Kit and method for detecting tp antibodies

Abstract

An enzyme label used in an immunodetection of Treponema pallidum (TP) antibody, the enzyme label being a fusion protein of a labeling enzyme and a TP antigenic protein. A kit for detecting a TP antibody and a method for detecting an antibody produced after infection with Treponema pallidum (TP) in a sample, the kit including a fusion protein of a labeling enzyme and a TP antigenic protein. The antigenic protein contains one or more of TP15 antigen, TP17 antigen, TP47 antigen, and TP45 antigen, or a fusion antigen of two or more thereof.

Description

TECHNICAL FIELD

[0001] The embodiments of the present application relate to the field of Treponema pallidum (TP) antibody detection, in particular to an enzyme label used in the immunodetection of Treponema pallidum (TP) antibody, especially in the double antigen sandwich method or capture method. BACKGROUND

[0002] Syphilis is a systemic, chronic and classic sexually transmitted disease caused by Treponema pallidum (TP) infection in human body, which can cause damage to multiple systems and multiple organs of human body, produce a variety of clinical manifestations, lead to tissue damage, dysfunction, and even endanger life. Syphilis can be transmitted through sexual contact, blood transmission and mother-to-child vertical transmission. At present, the Ministry of Health has listed syphilis as one of the four indicators that must be checked before blood transfusion, invasive and minimally invasive surgery, and prenatal care. Because of the complex and diverse clinical manifestations of syphilis, long course of disease and latent symptoms, diagnosis must be combined with epidemiological history, clinical manifestations and laboratory test results for comprehensive analysis and judgment, and immunodetection is one of the main methods for diagnosis and management of syphilis.

[0003] TP immunodetection methods include enzyme-linked immunosorbent assay, chemiluminescence immunoassay, gold labeling method, fluorescence immunoassay, etc. Among them, enzyme immunoassay techniques such as enzyme-linked immunosorbent assay and enzyme-catalyzed chemiluminescence immunoassay are common TP immunodetection methods using enzymes as reporter molecules. In the reaction mode of double antigen sandwich method, the labeled enzyme in enzyme immunoassay, the conjugate of antigenic substance, the antibody to be tested existing in the sample and the antigenic substance coated on the solid support form a sandwich structure, and the labeled enzyme in the sandwich structure is analyzed to obtain the qualitative or quantitative detection result of TP. In the reaction mode of capture method, the labeled enzyme in enzyme immunoassay, the conjugate of antigenic substance, the antibody to be tested existing in the sample and the anti-human IgG and anti-human IgM antibodies coated on the solid support form a complex, and the labeled enzyme in the complex is analyzed to obtain the qualitative or quantitative detection result of TP.

[0004] Generally, in the TP enzyme immunoassay based on double antigen sandwich method or capture method, a chemical activation cross-linking method is used to connect the labeled enzyme and the antigenic substance to form a cross-linking or conjugate. However, this connection process has the disadvantages of complicated operation and difficult control. In addition, this connection method makes the molecular molar ratio of enzyme and antigen uneven, resulting in a mixture of enzyme and antigen cross-linking products with different molecular molar ratios. Therefore, the mixture of enzyme and antigen cross-linking products prepared by using the chemical activation cross-linking method applied to the detection of Treponema pallidum antibody causes problems such as difficult production control of syphilis detection kit, large batch-to-batch difference of detection results, etc. SUMMARY

[0005] To solve the above problems, the embodiment of the present application provides an immunological detection kit for Treponema pallidum TP antibody, comprising a fusion protein of a TP antigenic protein and a labeling enzyme.

[0006] As used in the embodiments of the present application, "antigenic protein" or "antigenic substance" refers to a protein having immunoreactivity and being useful for TP immunological detection; it can be one TP antigen or a fragment thereof, and also can be a fusion antigen of two or more TP antigens or fragments thereof (or a chimeric protein of two or more TP antigens or fragments thereof).

[0007] In the embodiments of the present application, the antigenic protein contains one or more of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen, or a fusion antigen of two or more of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen.

[0008] As used in the embodiments of the present application, "TP antigen" refers to a substance having immunoreactivity and being useful for TP immunological detection, which is selected from a conserved protein of TP or a fragment thereof. Preferably, the TP antigen can be an outer membrane lipoprotein of Treponema pallidum having high immunoreactivity, and can be used as a diagnostic antigen of Treponema pallidum, for example, one or more of TP15, TP17 antigen, TP47 antigen and TP45 antigen, but is not limited thereto. In the embodiments of the present application, the TP antigen can exist in the form of one or more copies.

[0009] In some embodiments, the TP antigenic protein of the present application contains TP15 antigen, TP17 antigen and TP47 antigen.

[0010] In some embodiments, the TP antigenic protein of the present application is a chimeric protein containing multiple TP antigens, for example, a chimeric protein containing TP15 antigen and TP17, or a chimeric protein containing TP15 antigen and TP47 antigen, or a chimeric protein containing TP17 antigen and TP47 antigen, or a chimeric protein containing TP15 antigen, TP17 antigen and TP47 antigen.

[0011] In the case where the antigenic protein contains two or more TP antigens, these TP antigens can exist in any order. In exemplary embodiments, the antigenic protein of the present application can contain TP15 antigen and TP17 antigen in order from N-terminus to C-terminus; or can contain TP17 antigen TP15 antigen in order from N-terminus to C-terminus.

[0012] In specific embodiments, the TP antigenic protein of the present application contains, in order from N-terminus to C-terminus, a TP15 antigen, a TP17 antigen, and a TP47 antigen; or a TP15 antigen, a TP47 antigen, and a TP17 antigen; or a TP17 antigen, a TP15 antigen, and a TP47 antigen; or a TP17 antigen, a TP47 antigen, and a TP15 antigen; or a TP47 antigen, a TP15 antigen, and a TP17 antigen; or a TP47 antigen, a TP17 antigen, and a TP15 antigen.

