.

[0085] In some embodiments of the present invention, the capture site of the indicator molecule and the capture molecule are two halves of a binding pair, wherein the binding pair is selected from the following: - antigen and antibody or its antigen binding fragment; biotin and avidin, streptavidin, neutravidin or captavidin; immunoglobulin (or its appropriate domain) and protein A or G; carbohydrates and lectins; complementary nucleotide sequences; ligands and receptor molecules; hormones and hormone binding proteins; enzyme cofactors and enzymes; enzyme inhibitors and enzymes; cellulose binding domains and cellulose fibers; fixed aminophenylboronic acid and molecules carrying cis-diols; and xyloglucan and cellulose fibers and their analogs, derivatives and fragments.

[0086] In a specific embodiment of the invention, the binding pair consists of biotin and streptavidin. In a further embodiment of the invention, the capture site of the indicator molecule comprises an epitope and the capture molecule comprises an antibody that specifically binds to the epitope present at the first capture site. In the context of the present invention, the term antibody encompasses natural immunoglobulins from any species, chimeric antibodies, humanized antibodies, F(ab') 2 In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies. In some embodiments, the capture molecules include antibodies.

[0087] Another core component of the enzyme detection device, enzyme detection kit, enzyme detection material composition and method for detecting the presence of sialidase cleavage activity in a test sample described further herein is a binding molecule. The binding molecule is designed to specifically bind to the desialylated derivative of the indicator molecule formed after the sialylated group is cleaved from the indicator molecule by the sialidase activity present in the sample. The formation of the desialylated derivative reveals a novel binding site that can be specifically bound to the binding molecule. Therefore, in a preferred embodiment, the binding molecule cannot bind to the indicator molecule (at any perceptible or detectable level) unless and until the sialylated group has been cleaved from the indicator molecule. In an alternative embodiment, the binding molecule preferentially binds to the desialylated derivative relative to the sialylated peptide or indicator molecule. Therefore, compared with the sialylated form, the binding molecule has a higher affinity for the desialylated derivative.

[0088] Alternatively it is observed that in a preferred embodiment the binding molecule is able to bind specifically to the desialylated indicator molecule (fragment) and is unable to bind (at any appreciable or detectable level) to the intact (sialylated) indicator molecule.

[0089] The skilled artisan is well able to determine whether a particular binding molecule preferentially binds to the desialylated derivative relative to the sialylated form. This can be expressed, for example, as a relative dissociation constant. For example, in a particular embodiment, the dissociation constant for the binding interaction between the binding molecule and the desialylated derivative can be 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 500, 1000 times (or more) lower than the dissociation constant for the binding interaction between the binding molecule and the sialylated form.

[0090] In a specific embodiment, the binding molecule comprises an antibody. For the avoidance of doubt, the term antibody encompasses natural immunoglobulins from any species, chimeric antibodies, humanized antibodies, F(ab') 2 Fragments, Fab fragments, Fv fragments, sFv fragments and highly related molecules, such as molecules based on antibody domains that retain specific binding affinity (e.g., single domain antibodies). Antibodies can be monoclonal or polyclonal. The inventors have produced antibodies that only recognize desialylated derivatives of indicator molecules, and therefore unless and until cleavage of the sialylated group has occurred, the antibodies will not (to any significant extent) bind to the indicator molecule. Antibodies can be produced according to techniques known in the art. This may rely on desialylated derivatives of indicator molecules used when immunizing animals (e.g., sheep, rabbits or goats). Alternatively, animals can be immunized with desialylated peptides (i.e., without the need to connect capture sites). Polyclonal antibodies can be isolated from serum and affinity purified. Monoclonal antibodies can be produced using well-known and characteristic hybridoma technology.

[0091] Therefore, in a related aspect, the present invention also provides an antibody that can specifically bind to a desialylated derivative of a peptide of the present invention as defined herein or an indicator molecule of the present invention as defined herein. As will be understood by those skilled in the art based on the disclosure herein relative to a sialylated peptide or an indicator molecule, an antibody can preferentially bind to a desialylated derivative. Therefore, the antibody has a higher affinity for the desialylated derivative than the sialylated form. The technician can well determine whether a specific antibody preferentially binds to the desialylated derivative relative to the sialylated form. This can be expressed, for example, as a relative dissociation constant. For example, in a specific embodiment, the dissociation constant of the binding interaction between the antibody and the desialylated derivative can be 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 100 times, 200 times, 500 times, 1000 times (or more) lower than the dissociation constant of the binding interaction between the antibody and the sialylated form. In a specific embodiment, the antibody cannot bind to a sialylated peptide or an indicator molecule. That is, the antibody can only bind to the desialylated derivative after cleavage of the sialylation group has occurred.

[0092] Thus, in a preferred embodiment, the antibody can specifically bind to the following epitopes:

[0093] Cyc- D Ala-Ser[Gal]- DAla-Arg-PEG-biotin (SEQ ID NO: 11 - desialylated form of PEG-biotin); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule, Cyc- D Ala-Ser[Gal]- D Ala-Arg (desialylated form of SEQ ID NO: 11). Preferably, the antibody is incapable of binding (at any appreciable or detectable level) to the sialylated form of this molecule (Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg, i.e., SEQ ID NO: 11) binds.

[0094] In another embodiment, an antibody is provided that specifically binds to the following epitope:

[0095] Biotin-PEG-Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal]- D Ala-Arg-Phe- D Ser-Val (a desialylated form of biotin-PEG-Asp-SEQ ID NO: 12); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule. Preferably, the antibody is unable to bind (at any appreciable or detectable level) to a sialylated form of this molecule.

[0096] In further embodiments, antibodies are provided that specifically bind to the following epitopes:

[0097] Biotin-PEG-Asp-Arg- D Ala-BIP-Ser-Pro-Ser[Gal]- D Ala- D Asp-Ser (a desialylated form of biotin-PEG-Asp-SEQ ID NO: 13); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule. Preferably, the antibody is unable to bind (at any appreciable or detectable level) to the sialylated form of this molecule.

[0098] In further embodiments, antibodies are provided that specifically bind to the following epitopes:

[0099] Biotin-PEG-Asp-Ser-SEP- D Ala-Ile-Orn-Ser[Gal]- DAla-Nle-Glu (a desialylated form of biotin-PEG-Asp-SEQ ID NO: 14); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule. Preferably, the antibody is unable to bind (at any appreciable or detectable level) to the sialylated form of this molecule.

[0100] In further embodiments, antibodies are provided that specifically bind to the following epitopes:

[0101] Biotin-PEG-Asp- D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal]-BIP-Orn- D Ala-Glu-Ser (a desialylated form of biotin-PEG-Asp-SEQ ID NO: 15); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule. Preferably, the antibody is unable to bind (at any appreciable or detectable level) to the sialylated form of this molecule.

[0102] In further embodiments, antibodies are provided that specifically bind to the following epitopes:

[0103] Biotin-PEG-Asp-Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Val (a desialylated form of biotin-PEG-Asp-SEQ ID NO: 16); specifically an epitope present in this molecule, more specifically an epitope present in the Gal peptide portion of this molecule. Preferably, the antibody is unable to bind (at any appreciable or detectable level) to a sialylated form of this molecule.

[0104] In another embodiment, an antibody is provided, which has a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and / or the light chain CDR3 has SEQ ID NO: 6.

[0105] The antibody may have a heavy chain with SEQ ID NO:7 and / or a light chain with SEQ ID NO:8.

[0106] Preferably, the antibody can bind to the molecule Cyc- D Ala-Ser[Gal]- D Preferably, the antibody does not bind (at any appreciable or detectable level) to the epitope present in Ala-Arg. D Ala-Ser[Gal-Sial]- D Ala-Arg binding.

[0107] For the (desialylated) molecule Cyc- D Ala-Ser[Gal]- D Ala-Arg-PEG-oxime (SEQ ID NO: 11 - desialylated form of PEG-oxime), K of the antibody D It may be less than 100 nM, preferably less than 80, 60, 50, 40, 30, 20 or 10 nM, for example about 7.9 nM. For this molecule, its K on Can be about 54080M -1 s -1 ;

[0108] And K off About 0.000427s -1 .

[0109] The binding molecule can be directly or indirectly conjugated to the reporter molecule (i.e., labeled with it) to allow detection of the binding molecule and the indicator molecule. The reporter molecule can be any substance or part suitable for detection by any means available to those skilled in the art. Therefore, the reporter molecule is generally capable of generating or producing a signal. In certain embodiments of the present invention, the reporter molecule is selected from the following:-gold particles; chromophores; luminescent compounds; fluorescent molecules; radioactive compounds; visible compounds; liposomes or other vesicles containing signal-generating substances; electroactive species; or a combination of enzymes and their substrates. Suitable enzyme-substrate combinations used as reporting parts can be enzyme alkaline phosphatase and substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate. In a specific embodiment of the present invention, the reporter molecule is a gold particle.

[0110] In the present invention, it is also envisioned to indirectly label the binding molecule with a reporter molecule. Therefore, the reporter molecule can be connected to another binding molecule, which in turn binds to the binding molecule to provide a label. This indirect binding can be mediated by an adaptor that can bind to the binding molecule and the reporter molecule at the same time. As an illustrative embodiment, in the case where the binding molecule is an antibody, the indirect labeling can be mediated by another antibody that binds to the antibody binding molecule in a specific manner. Other antibodies can be directly labeled with reporter molecules, such as gold particles; chromophores; luminescent compounds; fluorescent molecules; radioactive compounds; visible compounds; liposomes or other vesicles containing signal-generating substances; electroactive species; or enzymes and combinations of substrates thereof. A suitable enzyme-substrate combination used as a reporter moiety can be an enzyme alkaline phosphatase and a substrate nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate. In a specific embodiment of the present invention, the reporter moiety is a gold particle.

[0111] In embodiments where gold particles are reported, an optical density of at least 4, preferably at least 5, 6 or 7, and most preferably at least or about 8, 9 or 10, of the gold particle-bound molecule conjugate should be used.

[0112] In embodiments of the invention where the reporter molecule is bound to the binding molecule via an adapter molecule, the adapter can be pre-complexed with the binding molecule prior to adding the test sample to the indicator molecule, provided that the adapter does not prevent the binding molecule from binding to the cleaved indicator molecule.

[0113] The adapter can be any material or molecule capable of mediating an indirect interaction between a binding molecule and a reporter molecule. In some embodiments, the adapter is streptavidin and the binding molecule comprises a biotin molecule. The adapter can also be an "adapter binding pair," wherein the binding pair comprises:

[0114] (i) a first member capable of binding to a binding molecule; and

[0115] (ii) a second member capable of binding to the first member of the pair and the reporter molecule. In certain embodiments of the invention, the detection zone of the indicator molecule comprises biotin, the first member of the adaptor binding pair is avidin or streptavidin, the second member of the adaptor binding pair is biotin, and the reporter molecule comprises a portion capable of binding to biotin.

[0116] In view of the foregoing, in a complementary aspect, desialylated derivatives of the peptides of the invention also form part of the invention and are used to generate binding molecules, in particular antibodies. Thus, the invention provides peptides for generating antibodies as defined herein, wherein the peptides are desialylated derivatives of the peptides according to the invention and the antibodies are capable of specifically binding to the desialylated derivatives of the peptides of the invention. Thus, in a particular embodiment, the peptide is:

[0117] (i) Cyc- D Ala-Ser[Gal]- D Ala-Arg (desialylated form of SEQ ID NO: 11);

[0118] (ii)Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal]- D Ala-Arg-Phe- D Ser-Val (desialylated form of SEQ ID NO: 12);

[0119] (iii) Asp-Arg- D Ala-BIP-Ser-Pro-Ser[Gal]- D Ala- D Asp-Ser (desialylated form of SEQ ID NO: 13);

[0120] (iv)Asp-Ser-SEP- D Ala-Ile-Orn-Ser[Gal]- D Ala-Nle-Glu (desialylated form of SEQ ID NO: 14);

[0121] (v)Asp- D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal]-BIP-Orn- D Ala-Glu-Ser (desialylated form of SEQ ID NO: 15); or

[0122] (vi)Asp-Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Va (desialylated form of SEQ ID NO: 16).

[0123] Including the sequence Cyc- D Ala-Ser[Gal]- D Peptides consisting essentially of, or consisting of Ala-Arg are particularly preferred.

[0124] In certain embodiments, the peptides can be conjugated to a carrier protein to increase immunogenicity and, therefore, antibody production in the host organism. For example, the peptides can be conjugated to keyhole limpet hemocyanin. The carrier protein can be located at the N-terminus or C-terminus of the peptide.

