BACTERIAL VAGINOSIS DIAGNOSTIC

Abstract

The invention provides a sialidase enzyme activity detection kit or device comprising: (i) an indicator molecule comprising a sialylated peptide and a capture site; (ii) a capture zone comprising capture molecules; and (iii) binding molecules capable of binding to the de-sialylated derivative of the indicator molecule. Also provided are methods of using the kits or devices, as well as specific indicator molecules and specific binding molecules.

Claims

1. An enzyme detection device or enzyme detection kit for detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the device or kit comprising: (i) an indicator molecule comprising (a) a peptide comprising the following sequence: TABLE-US-00015 (SEQ ID NO: 17) X.sub.1-X.sub.2-X.sub.3[Gal-Sial]-X.sub.4-X.sub.5 wherein: Sial is a sialyl group; X.sub.3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X.sub.3 is Ser; and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are independently selected from any amino acid provided that at least one, preferably at least two or three, of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a .sub.D-amino acid and/or a non-standard amino acid or a non-natural amino acid; and b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the sample; (ii) a capture zone to receive the test sample, wherein the capture zone comprises capture molecules capable of binding to the capture site of the indicator molecule, irrespective of whether or not the indicator molecule has been cleaved, in order to immobilise the indicator molecule; and (iii) binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred.

2. The device or kit of claim 1 wherein at least one of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is Ala, preferably wherein X.sub.1 and X.sub.2 are both Ala, and/or preferably wherein Ala is .sub.DAla or βAla.

3. The device or kit of claim 1, wherein at least one of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a charged amino acid, optionally wherein each charged amino acid is selected from Arg, .sub.DAsp and .sub.LOrn.

4. The device or kit of claim 1, wherein at least one of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a polar amino acid, optionally wherein each polar amino acid is selected from .sub.LSer, .sub.DSer and Thr.

5. The device or kit of claim 1, wherein the peptide comprises the following sequence: TABLE-US-00016 (SEQ ID NO: 10) X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.sub.10-X.sub.11-X.sub.12[Gal-Sial]-X.sub.13- X.sub.14-X.sub.15-X.sub.16-X.sub.17-X.sub.18-X.sub.19-X.sub.20 wherein: Sial is a sialyl group X.sub.1 is absent or Thr X.sub.2 is absent or .sub.DAla X.sub.3 is absent or Nle X.sub.4 is absent or Glu X.sub.5 is absent or .sub.DAla X.sub.6 is absent or Arg X.sub.7 is absent or selected from Glu, Arg, Ser, Nva, βAla X.sub.8 is absent or selected from .sub.DSer, βAla, SEP, Cyc X.sub.9 is absent or selected from Nva, BIP, .sub.DAla, βAla, Orn X.sub.10 is selected from Cyc, Ser, Ile, .sub.DAla, .sub.DSer X.sub.11 is selected from .sub.DAla, Pro, Orn, Nle X.sub.12 is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or SEP, preferably Ser X.sub.13 is selected from .sub.DAla, BIP, βAla X.sub.14 is selected from Arg, .sub.DAsp, Nle, Orn, Nva X.sub.15 is absent or selected from Phe, BIP, Ser, Glu, .sub.DAla, .sub.DSer X.sub.16 is absent or selected from .sub.DSer, Glu X.sub.17 is absent or selected from Val, Ser, Thr X.sub.18 is absent or Cha X.sub.19 is absent or .sub.DSer X.sub.20 is absent or Val.

6. The device or kit of claim 1, wherein the peptide comprises the following sequences: TABLE-US-00017 (i) (SEQ ID NO: 11) Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg (ii) (SEQ ID NO: 12) Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-Phe- .sub.DSer-Val (iii) (SEQ ID NO: 13) Arg-.sub.DAla-BIP-Ser-Pro-Ser[Gal-Sial]-.sub.DAla-.sub.DAsp-Ser (iv) (SEQ ID NO: 14) Ser-SEP-.sub.DAla-Ile-Orn-Ser[Gal-Sial]-.sub.DAla-Nle-Glu (v) (SEQ ID NO: 15) .sub.DAla-Arg-Nva-.sub.DSer-βAla-.sub.DAla-Nle-Ser[Gal-Sial]-BIP- Orn-.sub.DAla-Glu-Ser or (vi) (SEQ ID NO: 16) Thr-.sub.DAla-Nle-Glu-.sub.DAla-Arg-βAla-Cyc-Orn-.sub.DSer-Pro- Ser[Gal-Sial]-βAla-Nva-.sub.DSer-Glu-Thr-Cha-.sub.DSer-Val,

7. The device or kit of claim 1, wherein the peptide is biased for cleavage by one or more specific sialidases, optionally wherein the one or more specific sialidases are of bacterial origin, optionally wherein the bacteria are Prevotella, Bacteroides and/or Mobiluncus species and/or Gardnerella vaginalis.

8. The device or kit of claim 1, wherein the binding molecule is specific for the de-sialylated form of the peptide comprising SEQ ID NO: 11, 12, 13, 14, 15, or 16, preferably is specific for an epitope that is present in the peptide motif Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg and that is absent or cryptic in the corresponding sialylated peptide motif Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg.

9. The device or kit of claim 1, 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, preferably wherein the heavy chain has SEQ ID NO: 7 and/or the light chain has SEQ ID NO: 8.

10. The device or kit of claim 1, wherein the binding molecule is labelled with a reporter molecule, preferably a gold particle.

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

12. The device or kit of claim 1, wherein the capture site of the indicator molecule is attached to the peptide by a linker, optionally wherein the linker comprises a polyethylene glycol (PEG) moiety, optionally wherein the peptide is linked to a biotin group at its N- or C-terminus via a linker comprising a polyethylene glycol moiety.

13. The device or kit of claim 1, wherein the indicator molecule comprises the following structure: (i) Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin (ii) Biotin-PEG-Asp-Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-Phe-.sub.DSer-Val (iii) Biotin-PEG-Asp-Arg-.sub.DAla-BIP-Ser-Pro-Ser[Gal-Sial]-.sub.DAla-.sub.DAsp-Ser (iv) Biotin-PEG-Asp-Ser-Ser(PO.sub.3)-.sub.DAla-Ile-Orn-Ser[Gal-Sial]-.sub.DAla-Nle-Glu (v) Biotin-PEG-Asp-.sub.DAla-Arg-Nva-.sub.DSer-βAla-.sub.DAla-Nle-Ser[Gal-Sial]-BIP-Orn-.sub.DAla-Glu-Ser; or (vi) Biotin-PEG-Asp-Thr-.sub.DAla-Nle-Glu-.sub.DAla-Arg-βAla-Cyc-Orn-.sub.DSer-Pro-Ser[Gal-Sial]-βAla-Nva-.sub.DSer-Glu-Thr-Cha-.sub.DSer-Val.

