Treponema pallidum antibody detection kit and application thereof

Abstract

The application provides a treponema pallidum antibody detection kit and application thereof, and relates to the technical field of biology.The treponema pallidum antibody detection kit comprises: a first reagent comprising mixed treponema pallidum membrane polypeptide coated negative charge gelatin particles; a second reagent comprising negative charge gelatin particles; a sample diluent; wherein the mixed treponema pallidum membrane polypeptide coated negative charge gelatin particles are prepared by mixing negative charge gelatin particles coated by four kinds of synthetic treponema pallidum membrane polypeptides with amino acid sequences of SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 respectively.The kit adopts the negative charge gelatin particles coated by the four kinds of synthetic treponema pallidum membrane polypeptides, and greatly improves the sensitivity and specificity of treponema pallidum antibody detection.

Description

[0001] This application claims priority to Chinese Patent Application No. CN202211702576.3, filed on December 28, 2022, entitled "Treponema pallidum Antibody Detection Kit and Its Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of biotechnology, and in particular to a Treponema pallidum antibody detection kit and its application. Background Technology

[0003] Syphilis is a chronic, complex infectious disease caused by Treponema pallidum. Its course presents with different clinical symptoms at different stages, typically beginning with an ulcer called a chancre, followed by a syphilitic rash after a long incubation period. If left untreated, infection can eventually lead to cardiovascular disease and neurosyphilis, and may also result in intrauterine infection. Since the discovery in 1905 that syphilis is caused by the pathogenic bacterium Treponema pallidum, serological testing methods have been developed. Among these methods is the detection of anticardiolipin antibodies using phospholipid antigens such as cardiolipin and lecithin, but these methods frequently produce false-positive results.

[0004] Currently, the specific detection targets for Treponema pallidum (TP) are mainly membrane proteins and several lysozymes. The genes encoding these membrane proteins and lysozymes are 12 genes in the TP repetitive gene family, of which at least 9 genes express proteins considered to be major immunogens. Among these proteins, five proteins with molecular weights of 15kD (TpN15), 17kD (TpN17), 37kD (TpN37), 45kD (TmpA), and 47kD (TpN47) have diagnostic value. TpN15 is relatively scarce in TP membrane proteins but has strong immunogenicity; TpN17 is abundant in TP membrane proteins and has been found in all stages of syphilis; TmpA varies greatly in syphilis patients undergoing treatment and holds promise for monitoring the effectiveness of syphilis treatment; TpN47 has strong immunogenicity, is abundant in the TP membrane, and is a protein component specific to the pathogen. Existing Treponema pallidum antibody detection kits use natural Treponema pallidum strains as detection antigens to detect Treponema pallidum antibodies, which has the problem of low sensitivity in detecting Treponema pallidum antibodies.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a Treponema pallidum antibody detection kit and its application, to solve the problems of low sensitivity and specificity of existing Treponema pallidum antibody in vitro detection reagents. This invention's kit uses four different specific sequences of Treponema pallidum membrane polypeptides conjugated with negatively charged gelatin particles, further improving the sensitivity and specificity of the detection results.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In one aspect, the present invention provides a Treponema pallidum antibody detection kit, comprising:

[0009] The first reagent comprises a mixture of gelatin particle complexes;

[0010] The second reagent includes the negatively charged gelatin particles;

[0011] The gelatin particle complex mixture comprises: gelatin particle complex 1 obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 1 of amino acid sequence SEQ ID No. 1; gelatin particle complex 2 obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 2 of amino acid sequence SEQ ID No. 2; gelatin particle complex 3 obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 3 of amino acid sequence SEQ ID No. 3; and gelatin particle complex 4 obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 4 of amino acid sequence SEQ ID No. 4.

[0012] The negatively charged gelatin particles used in the first and second reagents are the same.

[0013] Furthermore, in the gelatin particle complex 1, the gelatin particle complex 2, the gelatin particle complex 3, and the gelatin particle complex 4, the final feed ratio of the Treponema pallidum membrane polypeptide to the coated negatively charged gelatin particles is 0.08 mg / mL, 0.1 mg / mL, 0.1 mg / mL, and 0.08 mg / mL, respectively.

[0014] Furthermore, the negatively charged gelatin particles are charged through activation with citric acid;

[0015] Preferably, the negatively charged gelatin particles in the second reagent are lyophilized agents.