[0125] In view of the foregoing, the present invention further provides an enzyme detection device, an enzyme detection kit, an enzyme detection material composition and a method for detecting the presence of sialidase cleavage activity in a test sample incorporating an indicator molecule, a capture molecule and a binding molecule as defined above. The use of a binding molecule such as an antibody that binds only to a desialylated derivative of the indicator molecule and not to the uncleaved indicator molecule enables the detection of low concentrations of sialidase activity in a test sample.

[0126] Therefore, in a further aspect, the present invention provides an enzyme detection device, an enzyme detection kit or an enzyme detection material composition for detecting the presence of cleavage activity of sialidase in a test sample, the device comprising:

[0127] (i) an indicator molecule as defined herein;

[0128] (ii) a capture zone for receiving the test sample, wherein the capture zone comprises a capture molecule as defined herein that is capable of binding to the capture site of the indicator molecule regardless of whether the indicator molecule has been cleaved, so as to immobilize the indicator molecule; and

[0129] (iii) a binding molecule as defined herein, which is capable of binding to a desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred.

[0130] The present invention further provides a method for detecting the presence or absence of cleavage activity of sialidase in a test sample, the method comprising:

[0131] (i) contacting an indicator molecule as defined herein with said test sample;

[0132] (ii) adding to said test sample a binding molecule as defined herein, said binding molecule being capable of binding to the desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred;

[0133] (iii) capturing the desialylated derivative of the indicator molecule at the capture zone by binding of a capture molecule in the capture zone to the capture site, the capture molecule being able to bind to the capture site regardless of whether the indicator molecule has been cleaved; and

[0134] (iv) detecting cleavage of the sialylation group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

[0135] The inventors have shown that the devices, kits, compositions of matter and methods of the present invention have particular application in the field of BV diagnosis. Therefore, the present invention further provides a method for diagnosing bacterial vaginosis in a test sample by detecting the cleavage activity of sialidase in the sample, the method comprising:

[0136] (i) contacting an indicator molecule as defined herein with said test sample;

[0137] (ii) adding to said test sample a binding molecule as defined herein, said binding molecule being capable of binding to the desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred;

[0138] (iii) capturing the desialylated derivative of the indicator molecule at the capture zone by binding of a capture molecule as defined herein in the capture zone to the capture site, the capture molecule being able to bind to the capture site regardless of whether the indicator molecule has been cleaved or not; and

[0139] (iv) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone, wherein an increased level of cleavage compared to a control is diagnostic for bacterial vaginosis.

[0140] In certain embodiments, the indicator molecule may be (pre)immobilized in the capture zone by a capture molecule (i.e., before contact with the test sample). Therefore, the present invention also provides a method for detecting the presence or absence of cleavage activity of a sialidase in a test sample, the method comprising:

[0141] (i) adding the test sample to a capture zone comprising capture molecules which bind to the capture sites of indicator molecules as defined herein, wherein the capture molecules remain bound to the capture sites irrespective of whether cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample occurs;

[0142] (ii) adding a binding molecule as defined herein, which is capable of binding to the desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred;

[0143] (iii) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

[0144] Similarly, the present invention also provides a method for diagnosing bacterial vaginosis in a test sample by detecting the cleavage activity of sialidase in the sample, the method comprising:

[0145] (i) adding the test sample to a capture zone comprising a capture molecule as defined herein, which is bound to the capture site of an indicator molecule as defined herein, wherein the capture molecule remains bound to the capture site regardless of whether cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample occurs;

[0146] (ii) adding a binding molecule as defined herein, which is capable of binding to the desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred;

[0147] (iii) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone, wherein an increased level of cleavage compared to a control is diagnostic for bacterial vaginosis.

[0148] Thus, for those embodiments in which the indicator molecules are (pre)fixed in the capture zone by the capture molecules (i.e., before contact with the test sample), the indicator molecules may be added to the capture zone prior to step (i) of the method, such that the indicator molecules are bound in the capture zone by the capture molecules. The addition of the binding molecules may be performed simultaneously or after the test sample is added to the capture zone. Thus, steps (i) and (ii) of the method may occur sequentially or simultaneously.

[0149] In the context of the methods provided herein, the step of "adding" the binding molecule to the test sample is understood to encompass any step of contacting the binding molecule with the test sample. Therefore, this step can encompass the step of applying the test sample to a device comprising the binding molecule. The binding molecule can be in solution, or can be on a carrier. For example, the binding molecule can be dried or otherwise impregnated into or on a solid phase carrier, which can be a "conjugate pad" discussed elsewhere herein. In a preferred embodiment, the test sample is contacted with a solid phase carrier of the binding molecule that has been dried thereon. The liquid included in the test sample and / or the liquid added to the carrier dissolves the binding molecule.

[0150] In embodiments where the binding molecules are dried or otherwise impregnated into or onto a solid support, the solid support preferably comprises or consists of glass fibers, polyester fibers, or a material having similar properties. The solid support should preferably have one or more of the following properties: a basic weight of about 75 g / m 2 ; Caliper of about 0.38-0.43 mm; Wicking rate of about 3-5 (s / 2 cm); and / or water absorption of about 63-79 mg / cm 2 . Therefore, the conjugate pad can have these properties. Similarly, the sample pad can have these properties.

[0151] Although in preferred embodiments, the binding molecule cannot bind to the indicator molecule unless and until the sialylated group is cleaved from the indicator molecule by a sialidase present in the sample, in some embodiments, the binding molecule preferentially binds to the desialylated derivative of the indicator molecule relative to the sialylated form as described elsewhere herein. Thus, some degree of binding to the sialylated indicator molecule may occur (which may be considered background signal in some cases). However, since the binding molecule will preferentially bind to the desialylated derivative, the signal generated in the sample comprising the desialylated derivative is much greater than in a sample in which the sialylated indicator molecule is not cleaved or the indicator molecule is not present.

[0152] In order to take into account the background level of sialidase activity (if any), the method generally involves comparing the cleavage level measured in the test sample with a control. Generally, the control represents the corresponding sialidase activity level in healthy subjects. "Healthy subjects" means subjects who do not suffer from BV. The control can be in a corresponding test sample taken from a matched healthy control. Alternatively, the control can be a threshold level of sialidase activity set by determining the sialidase activity in a series of healthy and sick patients. Suitable methods for setting the threshold are well known to those skilled in the art. The threshold can be mathematically derived from the training set of patient data. The scoring threshold is therefore separated from the test sample according to the presence or absence of BV. The explanation of this amount, i.e., the cut-off threshold, can be derived from a group of patients with known results in the development or training stage. Therefore, the threshold can be fixed before the claimed method is performed from the training data by methods known to those skilled in the art.

[0153] For example, as demonstrated in the Examples, a cubic reader can be used, such as an Optricon reader from OpTricon GmbH (ChembioDiagnostic systems), Schwarzschildstrasse 1, D-12489, Berlin, Germany. For example, in the assay used in the Examples, a cubic reading of less than 10 is associated with the absence of a visual signal and is considered a negative result, and thus indicates the absence of bacterial vaginosis; and a cubic reading of at least 30 units is associated with a strong visual signal and is considered a positive result, and thus indicates the presence of bacterial vaginosis.

[0154] Cubic readings of 10-20 units are associated with a weak visual signal and are considered to be results that may indicate the absence of established bacterial vaginosis, but may indicate low levels of infection, eg, early bacterial vaginosis.

[0155] In certain embodiments, the sialidase to be detected is derived from Prevotella, Bacteroides and / or Mobiluncus and / or Gardnerella vaginalis.

[0156] The enzyme detection device and composition of matter of the present invention can be supplied in a form ready for immediate use. Alternatively, the essential components can be provided as a complete kit, optionally together with suitable reagents and / or instructions for assembly of the enzyme detection device. Therefore, in another aspect, the present invention provides an enzyme detection kit for detecting the presence of cleavage activity of sialidase in a test sample, the kit comprising:

[0157] (i) an indicator molecule as defined herein, for addition to the test sample;

[0158] (ii) a capture molecule as defined herein, which is capable of binding to the capture site of the indicator molecule regardless of whether the indicator molecule has been cleaved;

[0159] (iii) a solid phase carrier, to which the capture molecule can be connected to form a capture zone for receiving the test sample; and

[0160] (iv) a binding molecule as defined herein, which is capable of binding to a desialylated derivative of said indicator molecule, wherein said binding molecule is incapable of binding to said indicator molecule unless and until cleavage of said sialylation group from said indicator molecule by a sialidase present in said sample has occurred.

[0161] In a related aspect, the present invention also provides a use of an enzyme detection device or composition of matter as described and defined herein for diagnosing BV in a test sample. Similarly, the present invention also provides a use of a method as described and defined herein for diagnosing BV in a test sample. The present invention further provides a use of an enzyme detection kit as described and defined herein for diagnosing BV in a test sample.

[0162] In certain embodiments, the present invention can be performed in a lateral flow or vertical flow device. Therefore, generally, the present invention relies on a certain form of solid phase carrier. The solid phase carrier can define the liquid flow path of the sample. In a specific embodiment, the solid phase carrier includes a chromatographic medium or a capillary flow device. In certain embodiments, the present invention can be provided in the form of a test strip.

[0163] In a specific embodiment of the present invention, the capture zone is formed on a solid phase carrier. It encompasses any carrier that can be connected to the capture molecule to form a capture zone. The solid phase carrier can take the form of, for example, beads (e.g., agarose or agarose beads) or holes (e.g., in a microplate). Therefore, in certain embodiments, the device includes a solid phase carrier, and the capture molecule is connected to the solid phase carrier to form a capture zone. In the case of the kit of the present invention, a solid phase carrier that is not connected to the capture molecule can be provided. In those embodiments, the user of the kit can fix the capture molecule on the solid phase carrier to form a capture zone before using the device with the test sample. Therefore, the kit can also include a device for fixing the capture molecule on the solid phase carrier. The fixing device can include any suitable reagent to allow the capture zone to be formed. The solid phase carrier can be preformed in a suitable fixing manner. For example, the solid phase carrier can include a biotin molecule that is arranged to interact with an avidin (e.g., streptavidin) molecule that forms (a part of) the capture molecule. Of course, as discussed herein and as will be readily appreciated by those skilled in the art, other binding pair interactions may be used to immobilize the capture molecules on the solid support to form the capture zone.

[0164] The capture zone can be defined by fixing therein or thereon a capture molecule capable of binding to the capture site of the indicator molecule. The immobilization of the capture molecule can be achieved by any suitable means. Where the device is a flow device comprising a chromatographic medium, the capture molecule can be fixed by direct binding to the medium, or indirectly by binding to a carrier molecule (such as a protein) associated or bound to the medium.

[0165] In other embodiments, the solid phase carrier further includes a sample application zone to which a sample is applied. The sample application zone can be pre-loaded with an indicator molecule so that when the test sample is applied, any enzyme in the sample acts on the cleavage site of the indicator molecule in the sample application zone. The sample application zone can contain a barrier that keeps the sample in a predetermined time period in the sample application zone. This allows the sample to interact with the indicator molecule for a sufficiently long duration to reach a measurable cleavage level. As will be readily appreciated by those skilled in the art, this can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 60 minutes or longer, depending on the sialidase to be detected. About 5-15 minutes, 5-10 minutes or about 5 minutes are preferred. After this time period, the barrier may be degraded by the sample or otherwise removed, thereby allowing the sample to continue to flow through the device. Alternatively, the test sample and the indicator molecule can be pre-mixed or pre-incubated before the mixture is added to the device such as the sample application zone. In some embodiments, the sample application zone may be omitted in the presence of a sample or indicator molecule. However, in the case where the test sample and indicator molecule can be pre-mixed or pre-incubated, the sample application zone may be omitted. Here, it may be possible to add the mixture directly to the capture zone to allow the indicator molecule to be fixed by interaction with the capture molecule. In some embodiments, the test sample may be applied to a chromatographic medium upstream of the capture zone so that it is drawn through the capture zone, for example, by capillary action. The chromatographic medium may be made of any material through which a fluid can pass, such as a fluid channel or a porous membrane. In certain embodiments of the invention, the chromatographic medium comprises a strip or membrane, for example, a nitrocellulose strip or membrane.

[0166] Indicator molecules can be present on a carrier, and the carrier should be an inert material and preferably porous. It can be a suitable polymer, such as polytetrafluoroethylene. For example, the carrier can be an inert porous membrane disk. The indicator molecules can, for example, have been freeze-dried onto the carrier. In a preferred embodiment, the test sample and the indicator molecules can be pre-mixed or pre-incubated before the mixture is added to the device. This can involve contacting the sample with a carrier comprising the indicator molecules, preferably in a freeze-dried form. This can be completed, for example, in a container such as a tube. Preferably, the sample and the indicator molecules are incubated for at least 3, 4 or 5 minutes and no more than 30 minutes, preferably incubated for about 5-15 minutes, 5-10 minutes or about 5 minutes, for example, 3-7 minutes or 4-6 minutes.