14. The device or kit of claim 1, wherein the indicator molecule comprises (i) a peptide comprising the sequence Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg; and (ii) a capture site that comprises a biotin molecule or an oxime moiety which is optionally linked to the N- or C-terminus of the peptide via a linker comprising a polyethylene glycol moiety; and wherein the binding molecule is: (a) an antibody specific for the de-sialylated form of the peptide comprising SEQ ID NO: 11, 12, 13, 14, 15, or 16, preferably is specific for an epitope that is present in the peptide motif Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg and that is absent or cryptic in the corresponding sialylated peptide motif Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg; or (b) 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, preferably wherein the heavy chain has SEQ ID NO: 7 and/or the light chain has SEQ ID NO: 8; and preferably is labelled with a reporter molecule, most preferably a gold particle.

15. A composition comprising: (a) an antibody specific for the de-sialylated form of the peptide comprising SEQ ID NO: 11, 12, 13, 14, 15, or 16, preferably specific for an epitope that is present in the peptide motif Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg and that is absent or cryptic in the corresponding sialylated peptide motif Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg; or (b) 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, preferably wherein the heavy chain has SEQ ID NO: 7 and/or the light chain has SEQ ID NO: 8; or (c) an indicator molecule suitable for use in detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the indicator molecule comprising a capture site and a peptide comprising: (i) Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin; (ii) Biotin-PEG-Asp-Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-Phe-.sub.DSer-Val; (iii) Biotin-PEG-Asp-Arg-.sub.DAla-BIP-Ser-Pro-Ser[Gal-Sial]-.sub.DAla-.sub.DAsp-Ser; (iv) Biotin-PEG-Asp-Ser-Ser(PO.sub.3)-.sub.DAla-Ile-Orn-Ser[Gal-Sial]-.sub.DAla-Nle-Glu; (v) Biotin-PEG-Asp-.sub.DAla-Arg-Nva-.sub.DSer-βAla-.sub.DAla-Nle-Ser[Gal-Sial]-BIP-Orn-.sub.DAla-Glu-Ser; or (vi) Biotin-PEG-Asp-Thr-.sub.DAla-Nle-Glu-.sub.DAla-Arg-βAla-Cyc-Orn-.sub.DSer-Pro-Ser[Gal-Sial]-βAla-Nva-.sub.DSer-Glu-Thr-Cha-.sub.DSer-Val; or (d) an indicator molecule suitable for use in detecting the presence in a test sample of cleavage activity of a sialidase enzyme, the indicator molecule comprising a capture site and a peptide comprising: X.sub.1-X.sub.2-X.sub.3[Gal-Sial]-X.sub.4-X.sub.5  wherein:  Sial is a sialyl group;  X.sub.3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X.sub.3 is Ser; and  X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are independently selected from any amino acid provided that at least one, preferably at least 2 or 3, of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a .sub.D-amino acid and/or a non-standard amino acid or a non-natural amino acid; or (e) a peptide comprising SEQ ID NO: 11, 12, 13, 14, 15 or 16.

16.-21. (canceled)

22. A method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme, the method comprising: (i) bringing an indicator molecule into contact with the test sample, wherein the indicator molecule comprises (a) a peptide comprising the following sequence: TABLE-US-00018 (SEQ ID NO: 17) X.sub.1-X.sub.2-X.sub.3[Gal-Sial]-X.sub.4-X.sub.5 wherein: Sial is a sialyl group; X.sub.3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X.sub.3 is Ser; and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are independently selected from any amino acid provided that at least one, preferably at least two or three, of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a .sub.D-amino acid and/or a non-standard amino acid or a non-natural amino acid; and b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the test sample; (ii) adding to the test sample binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred; (iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and (iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

23.-26. (canceled)

27. The method of claim 22, wherein the binding molecule is selected from: (a) an antibody specific for the de-sialylated form of the peptide comprising SEQ ID NO: 11, 12, 13, 14, 15, or 16, preferably specific for an epitope that is present in the peptide motif Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg and that is absent or cryptic in the corresponding sialylated peptide motif Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg; or (b) 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, preferably wherein the heavy chain has SEQ ID NO: 7 and/or the light chain has SEQ ID NO: 8.

28. The composition of claim 15 comprising an antibody, wherein the antibody comprises a reporter molecule.

29. The composition of claim 28, wherein the reporter molecule comprises a gold particle.

30. The composition of claim 15 comprising an indicator molecule, wherein the indicator molecule comprises Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg.

31. A method for detecting the presence or absence in a test sample of cleavage activity of a sialidase enzyme using the detection device or the enzyme detection kit of claim 1, the method comprising: (i) bringing an indicator molecule into contact with the test sample, wherein the indicator molecule comprises (a) a peptide comprising the following sequence: TABLE-US-00019 (SEQ ID NO: 17) X.sub.1-X.sub.2-X.sub.3[Gal-Sial]-X.sub.4-X.sub.5 wherein: Sial is a sialyl group; X.sub.3 is an amino acid comprising a glycosyl acceptor group and is selected from Ser, Thr, Tyr, Hyl, Hyp, Asn, Arg or phosphoserine (SEP), preferably wherein X.sub.3 is Ser; and X.sub.1, X.sub.2, X.sub.4 and X.sub.5 are independently selected from any amino acid provided that at least one, preferably at least two or three, of X.sub.1, X.sub.2, X.sub.4 and X.sub.5 is a .sub.D-amino acid and/or a non-standard amino acid or a non-natural amino acid; and b) a capture site which remains intact following cleavage of the sialyl group from the indicator molecule by a sialidase enzyme present in the test sample; (ii) adding to the test sample binding molecules capable of binding to the de-sialylated derivative of the indicator molecule, wherein the binding molecules are incapable of binding to the indicator molecule unless and until cleavage of the sialyl group from the indicator molecule by sialidase enzyme present in the sample has occurred; (iii) capturing the de-sialylated derivative of the indicator molecule at a capture zone through binding of capture molecules in the capture zone to the capture site, said capture molecules being able to bind to the capture site irrespective of whether or not the indicator molecule has been cleaved; and (iv) detecting cleavage of the sialyl group from the indicator molecule by determining binding of the binding molecules to the de-sialylated derivative of the indicator molecule captured in the capture zone.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0107] The invention will now be described by way of example with respect to the accompanying drawings in which:

[0108] FIG. 1 is a schematic view of one format of the assay in accordance with the invention. The format relies upon 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) that binds to the indicator molecule only after cleavage (6) has occurred. The indicator molecule Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin (SEQ ID NO: 11-PEG-Biotin; also termed “MOL600c” herein) is shown by way of example.