[0016] Furthermore, the second reagent also includes a dissolving solution, which is a first phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L. The first phosphate buffer solution contains 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate. The negatively charged gelatin particles have a volume percentage of 5% in the dissolving solution.

[0017] Furthermore, in the gelatin particle complex mixture, the volume ratio of gelatin particle complex 1, gelatin particle complex 2, gelatin particle complex 3 and gelatin particle complex 4 is 1:1:1:1.

[0018] Furthermore, the first reagent is a lyophilized agent; the Treponema pallidum antibody detection kit also includes a dissolving solution; the dissolving solution is a first phosphate buffer with a pH of 7.0 and a concentration of 0.01 mol / L, the first phosphate buffer containing 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate.

[0019] Furthermore, the first reagent also includes a dissolving solution, which is a first phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L. The first phosphate buffer solution contains 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate. The gelatin particle complex mixture has a volume percentage of 5% in the dissolving solution.

[0020] Furthermore, the kit also includes a sample diluent.

[0021] Preferably, the sample diluent is a second phosphate buffer with pH 7.0 and a concentration of 0.01 mol / L, wherein the second phosphate buffer comprises 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and a non-pathogenic Treponema pallidum solution with a volume percentage of 0.002%.

[0022] If the sample concentration is too high, a sample diluent can be used to dilute the sample so that the concentration is within the test range.

[0023] In another aspect, the present invention provides the use of the kit in the in vitro detection of Treponema pallidum antibodies for non-disease diagnostic purposes.

[0024] Furthermore, the kit is used for the quantitative detection of Treponema pallidum antibodies in vitro.

[0025] In one embodiment, the kit of the present invention is suitable for detecting samples including serum or plasma.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] The Treponema pallidum antibodies produced in the blood of syphilis patients are a mixture of antibodies (polyclonal antibodies) targeting different Treponema pallidum antigenic epitopes. The types and proportions of antibodies produced vary from individual to individual. Currently, commercially available natural Treponema pallidum strains only have some antigenic determinants fully exposed, making it highly susceptible to false negatives when using a single antigen for immunological testing, resulting in low sensitivity.

[0028] The Treponema pallidum antibody detection kit provided in this invention, through the combined use of Treponema pallidum membrane polypeptide 1 with amino acid sequence SEQ ID No. 1, Treponema pallidum membrane polypeptide 2 with amino acid sequence SEQ ID No. 2, Treponema pallidum membrane polypeptide 3 with amino acid sequence SEQ ID No. 3, and Treponema pallidum membrane polypeptide 4 with amino acid sequence SEQ ID No. 4, covers as many specific and effective antigenic epitopes as possible.

[0029] In the gelatin particle complex 1, gelatin particle complex 2, gelatin particle complex 3, and gelatin particle complex 4, the preferred final feed ratios of the Treponema pallidum membrane polypeptides (Treponema pallidum membrane polypeptide 1, Treponema pallidum membrane polypeptide 2, Treponema pallidum membrane polypeptide 3, and Treponema pallidum membrane polypeptide 4, respectively) to the coated negatively charged gelatin particles are 0.08 mg / mL, 0.1 mg / mL, 0.1 mg / mL, and 0.08 mg / mL, respectively. Using this ratio to replace the natural Treponema pallidum strain coating carrier, the sensitivity is 97.5%, the specificity is 97.5%, the false positive rate is 2.5%, and the false negative rate is 2.5%. The commercially available kits prepared with vectors coated with natural Treponema pallidum strains (at a final feed ratio of 0.15 mg / mL) have a false positive rate of 7.5% and a false negative rate of 12.5%. The Treponema pallidum antibody detection kit provided in this invention reduces the false positive rate by 66.7% and the false negative rate by 80%, significantly improving the effectiveness and further enhancing the sensitivity and specificity of Treponema pallidum antibody detection in the serum of different patients. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 The sample detection reaction image provided in the embodiment of the present invention;

[0032] Figure 2 The reaction image provided in this embodiment of the invention shows a sample detection result of (-).

[0033] Figure 3 The reaction image provided in this embodiment of the invention is a sample detection result of (±).

[0034] Figure 4 The reaction image provided in this embodiment of the invention shows a sample detection result of (+).

