NOVEL IMMUNOASSAY FORMAT FOR MEASURING TOTAL ANTIBODIES

Abstract

Disclosed are a novel immunoassay format design for determining a total antibody, and a kit accordingly provided for detecting antibodies of a pathogen or pathogens of infectious diseases within a human blood sample, wherein the kit comprises: a first reagent containing at least one antigen coated on a solid phase support and an anti-human IgM antibody coated on a solid phase support; and a second reagent containing at least one labelled antigen and a labelled anti-human IgG antibody, wherein at least one antigen of the at least one antigen coated on a solid phase support and at least one antigen of the at least one labelled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. The kit can overcome the disadvantages caused by detection principles while retaining the advantages of each detection principle. In addition, also provided is a new method for detecting an antibody produced after the infection of a pathogen or pathogens in a sample.

Claims

1-25. (canceled)

26. A kit for detecting antibodies against a pathogen or pathogens for infectious diseases in a human blood sample, comprising: a first reagent containing at least one antigen and an anti-human IgM antibody, the antigen and the anti-human IgM antibody are coated on a solid phase support, and a second reagent containing at least one labelled antigen and a labelled anti-human IgG antibody, wherein the at least one antigen coated on the solid phase support and the at least one labelled antigen are capable of binding to a same IgG antibody or a same IgM antibody in the sample.

27. The kit of claim 26, further comprising a third reagent, wherein the third reagent contains a blocking agent.

28. The kit of claim 27, wherein the blocking agent contains one or more components selected from a group consisting of: skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG and surfactant.

29. The kit of claim 26, further comprising a fourth reagent, wherein the fourth reagent contains a reducing agent, preferably, the reducing agent contains one or more components selected from a group consisting of: DTT and β-mercaptoethanol.

30. The kit of claim 29, wherein the reducing agent contains one or more components selected from a group consisting of: DTT and β-mercaptoethanol.

31. The kit of claim 26, wherein the kit is used for detection of an antibody produced in a subject infected by a pathogen or pathogens, wherein the pathogen is a virus or a spirochete or other microorganisms

32. The kit of claim 31, wherein the kit is used for detection of an antibody produced in a subject infected by HCV; the at least one antigen coated on the solid phase support is selected from a group consisting of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen, which are coated on the solid phase support respectively; the at least one labelled antigen is selected from a group consisting of respectively labelled HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen.

33. The kit of claim 32, wherein the at least one antigen coated on the solid phase support comprises HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen, which are coated on the solid phase support respectively; the at least one labelled antigen comprises respectively labelled HCV core antigen and HCV NS3 antigen.

34. The kit of claim 33, wherein the at least one antigen coated on the solid phase support comprises HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen, which are coated on the solid phase support respectively; the at least one labelled antigen comprises respectively labelled HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen.

35. The kit of claim 31, wherein the kit is used for detection of an antibody produced in a subject infected by Treponema pallidum; the at least one antigen coated on the solid phase support is selected from a group consisting of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen, which are coated on the solid phase support respectively; the at least one labelled antigen is selected from a group consisting of respectively labelled TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen.

36. The kit of claim 35, wherein the at least one antigen coated on the solid phase support comprises TP15 antigen, TP17 antigen and TP47 antigen, which are coated on the solid phase support respectively; the at least one labelled antigen comprises respectively labelled TP15 antigen, TP17 antigen and TP47 antigen.

37. The kit of claim 26, wherein the antigen is present in a form of a polymer, an antigen fragment or a peptide.

38. The kit of claim 26, wherein an enzyme is used for labelling the antigen or antibody of the second reagent, and the kit further comprises a reaction substrate for the enzyme, preferably, the reaction substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)-phenyl-1,2-dioxetane.

39. The kit of claim 26, wherein a concentration of the anti-human IgG antibody in the second reagent is 3-30 times a concentration of the anti-human IgM antibody in the first reagent.

40. The kit of claim 39, where the concentration of the anti-human IgG antibody in the second reagent is 10 times the concentration of the anti-human IgM antibody in the first reagent.