[0109] FIG. 2 is a schematic view of an enzyme detection device in accordance with the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of sialidase activity.

[0110] FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of sialidase activity in the test sample are increased.

[0111] FIG. 4 is a schematic view of an enzyme detection device in accordance with the present invention. The figure specifies the exact longitudinal dimensions and position of each of the card components.

[0112] FIG. 5 is a schematic view of the two conjugation chemistries used to couple peptides of the invention to Keyhole Limpet Hemocyanin (KLH): (A) cysteine/maleimide coupling; (B) hydrazine/benzaldehyde coupling.

[0113] FIG. 6 shows some of the non-standard and non-natural amino acids employed during the design of the peptides of the invention.

[0114] FIG. 7 is a schematic view summarising the synthetic approach used to produce peptides of the invention.

[0115] FIG. 8 shows the production of a sialylated peptide of the invention via an enzymatic route using transialidase and fetuin (a sialic acid donor).

[0116] FIG. 9 shows various peptides and peptide-protein conjugates synthesised as part of the invention. (A) Tabular summary of the peptides and peptide-protein conjugates synthesised. (B) Schematic overview of the peptides and peptide-protein conjugates synthesised.

[0117] FIG. 10 shows the initial ELISA results following immunisation of sheep with KLH-peptide conjugates of the invention. (A) a schematic of the detection method; (B)-(D) the results from the first bleeds, indicating that all sheep responded to the immunisations. Numbers 1056, 1057, 1058, 1059, 1062, 1063, 1064, 1065, 1066 and 1067 designate antisera from a specific immunised sheep respectively.

[0118] FIG. 11A-C shows ELISA results from a second bleed of the sheep described in FIG. 10. Antisera showed an increase in response in the second bleeds compared to the first bleeds

[0119] FIG. 12A-B shows all three bleeds compared for sheep CF1062-1067.

[0120] FIG. 13 shows ELISA results when antisera CF1064 was evaluated against MOL136 and MOL136c. (A) a schematic of the detection method; (B) ELISA results showing significant specificity to the galactosyl (i.e. de-sialylated) peptide MOL136 in preference to the sialylated peptide MOL136c.

[0121] FIG. 14 shows detection of the de-sialylated peptide MOL136 using gold-labelled CF1064 via lateral flow assay with little/no signal observed in relation to the sialylated peptide MOL136c nor the negative control (in which no peptide was included in the sample).

[0122] FIG. 15 shows detection of the de-sialylated peptide MOL136 using gold-labelled CF1064 and CF1065 via lateral flow assay with little/no signal observed in relation to the sialylated peptide MOL136c nor the negative control (in which no peptide was included in the sample).

[0123] FIG. 16 shows a lateral flow assay comparing MOL136 and MOL136c binding of gold-labelled CF1064 in the presence of healthy vaginal swab extracts.

[0124] FIG. 17 shows a lateral flow assay of MOL136c in the presence of 25 U/ml sialidase.

[0125] FIG. 18 shows lateral flow data showing a comparison between different affinity purified antisera fractions. (A) Graphical representation of line intensities; (B) lateral flow test strips as observed: (1)—Gold-sensitised affinity purified polyclonal antibody, (2)—Gold-sensitised affinity purified polyclonal antibody with cross-reactive antibodies removed, (3) Gold-sensitised cross-reactive antibodies from affinity purification.

[0126] FIG. 19 shows (A) a comparison of MOL136, MOL136c, MOL600 and MOL600c binding by ELISA and (B) lateral flow experiments showing marked improvement of performance as compared with MOL136/136c (refer to FIG. 18).

[0127] FIG. 20 shows stability data for heat dried MOL600c in polypropylene tubes at different storage temperatures and with two different heat drying methods: a speed vac and a heat block method. The measurement in ELISA are normalised to percentage desialylation as this would result in recognition by the antibody and increase in signal.

[0128] FIG. 21 shows typical data for MOL600c batches. (A) an analytical HPLC trace; (B) positive ion electrospray MS confirmation.

[0129] FIG. 22 is a schematic showing the column methods used for antibody purification with different sepharose matrices. The orange tags shown in respect of MOL600 and MPL600c represent biotin. The purple tag shown in respect of MOL615 represents an oxime group.

[0130] FIG. 23 shows stability data of lateral flow cassettes in storage over 12 weeks at 37° C.

[0131] FIG. 24 shows a lateral flow standard curve of sialidase concentration incubated for 5 minutes with peptide MOL600c. The test lines were quantified by using the Cube Reader.

[0132] FIG. 25 shows lateral flow testing of a healthy volunteer vaginal sample on a flock swab. The sample was extracted in 1 ml of sample buffer and split five ways. (A) sample but no peptide; (B) sample with MOL600c; (C) sample with MOL600c and sialidase at 500 U/ml for 5 minutes; (D) sample with MOL600c spun down; (E) sample with MOL600c and the same sialidase incubation spun down.

[0133] FIG. 26 shows the chemical formula of MOL616.

[0134] FIG. 27 shows results of Example 13 showing the specificity of antibody 125.1 for MOL600 compared to the sialylated form, MOL600c.

[0135] FIG. 28 shows a comparison of assays A and B using a range of different sialidase concentrations. For each sialidase concentration, the left bar represents the cube reading for assay B and the right bar represents the cube reading for assay A.

[0136] FIG. 29 shows a comparison of assays A and B using a range of different sialidase concentrations and read times. For each sialidase concentration, the bar represent from left to right (i) the cube reading for assay A after 5 minute read time; (ii) the cube reading for assay A after 10 minute read time; (iii) the cube reading for assay B after 5 minute read time; and (iv) the cube reading for assay B after 10 minute read time.

[0137] FIG. 30 shows a comparison of assays A, A′, B and B′ using a range of different sialidase concentrations, indicator concentrations and read times. For each sialidase concentration, the bar represent from left to right (i) the cube reading for assay B with 1 μg/ml indicator molecule per disc and 5 minutes read time; (ii) the cube reading for assay B with 3 μg/ml indicator molecule per disc and 5 minutes read time; (iii) the cube reading for assay A with 1 μg/ml indicator molecule per disc and 10 minutes read time; and (iv) the cube reading for assay A with 3 μg/ml indicator molecule per disc and 10 minutes read time.

[0138] FIG. 31 shows the sequences of antibody 125.1 indicating the CDR and framework regions.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0139] FIG. 1 is a schematic view of one format of the assay in accordance with the invention. The format relies upon 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) that binds to the indicator molecule only after cleavage (6) has occurred. The indicator molecule Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin (also termed “MOL600c” herein) is shown by way of example.