[0013] In embodiments of the present application, each TP antigen can be directly connected or can be connected via a Linker, as long as the structure and activity of each TP antigen are not affected when the TP antigens are connected. In embodiments of the present application, a flexible linker, such as a flexible (Gly4Ser) n , GGGS, GGSGGGSG, etc.

[0014] In embodiments of the present application, “enzyme”, “enzyme for labeling” can be used interchangeably, and refer to an enzyme used in enzyme immunoassay. For example, it can be an enzyme used in enzyme-linked immunosorbent assay and enzyme chemiluminescent immunoassay.

[0015] In some embodiments, the enzyme of the present application can be alkaline phosphatase (EC 3.1.3.1), which can catalyze, for example, the hydrolysis of nitrophenyl phosphate (PNP), β-glycerophosphate sodium, naphthyl phosphate, 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxetane (AMPPD), and other colorimetric substrates and chemiluminescent substrates containing phosphate groups.

[0016] The alkaline phosphatase of embodiments of the present application can be naturally occurring, artificially synthesized, or produced by genetic engineering. In addition, the alkaline phosphatase of embodiments of the present application can be modified, such as surface glycosylation treatment or deglycosylation treatment of alkaline phosphatase.

[0017] As for the source of alkaline phosphatase, embodiments of the present application are not particularly limited, as long as enzyme immunoassay can be achieved. Exemplary alkaline phosphatases can be derived from bacteria, such as Escherichia coli; mammals, such as a cow (e.g., Genebank: AF052227.1 (source https: / / www.ncbi.nlm.nih.gov / )) or a human (e.g., Genebank: M12551.1); a shrimp; but are not limited thereto.

[0018] In some embodiments, the enzyme of the present application can be horseradish peroxidase (EC 1.11.1.7), which has an iron porphyrin as a cofactor and can catalyze the polymerization of phenol, aniline, and their substitutes in the presence of hydrogen peroxide, and is widely distributed in the plant kingdom, with the highest content in horseradish.

[0019] The horseradish peroxidase of the application embodiments can be naturally occurring, artificially synthesized, or produced by genetic engineering. In addition, the horseradish peroxidase of the application embodiments can be modified.

[0020] In some embodiments, the enzyme of the application also includes mutants thereof. The mutants of the enzyme of the application embodiments have greater than 80%, optionally greater than 85%, greater than 90%, greater than 95%, greater than 98%, or greater than 99% sequence homology compared to the wild type. An exemplary mutant of alkaline phosphatase can be GeneBank: M29670.1 (source https: / / www.ncbi.nlm.nih.gov / ), but the application embodiments are not limited thereto. An exemplary mutant of horseradish peroxidase can be GnenBank: XM_018585035.1, but the application embodiments are not limited thereto.

[0021] The determination of sequence homology is made by comparing the test sequence to a reference sequence, with the wild type sequence serving as the reference sequence. Then, the sequence homology of the test sequence relative to the reference sequence is calculated using a sequence comparison algorithm. Two examples of algorithms that are suitable for determining sequence homology are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through NCBI.

[0022] In the application embodiments, the enzyme can be fused to any position of the antigenic protein. For example, the enzyme can be fused at the N-terminus of the TP antigenic protein; or, the enzyme can be fused at the C-terminus of the antigenic protein; or, the enzyme can be fused between any two adjacent antigens in the antigenic protein.

[0023] In some embodiments, the fusion protein of the TP antigenic protein and the enzyme contains, in order from N-terminus to C-terminus, the enzyme, the TP15 antigen, the TP17 antigen, and the TP47 antigen.

[0024] In some embodiments, the fusion protein of the TP antigenic protein and the enzyme contains, in order from N-terminus to C-terminus, the TP15 antigen, the TP17 antigen, the TP47 antigen, and the enzyme.

[0025] In some embodiments, the fusion protein of the TP antigenic protein and the enzyme contains, in order from N-terminus to C-terminus, the TP15 antigen, the TP17 antigen, the enzyme, and the TP47 antigen.

[0026] In specific embodiments, the fusion protein of the present application comprises, from N-terminus to C-terminus, alkaline phosphatase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and alkaline phosphatase; or TP15 antigen, TP17 antigen, alkaline phosphatase and TP47 antigen; or horseradish peroxidase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and horseradish peroxidase; or TP15 antigen, TP17 antigen, horseradish peroxidase and TP47 antigen.

[0027] In some embodiments, the immunodetection kit for Treponema pallidum TP antibody can comprise at least one of a fusion protein of a labeling enzyme and TP15 antigen, a fusion protein of a labeling enzyme and TP17 antigen, and a fusion protein of a labeling enzyme and TP47 antigen. In specific embodiments, the immunodetection kit comprises a fusion protein of a labeling enzyme and TP15 antigen, a fusion protein of a labeling enzyme and TP17 antigen, and a fusion protein of a labeling enzyme and TP47 antigen.

[0028] In specific embodiments, the immunodetection kit can comprise at least one of a fusion protein of alkaline phosphatase and TP15 antigen, a fusion protein of alkaline phosphatase and TP17 antigen, and a fusion protein of alkaline phosphatase and TP47 antigen, or at least one of a fusion protein of horseradish peroxidase and TP15 antigen, a fusion protein of horseradish peroxidase and TP17 antigen, and a fusion protein of horseradish peroxidase and TP47 antigen.

[0029] In the embodiments of the present application, the enzyme can be directly connected to the antigenic protein or can be connected through a linker, as long as the connection of the enzyme and the antigenic protein ensures that their respective structures and activities are not affected. In the embodiments of the present application, a flexible linker is used, such as a flexible (Gly4Ser) n , GGGS, GGSGGGSG, etc.

[0030] In the embodiments of the present application, "fusion protein" refers to a fusion protein of an enzyme and an antigenic protein. For example, it can be represented as enzyme + TP15 + TP17 + TP47, in which case it refers to a fusion protein of enzyme, TP15 antigen, TP17 antigen and TP47 antigen from N-terminus to C-terminus.