41. A method for detecting an antibody produced after infection of a pathogen or pathogens in a sample, comprising the following steps: providing a sample having IgM and/or IgG antibody to be detected; mixing and reacting the sample with a first reagent and a second reagent, wherein the first reagent has at least one antigen and an anti-human IgM antibody, the antigen and the anti-human IgM antibody are coated on a solid phase support; the second reagent has at least one labelled antigen and a labelled anti-human IgG antibody; the antigen on the solid phase support, the IgM and/or IgG antibody to be detected and the labelled antigen form a first complex; the antigen on the solid phase support, the IgG antibody to be detected and the labelled anti-human IgG antibody form a second complex if the sample has IgG antibody to be detected; and the anti-human IgM antibody on the solid phase support, the IgM antibody to be detected and labelled antigen form a third complex if the sample has IgM antibody to be detected; washing a solution obtained after reaction; acquiring a signal value of the first complex and at least one of the second and third complexes; and obtaining a detection result of the antibody.

42. The method of claim 41, wherein the sample is mixed with the first reagent and incubated for a predetermined period of time, and then the second reagent is added and incubated for another period of time.

43. The method of claim 41, wherein the pathogen is a virus or a spirochete, or other microorganisms; preferably, the virus is HIV or HCV.

44. The method of claim 43, wherein the pathogen is HCV; the at least one antigen coated on a solid phase support comprises at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on a solid phase support respectively; the at least one labelled antigen comprises at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen labelled respectively.

45. The method of claim 43, wherein the pathogen is Treponema pallidum; the at least one antigen coated on a solid phase support comprises at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen coated on a solid phase support respectively; the at least one labelled antigen comprises at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen labelled respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 is a schematic diagram of the reaction principle of the kit of the disclosure.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] The technical solutions of the embodiments of the disclosure will be described below clearly and completely in conjunction with the embodiments and accompanying drawings of the disclosure. The embodiments described are merely some of the embodiments of the disclosure rather than all the embodiments. Based on the embodiments in the disclosure, all the other embodiments that would have been obtained by those of ordinary skill in the art without any inventive effort shall fall within the scope of protection of the disclosure.

[0082] Reagent Preparation

[0083] First Reagent Ra:

[0084] A volume of V1/D.sub.x (x=1, 2 . . . ) (mL) of “magnetic beads coated with antigen” and “magnetic beads coated with anti-human IgM antibody” were respectively measured off using a pipette or a cylinder and added to a magnetic bead-coated tube to replace a supernatant; that is, after the magnetic separation was completed, the supernatant was suctioned off, and then an equal volume of a magnetic bead coating diluent was added and mixed evenly; the magnetic beads were mixed evenly and then added to a liquid preparation bottle and diluted to V1 (mL) volume by the magnetic bead coating diluent; and the mixture was stirred until the magnetic bead suspension was completely mixed to obtain the first reagent Ra; D.sub.x is the dilution degree of “magnetic bead coated e with antigen” and “magnetic bead coated with anti-human IgM antibody”; and the magnetic bead coating diluent is a conventional diluent with buffering capacity and contains a protein and a surfactant.

[0085] Second Reagent Rb:

[0086] A certain volume of a diluent for an enzyme-labelled conjugate was measured off using a suitable cylinder and added to a liquid preparation bottle, and V2/D.sub.y (y=1, 2 . . . ) (mL) volume of “antigen-enzyme label conjugate” and “anti-human IgG antibody-enzyme label conjugate” were measured off using a pipette or a cylinder and added to the liquid preparation bottle and diluted to V2 (mL) volume; the solution was stirred with a stirrer to fully dissolve and mix evenly; the resulting solution was sealed, then placed at 22° C. and left to equilibrate for 23 hours to 25 hours; after the equilibration was completed, the prepared solution was filtered using a suitable filter with a pore size of 0.22 μm, and the filtrate was collected to obtain the second reagent Rb; D.sub.y is the dilution degree of each “antigen-enzyme label conjugate” and “anti-human IgG antibody-enzyme label conjugate”; and the diluent of the enzyme label conjugate is a conventional diluent with buffering capacity and contains a protein and a surfactant.

[0087] Third Reagent Rc:

[0088] a diluent has buffering capacity and contains a blocking agent and a surfactant.

[0089] Fourth Reagent Rd:

[0090] a diluent has buffering capacity and contains a reducing agent.