[0140] In the format shown, the capture molecule (2) is streptavidin. Here, the capture molecule (2) binds to a biotin capture site (3) within the indicator molecule.

[0141] As shown in FIG. 1A, once the indicator molecule of the invention is added to a test sample, sialidase enzyme present in the sample specifically recognises the Gal-Sial cleavage site (4) and cleaves the sialyl group from the indicator molecule (6).

[0142] As shown in FIG. 1B, this cleavage event (6) produces a binding site for the specific antibody binding molecule (5). The binding molecule (5) is unable to bind to the indicator molecule until cleavage (6) has occurred. Thus, the antibody binding molecule (5) binds to the amino acid sequence Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg produced as a result of cleavage of the sialyl group. The antibody binding molecule (5) does not bind to the Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg sequence prior to cleavage (not shown).

[0143] FIG. 2 is a schematic view of an enzyme detection device in accordance with the present invention and shows operation of the device in the absence (FIG. 2A) or presence (FIG. 2B) of sialidase activity. The test strip includes an adhesive liner (1) upon 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. This is laid partially overlapping the conjugate pad (3), which is impregnated with the labelled binding molecules (7). In alternative embodiments, the labelled binding molecules may be impregnated in the sample application zone and this removes the need for a separate conjugate pad. The conjugate pad (3) is in fluid connection with a nitrocellulose membrane (4). The nitrocellulose membrane (4) contains immobilized streptavidin molecules (5) which define a capture zone. The membrane (4) further contains immobilized further binding molecules (6) downstream of the capture zone which bind to further labelled molecules (11) which pass through the device with the sample and form a separate control zone. Alternatively, the immobilised further binding molecules may bind to labelled binding molecules (7). The device optionally further comprises an absorbent pad (8) to absorb any test sample and reagents reaching the end of the device.

[0144] In use, the indicator molecule (9) is added to the test sample prior to bringing the test sample into contact with the sample application zone (8) of the device. As shown in FIG. 2A, in the absence of sialidase activity in the test sample, the sialyl group is not cleaved from the indicator molecule (9). Upon sample flow into the conjugate pad (3), the binding molecules (7) are unable to bind to the indicator molecule (9) because cleavage of the sialyl group has not occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are not immobilized at the capture zone because they cannot bind to the indicator molecules (9). Accordingly, the labelled binding molecules flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, absence of sialidase activity is displayed as a signal only at the control zone, but not at the capture zone. Excess sample, potentially containing labelled binding molecules (7), flows into the absorbent pad (8).

[0145] As shown in FIG. 2B, in the presence of sialidase activity in the test sample, the sialyl group is cleaved from the indicator molecule (9). Upon sample flow into the conjugate pad (3), the binding molecules (7) are able to bind to the indicator molecule (9) because cleavage of the sialyl group has occurred. The indicator molecules become bound at the capture zone via the interaction between streptavidin (5) and the biotin capture site (10) of the indicator molecule (9). The labelled binding molecules (7) are immobilized at the capture zone due to binding to the de-sialylated indicator molecules (9) at the cleavage site. Due to the relative excess of labelled binding molecule (7) to binding sites at the capture zone some labelled binding molecules (7) still flow through to the control zone and beyond. Further labelled molecules (11) also pass through the device to the control zone where they are immobilized by binding to the immobilized further binding molecules (6). Thus, presence of sialidase activity is displayed as a signal both at the capture zone and the control zone. Excess sample flows into the absorbent pad (8).

[0146] It should be noted that the control zone is optional. The presence or absence of sialidase activity in the sample can be monitored solely based upon the presence or absence of a corresponding signal at the capture zone.

[0147] FIG. 3 shows the visual read-out of the assay (shown in FIG. 2) as levels of sialidase activity in the test sample are increased. As can readily be seen, the signal at the control zone (1) is constant as sialidase amounts increase. In contrast, as sialidase amounts increase, the signal at the capture zone (2) also increases. This is due to cleavage of the sialyl group from the indicator molecule at the cleavage site by sialidase activity. This reveals a binding site, enabling binding of the binding molecules which is detected at the capture zone (2) via interaction between capture molecules defining the capture zone and the capture site of the indicator molecules.

[0148] FIG. 4 is a schematic view of one specific enzyme detection device in accordance with the present invention. The table below provides a legend for the figure and specifies the exact longitudinal dimensions and position of each of the card components in this particular embodiment. Of course, the dimensions and positions may be varied as would be readily understood by one skilled in the art.

TABLE-US-00006 Position from Component Size Datum point Backing card (1) 60 mm 0 mm Nitrocellulose Membrane (2) 25 mm 20 mm  Conjugate Pad (3) 17 mm 5 mm Sample Pad (4) 10 mm 0 mm Absorbent Pad (5) 22 mm 38 mm 

[0149] The invention will be further understood with reference to the following experimental examples.

EXAMPLES

Example 1: Assay Chemistry Principle and Experimental Design

[0150] In summary, the principle works on the basis of antibody recognition of the chemical product of the sialidase reaction, where the chemical substrate is a peptide designed specifically to react with sialidase and the product of the reaction is then recognised by the antibody raised to that synthetic product. As shown in FIGS. 1 and 2, a glycopeptide containing sialic acid and with a biotin tag is provided. When contacted with a test sample containing sialidase activity, the sialyl group is cleaved from the glycopeptide by sialidase to expose the pendant galactosyl group on the peptide. An antibody raised against the de-sialylated product then specifically binds the cleaved product. By using an antibody-gold conjugate and a lateral flow strip with a streptavidin test line the presence of the de-sialylated product can be detected as a red line and a direct measure of the sialidase activity in a sample.

[0151] Five peptides were originally designed and the subsequent antibodies raised were evaluated and profiled as to their performance in a lateral flow assay. It was shown that under optimised conditions that some of the antibodies outperformed others in terms of their cross reactivity to the substrate peptide and their overall signal levels in the assay. In evaluations described below for the feasibility phase of the project the peptides were re-evaluated and the peptide/antibody combination with best performance was taken forward into further development.

Example 2: Peptide Design

[0152] In the design of the candidate peptide sequences a number of factors were taken into account:

Size

[0153] As a general rule, molecules with molecular weight (MW) less than 5000 daltons are unlikely to stimulate a good immune response in a host organism. The peptides were planned as relatively short sequences with the intention of conjugating to KLH (Keyhole Limpet Hemocyanin) which results in a much more effective immunogen. A range of different lengths (9-20 amino acids) were proposed to cover any potential variation in performance.