[0031] The fusion protein of an enzyme and a TP antigenic protein can be prepared by conventional recombinant expression technology. In the embodiments of the present application, the recombinant expression technology can be prokaryotic expression technology, such as Escherichia coli expression technology, etc.; and eukaryotic expression technology, such as yeast expression technology and insect cell expression technology, etc.

[0032] The skilled in the art can understand that the kit of the embodiments of the present application can also include other reagents or components for determining TP antibodies based on the double antigen sandwich method or the capture method, for example, a solid phase support coated with TP antigenic proteins (the TP antigenic proteins coated on the solid phase support and the TP antigenic proteins in the fusion protein can bind to the same TP antibodies in the sample) or a solid phase support coated with anti-human IgM antibodies and anti-human IgG antibodies; calibrators for drawing a standard curve; quality control samples for quality control; a substrate solution for chemiluminescence reaction; and / or a washing buffer and a sample diluent, etc.

[0033] In some embodiments, the kit includes a solid phase support coated with a fusion antigen containing TP15 antigen, TP17 antigen and TP47 antigen; or includes a solid phase support coated with TP15 antigen, a solid phase support coated with TP17 antigen and a solid phase support coated with TP47 antigen.

[0034] In another aspect, the embodiments of the present application also relate to the use of a fusion protein of an enzyme and a TP antigenic protein in the preparation of an immunological detection kit for detecting TP. The immunological detection kit includes:

[0035] a first reagent containing a solid phase coating, the solid phase coating being coated with a first ligand capable of binding to TP antibodies in a sample;

[0036] a second reagent containing an enzyme label, the enzyme label being a second ligand fused with an enzyme, wherein the second ligand is a TP antigenic protein and is capable of binding to the TP antibodies bound by the first ligand.

[0037] In a specific embodiment, TP is detected based on the double antigen sandwich method or the capture method.

[0038] In one variant of the embodiments of the present application, an immunological detection kit for detecting TP is provided, including:

[0039] a first reagent containing a solid phase coating, the solid phase coating being a solid phase support coated with a TP antigenic protein (a first ligand);

[0040] a second reagent containing an enzyme label, the enzyme label being a TP antigenic protein (a second ligand) fused with an enzyme, wherein the TP antigenic protein coated on the solid phase support and the TP antigenic protein in the fusion protein can bind to the same TP antibodies in a sample; and

[0041] a description of detecting based on the double antigen sandwich method.

[0042] Those skilled in the art can understand that the types of the TP antigenic proteins coated on the solid support in the first reagent and the TP antigenic proteins fused in the fusion protein in the second reagent are the same, but the sources or the connection modes or the connection orders between the antigens can be the same or different. For example, the TP antigenic proteins coated on the solid support can be a fusion antigen of TP15 antigen, TP17 antigen, and TP47 antigen connected in sequence, and the TP antigenic proteins fused in the fusion protein in the second reagent can be a fusion antigen of TP17 antigen, TP47 antigen, and TP17 antigen connected in sequence.

[0043] In addition, in the embodiments of the present application, the TP antigenic proteins coated on the solid support can be the TP antigenic proteins as described above, or can be TP antigenic proteins connected in other manners, for example, chemically connected.

[0044] In the embodiments of the present application, the TP antibody refers to an antibody produced in the body of a subject after TP infection, for example, Anti-TP IgG antibody and Anti-TP IgM antibody.

[0045] In another variant of the embodiments of the present application, an immunodetection kit for detecting TP is provided, comprising:

[0046] The first reagent contains a solid phase coating, and the solid phase coating is a solid support coated with anti-human IgM antibody and anti-human IgG antibody (first ligand);

[0047] The second reagent contains an enzyme label, and the enzyme label is a fusion protein of an enzyme and a TP antigenic protein (second ligand); and

[0048] The specification describes detection based on the capture method.

[0049] As used in the embodiments of the present application, the "solid support" refers to a solid surface to which an antigen or an antibody can be attached. The solid support used in the present application is not particularly limited, and any solid support that can be used for immunodetection can be used in the present application. Exemplary solid supports can be magnetic beads (such as superparamagnetic microspheres), enzyme-labeled plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or microchips, but the embodiments of the present application are not limited thereto.

[0050] In the embodiments of the present application, the solid phase coating can be present in a conventional diluent containing proteins and surfactants and having buffering capacity.

[0051] In the embodiments of the present application, the enzyme label can be present in a conventional diluent containing proteins and surfactants and having buffering capacity.

[0052] In the embodiments of the present application, the fusion protein may, for example, be present in the second reagent at a concentration of about 100 ng / mL to about 300 ng / mL.

[0053] In specific embodiments, the kit of the present application can further comprise a third reagent comprising a blocking agent and a surfactant. For example, the blocking agent is selected from one or more of the group consisting of skim milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, a surfactant (e.g., Tween-20, Tween-80, Triton X-100, etc.).

[0054] In specific embodiments, the kit of the present application can further comprise a fourth reagent comprising a reducing agent. For example, the reducing agent is selected from one or more of the group consisting of DTT, β-mercaptoethanol.

[0055] In the embodiments of the present application, the blocking agent and the surfactant can be dissolved in a conventional diluent with buffering capacity; the reducing agent can be dissolved in a conventional diluent with buffering capacity.

[0056] In some embodiments, the kit can further comprise a reaction substrate for the labeling enzyme, preferably the reaction substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxacyclohexane.

[0057] Unless specifically stated, the terms "first", "second", "third", and "fourth" and the like in the embodiments of the present application are used only to distinguish one element from another, and do not necessarily indicate important or order among the elements.