[0091] Pathogen Detection Method

[0092] Step 1: the sample, the third reagent, the fourth reagent and the first reagent were added to a reaction tube and incubated at 37° C. for 10 minutes, so that the antigen and anti-human IgM antibody, which were coated on the solid phase of magnetic beads, were fully bound to IgG and IgM antibodies in the sample; and after incubation was complete, the solid phase of magnetic beads was placed in a magnetic field, attracted and adsorbed, the substances bound to the solid phase of magnetic beads were retained, and other unbound substances were washed off.

[0093] Step 2: the third reagent and the second reagent were added to the reaction tube; after the mixture was incubated at 37° C. for 10 minutes, the antigen and anti-human IgG antibody on the enzyme label conjugate were bound to the IgG and IgM antibodies captured on the magnetic beads to form a sandwich complex; and after incubation in the reaction tube was completed, the complex was attracted and adsorbed by the magnetic field, and other unbound substances were washed off.

[0094] Step 3: a chemiluminescent substrate was added to the reaction tube to generate chemiluminescence; and the number of photons produced by the reaction was then measured by a photomultiplier to obtain a chemiluminescence value of the sample.

Example 1 Preparation of HCV Antibody Detection Kit

[0095] The kits used in the example were prepared as described in Table 1 below and “Reagent preparation”.

TABLE-US-00001 TABLE 1 First reagent Second reagent Third reagent Fourth reagent Kit (V1 = 8 mL) (V2 = 8 mL) (15 mL) (8 mL) Kit 1-1 Magnetic bead coated HCV core antigen 1% BSA 10 mM DTT with HCV core antigen labelled with ALP 0.05% Tween (D = 100); (D = 500); −20 Magnetic bead coated HCV NS3 antigen 3% skimmed with HCV NS3 antigen labelled with ALP milk powder (D = 100); (D = 500); Magnetic bead coated HCV NS4 antigen with HCV NS4 antigen labelled with ALP (D = 200); (D = 800); Magnetic bead coated HCV NS5 antigen with HCV NS5 antigen labelled with ALP (D = 400); (D = 1000); Magnetic bead coated Anti-human IgG with anti-human IgM antibody labelled antibody (D = 100). with ALP (D = 300). Kit 1-2 Magnetic bead coated Anti-human IgG ″ ″ with HCV core antigen antibody labelled (D = 100); with ALP (D = 300). Magnetic bead coated with HCV NS3 antigen (D = 100); Magnetic bead coated with HCV NS4 antigen (D = 200); Magnetic bead coated with HCV NS5 antigen (D = 400). Kit 1-3 Magnetic bead coated HCV core antigen ″ ″ with HCV core antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS3 antigen with HCV NS3 antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS4 antigen with HCV NS4 antigen labelled with ALP (D = 200); (D = 800); Magnetic bead coated HCV NS5 antigen with HCV NS5 antigen labelled with ALP (D = 400). (D = 1000). Kit 1-4 Magnetic bead coated HCV core antigen ″ ″ with anti-human IgM labelled with ALP antibody (D = 100). (D = 500); HCV NS3 antigen labelled with ALP (D = 500); HCV NS 4 antigen labelled with ALP (D = 800); HCV NS5 antigen labelled with ALP (D = 1000). Kit 1-5 Magnetic bead coated HCV core antigen ″ ″ with HCV core antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS3 antigen with HCV NS3 antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS4 antigen with HCV NS4 antigen labelled with ALP (D = 200); (D = 800); Magnetic bead coated HCV NS5 antigen with HCV NS5 antigen labelled with ALP (D = 400). (D = 1000); Anti-human IgG antibody labelled with ALP (D = 300). Kit 1-6 Magnetic bead coated HCV core antigen ″ ″ with HCV core antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS3 antigen with HCV NS3 antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS4 antigen with HCV NS4 antigen labelled with ALP (D = 200); (D = 800); Magnetic bead coated HCV NS5 antigen with HCV NS5 antigen labelled with ALP (D = 400); (D = 1000). Magnetic bead coated with anti-human IgM antibody (D = 100). Kit 1-7 Magnetic bead coated HCV core antigen ″ ″ with HCV core antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated HCV NS3 antigen with HCV NS3 antigen labelled with ALP (D = 100); (D = 500); Magnetic bead coated Anti-human IgG with HCV NS4 antigen antibody labelled (D = 200); with ALP (D = 300). Magnetic bead coated with HCV NS5 antigen (D = 400); Magnetic bead coated with anti-human IgM antibody (D = 100).