Conjugation Chemistry

[0154] Two different conjugation chemistries were used. For two of the peptides a cysteine label was incorporated into the structure so that it could be conjugated to carrier proteins using a standard maleimide based chemistry. For the other three peptides a relatively new hydrazine based chemistry was used which is a more controllable process than cysteine chemistry as there is less risk of oxidation of the peptide prior to coupling and the extent of coupling can be easily monitored by UV absorbance. An overview of these conjugation chemistries is shown in FIG. 5.

Position of the Galactose Sugar

[0155] The aim was to obtain antibodies with very high affinity to the galactose sugar and surrounding regions of the peptide. With this in mind it was decided to position the sugar centrally in the structure so that it was well flanked by other distinctive peptide features. This approach would hopefully minimise peripheral binding antibodies which lacked interaction with the sugar moiety.

Structural Diversity

[0156] A range of amino acids were used in constructing the various peptide sequences. By combining charged, hydrophilic and hydrophobic groups along the sequence diverse topologies would be promoted. “Hinge groups” such as β-Alanine were also employed to allow additional degrees of freedom in the overall structural fold. In addition to this unnatural amino acids were employed to add to the sequence diversity and to promote an immune response. To reduce susceptibility to proteases some D-amino acids were also incorporated. Some of the non-standard and non-natural amino acids employed are shown in FIG. 6.

[0157] After consideration of these factors the following peptide sequences were chosen as putative epitopes.

TABLE-US-00007 Peptide sequence Designated name ---Arg-.sub.DAla-Bip-Ser-Pro-Ser(β-Gal)-.sub.DAla-.sub.DAsp-Ser---- MOL133 (Desialylated SEQ ID NO: 13) ---Ser-SEP-.sub.DAla-Ile-Orn-Ser(β-Gal)-.sub.DAla-Nle-Glu---- MOL134 (Desialylated SEQ ID NO: 14) ---.sub.DAla-Arg-Nva-.sub.DSer-βAla-.sub.DAla-Nle-Ser(β-Gal)-Bip-Orn-.sub.DAla- MOL135 Glu-Ser---- (Desialylated SEQ ID NO: 15) ---Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser(β-Gal)-.sub.DAla-Arg-Phe-.sub.DSer-Val--- MOL136 (Desialylated SEQ ID NO: 12) ---Thr-.sub.DAla-Nle-Glu-.sub.DAla-Arg-βAla-Cyc-Orn-.sub.DSer-Pro-Ser MOL137 (β-Gal)-βAla-Nva-.sub.DSer-Glu-Thr-Cha-.sub.DSer-Val---- (Desialylated SEQ ID NO: 16)

[0158] Spacers, conjugation groups and biotin labels were added to the C or N termini.

Example 3: Peptide Synthesis

[0159] Galactose-labelled peptides were synthesised using solid phase chemistry methods on an automated microwave synthesiser. All peptides were purified using reverse phase HPLC and characterised by Electrospray LCMS. To label the galactose moiety with sialic acid an enzymatic route was employed using recombinant transialidase from T. cruzi (TcTS) and fetuin as a sialic acid donor. FIGS. 7 and 8 summarise the synthetic approach used.

[0160] As well as the relevant peptide immunogens for conjugation to carrier proteins, two biotinylated derivatives of each sequence were also synthesised, one labelled with sialic acid. These biotinylated derivatives would form the basis for the lateral flow assay format. FIG. 9 shows the peptides synthesised for this work according to the various immunisations. Peptides labelled with sialic acid are designated with a ‘c’; e.g. MOL136c. Peptides which lack the sialyl group lack this designation (e.g. MOL136).

Example 4A: Antibody Generation

Immunisations

[0161] All five peptide immunogens were coupled to KLH and BSA using the appropriate coupling chemistries (see FIG. 9). The KLH conjugates were submitted to Micropharm Ltd for immunisation into ten sheep (two per peptide). The program was initiated with a dose of 1 mg of KLH-peptide in PBS. Three further boosters of 0.5 mg were given over a period of three months. Three separate bleeds were taken during this period (one sample and two production bleeds) and supplied to Mologic. BSA conjugates were retained to carry out the screening of the various bleeds.

Initial ELISA Screens

[0162] Polystyrene high binding multi-well plates were sensitized with the appropriate BSA conjugate and non-conjugated BSA as a control. Sera samples were incubated in the BSA blocked wells at various dilutions and further bound with a secondary anti-sheep antibody conjugated to alkaline phosphatase (AP). Incubations with para-Nitrophenylphosphate (pNPP) AP substrate indicated the presence of any binding. In all cases buffer, BSA and pre-bleed controls resulted in no background binding to the plate. FIG. 10A shows a schematic of the detection method. FIGS. 10B-10D show the results from the first bleeds, indicating that all sheep responded to the immunisations. Sera dilutions at 1/1000 gave high signal whilst controls of BSA were negative, confirming peptide specific response. Pre-bleed controls were also negative.

[0163] As further bleeds became available, these were also analysed by ELISA showing an enhancement of response over time. FIG. 11 illustrates the relationship between the first two bleeds for all the sheep while FIG. 12 shows all three bleeds compared for sheep CF1062-1067. Interestingly the third bleeds appear plateaued or even reduced in terms of their overall response to antigen. Nevertheless in some cases a binding response was detected at 1/1000000 dilution suggesting a polyclonal response highly sensitive to the peptide. Despite the slightly lower response in the third bleeds, these were preferred for affinity purifications as they were the most likely to contain a sub-population of highly specific antibodies.

Example 4B: Antisera CF1064 and CF1065 and Detection of MOL136 and MOL136c

Immunogen: KLH-MOL123A

[0164] Biotinylated Peptides: MOL136 (galactosyl); MOL136c (sialyl)

[0165] Both sheep responded well to immunisation with KLH-MOL123A with CF1064 giving a slightly stronger response overall (see FIG. 11B). In ELISA format (a schematic of which is shown in FIG. 13A), binding of CF1064 was evaluated against MOL136 and MOL136c, showing significant specificity to the galactosyl (i.e. de-sialylated) peptide MOL136 in preference to the sialylated peptide MOL136c (FIG. 13B). Thus, excellent discrimination of the two peptides by antisera CF1064 is demonstrated.