[0167] The skilled artisan will be able to determine appropriate amounts and concentrations of the various components, but by way of example, for a single assay or in a single kit, the indicator molecule is preferably present in an amount of at least 30, 40, 50, 60, 70, 80, 90 or 100 ng, more preferably at least or about 110, 120, 130, 140, 150, 160, 170, 180 or 190 ng.

[0168] If the sialylated group is cleaved from the indicator molecule by the sialidase present in the sample, the binding molecule must be provided in the device in a manner that allows interaction with the indicator molecule. Therefore, the binding molecule can be pre-mixed with the indicator molecule before being applied to the device. This can be before or after the indicator molecule has been mixed with the test sample. It is preferably after avoiding that the binding molecule may have any influence on the enzymatic activity (in the test sample) at the cleavage site of the indicator molecule. The binding molecule can be preferably provided on or in the device at any point upstream of the capture zone so that the binding molecule encounters the test sample and the indicator molecule before the indicator molecule is fixed (by the interaction between the capture site of the indicator molecule and the capture molecule that defines the capture zone). Alternatively, the binding molecule can be added to the capture zone after the test sample and the indicator molecule are added to the capture zone. This ensures that any indicator molecule will have been fixed at the capture zone, thereby (in the case of the cracked (i.e., desialylated) indicator molecule) providing a binding site for the binding molecule to generate a signal.

[0169] The solid phase carrier may further include a control zone, which is located downstream of the capture zone relative to the sample flow and the sample application zone (if present), and the control zone contains additional binding molecules (referred to herein as "control detection binding agents"), which bind to the binding molecules to indicate that the determination was successfully completed using the device. Alternatively, additional binding molecules (control detection binding agents) can be combined with additional molecules (referred to herein as "control detection molecules"), which are added to the sample or device and flow through the device with the sample. Additional molecules (control detection molecules) can be directly or indirectly labeled with reporter molecules defined herein. For ease of detection, preferably, the reporter molecules are the same as the reporter molecules connected to the binding molecules, although they may be different.

[0170] The control zone is spatially separated from the capture zone, for example, if the reporter is combined or fixed in each corresponding zone, two separated test lines are produced. This control zone is used to confirm that the test sample including the binding molecule has passed through the entire device, and confirms that the device is operating correctly. Regardless of whether there is sialidase activity in the sample, it is expected that there is a positive signal at the control zone. Based on the properties of the binding molecule combined with the cleavage site of the indicator molecule or based on the properties of the other molecules added to the sample, select other binding molecules. Binding molecules and other binding molecules or other molecules and other binding molecules can form a binding pair as defined herein. For example, if the binding molecule is a species-specific antibody (for example, sheep antibody), the other binding molecule can be an anti-species antibody (for example, anti-sheep antibody). For example, the control detection molecule can be a chicken IgY antibody, and the control detection binding agent can be an anti-chicken IgY antibody, or vice versa. Alternatively, if the other molecule is an antibody from a different species, for example, chicken or goat, the other binding molecule can be an appropriate anti-species antibody. This allows the binding molecule or other molecules to be fixed at the control zone by specific interactions. The additional binding molecule may be immobilized in the control zone by any suitable means, for example, by covalent or non-covalent interactions.

[0171] According to all aspects of the invention, the test sample can be a vaginal sample, optionally a vaginal swab. The test sample can be collected by any suitable means and in any form suitable for use with the present invention. Typically, a sterile swab is used to collect the sample. In addition, as part of obtaining the test sample from its original source, one or more treatment or pretreatment steps can be performed on the sample before testing using the present invention. In one embodiment, the sample can be processed to produce a solution or suspension for testing. In addition, in certain embodiments, the test sample can be stored, for example, frozen at about -20°C, as a way to preserve the sample for any given length of time before testing using the present invention.

[0172] It should be noted that the present invention is generally performed in vitro based on isolated samples. In some embodiments, for example, the methods of the present invention may comprise the step of obtaining a sample for testing, for example, using a sterile swab.

[0173] In view of the above, the present invention may be further defined by the following numbered clauses:

[0174] 1. A peptide comprising the following sequence:

[0175] X 1 -X 2 -X 3 [Gal-Sial]-X 4 -X 5

[0176] in:

[0177] (i) Sial is a sialylation group;

[0178] (ii)X 3 is a natural or unnatural amino acid that includes a glycosyl acceptor group; and

[0179] (iii)X 1 , X 2 , X 4 and X 5 are independently selected from any amino acid, provided that X 1 , X 2 , X 4 and X 5 At least one of D - amino acids and / or non-standard amino acids or unnatural amino acids.

[0180] 2. The peptide according to item 1, wherein X 3 Selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP).

[0181] 3. The peptide according to clause 2, wherein X 3 It's Ser.

[0182] 4. A peptide according to any one of clauses 1 to 3, wherein X 1 , X 2 , X 4 and X 5 At least two, three or all four of D - amino acids and / or non-standard amino acids or unnatural amino acids.

[0183] 5. A peptide according to any one of clauses 1 to 4, wherein X 1 , X 2 , X 4 and X 5 Includes at least two, three or four different amino acids.

[0184] 6. A peptide according to any one of clauses 1 to 5, wherein X 1 , X 2 , X 4 and X 5 At least one of the amino acids is a hydrophobic amino acid.

[0185] 7. A peptide according to any one of clauses 1 to 6, wherein X 1 , X 2 , X 4 and X 5 At least one of them is Ala.

[0186] 8. A peptide according to any one of clauses 1 to 7, wherein X 1 and X 2 All are Ala.

[0187] 9. A peptide according to clause 7 or 8, wherein Ala is D Ala or βAla.

[0188] 10. A peptide according to any one of clauses 1 to 9, wherein X 1 , X 2 , X 4 and X 5 At least one of the amino acids is a charged amino acid.

[0189] 11. The peptide according to clause 10, wherein each charged amino acid is selected from Arg, D Asp and L Orn.

[0190] 12. A peptide according to any one of clauses 1 to 11, wherein X 1 , X 2 , X 4 and X 5 At least one of the amino acids is a polar amino acid.

[0191] 13. A peptide according to clause 12, wherein each polar amino acid is selected from L Ser, D Ser and Thr.

[0192] 14. A peptide comprising the following sequence:

[0193] X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 -X 12 [Gal-Sial]-X 13 -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20

[0194] Where: Sial is the sialylation group; X 1Does not exist or is Thr;X 2 Does not exist or D Ala;X 3 does not exist or is Nle;

[0195] X 4 Does not exist or is Glu;X 5 Does not exist or D Ala;X 6 Does not exist or is Arg;X 7 Not present or selected from Glu, Arg, Ser, Nva, βAla; X 8 Does not exist or is selected from D Ser, D Ala, SEP, Cyc;

[0196] X 9 Not present or selected from Nva, BIP, D Ala, βAla, Orn;

[0197] X 10 Selected from Cyc, Ser, Ile, D Ala, D Ser;X 11 Selected from D Ala, Pro, Orn, Nle;

[0198] X 12 Selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or SEP;

[0199] X 13 Selected from D Ala, BIP, βAla; X 14 Selected from Arg, D Asp, Nle, Orn, Nva;

[0200] X 15 Not present or selected from Phe, BIP, Ser, Glu, D Ala, D Ser;

[0201] X 16 Does not exist or is selected from D Ser, Glu;

[0202] X 17 Not present or selected from Val, Ser, Thr; X 18 Does not exist or is Cha;

[0203] X 19 Does not exist or DSer;X 20 Does not exist or is Val.

[0204] 15. The peptide according to clause 14, wherein X 12 It's Ser.

[0205] 16. A peptide according to any one of clauses 1 to 15, wherein the peptide comprises the following sequence:

[0206] (i) Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg;

[0207] (ii)Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-Phe- D Ser-Val;

[0208] (iii) Arg- D Ala-BIP-Ser-Pro-Ser[Gal-Sial]- D Ala- D Asp-Ser;

[0209] (iv)Ser-SEP- D Ala-Ile-Orn-Ser[Gal-Sial]- D Ala-Nle-Glu;

[0210] (v) D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal-Sial]-BIP-Orn- D Ala-Glu-Ser; or

[0211] (vi)Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal-Sial]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Val.

[0212] 17. A peptide according to any one of clauses 15 or 16, wherein Nle, Nva, Orn and / or Cha are LNle, L Nva, L Orn and L Cha.

[0213] 18. A peptide according to any one of clauses 1 to 17, wherein the peptide is preferentially cleaved by one or more specific sialidases.

[0214] 19. The peptide according to clause 18, wherein the one or more specific sialidases are of bacterial origin.

[0215] 20. The peptide according to clause 19, wherein the bacterium is Prevotella, Bacteroides and / or Mobiluncus and / or Gardnerella vaginalis.

[0216] 21. An indicator molecule for use in detecting the presence of sialidase cleavage activity in a test sample, the indicator molecule comprising:

[0217] a) a peptide as defined in any one of clauses 1 to 20; and

[0218] b) A capture site which remains intact after cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample.

[0219] 22. An indicator molecule according to clause 21, wherein the capture site comprises a biotin molecule or an oxime moiety.

[0220] 23. An indicator molecule according to clause 21 or 22, wherein the capture site may be located at the N-terminus or the C-terminus of the peptide.

[0221] 24. The indicator molecule according to any one of clauses 21 to 23, wherein the capture site is connected to the peptide via a linker.

[0222] 25. The indicator molecule according to clause 24, wherein the linker comprises a polyethylene glycol (PEG) moiety.

[0223] 26. An indicator molecule according to clause 25, wherein the peptide is linked to a biotin group at its N-terminus or C-terminus via a linker comprising, consisting essentially of or consisting of a polyethylene glycol moiety.

[0224] 27. An indicator molecule according to any one of clauses 21 to 26, wherein the indicator molecule comprises the following structure:

[0225] (i) Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin;

[0226] (ii) Biotin-PEG-Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-Phe- D Ser-Val;

[0227] (iii) Biotin-PEG-Asp-Arg- D Ala-BIP-Ser-Pro-Ser[Gal-Sial]- D Ala- D Asp-Ser;

[0228] (iv) Biotin-PEG-Asp-Ser-Ser(PO 3 )- D Ala-Ile-Orn-Ser[Gal-Sial]- D Ala-Nle-Glu;

[0229] (v) Biotin-PEG-Asp- D Ala-Arg-Nva- D Ser-βAla- D Ala-Nle-Ser[Gal-Sial]-BIP-Orn- D Ala-Glu-Ser; or

[0230] (vi) Biotin-PEG-Asp-Thr- D Ala-Nle-Glu- D Ala-Arg-βAla-Cyc-Orn- D Ser-Pro-Ser[Gal-Sial]-βAla-Nva- D Ser-Glu-Thr-Cha- D Ser-Val.

[0231] 28a. An antibody capable of specifically binding to a desialylated derivative of a peptide according to any one of clauses 1 to 20, or an indicator molecule according to any one of clauses 21 to 27, wherein the antibody preferentially binds to the desialylated derivative over the sialylated peptide or indicator molecule.

[0232] 28b. An antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and / or the light chain CDR3 has SEQ ID NO: 6.

[0233] 28c. An antibody having a heavy chain with SEQ ID NO: 7 and / or having a light chain with SEQ ID NO: 8.

[0234] Any reference to "Clause 28" shall be construed as covering Clauses 28a, 28b and 28c.

[0235] 29. The antibody according to clause 28, wherein the antibody can specifically bind to the following epitope: Cyc- D Ala-Ser[Gal]- D Ala-Arg-PEG-biotin, more specifically the epitope Cyc- D Ala-Ser[Gal]- D Ala-Arg.

[0236] 30. The antibody according to clause 28 or 29, wherein the antibody is conjugated to a reporter molecule.

[0237] 31. The antibody according to clause 30, wherein the reporter molecule is a gold particle.

[0238] 32. A peptide for use in the production of an antibody according to clause 28, wherein the peptide is a desialylated derivative of the peptide as defined in any one of clauses 1 to 20.

[0239] 33. A peptide for use according to clause 32, wherein the peptide is conjugated to a carrier protein to increase immunogenicity.

[0240] 34. A peptide for use according to clause 33, wherein the peptide is conjugated to keyhole limpet hemocyanin.

[0241] 35. An enzyme detection device, enzyme detection kit or enzyme detection material composition for detecting the presence of sialidase cleavage activity in a test sample, the device comprising:

[0242] (i) an indicator molecule as defined in any one of clauses 21 to 27;

[0243] (ii) a capture zone for receiving the test sample, wherein the capture zone comprises capture molecules capable of binding to the capture sites of the indicator molecules regardless of whether the indicator molecules have been cleaved, so as to immobilize the indicator molecules; and

[0244] (iii) a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred.