Example 2 Detection of Serum Panel of HCV Antibody

[0096] According to “Pathogen detection method”, three seroconversion panels (9041, 10165 and 10185, purchased from Zeptometrix) were tested, and the COI results for each sample are as shown in Table 2 below.

TABLE-US-00002 TABLE 2 Kit 1-1 Kit1-2 Kit 1-3 Kit 1-4 Kit 1-5 Kit 1-6 Kit 1-7 Sample COI COI COI COI COI COI COI 9041-1 0.17 0.33 0.11 0.16 0.13 0.08 0.13 9041-2 0.18 0.32 0.11 0.18 0.16 0.10 0.15 9041-3 0.15 0.36 0.10 0.15 0.14 0.09 0.16 9041-4 0.18 0.46 0.12 0.25 0.17 0.08 0.16 9041-5 27.04 49.47 3.26 0.49 19.15 4.18 14.33 9041-6 29.80 53.03 4.10 0.62 20.02 5.13 19.56 9041-7 35.73 56.95 4.27 0.82 22.51 5.20 23.10 9041-8 35.22 59.01 4.55 1.10 22.02 6.18 27.84 10165-1 0.33 0.15 0.14 0.16 0.12 0.09 0.24 10165-2 0.26 0.13 0.20 0.14 0.11 0.08 0.25 10165-3 0.26 0.13 0.14 0.15 0.10 0.09 0.25 10165-4 0.28 0.14 0.14 0.15 0.11 0.09 0.26 10165-5 1.04 0.47 0.15 0.42 0.19 0.10 1.01 10165-6 19.92 11.79 0.55 1.56 5.27 2.57 16.77 10165-7 22.51 14.23 1.79 3.07 6.36 4.71 19.03 10165-8 34.09 24.82 2.77 4.77 10.73 4.50 28.15 10165-9 38.55 29.13 4.80 4.64 12.33 5.30 30.24 10185-1 1.08 0.15 1.02 0.61 0.87 0.88 1.03 10185-2 24.95 10.73 1.22 0.76 10.77 1.00 20.48 10185-3 31.38 12.54 1.25 0.90 13.59 1.05 27.67 10185-4 36.47 14.34 1.07 0.71 16.48 0.87 31.52 10185-5 31.99 13.66 0.95 0.62 14.54 0.79 30.87

[0097] In the disclosure, COI (Cutoff index) refers to the relative luminescence unit (RLU) value of a test sample, that is, the ratio of a chemiluminescence signal value to a threshold value (Cutoff), where COI≥1 represents that the test sample is a reactive sample, and COI<1 represents that the test sample is a non-reactive sample. For qualitative detection methods, the threshold value (cutoff value) is a cut-off value for determining whether the test result is reactive or non-reactive.

[0098] It can be seen from Table 2 that kit 1-1 (i.e., the kit of the disclosure) has a comparable detection rate to kit 1-2 (indirect method) and kit 1-5 (double antigens sandwich+indirect method), a higher COI value and a more obvious gradient of detected samples; and kit 1-3 (double antigens sandwich), kit 1-4 (immunocapture method) and kit 1-6 (double antigens sandwich+immunocapture method) have fewer detected samples, most of which have lower COI (<5), and the gradient of the detected samples is not obvious. It can be seen that, in terms of serum panel sensitivity, the kit/method of the disclosure is superior to the kit/method based on the principle of the double antigens sandwich, the kit/method based on the principle of the immunocapture method, and the kit/method based on a combination of the two (double antigens sandwich+immunocapture method). In addition, kit 1-7 has the similar performance in terms of serum panel sensitivity to kit 1-1.

Example 3 Detection of HCV-IgM Antibody Positive Sample

[0099] According to “Pathogen detection method”, 31 HCV-IgM antibody positive samples (which are the samples treated with IgG/RF adsorption reagent (purchased from Oumeng, Germany) and then confirmed by Roche Anti-HCV kit) were tested, and the results are as shown in Table 3 below.