[0035] Figure 5 The reaction image provided in this embodiment of the invention shows a sample detection result of (++). Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The "final feed ratio" mentioned in this application refers to the feed ratio in the process of synthesizing Treponema pallidum membrane polypeptide and coating with negatively charged gelatin particles. For example, when the negatively charged gelatin particles coated with Treponema pallidum membrane polypeptide 1 of amino acid sequence SEQ No. 1 at a final feed ratio of 0.08 mg / mL represent the actual reaction ratio of Treponema pallidum membrane polypeptide 1 of amino acid sequence SEQ No. 1 and negatively charged gelatin particles after feed, it is that 1 mL of negatively charged gelatin particles reacts with 0.08 μg of Treponema pallidum membrane polypeptide 1 to form the negatively charged gelatin particles coated with Treponema pallidum membrane polypeptide 1 of amino acid sequence SEQ No. 1 at a final feed ratio of 0.08 mg / mL.

[0038] 1. Preparation of the first reagent:

[0039] The synthetic Treponema pallidum membrane polypeptides used in this invention include Treponema pallidum membrane polypeptide 1 with the amino acid sequence SEQ ID No. 1, Treponema pallidum membrane polypeptide 2 with the amino acid sequence SEQ ID No. 2, Treponema pallidum membrane polypeptide 3 with the amino acid sequence SEQ ID No. 3, and Treponema pallidum membrane polypeptide 4 with the amino acid sequence SEQ ID No. 4.

[0040] SEQ ID No. 1:

[0041] MRQNGAVPMISCSVRRRPRWEPQVGAAFLAFALLPVLASGRGMQAAVATAAGSSGSGSDG

[0042] KHPGKEQFLQFLIPSGGRYEYLGVSFTALADDASFFEANPAGSAGLSRGEVALFHHSQIH

[0043] DSHTETVSFARRTQNTGYGASVRAFSSESDLKSFFGGNSGGNKNGGHQGKQGKGFVAIAN

[0044] ASHTFCGQYRFKGVSFGCNFKMGFRKGKTDSHVTVAGDLGLRAAFSVAKNFGSNEPNMHV

[0045] GLVLKNAGISVKTNSCQVEHLNPAIAVGFAYRPVYAFLFSLGLQQTLTKRESPVCSVGFM

[0046] FFCTQHVTLLASAACEGGAYALSGGAEIRIGSFHLDMGYRYDQIFQAAHPHHVSVGLKWL

[0047] IPNGGTQADQALLVKESYLVGLRFYDQRRYQEAITAWQLTLRQDPGFEPAAEGIERARRF

[0048] LKLHEKLSLFDILN

[0049] SEQ ID No. 2:

[0050] MCGATACGARICKAERQEHCEGAVPYGYAHPFCTVLVLISLREGVYPFTHCGCLRSELCV

[0051] GSNGRCKPRAAPDALHMCLRGAPCGAELFPVKPCKRKNACGRIGPRWVKGLVTFHSHI

[0052] IIPREWLMGYGSSAEETSSTPHASGQRKVGFLSLRTKLALVFGLLAFVSGLVQGGILVVF

[0053] ARNSIVGEISSHLAGRARDTSSIVEGRIGALFQFLEGLARLEVLQGSSDRRRAQVDRLKK

[0054] EAFFNRDIARLAVVDLAGVLYGEDGRTHYVQDRKYFQRAVKGRCYVSAPYPSRSSDDMVI

[0055] TFSIPVYDEDRRVIAVLVADVIWTWLCDITGDFSVGGVGRIAVIDEVGTVVAHPRHEVVA

[0056] QQTNYIRLAKEDPATYASVAEFVEKVIKSDSTASHVFSYEGLEKIGSSAKMKSTGWTVVV

[0057] FVPVSEFMGPVYTLQNYLLAVGIIVVLFSLIVVYAVARKIVRPLRSTVRVLEDIAYGEGD

[0058] LTVRLPVVGGDEVSLLCQYFNQTMEKIRFAIATVGSSSDDMRRIGDELASNMTETASAVN

[0059] EITANIDGVKHRVDLQVSGVSEATDTVERIIKTIGNLNSSIETQAVNVAQSSSSVEQMVA

[0060] NIVSITQTLERSDEAVHSLAIATADGRDTLVSSGITQKISEESGSLLEASSVIQHIASQ

[0061] TNLLAMNAAIEAAHAGEAGKGFAVVADEIRKLAEESSTQGKTITETLKTLSVEIDTLSTS

[0062] SKAVEEQFDTIFRLSDQVRTMSRSLTEAMKEQSDGSREVLAAIKSINAATVDVKEGSADM

[0063] LKGGEVIAREMQRLDDLTSEIARSMNEMAAGAIENNAVHEVNEITQRSKQSISSLADEV

[0064] EKFKV

[0065] SEQ ID No.3:

[0066] MEFCVLLRKAARKEKGMLKKASAFLIASCCVMSLAWAQANDNWYEGKPISAISFEGLEYI

[0067] ARGQLDTIFSQYKGQKWTYELYLEILQKVYDLEYFSEVSPKAVPTDPEYQYVMLQFTVKE

[0068] RPSVKGIKMVGNSQIRSGDLLSKILLKKGDIYNEVKMKVDQESLRRHYLDQGYAAVKISC

[0069] EAKTEAGGVVVQFTIQEGKQTVVSRIQFKGNKAFTESVLKKVLSTQEARFLTSGVFKENA

[0070] LEADKAAVHSYYAERGYIDARVEGVAKTVDKKTDASRNLVTLTYTVVEGEQYRYGGVTIV

[0071] GNQIFSTEELQAKIRLKRGAIMNMVAFEQGFQALADAYFENGYTSNYLNKEEHRDTAEKT

[0072] LSFKITVVERERSHVEHIIIKGTKNTKDEVILREMLLKPGDVFSKSKFTDSLRNLFNLRY

[0073] FSSLVPDVRPGSEQDLVDIILNVEEQSTANVQFGVTFSGVGEAGTFPLSLFCQWEEKNFL

[0074] GKGNEISVNATLGSEAQSLKLGYVERWFLGSPLTVGFDFELTHKNLFVYRAGSYGNGLPH

[0075] PYTSREQWASSPGLAESFRLKYSRFESAIGAHTGYQWYPRYAVIRVNGGVDFRVVKNFYD

[0076] KDNNQPFDLTVKEQLNWTSINSFWTSVSFDGRDFAYDPSSGWFLGQRCTFNGLVPFLEKE

[0077] HSFRSDTKAEFYVTLLNYPVSAVWNLKFVLAFYTGVSVQTYYGRRKSENGKGNGVRSGAL

[0078] VIDGVLVGRGWSEDAKKNTGDLLLHHWIEFRWPLAHGIVSFDFFFDAAMVYNIESQSPNG

[0079] SSSASSSSSSSSSSSRTTSSEGLYKMSYGPGLRFTLPQFPLKLAFANTFTSPGGIPKTKK

[0080] NWNFVLSFTVNNL

[0081] SEQ ID No.4:

[0082] MVRMRRRRACSSGGACGCAAVRGARSFLSVRVLGMRIGMSALCLAPLFARTASLGAWSSQ

[0083] GGEVLGEVRARVPAHRRVRRAVSGTSVTPVVAMAAKTSEKQKGVGRRALSLRTGGRYEML

[0084] GLAFTALADDASFFEANAAGSAAFPYLLVGGFHFARVNQSHTDTIALVHSIGRTGYGFSA

[0085] SVQYPYLTMEGKAVGGVAIFNVAHRFLSAYRFKGISVGTNVKVGYRDSSAGGERNKKNQG

[0086] GKKHVVVTADIGLQGTWSVAKNFGSHEPNLWVGGTVKNVGLSVEVDASNSGSSMSGGRTV

[0087] HATNSSFILACAYQPIRWFLFGTGIEWKYNVQEFADNNRFRYGVAFLLLPVQYVAFGSNV

[0088] FLTGLASDIRASAGVEFKSTWVRVDLTYTYESDKDEHVISCGIAGFFNRDRRKHLEKEVY

[0089] TSYLRGLRHYDAQHYEEAIAEWRRTLQRAGSFEPAREGIERATKLLQLNRQVYDFHFLH

[0090] (1) Commercially available gelatin particles with specification C1GFV0000 were activated with citric acid to form negatively charged gelatin particles. The negatively charged gelatin particles were centrifuged to remove the supernatant, and resuspended in a 0.9% g / L sodium chloride solution at pH 7.0 until the volume percentage of negatively charged gelatin particles was 5%. After centrifugation for 15 min, the supernatant was removed, and the process was repeated 3 times. The particles were then resuspended in a 0.01 mol / L phosphate buffer at pH 7.0 until the volume percentage of negatively charged gelatin particles was 10%. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate.