[0058] The embodiments of the present application also relate to a method for detecting TP antibodies produced after infection with Treponema pallidum TP in a sample, comprising the following steps:

[0059] Mixing the sample and the solid support coated with the first ligand, so that the first ligand coated on the solid support is fully combined with the TP antibodies in the sample;

[0060] Washing the above mixture to remove unbound substances;

[0061] Adding an enzyme-labeled material with a second ligand to the above washed mixture and mixing, so that the second ligand in the enzyme-labeled material is combined with the TP antibodies combined on the solid support to form a sandwich complex, wherein the enzyme-labeled material is a fusion protein of a labeling enzyme and a TP antigenic protein;

[0062] Washing the above sandwich complex to remove unbound substances;

[0063] The chemiluminescent substrate is added to the above-mentioned washed sandwich complex, and the number of photons generated by the reaction is detected to obtain the chemiluminescent signal value of the sample.

[0064] In the embodiments of the present application, the first ligand is a TP antigenic protein, or is an anti-human IgG antibody and an anti-human IgM antibody.

[0065] In the embodiments of the present application, the fusion protein of the enzyme for labeling and the TP antigenic protein is obtained by recombinant expression, and is applied to a TP immunological detection kit based on a double-antigen sandwich or capture method mode. It should be noted that:

[0066] By using such a fusion protein to replace the linker between the enzyme and the antigenic substance produced by chemical activation cross-linking, the fusion protein of the enzyme for labeling and the TP antigenic protein in the kit has a uniform molar ratio of the enzyme for labeling to the TP antigenic protein, thereby avoiding the problem of large batch-to-batch difference in kit production and detection results.

[0067] On the other hand, compared with conventional double-antigen sandwich or capture method kits, the kit of the embodiments of the present application shows better discrimination between positive and negative samples, and has better sample compliance rate.

[0068] On the other hand, compared with conventional double-antigen sandwich or capture method kits, the kit of the embodiments of the present application shows better discrimination between positive and negative samples, and has better sample compliance rate.

[0069] On the other hand, compared with conventional double-antigen sandwich or capture method kits, the kit of the embodiments of the present application shows better discrimination between positive and negative samples, and has better sample compliance rate. DETAILED DESCRIPTION

[0070] The technical means in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0071] As described above, the fusion protein of the enzyme and the TP antigenic protein in the embodiments of the present application can be produced by recombinant expression technology.

[0072] An exemplary recombinant expression method can include the following steps:

[0073] i) transfecting or transforming a host cell with a nucleic acid molecule or an expression vector;

[0074] ii) culturing the host cell under conditions for expressing the fusion protein; and

[0075] iii) isolating the fusion protein.

[0076] In the above step, the nucleic acid molecule encodes the above-mentioned fusion protein; and the expression vector for preparing the fusion protein of the enzyme and the antigenic protein of TP by recombinant expression comprises the nucleic acid molecule encoding the above-mentioned fusion protein operably linked to an expression control sequence, and further comprises genetic elements for maintenance and propagation in respective host cells, such as a replication origin and / or a selection marker gene.

[0077] Preparation of fusion proteins

[0078] Production of the fusion protein using the E. coli expression system:

[0079] First step: confirmation of the target gene. According to the gene sequences and amino acid sequences of the two proteins to be fused, a suitable linker, such as (Gly4Ser)3, AEAAAKA or GGSG, is added between the two, and a His tag is added at the C-terminus of the fusion protein for purification, and the target gene sequence is preliminarily determined. Then, through codon optimization, the final target gene sequence suitable for the E. coli expression strain BL21(DE3) or Rosetta(DE3) is determined.

[0080] Second step: vector construction. A suitable restriction site is selected, and the target gene is integrated into a suitable expression vector pET28 or pCold TF.

[0081] Third step: transformation and screening of positive clone strains. The expression vector containing the target gene is transformed into E. coli by electroporation or heat shock transformation. According to the reporter gene on the expression vector, a suitable antibiotic is added, and the positive strains are screened.

[0082] Fourth step: expression and purification of the protein.

[0083] TP antigen nucleic acid sequence information: TP15, Protein ID: AAC45732.1; TP17, GeneBank: M74825.1; TP47, GeneBank: M88769.1 (source https: / / www.ncbi.nlm.nih.gov / ).

[0084] Reagent preparation method

[0085] First reagent Ra:

[0086] Using a pipette or a measuring cylinder, 3.5 mL of "magnetic beads coated with TP15+TP17+TP47 chimeric protein" is added to a magnetic bead coating tube to replace the supernatant, i.e. after magnetic separation, the supernatant is removed, then an equal volume (3.5 mL) of magnetic bead coating diluent is added, and mixed evenly; after mixing the magnetic beads, add to a preparation bottle containing 96.5 mL of magnetic bead coating diluent; stir until the magnetic bead suspension is completely mixed, to prepare the first reagent Ra, wherein the concentration of magnetic beads coated with TP15+TP17+TP47 chimeric protein is 0.5 mg / mL; the magnetic bead coating diluent is a conventional diluent with buffering capacity and contains proteins and surfactants.

[0087] Second reagent Rb:

[0088] Using a suitable measuring cylinder, 99 mL of tracer diluent is added to the preparation bottle, and using a pipette, 1 mL of "enzyme and TP antigenic protein fusion protein", or "chemically linked enzyme and TP antigenic protein conjugate", or "chemically linked enzyme and anti-human IgG and anti-human IgM conjugate" is added to the tracer diluent as an enzyme label; use a stirrer to stir the solution to dissolve and mix thoroughly; then use a suitable 0.22 μm pore size filter to filter the prepared solution, collect the filtrate, and prepare the tracer Rb, wherein the concentration of the above enzyme label is 150 ng / mL; the tracer diluent is a conventional diluent with buffering capacity and contains proteins and surfactants.

[0089] Third reagent Rc:

[0090] Diluent with buffering capacity and containing blocking agent BSA and surfactant Tween 20.

[0091] Fourth reagent Rd:

[0092] Diluent with buffering capacity and containing reducing agent DTT.