TABLE-US-00003 TABLE 3 Kit 1-1 Kit 1-2 Kit 1-3 Kit 1-4 Kit 1-5 Kit 1-6 Kit 1-7 Sample COI COI COI COI COI COI COI 1 2.77 0.59 1.14 5.33 1.12 2.39 2.25 2 1.94 0.82 1.10 2.55 1.15 1.05 1.58 3 2.41 0.83 1.11 4.36 1.10 1.80 1.16 4 2.18 0.62 1.02 3.62 1.21 1.62 1.77 5 6.76 0.99 1.00 8.99 1.13 7.00 5.44 6 1.95 0.53 1.26 3.24 1.25 1.41 1.59 7 2.73 0.77 1.16 5.35 1.12 2.36 2.21 8 2.21 0.57 1.23 3.76 1.20 1.66 1.80 9 2.36 0.81 1.15 4.12 1.11 1.73 1.92 10 1.93 0.93 1.11 2.74 1.08 1.11 1.57 11 6.80 1.47 1.15 8.31 1.21 6.84 5.47 12 2.61 1.43 0.95 5.16 1.08 2.24 2.12 13 5.59 1.45 0.85 7.73 0.96 5.48 4.50 14 1.86 0.68 0.94 2.81 0.89 1.20 1.52 15 2.32 0.81 0.88 4.42 0.88 1.91 1.19 16 2.36 1.06 1.02 4.53 1.04 2.04 1.92 17 1.93 1.39 0.93 3.11 1.05 1.04 1.57 18 2.55 0.81 0.99 4.86 0.89 2.11 2.07 19 3.91 0.88 0.93 3.32 0.86 1.23 3.16 20 1.67 0.88 1.08 2.51 1.06 1.01 1.37 21 1.58 0.70 0.87 2.17 0.82 0.83 1.29 22 2.14 1.24 0.87 3.74 0.89 1.49 1.74 23 1.50 0.62 0.97 2.32 0.89 0.89 1.23 24 2.69 0.70 0.88 2.78 0.87 1.09 2.18 25 1.66 0.89 1.03 2.63 0.98 1.03 1.36 26 2.62 0.75 0.90 2.39 0.84 0.82 2.13 27 1.47 0.79 0.99 2.28 0.93 0.83 1.21 28 4.64 0.82 0.92 1.73 1.96 0.63 3.74 29 3.56 0.72 1.14 2.13 1.05 0.92 2.88 30 1.58 1.22 0.84 2.61 0.84 0.96 1.09 31 2.84 1.03 1.03 5.50 1.01 2.38 2.30

[0100] It can be seen from Table 3 that, in the case of HCV-IgM positive sample detection, all the samples are detected by kit 1-1 (the kit of the disclosure) and kit 1-4 (immunocapture method); less than half of the samples are detected positive by kit 1-2 (indirect method) and kits 1-3 (double antigens sandwich), and the COI values are all relatively low, most of which are around 1; kit 1-5 (double antigens sandwich+indirect method) and kit 1-6 (double antigens sandwich+immunocapture method) have an increase in the detected values compared with kit 1-2 and kit 1-3, but there are still samples that failed to be detected. It can be seen that, in terms of the sensitivity of detecting HCV IgM positive samples, the kit/method of the disclosure is superior to the kit/method based on the principle of the indirect method, the kit/method based on the principle of the double antigens sandwich, and the kit/method based on a combination of the two (double antigens sandwich+indirect method); moreover, a combination of the immunocapture method and the double antigens sandwich method may reduce the detection rate of HCV IgM positive samples (the detection rate of combination 1-6 is weaker than that of combination 1-4). In addition, kit 1-7 has basically the same performance in terms of the detection rate of HCV-IgM antibody positive samples as kit 1-1.

Example 4 Detection of HCV Antibody Negative Sample

[0101] According to “Pathogen detection method”, 500 HCV antibody negative samples (from hospital diagnosis results) were tested, and the statistical results are as shown in Table 4 below.