[0091] (2) Treponema pallidum membrane polypeptide 1 with amino acid sequence SEQ ID No.1, Treponema pallidum membrane polypeptide 2 with amino acid sequence SEQ ID No.2, Treponema pallidum membrane polypeptide 3 with amino acid sequence SEQ ID No.3, Treponema pallidum membrane polypeptide 4 with amino acid sequence SEQ ID No.4, Treponema pallidum antigen, and natural Treponema pallidum strain were diluted to 10 mg / mL with phosphate buffer at pH 7.0 and a concentration of 0.01 mol / L. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate.

[0092] (3) Natural Treponema pallidum strains, Treponema pallidum membrane polypeptide 1, Treponema pallidum membrane polypeptide 2, Treponema pallidum membrane polypeptide 3, and Treponema pallidum membrane polypeptide 4 were coated with negatively charged gelatin particles at coating concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.15 mg / mL, and 0.2 mg / mL, respectively. After incubating in a water bath at 37°C for 2 hours, the supernatant was removed by centrifugation. The negatively charged gelatin particles coated with different antigens were resuspended in the same volume of the aforementioned buffer solution (pH 7.0, concentration 0.05 mol / L phosphate buffer containing 1 mg / mL casein, 0.05 mg / mL sodium chloride, 0.2 mg / mL trehalose, and 0.01 mg / mL Tween 80) until the volume percentage of negatively charged gelatin particles was 5%. The gelatin particles were dispensed into 0.3 mL vials and freeze-dried to prepare lyophilized products.

[0093] 2. Preparation of the second reagent:

[0094] Commercially available gelatin particles (C1GFV0000) were activated with citric acid to form negatively charged gelatin particles. The negatively charged gelatin particles were centrifuged to remove the supernatant, and resuspended in a 0.9% g / mL sodium chloride solution (pH 7.0) until the volume percentage of negatively charged gelatin particles reached 5%. After centrifugation for 15 min, the supernatant was removed, and this process was repeated three times. The particles were then resuspended in a 0.01 mol / L phosphate buffer (pH 7.0) until the volume percentage of negatively charged gelatin particles reached 5%. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate. The product was then freeze-dried to obtain a lyophilized product.

[0095] 3. Prepare sample diluent:

[0096] Prepare a second phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L. The second phosphate buffer solution contains 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.002% (v / v) commercially available non-pathogenic Treponema pallidum solution with specification 190801A.A.

[0097] 4. Preparation of the solution:

[0098] Prepare a first phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L, containing 0.05 mg / ml trehalose, 0.05 mg / ml sodium chloride, 0.005 mg / ml Proclin 300, and 0.005 mg / ml sodium deoxycholate.

[0099] 5. Preparation of Treponema pallidum antibody detection kit

[0100] Assemble each of the first reagent (0.3 mL / bottle) with the second reagent (0.3 mL / bottle), sample diluent (60.0 mL / bottle), and soluble solution (18 mL / bottle), and store at 2-8℃.

[0101] I. Detection of the final feed ratio of negatively charged gelatin particles coated with synthetic Treponema pallidum membrane peptides.

[0102] Experimental Example 1: Preparation and Detection of Negatively Charged Gelatin Particles Coated with Treponema pallidum Membrane Polypeptide 1

[0103] 1. Preparation of the first reagent

[0104] (1) Commercially available gelatin particles with specification C1GFV0000 were activated with citric acid to form negatively charged gelatin particles. The negatively charged gelatin particles were centrifuged to remove the supernatant, and resuspended in a 0.9% g / L sodium chloride solution at pH 7.0 until the volume percentage of negatively charged gelatin particles was 5%. After centrifugation for 15 min, the supernatant was removed, and the process was repeated 3 times. The particles were then resuspended in a 0.01 mol / L phosphate buffer at pH 7.0 until the volume percentage of negatively charged gelatin particles was 10%. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate.

[0105] (2) The Treponema pallidum membrane polypeptide 1 with the amino acid sequence SEQ ID No.1 was diluted to 10 mg / mL with phosphate buffer at pH 7.0 and a concentration of 0.01 mol / L. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate.