[0166] Similar properties were observed in lateral flow format. CF1064 was conjugated to gold particles in optimised conditions (15 μg/ml loading in 10 mM sodium borate buffer; pH 8.0) and assayed against MOL136 and MOL136c at a final concentration of 1.3 ng/ml (38 μg per strip). Thus, a capture zone was first formed on each lateral flow test strip using 1.5 mg/ml streptavidin. Then, 3 μl of each peptide (12.5 ng/ml peptide in PBST) was mixed with 10 μl of gold-labelled CF1064 and 15 μl of running buffer (0.5 M Tris at pH 7.5+3% BSA+0.5% Triton X-100) before each sample was run along a separate lateral flow test strip. A negative control in which no peptide was added was also run as a negative control. 15 μl of running buffer wash was then run along each lateral flow test strip. De-sialylated peptide MOL136 was clearly detected in the capture zone, observed as a visible line on the test strip. Conversely, little/no signal was observed in relation to the sialylated peptide MOL136c nor the negative control (see FIG. 14).

[0167] Similar results were observed using gold-labelled CF1065, with a higher signal level observed for CF1065 relative to CF1064 (see FIG. 15).

Example 5: Detection of MOL136 and MOL136c Using CF1064 in the Presence of Healthy Vaginal Swab Extracts

[0168] The procedure described in Example 4B was repeated in the presence of healthy vaginal swab extracts. Although the signal levels were slightly lower, the specificity to MOL136 was retained without any interference of the swab matrix (see FIG. 16).

Example 6: Detection of Sialidase Activity Using MOL136c and CF1064

[0169] The procedure described in Example 4B was repeated in the presence of 25 U/ml sialidase. A sample in which sialidase was absent was used as a negative control. A further negative control was run in which no peptide and no sialidase was included in the sample. As a positive control, MOL136 was used in place of MOL136c in the presence of 25 U/ml sialidase. A clear positive signal was observed in respect of MOL136c incubated in the presence of 25 U/ml sialidase in PBST for 5 minutes. The level of signal was similar in intensity to that of the positive control which shows the putative maximum signal. As expected, little/no signal was observed in respect of the negative controls (see FIG. 17).

[0170] Overall both CF1064 and CF1065 showed good specificity for the galactosyl (i.e. de-sialylated) peptide MOL136 and are both potential candidates for monoclonal screening.

Example 7: Peptide Development

[0171] After an evaluation of the peptides and antisera, it was shown that sera CF1064 and peptides MOL136 and MOL136c provided the most feasible combination for the test. Sheep CF1064 was immunised with the same peptide sequence as MOL136 but as a KLH conjugate using cysteine-maleimide chemistry in place of the biotin.

MOL136: biotin-PEG-Asp-Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg-Phe-.sub.DSer-Val-OH
MOL136c: biotin-PEG-Asp-Glu-.sub.DSer-Nva-Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-Phe-.sub.DSer-Val-OH

[0172] To purify the antisera, a method was devised using a streptavidin column to capture MOL136 and MOL136c. Antibody was then bound and eluted from the MOL136 column and absorbed to the MOL136c column to remove any peripheral binders picking up common epitopes between the two structures. By evaluating the purified antibody in lateral flow, the overall specificity to the original galactosyl peptide antigen and the degree of cross-reactivity to the sialylated peptide was measured (FIG. 18). This is important because it represents the amount of non-specific binding observed in a negative result in the prototype and, without an optical reader in use, would need to be reduced significantly so that the naked eye would not pick it up.

[0173] Purified and absorbed fraction performed best in the assay when compared to non-absorbed materials. Despite the significantly lower background signal, further method development was required to improve the performance by truncating the peptide structure.

[0174] To refine the peptide design and remove peripheral interactions of antibodies not binding the galactose moiety in the peptide, the original sequence MOL136 was truncated to a shorter sequence MOL600:

MOL600: NH.sub.2—Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg-PEG-Biotin
MOL600c: NH.sub.2—Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin

[0175] MOL600 and MOL600c were then assayed by ELISA and lateral showing a significant improvement in performance (see FIG. 19).

Example 8: MOL600c Formulation/Stability Studies and Scale-Up Production

[0176] For evaluation of how the MOL600c peptide could be formulated, a study was set up to look at drying conditions. The peptide was formulated in a drying buffer, air dried and stored at room temperature prior to use in the prototype format. Data is shown in FIG. 20 demonstrating the stability of peptide MOL600c stored in polypropylene tubes at different temperatures and with different drying methods. The peptides retained their functionality up to week 4 overall, however performance appears to decline at 8 weeks.

[0177] For manufacture, the peptide synthesis has been shown to be scalable and can be ramped up to manufacturing batches. The peptide is produced by solid phase synthesis on an automated system and then purified by HPLC. Two further chemical steps are required and a further final purification by HPLC to a specification of >95% purity. The product is confirmed by electrospray mass spectrometry. As only 12 ng of peptide is required per test, 1-2 mg batches of peptide are of adequate size. FIG. 21 shows typical analytical data of a completed batch of MOL600c.

Example 9: Antibody Purification Development

[0178] The purification process used with antisera CF1064 along with the optimised truncated peptide system was developed initially to work on streptavidin columns charged with MOL600 and an absorption column charged with MOL600c. Whilst this process produces effective antibody with good cross reactivity features, further improvements were required to improve consistency and reduce the number of manual steps for automation.

[0179] To explore a more refined method using a single pass, other column formats were tried and were shown to be feasible and with potential to be automated (FIG. 22). Two different columns types have been used and processes have varied from a single pass method to a post-absorption method where unwanted interactions are mopped up to reduce non-specific binding in the assay. The best option for manufacture is a single pass process and this can be achieved using the peptide MOL615 conjugated to carrier BSA and then derivatised on to an NHS sepharose column.

MOL615: NH.sub.2—Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg-PEG-Oxime

[0180] The antibody component in the prototype is formulated as a gold conjugate dried into a conjugate pad within the lateral flow assembly. Conditions have been established for gold conjugate stabilisation and further optimised. The amount of antibody calculated to be required per test is estimated at 85 ng which is sufficient to generate the required validation batches achieve CE Marking. In the long term additional polyclonal reagent will have to be developed and—as more permanent solution—a monoclonal. New immunisations have been started and strong antibody titres have already been identified to the peptide target (FIG. 10). Several options will be utilised for monoclonal generation, including commercial hybridoma technology and also in house Phage panning and fab generation (Mologic York and Scotia Biologics).

Example 10: Sample Buffer

[0181] Extraction of sample from the swab, dissolving of peptide and optimal digest conditions for sialidase are the principle requirements of the sample buffer. The following formulation was used:

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

[0182] The buffer has been used successfully to run devices in dry and also extract real samples in spiked recovery.

Example 11: Lateral Flow Device

[0183] A lateral flow device was constructed per FIG. 4.

[0184] A batch of lateral flow devices were evaluated for stability at 37° C. over 12 weeks. After 6 weeks, signals enhanced in value over earlier measurements however the shape of the curves and dynamic range appeared relatively consistent (see FIG. 23).