TABLE-US-00004 TABLE 4 Kit 1-1 Kit 1-2 Kit 1-3 Kit 1-4 Kit 1-5 Kit 1-6 Total number of 500 500 500 500 500 500 samples Number of false 1 3 1 5 2 3 positive samples False positive 0.2% 0.6% 0.2% 1% 0.4% 0.6% probability

[0102] It can be seen from Table 4 that in the case of HCV antibody negative sample detection, kit 1-1 (the kit the disclosure) and kit 1-3 (double antigens sandwich) have comparable false positive occurrence, which is 0.2%; combination 1-2 (indirect method) and kit 1-4 (immunocapture method) have higher false positive occurrence, which are 0.6% and 1%, respectively; kits combining two detection formats: kit 1-5 (double antigens sandwich+indirect method) and combination 1-6 (double antigens sandwich+immunocapture method) have reduced false positive occurrence compared with kit 1-2 (indirect method) and kit 1-4 (immunocapture method), but higher false positive occurrence than kit 1-1 (the kit of the disclosure) and kit 1-3 (double antigens sandwich). It can be seen that in terms of the specificity of detecting HCV antibody negative samples, the kit/method of the disclosure is superior to the kit/method based on the principle of the indirect method or the principle of the immunocapture method and the kit/method based on a combination of either the indirect method or the immunocapture method and the double antigens sandwich method.

Example 5 Preparation of Treponema pallidum Antibody Detection Kit

[0103] The kits used in the example were prepared as described in Table 5 below and “Reagent preparation”.

TABLE-US-00005 TABLE 5 First reagent Second reagent Third reagent Fourth reagent Kit (V1 = 8 mL) (V2 = 8 mL) (15 mL) (8 mL) Kit 2-1 Magnetic bead TP15 antigen 1% BSA 10 mM DTT coated with TP15 labelled with ALP 0.05% Tween antigen (D = 100); (D = 500); −20 Magnetic bead TP17 antigen 3% casein coated with TP17 labelled with ALP 0.001% SDS antigen (D = 100); (D = 500); Magnetic bead TP47 antigen coated with TP47 labelled with ALP antigen (D = 200); (D = 800); Magnetic bead Anti-human IgG coated with anti- antibody labelled human IgM with ALP (D = 300). antibody (D = 100). Kit 2-2 Magnetic bead Anti-human IgG ″ ″ coated with TP15 antibody labelled antigen (D = 100); with ALP (D = 300). Magnetic bead coated with TP17 antigen (D = 100); Magnetic bead coated with TP47 antigen (D = 200). Kit 2-3 Magnetic bead TP15 antigen ″ ″ coated with TP15 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP17 antigen coated with TP17 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP47 antigen coated with TP47 labelled with ALP antigen (D = 200). (D = 800). Kit 2-4 Magnetic bead TP15 antigen ″ ″ coated with anti- labelled with ALP human IgM (D = 500); antibody (D = 100). TP17 antigen labelled with ALP (D = 500); TP47 antigen labelled with ALP (D = 800). Kit 2-5 Magnetic bead TP15 antigen ″ ″ coated with TP15 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP17 antigen coated with TP17 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP47 antigen coated with TP47 labelled with ALP antigen (D = 200). (D = 800); Anti-human IgG antibody labelled with ALP (D = 300). Kit 2-6 Magnetic bead TP15 antigen ″ ″ coated with TP15 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP17 antigen coated with TP17 labelled with ALP antigen (D = 100); (D = 500); Magnetic bead TP47 antigen coated with TP47 labelled with ALP antigen (D = 200); (D = 800). Magnetic bead coated with anti- human IgM antibody (D = 100).

Example 6 Detection of Serum Panel of Treponema pallidum Antibody

[0104] According to “Pathogen detection method”, two serum panels (0615-0017 and 0820-0300, purchased from SeraCare) were tested, and the results are shown in Table 6 below.