[0106] (3) The syphilis spirochete membrane polypeptide 1 was coated with negatively charged gelatin particles at coating concentrations of 0.01 mg / mL, 0.02 mg / mL, 0.04 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.15 mg / mL, and 0.2 mg / mL. After incubating in a water bath at 37°C for 2 hours, the supernatant was removed by centrifugation. The negatively charged gelatin particles coated with different antigens were resuspended in the same volume of the buffer solution described above, which is a phosphate buffer solution with a pH of 7.0 and a concentration of 0.05 mol / L (containing 1 mg / mL casein, 0.05 mg / mL sodium chloride, 0.2 mg / mL trehalose, and 0.01 mg / mL Tween 80). The volume percentage of negatively charged gelatin particles was 5%. The gelatin particles were then dispensed into 0.3 mL vials and freeze-dried to prepare lyophilized products.

[0107] 2. Preparation of the second reagent:

[0108] Commercially available gelatin particles with specification C1GFV0000 were activated with citric acid to form negatively charged gelatin particles. The negatively charged gelatin particles were centrifuged to remove the supernatant, and resuspended in a 0.9% g / mL sodium chloride solution (pH 7.0) until the volume percentage of negatively charged gelatin particles reached 5%. After centrifugation for 15 min, the supernatant was removed, and this process was repeated three times. The particles were then resuspended in a 0.01 mol / L phosphate buffer (pH 7.0) until the volume percentage of negatively charged gelatin particles reached 5%. The phosphate buffer contained 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate. The product was then freeze-dried to obtain a lyophilized product.

[0109] 3. Prepare sample diluent:

[0110] Prepare a second phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L. The second phosphate buffer solution contains 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.002% (v / v) commercially available non-pathogenic Treponema pallidum solution with specification 190801A.A.

[0111] 4. Preparation of the solution:

[0112] Prepare a first phosphate buffer solution with a pH of 7.0 and a concentration of 0.01 mol / L, containing 0.05 mg / ml trehalose, 0.05 mg / ml sodium chloride, 0.005 mg / ml Proclin 300, and 0.005 mg / ml sodium deoxycholate.

[0113] 5. Preparation of Treponema pallidum antibody detection kit

[0114] Each of the first reagents is assembled with the second reagent, 60.0 mL / bottle of sample diluent, 18 mL / bottle of dissolving solution, and positive control serum containing rabbit immune serum against Treponema pallidum to form a Treponema pallidum antibody detection kit, which is stored at 2-8℃.

[0115] 6. First sample test

[0116] (1) Forty positive and forty negative treponemal antibody serum samples were tested using the prepared reagent. 0.6 mL of the solution was mixed with the first reagent for reconstitution. 0.6 mL of the solution was then mixed with the second reagent for reconstitution.

[0117] (2) Add 25 μL of sample to the first well of a 96-well plate, then add 100 μL of sample diluent to dilute the sample to a dilution factor of 1:5. Take 25 μL of the liquid from the first well and add it to the second well, then add 25 μL of sample diluent to further dilute the sample to a dilution factor of 1:10. Take 25 μL of the liquid from the second well and add it to the third well, then add 25 μL of sample diluent to further dilute the sample to a dilution factor of 1:20. Take 25 μL of the liquid from the third well and add it to the fourth well, then add 25 μL of sample diluent to further dilute the sample to a dilution factor of 1:40. Add 25 μL of the reconstituted second reagent to the third well to further dilute the sample in the third well to a dilution factor of 1:40. Add 25 μL of the reconstituted first reagent to the fourth well to further dilute the sample in the fourth well to a dilution factor of 1:80. Mix the liquid in the 96-well plate with a plate mixer and let it stand horizontally at room temperature (15-30℃) for 2 hours. Record and observe the reaction images in the 96-well plate against a white background.

[0118] (3) Reaction diagram as follows Figure 1 As shown, when particles in a well of a 96-well plate aggregate in a button-like shape, exhibiting a uniform and smooth circular outer edge, the result in that well is determined to be (-), and the reaction image is as follows. Figure 2 As shown. When particles in a well of a 96-well plate form small rings with uniform and smooth outer edges, the result in that well is determined to be (±). The reaction image is shown below. Figure 3 As shown in the figure. When the particle ring in a well of a 96-well plate becomes significantly larger, and its outer edge is unevenly and randomly aggregated around it, the result in that well is determined to be (+), and the reaction image is shown in the figure. Figure 4 As shown. When uniform aggregation occurs in a well of a 96-well plate, and the aggregated particles extend in a film-like manner across the bottom, the result in that well is determined to be (++), and the reaction image is as follows. Figure 5As shown. If the reaction image in the third well (final sample dilution 1:40) is negative and the reaction image in the fourth well (final sample dilution 1:80) is positive, the sample is considered positive. If the reaction image in the fourth well (final sample dilution 1:80) is negative, the sample is considered negative. If the reaction image in the third well (final sample dilution 1:40) is negative and the reaction image in the fourth well (final sample dilution 1:80) is ±, the sample should be retested.