[0245] 36. An enzyme detection device, enzyme detection kit or enzyme detection composition of matter according to clause 35, wherein the device, kit or composition of matter comprises a solid support linked to the capture molecule to form the capture zone.

[0246] 37. The enzyme detection device, enzyme detection kit or enzyme detection material composition according to clause 36, wherein the solid phase carrier further comprises a sample application area to which the sample is applied.

[0247] 38. An enzyme detection device, enzyme detection kit or enzyme detection material composition according to any one of clauses 36 to 37, wherein the solid phase carrier further includes a control zone, which is located downstream of the capture zone relative to the sample application zone, and the control zone contains additional binding molecules, which bind to the binding molecules to indicate that the determination using the device, kit or material composition is successfully completed.

[0248] 39. An enzyme detection device, enzyme detection kit or enzyme detection material composition according to any one of clauses 36 to 38, wherein the solid phase carrier comprises a chromatographic medium.

[0249] 40. An enzyme detection device, an enzyme detection kit or an enzyme detection material composition according to any one of clauses 35 to 39, wherein the indicator molecule is immobilized in the capture zone via the capture site that binds to the capture molecule.

[0250] 41. A method for detecting the presence or absence of cleavage activity of a sialidase in a test sample, the method comprising:

[0251] (i) contacting an indicator molecule as defined in any one of clauses 21 to 27 with the test sample;

[0252] (ii) adding to the test sample a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred;

[0253] (iii) capturing the desialylated derivative of the indicator molecule at the capture zone by binding of a capture molecule in the capture zone to the capture site, the capture molecule being able to bind to the capture site regardless of whether the indicator molecule has been cleaved; and

[0254] (iv) detecting cleavage of the sialylation group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

[0255] 42. A method for detecting the presence or absence of cleavage activity of a sialidase in a test sample, the method comprising:

[0256] (i) adding the test sample to a capture zone comprising capture molecules which bind to the capture sites of indicator molecules as defined in any one of clauses 21 to 27, wherein the capture molecules remain bound to the capture sites irrespective of whether cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample occurs;

[0257] (ii) adding a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred;

[0258] (iii) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

[0259] 43. The method according to clause 42, wherein before step (i), the indicator molecules are added to the capture zone such that the indicator molecules are bound in the capture zone by the capture molecules.

[0260] 44. A method for diagnosing bacterial vaginosis in a test sample by detecting the cleavage activity of sialidase in the sample, the method comprising:

[0261] (i) contacting an indicator molecule as defined in any one of clauses 21 to 27 with the test sample;

[0262] (ii) adding to the test sample a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred;

[0263] (iii) capturing the desialylated derivative of the indicator molecule at the capture zone by binding of a capture molecule in the capture zone to the capture site, the capture molecule being able to bind to the capture site regardless of whether the indicator molecule has been cleaved; and

[0264] (iv) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone, wherein an increased level of cleavage compared to a control is diagnostic for bacterial vaginosis.

[0265] 45. A method for diagnosing bacterial vaginosis in a test sample by detecting the cleavage activity of sialidase in the sample, the method comprising:

[0266] (i) adding the test sample to a capture zone comprising capture molecules which bind to the capture sites of indicator molecules as defined in any one of clauses 21 to 27, wherein the capture molecules remain bound to the capture sites irrespective of whether cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample occurs;

[0267] (ii) adding a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred;

[0268] (iii) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone, wherein an increased level of cleavage compared to a control is diagnostic for bacterial vaginosis.

[0269] 46. ​​The method according to clause 45, wherein before step (i), the indicator molecules are added to the capture zone such that the indicator molecules are bound in the capture zone by the capture molecules.

[0270] 47. A method according to any one of clauses 41 to 46, wherein the sialidase is derived from Prevotella, Bacteroides and / or Mobiluncus and / or Gardnerella vaginalis.

[0271] 48. The method according to any one of clauses 41 to 47, wherein the method is performed using an apparatus, a kit or a composition of matter according to any one of clauses 35 to 40.

[0272] 49. A method according to any one of clauses 41, 44 or 47, wherein the method is performed using a device, kit or composition of matter according to any one of clauses 35 to 39, and further wherein the test sample and indicator molecules are mixed before contacting the test sample with the device, kit or composition of matter.

[0273] 50. An enzyme detection kit for detecting the presence of cleavage activity of a sialidase in a test sample, the kit comprising:

[0274] (i) an indicator molecule as defined in any one of clauses 21 to 27, which is for addition to the test sample;

[0275] (ii) a capture molecule, which is capable of binding to the capture site of the indicator molecule regardless of whether the indicator molecule has been cleaved;

[0276] (iii) a solid phase carrier, to which the capture molecule can be connected to form a capture zone for receiving the test sample; and

[0277] (iv) a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred.

[0278] 51. An enzyme detection kit according to clause 50, wherein the kit further comprises an additional binding molecule (control detection binder) which is capable of binding to the binding molecule.

[0279] 52. An enzyme detection kit according to clause 51, wherein the solid phase carrier further comprises a control zone, which is located downstream of the capture zone relative to the sample application zone, and the additional binding molecule (control detection binder) can be connected to the control zone.

[0280] 53. An enzyme detection device, an enzyme detection kit or an enzyme detection material composition according to any one of clauses 35 to 40, a method according to any one of clauses 41 to 49 or an enzyme detection kit according to any one of clauses 50 to 52, wherein the binding molecule comprises an antibody as defined in any one of clauses 28 to 31.

[0281] 54. An enzyme detection device, an enzyme detection kit or an enzyme detection material composition according to any one of clauses 35 to 40 or 53, a method according to any one of clauses 41 to 49 or 53 or an enzyme detection kit according to any one of clauses 50 to 53, wherein the capture site comprises a biotin molecule and the capture zone comprises a streptavidin molecule.

[0282] 55. Use of an enzyme detection device, an enzyme detection kit or an enzyme detection material composition according to any one of clauses 35 to 40, 53 or 54, a method according to any one of clauses 41 to 49, 53 or 54 or an enzyme detection kit according to any one of clauses 50 to 54 for diagnosing bacterial vaginosis in a test sample.

[0283] 56. An indicator molecule according to any one of clauses 21 to 27, an enzyme detection device, an enzyme detection kit or an enzyme detection material composition according to any one of clauses 35 to 40, 53 or 54, a method according to any one of clauses 41 to 49, 53 or 54, an enzyme detection kit according to any one of clauses 50 to 54 or used in clause 55, wherein the test sample is a vaginal sample, optionally a vaginal swab. BRIEF DESCRIPTION OF THE DRAWINGS

[0284] The present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0285] Figure 1 is a schematic diagram of one format of an assay according to the invention. The format relies on the following basic components: a solid support (1); a capture molecule (2); an indicator molecule comprising a capture site (3) and a peptide of the invention, the peptide comprising a Gal-Sial cleavage site (4); and a binding molecule (5) which binds to the indicator molecule only after cleavage (6) has occurred. D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin (SEQ ID NO: 11-PEG-biotin; also referred to herein as "MOL600c") is shown by way of example.

[0286] FIG. 2 is a schematic diagram of an enzyme detection device according to the present invention, and shows that in the absence of ( Figure 2A) or exists( Figure 2B ) Operation of the device in the presence of sialidase activity.

[0287] Figure 3 Shown is the visual readout of the assay (as shown in Figure 2) as the level of sialidase activity in the test samples increases.

[0288] Figure 4 Schematic diagram of an enzyme detection device according to the present invention. This figure specifies the exact longitudinal dimensions and position of each card component in the card assembly.

[0289] Figure 5 Schematic representation of two conjugation chemistries used to couple the peptides of the invention to keyhole limpet hemocyanin (KLH): (A) cysteine / maleimide coupling; (B) hydrazine / benzaldehyde coupling.

[0290] Figure 6 Some of the non-standard and unnatural amino acids employed during the design of the peptides of the invention are shown.

[0291] Figure 7 is a schematic diagram summarizing the synthetic methods used to produce the peptides of the present invention.

[0292] Figure 8 The production of sialylated peptides of the invention via an enzymatic pathway using transialidase and fetuin (sialic acid donor) is shown.

[0293] Figure 9 shows various peptides and peptide-protein conjugates synthesized as part of the present invention. (A) Tabular summary of the peptides and peptide-protein conjugates synthesized. (B) Schematic overview of the peptides and peptide-protein conjugates synthesized.

[0294] Figure 10 shows the initial ELISA results after immunization of sheep with KLH-peptide conjugates of the present invention. (A) Schematic diagram of the detection method; (B)-(D) Results of the first bleed, indicating that all sheep responded to the immunization. Numbers 1056, 1057, 1058, 1059, 1062, 1063, 1064, 1065, 1066 and 1067 represent antisera from specific immunized sheep, respectively.

[0295] Fig.11A -C shows the ELISA results of the sheep second bleed described in Figure 10. There was an increase in the antisera responding in the second bleed relative to the first bleed.

[0296] Fig. 12A -B shows a comparison of all three bleeds from sheep CF1062-1067.

[0297] Fig.13ELISA results are shown when antiserum CF1064 was evaluated against MOL136 and MOL136c. (A) Schematic diagram of the detection method; (B) ELISA results show that the specificity for the galactosyl (ie, desialylated) peptide MOL136 is significantly preferential to the sialylated peptide MOL136c.

[0298] Fig.14 Detection of the desialylated peptide MOL136 by lateral flow assay using gold labeled CF1064 is shown, with little / no signal observed relative to the sialylated peptide MOL136c or a negative control (no peptide included in the sample).

[0299] Fig.15 Detection of the desialylated peptide MOL136 by lateral flow assay using gold labeled CF1064 and CF1065 is shown, with little / no signal observed relative to the sialylated peptide MOL136c or a negative control (no peptide included in the sample).

[0300] Fig.16 Shown is a lateral flow assay comparing the binding of MOL136 and MOL136c to gold-labeled CF1064 in the presence of healthy vaginal swab extract.

[0301] Fig.17 Shown is a lateral flow assay of MOL136c in the presence of 25 U / ml sialidase.

[0302] Figure 18 shows lateral flow data showing comparison between different affinity purified antiserum fractions. (A) Graphical representation of line intensity; (B) Observed lateral flow test strips: (1) - gold sensitive affinity purified polyclonal antibody, (2) - gold sensitive affinity purified polyclonal antibody, where cross-reactive antibodies were removed, (3) gold sensitive cross-reactive antibodies from affinity purified.

[0303] Figure 19 shows (A) a comparison of MOL136, MOL136c, MOL600 and MOL600c binding by ELISA and (B) a lateral flow assay showing significant improvement in performance compared to MOL136 / 136c (see Figure 18).

[0304] Fig. 20 Stability data for heat-dried MOL600c in polypropylene tubes at different storage temperatures and with two different heat drying methods are shown: rapid vacuum and heat block methods. Measurements in the ELISA were normalized to the percentage of desialylation, as this will result in recognition by the antibody and increase the signal.

[0305] Figure 21 shows typical data for batch MOL600c. (A) HPLC trace analysis; (B) positive ion electrospray MS confirmation.

[0306] Fig. 22 Schematic diagram showing the column method for antibody purification using different agarose matrices. The orange labels shown for MOL600 and MPL600c represent biotin. The purple labels shown for MOL615 represent oxime groups.

[0307] Fig.23 Stability data for lateral flow cassettes stored at 37°C over 12 weeks are shown.

[0308] Fig.24 A lateral flow standard curve of sialidase concentration incubated with peptide MOL600c for 5 minutes is shown. The test line was quantified using a cubic reader.

[0309] Fig.25 A lateral flow test of a healthy volunteer vaginal sample on a swarm swab is shown. The sample was extracted in 1 ml of sample buffer and split in five ways. (A) Sample without peptide; (B) Sample with MOL600c; (C) Sample with MOL600c and treated with 500 U / ml sialidase for 5 minutes; (D) Sample centrifuged with MOL600c; (E) Sample with MOL600c and the same sialidase incubation centrifuged.

[0310] Fig.26 The chemical formula of MOL616 is shown.

[0311] Fig. 27 The results of Example 13 showing the specificity of antibody 125.1 for MOL600 relative to the sialylated form MOL600c are shown.

[0312] Fig.28 Shown is a comparison of assays A and B using a range of different sialidase concentrations. For each sialidase concentration, the left bar represents the cubic readout for assay B, and the right bar represents the cubic readout for assay A.

[0313] Fig.29 A comparison of assays A and B using a range of different sialidase concentrations and read times is shown. For each sialidase concentration, the bars represent from left to right (i) the cubic read of assay A after a 5 minute read time; (ii) the cubic read of assay A after a 10 minute read time; (iii) the cubic read of assay B after a 5 minute read time; and (iv) the cubic read of assay B after a 10 minute read time.