Example 12: Assay Optimisation

[0185] To satisfy a feasible performance, specification factors such as length of assay time, sensitivity of response to the marker, line signal strength and quality of standard curve were evaluated.

[0186] The assay has been shown to work with an incubation time of 5 minutes. This would keep the total time of the assay within 15 minutes from swab sample collection to the reading of the result. In the in-vitro diagnostic industry, 15 minutes is generally the norm as an upper limit for a point of care rapid test.

[0187] The full relevant range of the marker (sialidase) is believed to be from 4 U/ml to 250 U/ml. This is based on the literature by estimating the spread of sample data sets in relation to a fluorescent sialidase reference assay (Marconi et. al., European Journal of Obstetrics and Gynaecology and Reproductive Biology, 2013, vol. 167, pages 205-209). There are assumptions made in this approach as in the BV condition a number of different phenotypes (bacterial sialidases) will be present in the sample as about 20 different bacterial species have been associated with the condition. The assay has been tested across this range of sialidase in buffer and a standard curve representing this range has been achieved in lateral flow (FIG. 24).

[0188] The assay has also been tested on real healthy samples and signal has been recovered by spiking in sialidase. Whether a sample is spun down or not has been shown not to make a difference. The appearance of the test line was not adversely affected by the presence of sample matrix (FIG. 25). For the sample to be of sufficient mobility, 0.5 ml of diluent sample buffer was found to be too concentrated as the samples retained too much viscosity. Currently 1 ml is considered the appropriate amount of buffer to use.

Example 13 Further Antibody Generation

[0189] The following peptide, designated MOL616, was chosen to generate further antibodies: Dpr(AOA)-dSer-Nva-Cyc-dAla-Ser(Gal)-dAla-Arg-Phe-dSer-Val-NH.sub.2.

[0190] The chemical formula of MOL616 is shown in FIG. 26.

[0191] The peptide was coupled to KLH, yielding an immunogen denoted KLH-MOL616. Rabbits (Oryctolagus cuniculus) were immunised with KLH-MOL616 and splenocytes were captured and screened with a truncated version of this peptide (MOL615, conjugated to BSA). Counter screens were carried out with the sialylated form of peptide MOL615, MOL615c. The screening process involved a multi-clone screen followed by a subclone screen to yield an antibody specific for the de-sialylated form.

[0192] This process led to the generation of a monoclonal antibody denoted antibody 125.1. This antibody is specific for an epitope present in the de-sialidated molecule Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg-PEG-Oxime, more particularly for an epitope present in the de-sialidated peptide Cyc-.sub.DAla-Ser[Gal]-.sub.DAla-Arg.

[0193] MOL615 was biotinylated to yield MOL600 (sequence provided in Example 7) and antibody binding affinity and kinetics was measured using bio-inferometry. Briefly, MOL600 or MOL600c was immobilised on a biosensor by streptavidin/biotin interaction. Binding of the antibody was detected by a shift in the reflected light interference.

[0194] Using different concentrations of antibody 125.1 (1, 2, 4, 8 or 16 nM respectively) the following binding affinity and kinetics were determined for antibody 125.1: K.sub.D=7.9 nM; K.sub.on=54080M.sup.−1s.sup.−1, K.sub.off=0.000427 s.sup.−1

[0195] The binding was clearly specific for MOL600 compared to the sialylated form, MOL600c, as shown in FIG. 27.

[0196] The antibody was sequenced and determined to have a Gamma Heavy chain and a Kappa Light chain. The sequences of this antibody are shown in FIG. 31 and listed below.

TABLE-US-00008 Antibody-Heavy-Chain (SEQ ID NO: 7) QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYSMDWVRQAPGKGLEWVGGI TTTLHTFYATWAKGRFTISKTSSTTVDLKMTSLTTDDAATYFCARGGSSV IWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVT VTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATN TKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCV VVDVSEDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRWSTLPIAHEDW LRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSV SLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVP TSEWQRGDVFTCSVMHEALHNHYTQKSISRSPG Antibody-Light-Chain (SEQ ID NO: 8) ALVMTQTPSPVSAAVGGTVTISCQSSQSVYGNNHLNWHQQKRGQPPKQLI GSASRLASGVPSRFKGSGSGTQFTLTISGVQCDDAATYYCQGSYYNGAWY VAFGGGTELEIKRDPVAPSVLLFPPSKEELTTGTATIVCVANKFYPSDIT VTWKVDGTTQQSGIENSKTPQSPEDNTYSLSSTLSLTSAQYNSHSVYTCE VVQGSASPIVQSFNRGDC Heavy Chain CDR sequences CDR1 (SEQ ID NO: 1) GFSLSSY CDR2 (SEQ ID NO: 2) TTTLH CDR3 (SEQ ID NO: 3) GGSSVI Light Chain CDR sequences CDR1 (SEQ ID NO: 4) QSSQSVYGNNHLN CRD2 (SEQ ID NO: 5) SASRLAS CDR3 (SEQ ID NO: 6) QGSYYNGAWYVA

Notable Observations:

[0197] An additional O-linked glycosylation site was detected in the heavy chain constant region between positions 213 and 216. From the gel image, the form that is glycosylated at this site is similar in abundance to the unglycosylated form.

[0198] N-linked glycosylation was detected on heavy chain constant region N@285.

[0199] Pyro-Glu(Q) modification observed at N-terminal of heavy chain.

[0200] Elimination of C-terminal lysine from regular constant region sequence observed on heavy chain.

Example 14 Further Assay Optimisations

[0201] For further optimization, two different sialidase activity assays (A and B, see below) were compared. The assay set up was essentially as described in connection with FIG. 2.

[0202] Test antibodies were conjugated to gold and dried onto a carrier, referred to herein as a “conjugate pad”. The test antibody in assay A was “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 denoted “125.1” (see Example 13).

[0203] 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.

[0204] Capture molecules (polystreptavidin) were immobilised on a nitrocellulose membrane to form a capture line. A separate control capture line was formed. For assay B, this was Anti-Chicken IgY antibodies (supplied by Lampire, product code 7455207).

[0205] The conjugate pad was laminated onto the nitrocellulose as shown in FIG. 2.

[0206] The test indicator molecule, NH.sub.2—Cyc-.sub.DAla-Ser[Gal-Sial]-.sub.DAla-Arg-PEG-Biotin (MOL600c), was prepared and 1 μg/ml or 3 μg/ml was freeze dried onto an inert porous material (a Porex disc) to yield an indicator substrate disc comprising 36 ng indicator molecule per disc or 108 ng indicator molecule per disc respectively.