[0093] Double antigen sandwich assay method

[0094] First step: add the sample, the fourth reagent and the first reagent to the reaction tube, incubate at 37°C for 10 minutes, so that the TP antigens coated on the magnetic bead solid phase can fully bind with Anti-TP IgG and Anti-TP IgM antibodies in the sample; after incubation, the magnetic bead solid phase is attracted to the magnetic field, the substances bound to the magnetic bead solid phase are retained, and other unbound substances are removed by washing.

[0095] Second step: Add the third reagent and the second reagent into the reaction tube and mix well; incubate at 37°C for 10 minutes, and the TP antigen on the enzyme label binds to the Anti-TP IgG and Anti-TP IgM antibodies captured on the magnetic beads to form a sandwich complex. After the incubation in the reaction tube is completed, the complex is attracted by the magnetic field, and other unbound substances are removed by washing.

[0096] Third step: Add the chemiluminescent substrate into the reaction tube to generate chemiluminescence. The number of photons generated by the reaction is measured by a photomultiplier tube to obtain the chemiluminescence signal value of the sample.

[0097] In the embodiments of the present application, the COI (Cutoff index) is the ratio of the chemiluminescence signal value (RLU) of the sample to the threshold value (Cutoff value), wherein COI≥1 indicates that the sample is a positive sample, and COI<1 indicates that the sample is a negative sample. For a qualitative detection method, the threshold value (cutoff value) is the dividing value for judging the test result as positive or negative.

[0098] In the embodiments of the present application, the negative coincidence rate refers to the proportion of the number of samples judged as negative by the test method of the embodiments of the present application to the actual negative samples participating in the evaluation, and the positive coincidence rate refers to the proportion of the number of samples judged as positive by the test method of the embodiments of the present application to the actual positive samples participating in the evaluation; the true positive and negative results of the samples come from the hospital diagnosis results.

[0099] Example 1

[0100] To compare the TP detection effect of the fusion protein of the enzyme and the TP antigenic protein in the embodiments of the present application with that of the conjugate of the enzyme and the antigenic protein produced by chemical cross-linking, the following three reagent combinations are prepared.

[0101] Combination 1: Use the TP15+TP17+TP47 chimeric protein (or fusion antigen) as the TP antigenic protein and alkaline phosphatase to prepare the second reagent Rb by using the above-described E. coli expression system, wherein the alkaline phosphatase (Genebank: AF052227.1 (source https: / / www.ncbi.nlm.nih.gov / )) in the fusion protein of the second reagent Rb is fused to the N terminus of the TP15+TP17+TP47 chimeric protein, and is prepared according to the above-mentioned “reagent preparation method”.

[0102] Combination 2: Use the conjugate of the chemically linked alkaline phosphatase and the TP15+TP17+TP47 chimeric protein to prepare the second reagent Rb, and the rest is the same as combination 1.

[0103] Next, using combinations 1 and 2, 500 TP-negative samples and 500 TP-positive samples from a general hospital that were confirmed by diagnosis were tested according to the "double antigen sandwich detection method" described above. The results are shown in Table 1 below.

[0104] Table 1

[0105] Reagent combination Combination 1 Combination 2 Combination 3 (indirect method) Negative sample 1 (COI) 0.17 0.13 0.10 Negative sample 2 (COI) 0.15 0.14 0.21 Positive sample 1 (COI) 2.02 1.81 2.35 Positive sample 2 (COI) 12.16 7.96 9.64 Positive sample 3 (COI) 64.70 45.24 48.66 Negative coincidence rate (500 cases) 98.4％ 98.2％ 98.2％ Positive coincidence rate (500 cases) 99.2％ 98.6％ 98.4％

[0106] As can be seen from Table 1, the negative coincidence rate of combination 1 was 98.4%, and the positive coincidence rate was 99.2%, which was higher than that of the conventional double antigen sandwich method using chemical linkage (combination 2: negative coincidence rate 98.2%, positive coincidence rate 98.6%). This result shows that the use of alkaline phosphatase and the fusion protein of TP15+TP17+TP47 can avoid the damage to the activity of the antigen or alkaline phosphatase in the preparation process using chemical linkage in the conventional double antigen sandwich method. In the embodiments of the present application, the alkaline phosphatase in the fusion protein of alkaline phosphatase and TP15+TP17+TP47 and the antigen both maintained high activity.

[0107] In addition, in order to prove the activity of the TP15+TP17+TP47 chimeric protein used in the embodiments of the present application, reagent combination 3 was prepared: the second reagent Rb was prepared using a chemical linkage of alkaline phosphatase and an anti-human IgG and anti-human IgM conjugate, and the rest was the same as combination 1.

[0108] According to the indirect detection method, the above-mentioned combination 3 was used to test the above-mentioned 500 TP-negative samples and 100 TP-positive samples that were confirmed by diagnosis. The specific steps of the "indirect detection method" are as follows:

[0109] Step 1: Add the sample, the third reagent, the fourth reagent and the first reagent to the reaction tube, and incubate at 37°C for 10 minutes to allow the TP antigen coated on the magnetic bead solid phase to fully bind with the Anti-TP IgG and Anti-TP IgM antibodies in the sample; after incubation, the magnetic bead solid phase is attracted to the magnetic field, and the substances bound to the magnetic bead solid phase are retained, while other unbound substances are removed by washing.

[0110] Step 2: Add the third reagent and the second reagent to the reaction tube and mix well; incubate at 37°C for 10 minutes, and the anti-human IgG and anti-human IgM secondary antibodies on the enzyme label bind with the Anti-TP IgG and Anti-TP IgM antibodies captured on the magnetic beads to form a sandwich complex. After incubation in the reaction tube, the complex is attracted to the magnetic field, and other unbound substances are removed by washing.

[0111] Third step: add chemiluminescent substrate into the reaction tube to generate chemiluminescence. Then measure the number of photons generated by the reaction through a photomultiplier tube to obtain the chemiluminescence signal value of the sample.

[0112] The results are shown in Table 1. The indirect method test (combination 3) can better distinguish between negative and positive samples, indicating that the TP15+TP17+TP47 chimeric protein used in the embodiments of the present application has good activity.