[0314] Fig.30A comparison of assays A, A', B, and B' using a range of different sialidase concentrations, indicator concentrations, and read times is shown. For each sialidase concentration, the bars represent from left to right (i) the cubic readings for assay B with 1 μg / ml indicator molecule per plate and a read time of 5 minutes; (ii) the cubic readings for assay B with 3 μg / ml indicator molecule per plate and a read time of 5 minutes; (iii) the cubic readings for assay A with 1 μg / ml indicator molecule per plate and a read time of 10 minutes; and (iv) the cubic readings for assay A with 3 μg / ml indicator molecule per plate and a read time of 10 minutes.

[0315] Fig.31 The sequence of antibody 125.1 is shown with the CDR and framework regions indicated. DETAILED DESCRIPTION

[0316] Figure 1 is a schematic diagram of one format of an assay according to the invention. The format relies on the following basic components: a solid support (1); a capture molecule (2); an indicator molecule comprising a capture site (3) and a peptide of the invention, the peptide comprising a Gal-Sial cleavage site (4); and a binding molecule (5) which binds to the indicator molecule only after cleavage (6) has occurred. D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin (also referred to herein as "MOL600c") is shown by way of example.

[0317] In the form shown, the capture molecule (2) is streptavidin. Here, the capture molecule (2) binds to the biotin capture site (3) within the indicator molecule.

[0318] like Figure 1A As shown, once the indicator molecule of the present invention is added to the test sample, the sialidase present in the sample will specifically recognize the Gal-Sial cleavage site (4) and cleave the sialylated group (6) from the indicator molecule.

[0319] like Figure 1B As shown, this cleavage event (6) creates a binding site for the specific antibody binding molecule (5). The binding molecule (5) cannot bind to the indicator molecule until the cleavage (6) has occurred. Thus, the antibody binding molecule (5) binds to the amino acid sequence Cyc- D Ala-Ser[Gal]- D The antibody binding molecule (5) does not bind to Cyc- DAla-Ser[Gal-Sial]- D Ala-Arg sequence binding (not shown).

[0320] FIG. 2 is a schematic diagram of an enzyme detection device according to the present invention, and shows that in the absence of ( Figure 2A ) or exists( Figure 2B ) sialidase activity. The test strip comprises an adhesive pad (1) on which the other components of the device are assembled. From right to left, the sample application zone (2) is in the form of an absorbent pad. The sample application zone is partially overlapped and placed on a conjugate pad (3), which is impregnated with a labeled binding molecule (7). In an alternative embodiment, the labeled binding molecule can be impregnated in the sample application zone, and this eliminates the need for a separate conjugate pad. The conjugate pad (3) is fluidly connected to a nitrocellulose membrane (4). The nitrocellulose membrane (4) contains a fixed streptavidin molecule (5) that defines a capture zone. The membrane (4) further contains a fixed additional binding molecule (6) downstream of the capture zone, the additional binding molecule is combined with an additional labeled molecule (11), and the additional labeled molecule passes through the device together with the sample and forms a separate control zone. Alternatively, the fixed additional binding molecule can be combined with the labeled binding molecule (7). The device optionally further comprises an absorbent pad (8) to absorb any test sample and reagents that reach the end of the device.

[0321] In use, indicator molecules (9) are added to the test sample before the test sample is brought into contact with the sample application area (8) of the device. Figure 2A As shown, in the absence of sialidase activity in the test sample, the sialylated group is not cleaved from the indicator molecule (9). When the sample flows into the conjugate pad (3), the binding molecule (7) cannot bind to the indicator molecule (9) because the cleavage of the sialylated group does not occur. The indicator molecule binds at the capture zone through the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labeled binding molecule (7) is not fixed at the capture zone because it cannot bind to the indicator molecule (9). Therefore, the labeled binding molecule flows through the control zone and other areas. The additional labeled molecule (11) also passes through the device to the control zone, where it is fixed by binding to the fixed additional binding molecule (6). Therefore, the absence of sialidase activity is only displayed as a signal at the control zone, and not at the capture zone. Excess sample that may contain labeled binding molecules (7) flows into the absorption pad (8).

[0322] like Figure 2BAs shown, in the presence of sialidase activity in the test sample, the sialylated group is cleaved from the indicator molecule (9). When the sample flows into the conjugate pad (3), because the cleavage of the sialylated group has occurred, the binding molecule (7) can bind to the indicator molecule (9). The indicator molecule binds at the capture zone through the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labeled binding molecule (7) is fixed at the capture zone due to binding to the desialylated indicator molecule (9) at the cleavage site. Due to the relative excess of labeled binding molecules (7) relative to the binding sites at the capture zone, some labeled binding molecules (7) still flow through the control zone and other areas. Additional labeled molecules (11) also pass through the device to the control zone, where they are fixed by binding to the fixed additional binding molecules (6). Therefore, the presence of sialidase activity is displayed as a signal at both the capture zone and the control zone. Excess sample flows into the absorbent pad (8).

[0323] It should be noted that the control zone is optional.The presence or absence of sialidase activity in a sample can be monitored solely based on the presence or absence of a corresponding signal at the capture zone.

[0324] Figure 3 The visual reading of the assay (as shown in Figure 2) is shown as the level of sialidase activity in the test sample increases. It can be easily seen that the signal at the control zone (1) is constant as the amount of sialidase increases. In contrast, the signal at the capture zone (2) also increases as the amount of sialidase increases. This is due to the sialyl group being cleaved from the indicator molecule at the cleavage site by the sialidase activity. This reveals the binding site, enabling binding to a binding molecule that is detected at the capture zone (2) by the interaction between the capture molecule that defines the capture zone and the capture site of the indicator molecule.

[0325] Figure 4 Schematic diagram of a specific enzyme detection device according to the present invention. The following table provides a legend for this figure and specifies the exact longitudinal dimensions and positions of each card component in the card assembly of this particular embodiment.

[0326] Of course, as will be readily appreciated by those skilled in the art, the dimensions and locations may vary.

[0327] Components size Location of the reference point Backing Card(1) 60mm 0mm Nitrocellulose membrane (2) 25mm 20mm Conjugate Pad (3) 17mm 5mm Sample pads (4) 10mm 0mm Absorbent pads(5) 22mm 38mm

[0328] The present invention will be further understood with reference to the following experimental examples.

[0329] Examples

[0330] Example 1: Assay chemistry principles and experimental design

[0331] In summary, the principle works based on antibody recognition of the chemical product of the sialidase reaction, wherein the chemical substrate is a peptide specifically designed to react with the sialidase, and the reaction product is then recognized by an antibody produced against the synthetic product. As shown in Figures 1 and 2, a glycopeptide containing sialic acid and having a biotin tag is provided. When contacted with a test sample containing sialidase activity, the sialylated group is cleaved from the glycopeptide by the sialidase to expose the side galactosyl groups on the peptide. The antibody produced against the desialylated product then specifically binds to the cleaved product. By using an antibody-gold conjugate and a lateral flow strip with a streptavidin test line, the presence of the desialylated product can be detected as a red line, as well as the sialidase activity directly measured in the sample.

[0332] Five peptides were initially designed and the resulting antibodies were subsequently evaluated and their performance in the lateral flow assay was profiled. This showed that under optimized conditions, some of the antibodies outperformed the others in terms of cross-reactivity with the substrate peptide and overall signal level in the assay. In the evaluation described below for the project feasibility phase, the peptides were re-evaluated and the peptide / antibody combinations with the best performance were used for further development.

[0333] Example 2: Peptide Design

[0334] In the design of candidate peptide sequences, many factors were considered:

[0335] size

[0336] Typically, molecules with a molecular weight (MW) of less than 5000 Daltons are unlikely to stimulate a good immune response in a host organism. The peptides were planned to be relatively short sequences, intended to be conjugated to KLH (keyhole limpet hemocyanin) to produce more potent immunogens. A range of different lengths (9-20 amino acids) were proposed to cover any potential performance variations.

[0337] Conjugation Chemistry

[0338] Two different conjugation chemistries were used. For two of the peptides, a cysteine ​​label was incorporated into the structure, allowing it to be conjugated to a carrier protein using standard maleimide-based chemistry. For the other three peptides, a relatively new hydrazine-based chemistry was used, which is considered a more controllable process than cysteine ​​chemistry, as there is less risk of peptide oxidation prior to coupling, and the degree of coupling can be easily monitored by UV absorption. An overview of these conjugation chemistries is given in Figure 5 shown.

[0339] Location of galactose

[0340] The goal was to obtain antibodies with very high affinity for galactose and the surrounding regions of the peptide. With this in mind, it was decided to place the sugar centrally in the structure so that the sugar flanks were appropriately other unique peptide features. This approach is expected to minimize peripherally bound antibodies that lack interaction with the sugar moiety.

[0341] Structural diversity

[0342] A series of amino acids are used to construct various peptide sequences. By combining charged groups, hydrophilic groups and hydrophobic groups along the sequence, multiple topologies will be promoted. "Hinge groups" (such as β-alanine) are also used to allow additional degrees of freedom to be added in the overall structural folding. In addition, non-natural amino acids are also used to increase sequence diversity and promote immune response. In order to reduce the sensitivity to proteases, some D-amino acids are also incorporated. Figure 6 Some of the non-standard and unnatural amino acids employed are shown in FIG.

[0343] After considering these factors, the following peptide sequences were selected as putative epitopes.

[0344]

[0345] Spacers, conjugation groups, and biotin labels were added to the C-terminus or N-terminus.

[0346] Example 3: Peptide Synthesis

[0347] Galactose-labeled peptides were synthesized using solid phase chemistry on an automated microwave synthesizer. All peptides were purified using reverse phase HPLC and characterized by electrospray LCMS. To label the galactose moiety with sialic acid, an enzymatic pathway using transsialic acid (TcTS) from T. cruzi and fetuin as sialic acid donors was employed. Figure 7 and 8 The synthetic methods used are summarized.

[0348] And also synthesized related peptide immunogens conjugated to carrier proteins, two biotinylated derivatives of each sequence, one of which was labeled with sialic acid. These biotinylated derivatives will form the basis of the lateral flow assay format. Figure 9 shows the peptides synthesized for this work according to various immunoassays. Peptides labeled with sialic acid are designated with "c"; for example, MOL136c. Peptides lacking a sialylated group do not have this name (e.g., MOL136).

[0349] Example 4A: Antibody Generation

[0350] immunity

[0351] All five peptide immunogens were coupled to KLH and BSA using appropriate coupling chemistry (see Figure 9). The KLH conjugate was submitted to Micropharm Ltd for immunization of ten sheep (two per peptide). The procedure was initiated with PBS containing a 1 mg dose of KLH peptide. Three additional 0.5 mg of enhancer were given over a three month duration. Three separate bleeds (one sampling bleed and two production bleeds) were drawn during this period and supplied to Mologic. The BSA conjugate was retained for screening of various bleeds.

[0352] Initial ELISA screening

[0353] Polystyrene high binding multiwell plates were sensitized with appropriate BSA conjugates and non-conjugated BSA. Serum samples were incubated in BSA-blocked wells at various dilutions and further bound to secondary anti-sheep antibodies conjugated to alkaline phosphatase (AP). Incubation with p-nitrophenyl phosphate (pNPP) AP substrate indicated any binding. In all cases, buffer, BSA, and pre-bleeding controls resulted in no background binding to the plate. Fig. 10A A schematic diagram of the detection method is shown. Figures 10B-10D The results of the first bleed are shown, indicating that all sheep responded to the immunization. A high signal was produced when the serum dilution was 1 / 1000, while the BSA control was negative, thus confirming the specific response of the peptide. The first bleed control was also negative.

[0354] Along with can obtain other hemorrhagic thing, also analyzed these hemorrhagic things by ELISA, shown that as time goes by, reply and strengthen.Figure 11 has shown the relation between first two kinds of hemorrhagic things of all sheep, and Figure 12 shows the relation of all three kinds of hemorrhagic things compared with sheep CF1062-1067.What is interesting is, with regard to the overall response of described all sheep to antigen, the 3rd hemorrhagic thing seems to be in stable phase or even reduces.Yet in some cases, under 1 / 1000000 dilution, detected in conjunction with response, thereby show to the polyclonal response of peptide high sensitivity.Although reply is slightly low in the 3rd hemorrhagic thing, because described 3rd hemorrhagic thing most likely contains the subgroup of high specific antibody, therefore described 3rd hemorrhagic thing is preferred to described affinity purification.