[0207] Sialidase was tested at the following concentrations: 50, 12.5 and 1.56 U/ml. A negative standard using peptide disk and assay buffer was also run. Responses were measured against cube readings and a visual scale.

[0208] The following basic protocol was used: Ensure all solutions are at room temperature and mixed well.

[0209] Test each of the desired conditions with the 50, 12.5 and 1.56 U/ml sialidase buffer standards solutions.

[0210] Test all standards and the sialidase buffer in triplicate.

[0211] Add 1000 μl of sialidase standard to an extraction tube, together with an indicator substrate disc. Seal the tube by folding the lid and cap into place. Gently agitate the tube and allow to incubate for 5 minutes with the indicator substrate disc.

[0212] Add 4 drops from the extraction tube to the device to the sample pad.

[0213] Read at 5 or 10 minutes using the cube reader (Optricon reader from opTricon GmbH (Chembio Diagnostic systems) of Schwarzschildstrasse 1, D-12489 Berlin, Germany) and record test and control line signals.

[0214] Features of the test assays are shown below.

TABLE-US-00009 Assay A Assay B Conjugate Pad Millipore GFDX Ahlstrom 8951 Control Line Antibody Anti-sheep Antibody Anti-Chicken IgY Antibody Gold conjugate Test Only Test and Control Test gold conjugate 1064 purified MoI615 125.1 Monoclonal antibody Polyclonal Antibody Antibody Peptide Concentration 1 μg/ml (36 ng per 3 μg/ml (108 ng per disc) disc) Device Read Time 10 minutes 5 minutes Gold OD 5 4 Sample Pad MDI FR1 MDI FR1

[0215] (I) Comparison of Read Times

[0216] The desirable output is to have a 10-minute test, with a 5-minute incubation and then a 5-minute read time. Results were read at 5 and 10 minutes to investigate the effect of read time on results. Results are shown in FIG. 28. As can be seen, assay A requires a 10-minute read time to meet the correct response for the bottom standard where assay B only requires a 5-minute read time. Assay B does not seem to benefit significantly from a 10-minute read time; it meets the desirable output of a 10-minute test with a 5-minute read.

[0217] (ii) Comparison of Sensitivity

[0218] The results of assay B were read after 5 minutes and the results of assay A were read after 10 minutes. Results are shown in FIG. 29.

[0219] Assay B shows higher cube readings compared to assay A across the 50 U/ml, 12.5 U/ml and 1.56 U/ml standards and has lower cube readings for the 0 U/ml standard. Both A and B are giving the correct response for each of the standards and the % CV are within specification. Assay B is giving cube readings for the standards after a shorter device development time and with lower cube readings for the 0 standards. Thus, assay B is superior to assay A.

[0220] (iii) Effect of Peptide Concentration

[0221] Assays A and B were compared to each other and to variants with different peptide concentrations. Assay A′ corresponded to assay A, but with a peptide concentration of 3 μg/ml (108 ng per disc); Assay B′ corresponded to assay B, but with a peptide concentration of 1 μg/ml (108 ng per disc).

[0222] The results of assay B and B′ were read after 5 minutes and the results of assay A and A′ were read after 10 minutes. Results are shown in FIG. 30. Higher peptide concentrations gave better results and a peptide concentration of >1 μg/ml (>108 ng per disc) is preferred.

Conclusions:

[0223] The assay B has improved performance to the assay A in a shorter time because of the changes made to the antibody and the increase of the peptide concentration.

Example 15—Additional Assay Optimisations

Gold Optical Density

[0224] The effect of increasing the gold conjugate spraying concentration from OD4 to OD6 or OD8 was investigated, and it was found that while OD6 gold gave a slight increase in test line signal compared to OD4, OD8 gave double the test line signal of OD4 at 3.125 U/mL sialidase. Thus, using gold conjugation at an OD of about or at least 8 is advantageous.

Sample/Conjugate Pad

[0225] In a lateral flow assay device, such as the type schematically represented in FIG. 2, the sample pad should allow the sample to flow along the device to allow the sample to come into contact with the conjugate pad containing the binding molecules and subsequently come into contact with the capture molecules. Depending on the viscosity of the sample, some sample pad materials may be advantageous.

[0226] In order to assess the different sample pads, a synthetic vaginal fluid substitute was 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). The synthetic vaginal fluid was allowed to run through all of the sample pads. The time taken for the sample to run the length of the test strip was recorded (see Table below)

TABLE-US-00010 Time to start Time to run Sample Pad of window length of window 1. FR1 10 mm 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

[0227] Ahlstrom are glass fibre pads of different densities (8964, 6613 and 6615 respectively).

[0228] For further comparison, materials were made using two sample pads (FR1 and Ahlstrom 8964). Devices were run using a 4 point standard curve (0, 3.125, 12.5 and 50 U/mL sialidase in aqueous buffer solution), in order to determine whether the alternative sample pad had any effect on either specific or nonspecific signal. Samples run using the 8964 sample pad gave higher specific signal compared to the FR1, with no non-specific binding (NSB).

[0229] Using a set of clinical samples spiked with 6.25 U/ml sialidase it was determined that a fibreglass-type pad such as the Ahlstrom 8964 sample pad allows very reliable sample flow and enzyme detection.

Example 16—Diagnosis of Bacterial Vaginosis

[0230] Clinical samples were obtained and analysed for criteria such as clue cells to determine a Nugent score, on the basis of which the samples were classed as positive or negative for bacterial vaginosis. The samples were analysed using assay A or assay B (see Example 14).

[0231] The results are shown below, wherein se stands for sensitivity; sp stands for specificity; ppv stands for positive predicted value; and nvp stands for negative predicted value.

Assay A

[0232]

TABLE-US-00011 Nugent Nugent POS NEG TOTAL Assay A POS 25  0 25 Assay A NEG  2 25 27 TOTAL 27 25 52

TABLE-US-00012 se sp ppv npv estimate: 0.93 1 1 0.93 95% Cl: [0.76; 0.99] [0.86; 1] [0.86; 1] [0.76; 0.99] p-value: 5.65E−06 5.96E−08 5.96E−08 5.65E−06

Assay B

[0233]

TABLE-US-00013 NUGENT SCORE POS NEG TOTAL Assay B POS 27  0 27 Assay B NEG  0 25 25 TOTAL 27 25 52

TABLE-US-00014 se sp ppv npv estimate: 1 1 1 1 95% Cl: [0.87; 1] [0.86; 1] [0.87; 1] [0.86; 1] p-value: 1.49E−08 5.96E−08 1.49E−08 5.96E−08

[0234] Thus, both assays performed well at distinguishing between clinical samples that are positive or negative for bacterial vaginosis. Assay B was superior to assay A.

[0235] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.