[0113] Example 2

[0114] To compare the TP detection effect of the fusion protein of the enzyme and the TP antigenic protein of the embodiments of the present application with the conjugate of the enzyme and the antigenic protein produced by chemical cross-linking, the following three reagent combinations were prepared:

[0115] Combination 4: using the above-described E. coli expression system, using TP15+TP17+TP47 as the antigenic protein and horseradish peroxidase for fusion to prepare the second reagent Rb. The fusion protein of the second reagent Rb has horseradish peroxidase (GeneBank: KU504630.1 (source https: / / www.ncbi.nlm.nih.gov / )) located at the N-terminus of the antigenic protein, which is prepared according to the above-mentioned “reagent preparation method”.

[0116] Combination 5: using a conjugate of chemically linked horseradish peroxidase and TP15+TP17+TP47 chimeric protein to prepare the second reagent Rb, and the rest is the same as combination 4.

[0117] Next, using combinations 4 and 5, 500 TP-negative samples and 100 positive samples in Example 1 were tested according to the above-mentioned “double antigen sandwich detection method”. The results are shown in Table 2 below.

[0118] Table 2

[0119] Reagent combination Combination 4 Combination 5 Combination 6 (indirect method) Negative sample 1 (COI) 0.18 0.21 0.31

[0120] Negative sample 2 (COI) 0.23 0.16 0.25 Positive sample 1 (COI) 1.85 1.53 1.65 Positive sample 2 (COI) 8.77 5.45 6.50 Positive sample 3 (COI) 42.26 32.36 34.58 Negative coincidence rate (500 cases) 98.2％ 97.8％ 97.6％ Positive coincidence rate (500 cases) 98.6％ 98.4％ 97.8％

[0121] As shown in Table 2, the negative coincidence rate of combination 4 is 98.2%, and the positive coincidence rate is 98.6%, which is higher than that of the conventional double-antigen sandwich method using chemical linkage (combination 5: negative coincidence rate 97.8%, positive coincidence rate 98.4%). This result shows that the use of horseradish peroxidase and the fusion protein of TP15+TP17+TP47 can avoid the damage to the activity of the antigen or horseradish peroxidase in the preparation process using chemical linkage in the conventional double-antigen sandwich method. In the embodiment of the present application, the horseradish peroxidase and the antigen in the fusion protein of TP15+TP17+TP47 maintain high activity.

[0122] Similarly, in order to prove the activity of the TP15+TP17+TP47 chimeric protein used in the embodiment of the present application, reagent combination 6 was prepared: the second reagent Rb was prepared using a conjugate of chemically linked horseradish peroxidase and anti-human IgG and anti-human IgM, and the rest was the same as combination 4.

[0123] According to the above indirect detection method, the above-mentioned 500 TP-negative samples and 100 positive samples were tested using the above-mentioned combination 6. The results are shown in Table 1. The indirect method test (combination 6) can better distinguish the negative and positive samples, which shows that the TP15+TP17+TP47 chimeric protein used in the embodiment of the present application has good activity.

[0124] Example 3

[0125] In order to investigate the effect of the fusion position of the enzyme on the fusion protein of the embodiment of the present application, the following three reagent combinations were prepared:

[0126] Combination 1: using the above-described E. coli expression system, the TP15+TP17+TP47 chimeric protein was used as an antigenic protein and fused with alkaline phosphatase to prepare the second reagent Rb. The alkaline phosphatase in the fusion protein of the second reagent Rb is fused to the N-terminus of the TP15+TP17+TP47 chimeric protein, which is prepared according to the above-mentioned "reagent preparation method".

[0127] Combination 7: in the fusion protein of the second reagent Rb, the alkaline phosphatase is fused to the C-terminus of the TP15+TP17+TP47 chimeric protein, and the rest is the same as combination 1.

[0128] Combination 8: in the fusion protein of the second reagent Rb, the alkaline phosphatase is fused between TP17 and TP47 of the TP15+TP17+TP47 chimeric protein, and the rest is the same as combination 1.

[0129] Next, using combinations 1, 7 and 8, the above-described "double antigen sandwich detection method" was used to test 500 TP-negative samples and 500 TP-positive samples from Example 1, respectively, and the results are shown in Table 3 below.

[0130] Table 3

[0131] Reagent combination Combination 1 Combination 7 Combination 8 Negative sample 1 (COI) 0.17 0.12 0.16 Negative sample 2 (COI) 0.15 0.15 0.14 Positive sample 1 (COI) 2.02 2.24 2.11 Positive sample 2 (COI) 12.16 12.84 12.69 Positive sample 3 (COI) 64.70 60.08 58.66 Negative coincidence rate (500 cases) 98.4％ 98.2％ 98.4％ Positive coincidence rate (500 cases) 99.2％ 99.2％ 99.0％

[0132] As can be seen from Table 3, when alkaline phosphatase is fused to the N-terminus (combination 1), C-terminus (combination 7) and middle (combination 8) of the antigenic protein, both the negative coincidence rate and the positive coincidence rate of the samples can be high. In addition, the effect of fusing the enzyme in the middle of the TP antigenic protein is slightly worse than fusing the enzyme in the N and C termini of the TP antigenic protein. Although not wishing to be bound by theory, the inventors believe that in the case of fusing the enzyme in the middle of the TP antigenic protein, the active center of the enzyme is partially blocked by other protein parts.

[0133] Example 4

[0134] To investigate the effect of the fusion position of the enzyme on the fusion protein of the present application, the following three reagent combinations were prepared:

[0135] Combination 4: using the above-described E. coli expression system, the TP15+TP17+TP47 chimeric protein was used as the antigenic protein and was fused with horseradish peroxidase to prepare the second reagent Rb. The fusion protein of the second reagent Rb has horseradish peroxidase fused to the N-terminus of the TP15+TP17+TP47 chimeric protein, and was prepared according to the above-described "reagent preparation method".