[0355] Example 4B: Detection of Antisera CF1064 and CF1065 and MOL136 and MOL136c Immunogen: KLH-MOL123A

[0356] Biotinylated peptides: MOL136 (galactosylation); MOL136c (sialylation)

[0357] Both sheep responded well to KLH-MOL123A immunization and had a slightly stronger overall response to CF1064 (see Fig. 11B In the ELISA format (schematic diagram as shown in Fig.13 ), the binding of CF1064 to MOL136 and MOL136c was evaluated, showing a significant specificity for the galactosyl (ie, desialylated) peptide MOL136 ( Fig.13 B) Specificity in preference to the sialylated peptide MOL136c. Thus, excellent discrimination of the two peptides by antiserum CF1064 was demonstrated.

[0358] Similar properties were observed in the lateral flow format. CF1064 was conjugated to gold particles under optimal conditions (15 μg / ml loaded in 10 mM sodium borate buffer; pH 8.0) and measured for MOL136 and MOL136c at a final concentration of 1.3 ng / ml (38 pg per strip). Therefore, 1.5 mg / ml streptavidin was first used to form a capture zone on each lateral flow test strip. Then, 3 μl of each peptide (PBST containing 12.5 ng / ml peptide) was mixed with 10 μl gold-labeled CF1064 and 15 μl running buffer (0.5 M Tris + 3% BSA + 0.5% Triton X-100 at pH 7.5), and each sample was then run along a separate lateral flow test strip. Negative controls without added peptides were also run as negative controls. Then 15 μl running buffer was run along each lateral flow test strip to wash. The desialylated peptide MOL136 was clearly detected in the capture zone and observed as a visible line on the test strip. In contrast, little / no signal was observed compared to the sialylated peptide MOL136c or the negative control (see Fig.14 ).

[0359] Similar results were observed using gold-labeled CF1065, with higher signal levels observed for CF1065 relative to CF1064 (see Fig.15 ).

[0360] Example 5: Detection of MOL136 and MOL136c using CF1064 in the presence of healthy vaginal swab extract

[0361] The procedure described in Example 4B was repeated in the presence of healthy vaginal swab extracts. Although the signal level was slightly lower, the specificity for MOL136 was retained without any interference from the swab matrix (see Fig.16 ).

[0362] Example 6: Detection of sialidase activity using MOL136c and CF1064

[0363] The procedure described in Example 4B was repeated in the presence of 25U / ml sialidase. The sample without sialidase was used as a negative control. An additional negative control was run in which the sample did not contain peptide and sialidase. As a positive control, MOL136 was used instead of MOL136c in the presence of 25U / ml sialidase. A clear positive signal was observed for MOL136c incubated in PBST for 5 minutes in the presence of 25U / ml sialidase. The signal level was similar in intensity to that of the positive control, which showed an estimated maximum signal. As expected, little / no signal was observed for the negative control (see Fig.17 ).

[0364] Overall, both CF1064 and CF1065 showed good specificity for the galactosyl (ie, desialylated) peptide MOL136 and are potential candidates for monoclonal screening.

[0365] Example 7: Peptide Development

[0366] After evaluation of peptides and antisera, this showed that serum CF1064 and peptides MOL136 and MOL136c provided the most viable combinations tested. Sheep CF1064 were immunized with the same peptide sequence as MOL136, but using cysteine-maleimide chemistry instead of biotin as the KLH conjugate.

[0367] MOL136: Biotin-PEG-Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal]- D Ala-Arg-Phe- D Ser-Val-OH;

[0368] MOL136c: Biotin-PEG-Asp-Glu- D Ser-Nva-Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-Phe- D Ser-Val-OH.

[0369] In order to purify the antiserum, a method of capturing MOL136 and MOL136c using a streptavidin column was envisioned. The antibodies were then bound and eluted from the MOL136 column and then absorbed onto the MOL136c column to remove any peripheral binding agents, thereby picking up the common epitope between the two structures. By evaluating the purified antibodies in lateral flow, the overall specificity for the original galactosyl peptide antigen and the degree of cross-reaction with sialylated peptides were measured (Figure 18). This is important because it represents that the number of non-specific bindings observed in the prototype and with negative results without the use of an optical reader will need to be significantly reduced, making it impossible to pick up by the naked eye.

[0370] The purified and absorbed fractions performed best in the assay compared to the unabsorbed material. Although the background signal was significantly reduced, further method development is required to improve performance by truncating the peptide structure.

[0371] To further refine the peptide design and remove peripheral interactions of antibodies that do not bind to the galactose moiety in the peptide, the original sequence MOL136 was truncated to the shorter sequence MOL600:

[0372] MOL600: NH 2 -Cyc- D Ala-Ser[Gal]- D Ala-Arg-PEG-biotin;

[0373] MOL600c: NH 2 -Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-Biotin.

[0374] Then, MOL600 and MOL600c were tested by ELISA and showed significantly improved performance (see Figure 19).

[0375] Example 8: MOL600c Formulation / Stability Study and Scale-up

[0376] To evaluate how the MOL600c peptide was formulated, a study was set up to investigate drying conditions. The peptide was formulated in drying buffer, air dried and stored at room temperature before being used in prototype form. The data are available at Fig. 20 , showing the stability of peptide MOL600c stored in polypropylene tubes at different temperatures and different drying methods. The peptide retained its function throughout 4 weeks, however, the performance seemed to decline at 8 weeks.

[0377] For manufacturing, peptide synthesis has been shown to be scalable and can be ramped up to manufacturing batches. Peptides are produced by solid phase synthesis on an automated system and then purified by HPLC. Two additional chemical steps are required and further final purification by HPLC to a specification of >95% purity. The product is confirmed by electrospray mass spectrometry. Since only 12ng of peptide is required for each test, 1-2mg of peptide batches are of sufficient size. Figure 21 shows typical analytical data for a complete MOL600c batch.

[0378] Example 9: Antibody Purification Development

[0379] The purification process was initially developed for use with antiserum CF1064 and a truncated peptide system optimized to work on a streptavidin column loaded with MOL600 and an absorbance column loaded with MOL600c. Although this process produced potent antibodies with a good cross-reactivity profile, further improvements were needed to increase consistency and reduce the number of manual steps for automation.

[0380] To explore a more refined approach using a single pass, other column formats were tried and shown to be feasible and potentially automated ( Fig. 22 ). Two different column types have been used and the process has changed from a single pass approach to a post-absorption approach where unwanted interactions are eliminated to reduce non-specific binding in the assay. The best option for manufacturing is a single pass process and this can be achieved using peptide MOL615 conjugated to the carrier BSA and then derivatized onto an NHS agarose column.

[0381] MOL615: NH 2 -Cyc- D Ala-Ser[Gal]- D Ala-Arg-PEG-oxime.

[0382] The antibody component in the prototype is formulated into the gold conjugate dried into the conjugate pad in the lateral flow assembly. The conditions of gold conjugate stabilization have been established, and it has been further optimized. The amount of antibody calculated required for each test is estimated to be 85ng, which is enough to produce the required verification batch realizing CE marking. In the long run, other polyclonal reagents must be developed, and as a more permanent solution, monoclonal reagents must be developed. New immunity has begun, and strong antibody titers (Figure 10) have been identified for peptide targets. Several selections will be used for monoclonal production, including commercial hybridoma technology and also indoor phage panning and fab production (Molojik York and Scotia Biologics).

[0383] Example 10: Sample Buffer

[0384] Optimal conditions for sample extraction from the swab, peptide solubilization, and sialidase digestion are a matter of principle for the sample buffer. The following formulation was used:

[0385] 100 mM sodium acetate, 2 mM calcium chloride, pH 6.0, containing 0.1% BSA, 0.125% Tween-20, and 0.375% Triton X-100.

[0386] Buffers have been used successfully to run the device in dryness and also to extract actual samples with recoveries of spiked standards.

[0387] Example 11: Lateral flow device

[0388] according to Figure 4 A lateral flow device was constructed.

[0389] A batch of lateral flow devices was evaluated for stability over 12 weeks at 37°C. After 6 weeks, the signal values ​​increased compared to earlier measurements, however the shape and dynamic range of the curves appeared relatively consistent (see Fig.23 ).

[0390] Example 12: Assay Optimization

[0391] Specifications such as assay length, sensitivity of response to marker, line signal intensity, and standard curve quality were evaluated to meet feasible performance.

[0392] The assay has been shown to work within a 5 minute incubation time. This would keep the total time of the assay from swab sample collection to result reading to under 15 minutes. In the in vitro diagnostic industry, 15 minutes is often used as the upper limit for point-of-care rapid tests.

[0393] The entire relevant range of the marker (sialidase) is believed to be 4U / ml to 250U / ml. This is based on the literature obtained by estimating the propagation of sample data sets related to the fluorescent sialidase reference assay (Marconi et al., European Journal of Obstetrics and Gynaecology and Reproductive Biology, 2013, Vol. 167, pp. 205-209). Some assumptions were made in this method because in BV symptoms, since about 20 different bacterial species are associated with the symptoms, there will be a variety of different phenotypes (bacterial sialidase) in the sample. The assay has been tested across this range of sialidase in buffer, and a standard curve representing this range has been obtained with lateral flow ( Fig.24 ).

[0394] The assay has also been tested on actual healthy samples and the signal has been restored by incorporating sialidase. It has been shown that it has no effect whether the sample is centrifuged or not. The appearance of the test line is not adversely affected by the presence of sample matrix ( Fig.25 ). In order to make the sample sufficiently fluid, 0.5 ml of dilute sample buffer was found to be too concentrated as the sample retained too much viscosity. Currently 1 ml is considered to be the appropriate amount of buffer to use.

[0395] Example 13: Additional Antibody Generation

[0396] The following peptide, designated MOL616, was selected for generating additional antibodies:

[0397] Dpr(AOA)-dSer-Nva-Cyc-dAla-Ser(Gal)-dAla-Arg-Phe-dSer-Val-NH 2 .

[0398] The chemical formula of MOL616 is shown in Fig.26 middle.

[0399] The peptide was coupled to KLH, generating an immunogen called KLH-MOL616. Rabbits (Ornithogalum) were immunized with KLH-MOL616, and splenocytes were captured and screened with a truncated version of the peptide (MOL615, conjugated to BSA). Counter screening was performed with the sialylated form of the peptide MOL615, MOL615c. The screening process involved polyclonal screening followed by subclonal screening to generate antibodies specific for the desialylated form.

[0400] This process resulted in the production of a monoclonal antibody designated as antibody 125.1. This antibody is specific for the epitope Cyc- D Ala-Ser[Gal]- D Ala-Arg-PEG-oxime is specific, more specifically to the epitope Cyc- D Ala-Ser[Gal]- D Ala-Arg is specific.

[0401] MOL615 was biotinylated to generate MOL600 (sequence provided in Example 7), and biointerference measurement was used to measure antibody binding affinity and kinetics. Briefly, MOL600 or MOL600c was immobilized on the biosensor via streptavidin / biotin interactions. Binding of the antibody was detected by a shift in reflected light interference.

[0402] Using different concentrations of antibody 125.1 (1, 2, 4, 8 or 16 nM respectively), the following binding affinities and kinetics were determined for antibody 125.1:

[0403] K D =7.9nM; K on =54080M -1 s -1 ; K off =0.000427s -1 .

[0404] Binding was clearly specific for MOL600 compared to the sialylated form, MOL600c, as Fig. 27 shown.

[0405] The antibody was sequenced and determined to have a gamma heavy chain and a kappa light chain. The sequence of this antibody is shown in Fig.31 and listed below.

[0406] Antibody heavy chain

[0407] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYSMDWVRQAPGKGLEWVGGITTTLHTFYATWAKGRFTISKTSSTTVDLKMTSLTTDDAATYFCARGGSSVIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHEDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPG(SEQ ID NO:7)

[0408] Antibody light chain

[0409] ALVMTQTPSPVSAAVGGTVTISCQSSQSVYGNNHLNWHQQKRGQPPKQLIGSASRLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGSYYNGAWYVAFGGGTELEIKRDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDITVTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCEVVQGSASPIVQSFNRGDC

[0410] (SEQ ID NO:8)

[0411] Heavy chain CDR sequences

[0412] CDR1 GFSLSSY(SEQ ID NO:1)

[0413] CDR2 TTTLH(SEQ ID NO:2)

[0414] CDR3 GGSSVI(SEQ ID NO:3)

[0415] Light chain CDR sequences

[0416] CDR1 QSSQSVYGNNHLN(SEQ ID NO:4)

[0417] CRD2 SASRLAS (SEQ ID NO:5)

[0418] CDR3 QGSYYNGAWYVA(SEQ ID NO:6)

[0419] Noteworthy observations:

[0420] An additional O-linked glycosylation site was detected in the heavy chain constant region between positions 213 and 216. From the gel image, the glycosylated form at this site was similar in abundance to the unglycosylated form.

[0421] N-linked glycosylation was detected on N@285 of the heavy chain constant region.

[0422] A Pyro-Glu (Q) modification was observed at the N-terminus of the heavy chain.