TABLE-US-00006 TABLE 6 Kit 2-1 Kit 2-2 Kit 2-3 Kit 2-4 Kit 2-5 Kit 2-6 Sample COI COI COI COI COI COI 0615-0017-01 0.17 0.14 0.05 0.42 0.18 0.35 0615-0017-02 0.47 0.12 0.04 0.44 0.19 0.39 0615-0017-03 0.42 0.12 0.03 0.41 0.17 0.41 0615-0017-04 0.58 0.13 0.03 0.40 0.21 0.42 0615-0017-05 1.98 0.16 0.13 0.68 0.23 0.73 0615-0017-06 3.07 0.51 1.04 1.17 0.78 1.23 0615-0017-07 3.68 0.80 2.11 1.26 1.21 1.31 0615-0017-08 4.20 0.97 5.10 1.51 1.68 2.49 0615-0017-09 11.20 1.32 12.40 1.87 2.05 3.88 0820-0300-01 10.14 1.91 5.37 0.73 2.19 1.08 0820-0300-02 2.92 3.16 1.10 0.85 2.15 0.98 0820-0300-03 4.10 4.48 2.22 0.77 1.86 1.03 0820-0300-04 3.31 0.89 2.15 0.71 1.36 0.95 0820-0300-05 3.54 1.05 1.33 0.73 0.98 1.09 0820-0300-06 2.90 1.38 1.21 0.79 0.97 1.00 0820-0300-07 10.20 3.11 7.29 0.79 1.04 1.05 0820-0300-08 2.62 1.05 1.21 0.74 1.04 1.17 0820-0300-09 8.11 1.15 6.41 3.19 1.10 1.02 0820-0300-10 2.34 1.14 1.13 0.96 1.05 1.02 0820-0300-11 3.28 0.91 1.13 0.78 1.02 0.96 0820-0300-12 0.07 0.26 0.06 0.48 0.15 0.21 0820-0300-13 4.15 1.07 2.26 1.72 1.02 2.10 0820-0300-14 2.90 1.07 1.14 1.69 0.95 2.06 0820-0300-15 3.61 0.80 1.34 1.46 1.02 1.79 0820-0300-16 3.28 1.29 3.17 1.06 1.02 1.33 0820-0300-17 2.65 0.68 1.28 0.68 0.96 0.89 0820-0300-18 2.83 1.00 1.20 0.75 2.03 3.78 0820-0300-19 2.32 0.74 1.48 0.73 1.24 1.22 0820-0300-20 8.16 1.05 5.09 0.73 1.00 1.01

[0105] It can be obtained from the results in Table 6 that the detection rates of serum panels of kit 2-1 (i.e., the kit of the disclosure) and kit 2-3 (double antigens sandwich method) are comparable; however, kit 2-1 shows a higher COI value and a more obvious gradient of detected samples than kit 2-3; the detection rates of kit 2-2 (indirect method), kit 2-4 (immunocapture method), kit 2-5 (double antigens sandwich+indirect method) and kit 2-6 (double antigens sandwich+immunocapture method) are weaker than those of kits 2-1 and 2-3; and most of the detected samples of kit 2-2 (indirect method), kit 2-4 (immunocapture method), kit 2-5 (double antigens sandwich+indirect method) and kit 2-6 (double antigens sandwich+immunocapture method) have lower COI (around 1), and the gradient of the detected samples is not obvious. It can be seen that, in terms of serum panel sensitivity, the kit/method of the disclosure is superior to the method based on the principle of the indirect method, the kit/method based on the principle of the immunocapture method and the kit/method based on a combination of either the indirect method or the immunocapture method and the double antigens sandwich (double antigens sandwich+indirect method and double antigens sandwich+immunocapture method).

Example 7 Detection of Treponema pallidum Antibody-IgM Positive Sample

[0106] According to “Pathogen detection method”, 30 Treponema pallidum antibody-IgM positive samples (which are the samples treated with IgG/RF adsorption reagent (purchased from Oumeng, Germany) and then confirmed by Roche Treponema pallidum antibody kit) were tested, and the results are as shown in Table 7 below.