[0136] Combination 9: in the fusion protein of the second reagent Rb, horseradish peroxidase is fused to the C-terminus of the TP15+TP17+TP47 chimeric protein, and the rest is the same as combination 4.

[0137] Combination 10: in the fusion protein of the second reagent Rb, horseradish peroxidase is fused between TP17 and TP47 of the TP15+TP17+TP47 chimeric protein, and the rest is the same as combination 4.

[0138] Next, using combinations 4, 9 and 10, the above-described "double antigen sandwich detection method" was used to test 500 TP-negative samples and 500 TP-positive samples from Example 1, respectively, and the results are shown in Table 4 below.

[0139] Table 4

[0140] Reagent combination Combination 4 Combination 9 Combination 10 Negative sample 1 (COI) 0.18 0.20 0.20 Negative sample 2 (COI) 0.23 0.16 0.21 Positive sample 1 (COI) 1.85 1.92 1.80 Positive sample 2 (COI) 8.77 7.86 8.06 Positive sample 3 (COI) 42.26 39.68 40.33 Negative coincidence rate (500 cases) 98.2％ 98.2％ 98.2％ Positive coincidence rate (500 cases) 98.6％ 98.4％ 98.0％

[0141] As shown in Table 4, the horseradish peroxidase fused to the N-terminus (combination 4), C-terminus (combination 9) and middle (combination 10) of the antigenic protein can achieve high negative and positive coincidence rates. In addition, the enzyme fused to the middle of the TP antigenic protein is slightly less effective than the enzyme fused to the N- and C-termini of the TP antigenic protein. Although not wishing to be bound by theory, the inventors believe that in the case of enzyme fusion to the middle of the TP antigenic protein, the active center of the enzyme is partially blocked by other protein parts.

[0142] Example 5

[0143] To investigate the effect of enzyme fusion to individual antigens vs. enzyme fusion to chimeric proteins on the fusion proteins of the present application, the following four reagent combinations were prepared:

[0144] Combination 1: Using the above-described E. coli expression system, the TP15+TP17+TP47 chimeric protein was used as the antigenic protein and fused to alkaline phosphatase to prepare the second reagent Rb. The fusion protein of the second reagent Rb has alkaline phosphatase fused to the N-terminus of the TP15+TP17+TP47 chimeric protein, which was prepared according to the above "reagent preparation method".

[0145] Combination 11: The second reagent Rb contains three fusion proteins, namely the fusion protein of TP15 and alkaline phosphatase, the fusion protein of TP17 and alkaline phosphatase, and the fusion protein of TP47 and alkaline phosphatase, and the rest is the same as combination 1.

[0146] Combination 12: The second reagent Rb contains only the fusion protein of TP17 and alkaline phosphatase, with alkaline phosphatase fused to the N-terminus of TP17, and the rest is the same as combination 1.

[0147] Combination 13: The second reagent Rb contains three fusion proteins, namely the fusion protein of TP15 and horseradish peroxidase, the fusion protein of TP17 and horseradish peroxidase, and the fusion protein of TP47 and horseradish peroxidase, and the rest is the same as combination 1.

[0148] Next, using combinations 1 and 11-13, the 500 TP-negative samples and 500 positive samples from Example 1 were tested according to the above "double antigen sandwich detection method", and the results are shown in Table 5.

[0149] Table 5

[0150] Reagent combination Combination 1 Combination 11 Combination 12 Combination 13 Negative sample 1 (COI) 0.17 0.20 0.10 0.24 Negative sample 2 (COI) 0.15 0.18 0.11 0.19 Positive sample 1 (COI) 2.02 2.03 1.89 1.79 Positive sample 2 (COI) 12.16 10.64 9.35 6.73 Positive sample 3 (COI) 64.70 60.52 67.29 39.66 Negative coincidence rate (500 cases) 98.4％ 98.2％ 98.8％ 97.8％ Positive coincidence rate (500 cases) 99.2％ 98.8％ 98.2％ 97.6％

[0151] As shown in Table 5, enzyme fusion to individual antigens or enzyme fusion to chimeric proteins can achieve good negative and positive coincidence rates.

[0152] Example 6

[0153] To investigate the TP detection effect when using mutants of enzymes, the following reagent combinations were prepared:

[0154] Combination 1: Using the E. coli expression system described above, the TP15+TP17+TP47 chimeric protein was used as an antigenic protein and fused with wild-type alkaline phosphatase to configure the second reagent Rb. The wild-type alkaline phosphatase (gene derived from bovine small intestine) was fused at the N-terminus of the antigenic protein, and was prepared according to the above “reagent preparation method”.

[0155] Combination 14: Using mutant alkaline phosphatase (GeneBank: M29670.1 (source https: / / www.ncbi.nlm.nih.gov / )), and the rest is the same as combination 1.

[0156] Next, using combinations 1 and 14, 500 TP-negative samples and 500 positive samples in Example 1 were tested according to the above “double antigen sandwich detection method”, and the results are shown in Table 6.

[0157] Table 6

[0158] Reagent combination Combination 1 Combination 14 Negative sample 1 (COI) 0.17 0.18 Negative sample 2 (COI) 0.15 0.25 Positive sample 1 (COI) 2.02 5.95 Positive sample 2 (COI) 12.16 26.22 Positive sample 3 (COI) 64.70 157.72 Negative coincidence rate (500 cases) 98.4％ 98.2％ Positive coincidence rate (500 cases) 99.2％ 99.4％

[0159] As can be seen from Table 6, using wild-type alkaline phosphatase or mutant alkaline phosphatase can achieve good negative coincidence rate and positive coincidence rate of samples. In addition, compared with wild-type alkaline phosphatase, the detection resolution is better when using mutant alkaline phosphatase.

[0160] Example 7

[0161] To investigate the effect of different expression techniques on the TP detection effect of the present application, a eukaryotic expression system was further used to prepare a fusion protein of alkaline phosphatase and TP15+TP17+TP47.