[0423] The C-terminal lysine was eliminated from the regular constant region sequence observed on the heavy chain.

[0424] Example 14: Further assay optimization

[0425] For further optimization, two different sialidase activity assays were compared (A and B, see below). The assay setup was essentially as described in conjunction with FIG. 2 .

[0426] The test antibody was conjugated to gold and dried onto a support, referred to herein as the "conjugate pad". The test antibody in assay A was the "1064 purified Mol615 antibody", i.e., a polyclonal antibody purified from serum 1064 using Mol615. The test antibody in assay B was a monoclonal antibody referred to as "125.1" (see Example 13).

[0427] A control antibody was also conjugated to gold and dried onto the conjugate pad. For assay B this was chicken IgY supplied by Lampire, product code 7401403.

[0428] The capture molecule (polystreptavidin) was immobilized on a nitrocellulose membrane to form a capture line. A separate control capture line was formed. For assay B, this was an anti-chicken IgY antibody (supplied by Rampur, product code 7455207).

[0429] As shown in Figure 2, the conjugate pad was laminated onto nitrocellulose.

[0430] The test indicator molecule NH 2 -Cyc- DAla-Ser[Gal-Sial]- D Ala-Arg-PEG-biotin (MOL600c) and 1 μg / ml or 3 μg / ml were freeze-dried onto an inert porous material (Porex disks) to produce indicator substrate disks containing 36 ng of indicator molecules per disk or 108 ng of indicator molecules per disk, respectively.

[0431] Sialidase was tested at the following concentrations: 50, 12.5 and 1.56 U / ml. A negative standard using peptide disks and assay buffer was also run. Responses were measured for both cubic readouts and visual scales.

[0432] The following basic scheme was used:

[0433] Make sure all solutions are at room temperature and mixed thoroughly.

[0434] Each of the desired conditions was tested with 50, 12.5 and 1.56 U / ml sialidase buffer standard solutions.

[0435] All standards and sialidase buffer were tested in triplicate.

[0436] Add 1000 μl of sialidase standard to the extraction tube along with the indicator substrate disc. Seal the tube by folding the lid and snapping it into place. Agitate the tube gently and allow to incubate with the indicator substrate disc for 5 minutes.

[0437] Add 4 drops from the extraction tube to the sample pad of the device.

[0438] The test and control line signals were read and recorded at 5 or 10 minutes using a cubic reader (Optricon reader from opTricon GmbH (Chemical Biodiagnostic Systems), Schwarzschildstrasse 1, D-12489, Berlin, Germany).

[0439] Characteristics of the test assays are presented below.

[0440]

[0441] (i) Comparison of read times

[0442] The desired output is to have a 10 minute test, a 5 minute incubation, and a 5 minute read time. The results were read at 5 and 10 minutes to study the effect of the read time on the results. The results are shown in Fig.28As can be seen, assay A requires a 10-minute read time to meet the minimum standard for a correct response, while assay B only requires a 5-minute read time. There does not appear to be a significant benefit to assay B from the 10-minute read time; it meets the expected output of a 10-minute test and a 5-minute read.

[0443] (ii) Comparison of sensitivity

[0444] The results of assay B were read after 5 minutes and the results of assay A were read after 10 minutes. The results are shown in Fig.29 middle.

[0445] In the standards of 50 U / ml, 12.5 U / ml and 1.56 U / ml, the cubic readings of assay B were higher relative to assay A, and the cubic readings were lower for the standard of 0 U / ml. Both A and B gave the correct response for each of the standards, and the %CV was within the specification. After the device development time was reduced, assay B gave the cubic readings of the standards, and for the 0 standard, it gave a lower cubic reading. Therefore, assay B is superior to assay A.

[0446] (iii) Effect of peptide concentration

[0447] Assays A and B were compared to each other and to variants with different peptide concentrations. Assay A' corresponds to assay A, but with a peptide concentration of 3 μg / ml (108 ng per plate); Assay B' corresponds to assay B, but with a peptide concentration of 1 μg / ml (108 ng per plate).

[0448] The results of assays B and B' were read after 5 minutes, and the results of assays A and A' were read after 10 minutes. The results are shown in Fig.30 Higher peptide concentrations provide better results, and peptide concentrations >1 μg / ml (>108 ng per plate are preferred.

[0449] in conclusion:

[0450] Assay B had improved performance over assay A in a shorter time due to changes in antibodies and increased peptide concentration.

[0451] Example 15 - Additional Assay Optimization

[0452] Gold optical density

[0453] The effect of increasing the gold conjugate spray concentration from OD4 to OD6 or OD8 was studied and it was found that while OD6 gold slightly increased the test line signal compared to OD4, the test line signal at OD8 at 3.125 U / mL sialidase was twice that of OD4. Therefore, it is advantageous to use a gold conjugate at an OD of about or at least 8.

[0454] Sample / conjugate pad

[0455] In a lateral flow assay device, such as the type schematically represented in Figure 2, the sample pad should allow the sample to flow along the device to allow the sample to contact the conjugate pad containing the binding molecules and subsequently the capture molecules. Depending on the viscosity of the sample, some sample pad materials may be advantageous.

[0456] To evaluate different sample pads, synthetic vaginal fluid surrogates were prepared using guar gum and bovine mucin (0.25% guar gum, 0.125% mucin, 5% blue latex (Polysciences Inc., 15709, 2.6%, dissolved in water). Synthetic vaginal fluid was allowed to flow through all sample pads. The time it took for the sample to run the length of the test strip was recorded (see table below).

[0457] Sample pad Window startup time The length of time the window runs 1.FR1 10mm blood separator 01:20 02:20 2. GF-142 01:16 02:06 3.8964, Ahlstrom 01:17 02:07 4.6613, Ahlstrom 01:45 01:20 5.6615, Ahlstrom 02:20 01:30

[0458] Ahlstrom is a fiberglass mat with different densities (8964, 6613 and 6615 respectively).

[0459] For further comparison, material was prepared using two sample pads (FR1 and Ahlstrom 8964). The device was run using a 4-point standard curve (aqueous buffer containing 0, 3.125, 12.5, and 50 U / mL sialidase) to determine if the alternative sample pads had any effect on specific or nonspecific signal. Samples run using the 8964 sample pad had higher specific signal and no nonspecific binding (NSB) relative to FR1.

[0460] Using a panel of clinical samples spiked with 6.25 U / ml sialidase, it was determined that glass fiber-based pads (such as the Ahlstrom 8964 sample pad) allow for very reliable sample flow and enzyme detection.

[0461] Example 16 - Diagnosis of Bacterial Vaginosis

[0462] Clinical samples are obtained and analyzed for criteria such as clue cells to determine the Nugent score, based on which the sample is classified as positive or negative for bacterial vaginosis. The samples are analyzed using either Assay A or Assay B (see Example 14).

[0463] The results are shown below, where se stands for sensitivity; sp stands for specificity; ppv stands for positive predictive value; and nvp stands for negative predictive value.

[0464] Determination A

[0465]

[0466]

[0467] Assay B

[0468]

[0469]

[0470] Therefore, both assays performed well in differentiating between clinical samples that were positive or negative for bacterial vaginosis. Assay B was superior to Assay A.