TABLE-US-00007 TABLE 7 Kit 2-1 Kit 2-2 Kit 2-3 Kit 2-4 Kit 2-5 Kit 2-6 Sample COI COI COI COI COI COI 1 2.39 0.83 0.91 4.55 0.91 1.97 2 2.43 1.09 1.05 4.67 1.07 2.10 3 1.99 1.43 0.96 3.20 1.08 1.07 4 2.63 0.83 1.02 5.01 0.92 2.17 5 6.96 1.02 1.03 9.26 1.16 7.21 6 2.01 0.55 1.30 3.34 1.29 1.45 7 2.81 0.79 1.19 5.51 1.15 2.43 8 2.28 0.59 1.27 3.87 1.24 1.71 9 2.43 0.83 1.18 4.24 1.14 1.78 10 1.99 0.96 1.14 2.82 1.11 1.14 11 7.00 1.51 1.18 8.56 1.25 7.05 12 2.69 1.47 0.98 5.31 1.11 2.31 13 5.76 1.49 0.88 7.96 0.99 5.64 14 1.92 0.70 0.97 2.89 0.92 1.24 15 2.77 0.72 0.91 2.86 0.90 1.12 16 1.71 0.92 1.06 2.71 1.01 1.06 17 2.70 0.77 0.93 2.46 0.87 0.84 18 1.51 0.81 1.02 2.35 0.96 0.85 19 4.03 0.91 0.96 3.42 0.89 1.27 20 1.72 0.91 1.11 2.59 1.09 1.04 21 1.63 0.72 0.90 2.24 0.84 0.85 22 2.20 1.28 0.90 3.85 0.92 1.53 23 1.55 0.64 1.00 2.39 0.92 0.92 24 4.78 0.84 0.95 1.78 2.02 0.65 25 3.67 0.74 1.17 2.19 1.08 0.95 26 1.63 1.26 0.87 2.69 0.87 0.99 27 2.93 1.06 1.06 5.67 1.04 2.45 28 2.85 0.61 1.17 5.49 1.15 2.46 29 2.00 0.84 1.13 2.63 1.18 1.08 30 2.48 0.85 1.14 4.49 1.13 1.85

[0107] It can be seen from Table 7 that, in the case of Treponema pallidum-IgM positive sample detection, all the samples are detected by kit 2-1 (the kit of the disclosure) and kit 2-4 (immunocapture method); only about half of the samples are detected positive by kit 2-2 (indirect method) and kits 2-3 (double antigens sandwich), and the COI values are all relatively low, most of which are around 1; kit 2-5 (double antigens sandwich+indirect method) and kit 2-6 (double antigens sandwich+immunocapture method) have an increase in the detected number compared with kit 2-2 and kit 2-3, but there are still samples that failed to be detected. It can be seen that, in terms of the sensitivity for detecting Treponema pallidum antibody-IgM positive samples, the kit/method of the disclosure is superior to the method based on the principle of the indirect method, the kit/method based on the principle of the double antigens sandwich and the kit/method based on a combination of the two (double antigens sandwich+indirect method); moreover, a combination of the immunocapture method and the double antigens sandwich method may reduce the detection rate of Treponema pallidum antibody-IgM positive samples (the detection rate of kit 2-6 is weaker than that of kit 2-4). This conclusion is consistent with that in Example 3.

Example 8 Detection of Treponema pallidum Antibody Negative Samples

[0108] According to “Pathogen detection method”, 500 Treponema pallidum antibody negative samples (from hospital diagnosis results) were tested, and the statistical results are as shown in Table 8 below.

TABLE-US-00008 TABLE 8 Kit 2-1 Kit 2-2 Kit 2-3 Kit 2-4 Kit 2-5 Kit 2-6 Total number of 500 500 500 500 500 500 samples Number of false 0 1 0 2 1 1 positive samples False positive 0% 0.2% 0% 0.4% 0.2% 0.2% probability

[0109] It can be seen from Table 8 that in the case of Treponema pallidum antibody negative sample detection, neither kit 2-1 (the kit the disclosure) nor kit 2-3 (double antigens sandwich) have false positive samples; kit 2-2 (indirect method), kit 2-4 (immunocapture method) and kits combining the two detection formats, i.e., kit 2-5 (double antigens sandwich+indirect method) and kit 2-6 (double antigens sandwich+immunocapture method) all have a false positive occurrence of 0.2%-0.4%. It can be seen that in terms of the specificity for detecting Treponema pallidum antibody negative samples, the kit/method of the disclosure is superior to the kit/method based on the indirect method or the immunocapture method and the kit/method based on a combination of either the indirect method or the immunocapture method and the double antigens sandwich method.