[0162] The specific method for producing the fusion protein by the eukaryotic expression system is as follows:

[0163] First step: confirmation of target gene. Add a nucleotide sequence capable of encoding a linker short peptide between the two gene sequences of the two proteins to be fused, and add a His tag at the C-terminus of the fusion protein for purification. The target gene sequence is initially determined. Then, through codon optimization, the final target gene sequence suitable for the Pichia pastoris expression strain is determined. In the present application, the number of amino acids of the linker short peptide is greater than 8, especially greater than 10, for example, it can be (Gly4Ser)3 or AEAAAKEAAAKA, and the Pichia pastoris strain can be a methanol-inducible Pichia pastoris (X33).

[0164] Second step: vector construction. Select the appropriate enzyme cutting site, and integrate the target gene into the appropriate expression plasmid.

[0165] Third step: transformation and screening of positive clone strains. Through electroporation, the expression vector containing the target gene is transferred into Pichia pastoris cells or 293 cells. According to the reporter gene on the expression vector, add the appropriate antibiotic to screen the positive strains,

[0166] Fourth step: protein expression and purification.

[0167] According to the "reagent preparation method", combination 15 (Pichia pastoris) and combination 16 (293 cells) are respectively obtained.

[0168] Next, using combinations 1, 15 and 16, 500 TP negative samples and 500 positive samples in Example 1 are respectively tested according to the above-mentioned "double antigen sandwich detection method", and the results are shown in Table 7.

[0169] Table 7

[0170] Reagent combination Combination 1 Combination 15 Combination 16 Negative sample 1 (COI) 0.17 0.12 0.18 Negative sample 2 (COI) 0.15 0.16 0.12 Positive sample 1 (COI) 2.02 2.97 2.27 Positive sample 2 (COI) 12.16 14.86 10.28 Positive sample 3 (COI) 64.70 78.81 60.46 Negative coincidence rate (500 cases) 98.4％ 98.2％ 98.0％ Positive coincidence rate (500 cases) 99.2％ 99.2％ 99.0％

[0171] As can be seen from Table 7, the use of prokaryotic expression system and eukaryotic expression system to prepare fusion protein can achieve better negative coincidence rate and positive coincidence rate of samples.

Claims

1. A kit for detecting Treponema pallidum (TP) antibodies, comprising: a fusion protein of a labeling enzyme and TP antigenic proteins, wherein the labeling enzyme is alkaline phosphatase or horseradish peroxidase; wherein the fusion protein comprises, from N-terminal to C-terminal, alkaline phosphatase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and alkaline phosphatase; or TP15 antigen, TP17 antigen, alkaline phosphatase and TP47 antigen; or the fusion protein comprises, from N-terminal to C-terminal, horseradish peroxidase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and horseradish peroxidase; or TP15 antigen, TP17 antigen, horseradish peroxidase and TP47 antigen.

2. The kit of claim 1, wherein, The kit further comprises: a solid support coated with TP antigenic proteins, wherein the antigenic proteins coated on the solid support comprise TP15 antigen, TP17 antigen and TP47 antigen, and the TP antigenic proteins coated on the solid support and the TP antigenic proteins in the fusion protein can bind to the same TP antibodies in a sample; or a solid support coated with anti-human IgG antibodies and anti-human IgM antibodies.

3. The kit of claim 1 or 2, wherein, The kit further comprises: a solid support coated with a fusion antigen comprising TP15 antigen, TP17 antigen and TP47 antigen; or a solid support coated with TP15 antigen, a solid support coated with TP17 antigen and a solid support coated with a fusion antigen of TP47 antigen. 4.The kit of claim 1 or 2, further comprising a blocking agent.

5. The kit of claim 4, wherein, The blocking agent is selected from one or more of the group consisting of skimmed milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, surfactant. 6.The kit of claim 1 or 2, further comprising a reducing agent.

7. The kit of claim 6, wherein, The reducing agent is selected from one or more of the group consisting of DTT, β-mercaptoethanol.

8. The kit of claim 1 or 2, wherein, The kit further comprises a reaction substrate of the labeling enzyme.

9. The kit of claim 8, wherein, The reaction substrate is 3-(2-spiro adamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxolane.

10. The kit of claim 1 or 2, wherein, The fusion protein is produced via eukaryotic expression technology. 11.Use of a fusion protein of a labeling enzyme and TP antigenic proteins in the preparation of a kit for detecting antibodies produced after infection with Treponema pallidum (TP) in a sample, comprising the following: mixing the sample and a solid support coated with a first ligand, so that the first ligand coated on the solid support binds to the TP antibodies in the sample; washing the mixture to remove unbound substances; and detecting the bound antibodies. adding an enzyme label with a second ligand into the above-mentioned cleaned mixture and mixing, so that the second ligand in the enzyme label binds with the TP antibody bound on the solid support to form a sandwich complex, wherein the enzyme label is a fusion protein of a labeling enzyme and TP antigenic protein, and the labeling enzyme is alkaline phosphatase or horseradish peroxidase; cleaning the above-mentioned sandwich complex to remove unbound substances; adding a chemiluminescence substrate into the above-mentioned cleaned sandwich complex, detecting the photon number generated by the reaction to obtain a chemiluminescence signal value of the sample; wherein the fusion protein sequentially comprises, from N-terminal to C-terminal, alkaline phosphatase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and alkaline phosphatase; or TP15 antigen, TP17 antigen, alkaline phosphatase and TP47 antigen; or the fusion protein sequentially comprises, from N-terminal to C-terminal, horseradish peroxidase, TP15 antigen, TP17 antigen and TP47 antigen; or TP15 antigen, TP17 antigen, TP47 antigen and horseradish peroxidase; or TP15 antigen, TP17 antigen, horseradish peroxidase and TP47 antigen.

12. The use according to claim 11, wherein, the first ligand is TP antigenic protein, or is anti-human IgG antibody and anti-human IgM antibody.