[0471] The present invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. In addition, all aspects and embodiments of the present invention described herein are considered to be widely applicable and can be combined with any and all other consistent embodiments, including embodiments appropriately selected from other aspects of the present invention (including individually). Various publications are cited herein, and their disclosures are incorporated herein by reference in their entirety. Sequence Listing <110> Mologic Ltd <120> Bacterial vaginosis diagnosis <130> P148556WO00 <150> GB1812331.5 <151> 2018-07-27 <160> 17 <170> PatentIn Version 3.5 <210> 1 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 1 Gly Phe Ser Leu Ser Ser Tyr 1 5 <210> 2 <211> 5 <212> PRT <213> Rabbit <400> 2 Thr Thr Thr Leu His 1 5 <210> 3 <211> 6 <212> PRT <213> European rabbit <400> 3 Gly Gly Ser Ser Val Ile 1 5 <210> 4 <211> 13 <212> PRT <213> European rabbit <400> 4 Gln Ser Ser Gln Ser Val Tyr Gly Asn Asn His Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> European rabbit <400> 5 Ser Ala Ser Arg Leu Ala Ser 1 5 <210> 6 <211> 12 <212> PRT <213> European rabbit <400> 6 Gln Gly Ser Tyr Tyr Asn Gly Ala Trp Tyr Val Ala 1 5 10 <210> 7 <211> 434 <212> PRT <213> European rabbit <400> 7 Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Tyr Ser 20 25 30 Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 35 40 45 Gly Ile Thr Thr Thr Leu His Thr Phe Tyr Ala Thr Trp Ala Lys Gly 50 55 60 Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys Met 65 70 75 80 Thr Ser Leu Thr Thr Asp Asp Ala Ala Thr Tyr Phe Cys Ala Arg Gly 85 90 95 Gly Ser Ser Val Ile Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser 100 105 110 Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys Gly 115 120 125 Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 130 135 140 Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr Asn 145 150 155 160 Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr Ser 165 170 175 Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr Cys 180 185 190 Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val Ala 195 200 205 Pro Ser Thr Cys Ser Lys Pro Thr Cys Pro Pro Pro Glu Leu Leu Gly 210 215 220 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 225 230 235 240 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Glu 245 250 255 Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln Val 260 265 270 Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr Ile 275 280 285 Arg Val Val Ser Thr Leu Pro Ile Ala His Glu Asp Trp Leu Arg Gly 290 295 300 Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro Ile 305 310 315 320 Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys Val 325 330 335 Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val Ser 340 345 350 Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val Glu 355 360 365 Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro Ala 370 375 380 Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser Val 385 390 395 400 Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val Met 405 410 415 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg Ser 420 425 430 Pro Gly <210> 8 <211> 218 <212> PRT <213> European rabbit <400> 8 Ala Leu Val Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly 1 5 10 15 Gly Thr Val Thr Ile Ser Cys Gln Ser Ser Gln Ser Val Tyr Gly Asn 20 25 30 Asn His Leu Asn Trp His Gln Gln Lys Arg Gly Gln Pro Pro Lys Gln 35 40 45 Leu Ile Gly Ser Ala Ser Arg Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Gly Val 65 70 75 80 Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Ser Tyr Tyr Asn 85 90 95 Gly Ala Trp Tyr Val Ala Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys 100 105 110 Arg Asp Pro Val Ala Pro Ser Val Leu Leu Phe Pro Pro Ser Lys Glu 115 120 125 Glu Leu Thr Thr Gly Thr Ala Thr Ile Val Cys Val Ala Asn Lys Phe 130 135 140 Tyr Pro Ser Asp Ile Thr Val Thr Trp Lys Val Asp Gly Thr Thr Gln 145 150 155 160 Gln Ser Gly Ile Glu Asn Ser Lys Thr Pro Gln Ser Pro Glu Asp Asn 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Ser Leu Thr Ser Ala Gln Tyr Asn 180 185 190 Ser His Ser Val Tyr Thr Cys Glu Val Val Gln Gly Ser Ala Ser Pro 195 200 205 Ile Val Gln Ser Phe Asn Arg Gly Asp Cys 210 215 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(5) <223> X may be an amino acid, provided that at least one of X1, X2, X4 or X5 is a D amino acid and / or non-standard or unnatural amino acids <220> <221> MOD_RES <222> (3) <223> Amino acids have a Gal-Sial group <400> 9 Xaa Xaa Xaa Xaa Xaa 1 5 <210> 10 <211> 20 <212> PRT <213> ARTIFICIAL <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1) <223> X does not exist or is Thr <220> <221> MISC_FEATURE <222> (2) <223> X is absent or is Ala in the D form <220> <221> MISC_FEATURE <222> (3) <223> X is absent or is norleucine <220> <221> MISC_FEATURE <222> (4) <223> X does not exist or is Glu <220> <221> MISC_FEATURE <222> (5) <223> X is absent or is Ala in the D form <220> <221> MISC_FEATURE <222> (6) <223> X does not exist or is Arg <220> <221> MISC_FEATURE <222> (7) <223> X is absent or selected from Glu, Arg, Ser, valeric acid, β-Ala <220> <221> MISC_FEATURE <222> (8) <223> X is absent or selected from the group consisting of D-form Ser, D-form Ala, phosphoserine, 1-Aminocyclohexane-carboxylic acid <220> <221> MISC_FEATURE <222> (9)..(9) <223> X is absent or selected from valeric acid, 2'-(aminomethyl)biphenyl-2-carboxylic acid, D-form of Ala, β-Ala, ornithine <220> <221> MISC_FEATURE <222> (10)..(10) <223> X is selected from 1-aminocyclohexane-carboxylic acid, Ser, Ile, D-form Ala, D-form Ser <220> <221> MISC_FEATURE <222> (11)..(11) <223> X is selected from the group consisting of Ala, Pro, ornithine, norleucine in D form <220> <221> MISC_FEATURE <222> (12)..(12) <223> X is selected from Ser, Thr, Tyr, hydroxylysine, hydroxyproline, Asn, Arg or Phosphoserine; and has a Gal-Sial group <220> <221> MISC_FEATURE <222> (13) <223> X is selected from the group consisting of D-form Ala, 2'-(aminomethyl)biphenyl-2-carboxylic acid, β-Ala <220> <221> MISC_FEATURE <222> (14)..(14) <223> X is selected from Arg, D-form of Asp, norleucine, ornithine, valerine <220> <221> MISC_FEATURE <222> (15) <223> X is absent or selected from Phe, 2'-(aminomethyl)biphenyl-2-carboxylic acid, Ser, Glu, D-form of Ala, D-form of Ser <220> <221> MISC_FEATURE <222> (16) <223> X is absent or selected from Ser, Glu in D form <220> <221> MISC_FEATURE <222> (17) <223> X is absent or selected from Val, Ser, Thr <220> <221> MISC_FEATURE <222> (18) <223> X is absent or is cyclohexylalanine <220> <221> MISC_FEATURE <222> (19)..(19) <223> X does not exist or is a Ser of the D form <220> <221> MISC_FEATURE <222> (20)..(20) <223> X does not exist or is Val <400> 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 <210> 11 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (1) <223> X is 1-aminocyclohexane-carboxylic acid <220> <221> MOD_RES <222> (2)..(4) <223> Each alanine is in the D form <220> <221> MOD_RES <222> (3) <223> Serine has a Gal-Sial group <400> 11 Xaa Ala Ser Ala Arg 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (2) <223> Serine is in the D form <220> <221> MISC_FEATURE <222> (3) <223> X is valeric acid <220> <221> MISC_FEATURE <222> (4) <223> X is 1-aminocyclohexane-carboxylic acid <220> <221> MISC_FEATURE <222> (5)..(7) <223> Each alanine is in the D form <220> <221> MOD_RES <222> (5) <223> Serine has a Gal-Sial group <220> <221> MISC_FEATURE <222> (10)..(10) <223> Serine is in the D form <400> 12 Glu Ser Xaa Xaa Ala Ser Ala Arg Phe Ser Val 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (2)..(7) <223> Each alanine is in the D form <220> <221> MISC_FEATURE <222> (3) <223> X is 2'-(aminomethyl)biphenyl-2-carboxylic acid <220> <221> MOD_RES <222> (6) <223> Serine has a Gal-Sial group <220> <221> MISC_FEATURE <222> (8) <223> Asp is in the D form <400> 13 Arg Ala Xaa Ser Pro Ser Ala Asp Ser 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MOD_RES <222> (2) <223> Serine is phosphoserine <220> <221> MOD_RES <222> (3)..(7) <223> Each alanine is in the D form <220> <221> MISC_FEATURE <222> (5) <223> X is ornithine <220> <221> MOD_RES <222> (6) <223> Serine has a Gal-Sial group <220> <221> MISC_FEATURE <222> (8) <223> X is norleucine <400> 14 Ser Ser Ala Ile Xaa Ser Ala Xaa Glu 1 5 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1) <223> Alanine in D form <220> <221> MISC_FEATURE <222> (3) <223> x is valeric acid <220> <221> MISC_FEATURE <222> (4) <223> Serine is in the D form <220> <221> MISC_FEATURE <222> (5) <223> x is beta-alanine <220> <221> MISC_FEATURE <222> (6)..(11) <223> Each alanine is in the D form <220> <221> MISC_FEATURE <222> (7) <223> x is norleucine <220> <221> MOD_RES <222> (8) <223> Serine has a Gal-Sial group <220> <221> MISC_FEATURE <222> (9)..(9) <223> X is 2'-(aminomethyl)biphenyl-2-carboxylic acid <220> <221> MISC_FEATURE <222> (10)..(10) <223> X is ornithine <400> 15 Ala Arg Xaa Ser Xaa Ala Xaa Ser Xaa Xaa Ala Glu Ser 1 5 10 <210> 16 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (2)..(5) <223> Each alanine is in the D form <220> <221> MISC_FEATURE <222> (3) <223> X is norleucine <220> <221> MISC_FEATURE <222> (7) <223> X is β-alanine <220> <221> MISC_FEATURE <222> (8) <223> X is 1-aminocyclohexane-carboxylic acid <220> <221> MISC_FEATURE <222> (9)..(9) <223> X is ornithine <220> <221> MISC_FEATURE <222> (10)..(10) <223> Serine is in the D form <220> <221> MOD_RES <222> (12)..(12) <223> Serine has a Gal-Sial group <220> <221> MISC_FEATURE <222> (13) <223> X is β-alanine <220> <221> MISC_FEATURE <222> (14)..(14) <223> X is valeric acid <220> <221> MISC_FEATURE <222> (15)..(19) <223> Serine is in the D form <220> <221> MISC_FEATURE <222> (18) <223> X is cyclohexylalanine <400> 16 Thr Ala Xaa Glu Ala Arg Xaa Xaa Xaa Ser Pro Ser Xaa Xaa Ser Glu 1 5 10 15 Thr Xaa Ser Val 20 <210> 17 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> MISC_FEATURE <222> (1) <223> X can be any amino acid, provided that at least one of X1, X2, X4 and X5 is D amino acids and / or non-standard amino acids or unnatural amino acids. <220> <221> MISC_FEATURE <222> (2) <223> X can be any amino acid, provided that at least one of X1, X2, X4 and X5 is D amino acids and / or non-standard amino acids or unnatural amino acids. <220> <221> MISC_FEATURE <222> (3) <223> X is an amino acid including a Gal-Sial group and is selected from Ser, Thr, Tyr, Hydroxylysine, hydroxyproline, Asn, Arg, or phosphoserine <220> <221> MISC_FEATURE <222> (4) <223> X can be any amino acid, provided that at least one of X1, X2, X4 and X5 is D amino acids and / or non-standard amino acids or unnatural amino acids. <220> <221> MISC_FEATURE <222> (5) <223> X can be any amino acid, provided that at least one of X1, X2, X4 and X5 is D amino acids and / or non-standard amino acids or unnatural amino acids. <400> 17 Xaa Xaa Xaa Xaa Xaa 1 5

Claims

1. An enzyme detection kit for detecting the presence of sialidase cleavage activity in a test sample, the kit comprising: include: (i) an indicator molecule, the indicator molecule comprising: (a) A peptide having the following sequence: Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg (SEQ ID NO: 11); and (b) a capture site that remains intact after cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample; (ii) a capture zone for receiving the test sample, wherein the capture zone comprises a capture molecule capable of binding to the capture site of the indicator molecule regardless of whether the indicator molecule has been cleaved, so as to immobilize the indicator molecule; and (iii) a binding molecule capable of binding to a desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred; and wherein the binding molecule is an antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO:

6.

2. The kit of claim 1, wherein the peptide is preferentially cleaved by one or more specific sialidases.

3. The kit according to claim 2, wherein the one or more specific sialidases are of bacterial origin.

4. The kit according to claim 3, wherein the bacteria are Prevotella species, Bacteroides species and / or Mobiluncus species and / or Gardnerella vaginalis Gardnerella vaginalis ).

5. The kit of claim 1, wherein the binding molecule is specific for a desialylated form of a peptide consisting of the sequence: (i)Cyc- D Only-Ser[Gal-False]- D Ala-Arg(SEQ ID NO: 11)。 6. The kit according to claim 1, wherein the heavy chain of the binding molecule has SEQ ID NO: 7 and / or the light chain of the binding molecule has SEQ ID NO:

8.

7. The kit according to claim 1, wherein the binding molecule is labeled with a reporter molecule.

8. The kit according to claim 1, wherein the capture site of the indicator molecule comprises or consists of a biotin molecule or an oxime moiety; and / or is at the N-terminus or C-terminus of the peptide.

9. The kit according to claim 1, wherein the capture site of the indicator molecule is connected to the peptide via a linker.

10. The kit of claim 9, wherein the linker comprises a polyethylene glycol (PEG) moiety.

11. The kit according to claim 10, wherein the peptide is linked to a biotin group at its N-terminus or C-terminus via a linker comprising or consisting of a polyethylene glycol moiety.

12. The kit according to claim 1, wherein the indicator molecule has the following structure composition: (i)Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg-PEG-Biotin.

13. The kit according to claim 1, wherein the indicator molecule comprises (i) a peptide comprising the sequence Cyc- D Ala-Ser[Gal-Sial]- D Ala-Arg or consisting thereof; and (ii) a capture site comprising or consisting of a biotin molecule or an oxime moiety; and in: The heavy chain of the binding molecule has SEQ ID NO: 7 and / or the light chain of the binding molecule has SEQ ID NO: 8, or The binding molecule is an antibody specific for the desialylated form of a peptide consisting of the following sequence: (i)Cyc- D Only-Ser[Gal-False]- D Ala-Arg(SEQ ID NO: 11)。 14. The kit according to claim 13, wherein the capture site is connected to the N-terminus or C-terminus of the peptide via a linker, the linker comprising or consisting of a polyethylene glycol moiety.

15. An antibody capable of specifically binding to: (i) a desialylated derivative of a peptide as defined in any one of claims 1 to 4; or (ii) an indicator molecule as defined in any one of claims 1 to 4 or 8 to 14, wherein the antibody preferentially binds to the desialylated derivative over the sialylated peptide or indicator molecule; and wherein the antibody has a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO:

6.

16. The antibody according to claim 15, wherein the antibody is as defined in claim 5, 6 and / or 7.

17. An indicator molecule suitable for use in detecting the presence of sialidase cleavage activity in a test sample, the indicator molecule include: a) A peptide as defined in any one of claims 1 to 4; as well as b) A capture site which remains intact after cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample.

18. The indicator molecule according to claim 17, wherein the indicator molecule is as defined in claim 12.

19. A peptide consisting of the following sequence composition: (i)Cyc- D Only-Ser[Gal-False]- D Ala-Arg(SEQ ID NO: 11)。 20. A peptide for use in producing the antibody according to claim 15 or 16, wherein the peptide is a desialylated derivative of the peptide as defined in any one of claims 1 to 4.

21. A method for detecting the presence or absence of cleavage activity of a sialidase in a test sample, the method include: (i) contacting an indicator molecule as defined in any one of claims 1 to 4 or 8 to 14 with the test sample; (ii) adding to the test sample a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample has occurred, and wherein the binding molecule is an antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO: 6; (iii) capturing the desialylated derivative of the indicator molecule at the capture zone by binding of a capture molecule in the capture zone to the capture site, the capture molecule being able to bind to the capture site regardless of whether the indicator molecule has been cleaved; as well as (iv) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

22. A method for detecting the presence or absence of cleavage activity of a sialidase in a test sample, the method include: (i) adding the test sample to a capture zone comprising capture molecules which bind to the capture sites of indicator molecules as defined in any one of claims 1 to 4 or 8 to 14, wherein the capture molecules remain bound to the capture sites irrespective of whether cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample occurs; (ii) adding a binding molecule capable of binding to the desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylated group from the indicator molecule by a sialidase present in the sample has occurred, and wherein the binding molecule is an antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO: 6; (iii) detecting cleavage of the sialylated group from the indicator molecule by determining binding of the binding molecule to the desialylated derivative of the indicator molecule captured in the capture zone.

23. An enzyme detection kit for detecting the presence of sialidase cleavage activity in a test sample, the kit include: (i) an indicator molecule as defined in any one of claims 1 to 4; (ii) a capture molecule, which is capable of binding to the capture site of the indicator molecule regardless of whether the indicator molecule has been cleaved; (iii) a solid phase carrier, the capture molecule being capable of being linked to the solid phase carrier or being linked to the solid phase carrier to form a capture zone for receiving the test sample; as well as (iv) a binding molecule capable of binding to a desialylated derivative of the indicator molecule, wherein the binding molecule is incapable of binding to the indicator molecule unless and until cleavage of the sialylation group from the indicator molecule by a sialidase present in the sample has occurred, and wherein the binding molecule is an antibody having a heavy chain with 3 CDRs and a light chain with 3 CDRs, wherein the heavy chain CDR1 has SEQ ID NO: 1; the heavy chain CDR2 has SEQ ID NO: 2; the heavy chain CDR3 has SEQ ID NO: 3; the light chain CDR1 has SEQ ID NO: 4; the light chain CDR2 has SEQ ID NO: 5; and the light chain CDR3 has SEQ ID NO:

6.

24. The kit according to claim 23, wherein the kit include: (v) extraction tubes and / or buffer; and / or (vi) Control assay molecules and control assay binders.

Images