[0119] 7. Second test of the sample

[0120] Samples that tested positive in the first test of sample 6 were subjected to a second test. The testing process is as follows:

[0121] (1) Add 25 μL of the positive sample to the first well of a 96-well plate, then add 100 μL of sample diluent to dilute the sample to a dilution factor of 1:5. Take 25 μL of the liquid from the first well and add it to the second well, then add 25 μL of sample diluent to dilute the sample to a dilution factor of 1:10. Take 25 μL of the liquid from the second well and add it to the third well, then add 25 μL of sample diluent to dilute the sample to a dilution factor of 1:20. Take 25 μL of the liquid from the third well and add it to the fourth well, then add 25 μL of sample diluent to dilute the sample to a dilution factor of 1:40. Take 25 μL of the liquid from the fourth well and add it to the fifth well, then add 25 μL of sample diluent to dilute the sample to a dilution factor of 1:80. Add 25 μL of the liquid from well 5 to well 6, then add 25 μL of sample diluent to well 6 to further dilute the sample to a dilution factor of 1:160. Add 25 μL of the liquid from well 6 to well 7, then add 25 μL of sample diluent to well 5 to further dilute the sample to a dilution factor of 1:320. Add 25 μL of the liquid from well 7 to well 5, then add 25 μL of sample diluent to well 8 to further dilute the sample to a dilution factor of 1:640. Add 25 μL of the liquid from well 8 to well 9, then add 25 μL of sample diluent to well 9 to further dilute the sample to a dilution factor of 1:1280. Add 25 μL of the liquid from well 9 to well 10, then add 25 μL of sample diluent to well 10 to further dilute the sample to a dilution factor of 1:2560. Add 25 μL of the liquid from well 10 to well 11, then add 25 μL of sample diluent to well 11 to further dilute the sample to a dilution factor of 1:5120. Add 25 μL of the liquid from well 11 to well 12, then add 25 μL of sample diluent to well 12 to further dilute the sample to a dilution factor of 1:10240.

[0122] (2) Add 25 μL of the reconstituted second reagent to the third well to further dilute the sample in the third well to a dilution factor of 1:40. Add 25 μL of the reconstituted first reagent to the fourth well to further dilute the sample in the fourth well to a dilution factor of 1:80. Add 25 μL of the reconstituted first reagent to the fifth well to further dilute the sample in the fifth well to a dilution factor of 1:160. Add 25 μL of the reconstituted first reagent to the sixth well to further dilute the sample in the sixth well to a dilution factor of 1:320. Add 25 μL of the reconstituted first reagent to the seventh well to further dilute the sample in the seventh well to a dilution factor of 1:640. Add 25 μL of the reconstituted first reagent to the eighth well to further dilute the sample in the eighth well to a dilution factor of 1:1280. Add 25 μL of the reconstituted first reagent to well 9 to further dilute the sample to a dilution factor of 1:2560. Add 25 μL of the reconstituted first reagent to well 10 to further dilute the sample to a dilution factor of 1:5120. Add 25 μL of the reconstituted first reagent to well 11 to further dilute the sample to a dilution factor of 1:10240. Add 25 μL of the reconstituted first reagent to well 12 to further dilute the sample to a dilution factor of 1:20480.

[0123] (3) Mix the liquid in the 96-well plate using a plate mixer, and let it stand horizontally at room temperature (15-30℃) for 2 hours. Record and observe the reaction image in the 96-well plate against a white background. The final dilution factor when the reaction image is (+) is taken as the antibody titer to complete the quantitative detection of Treponema pallidum antibodies in the positive samples. Use positive control serum to detect antibody titer according to the above procedure. In this example, the antibody titer of the positive control serum is 1:320 (final dilution factor). The detection result of the positive control serum should be within ±1 well. The detection results of Treponema pallidum antibodies for 40 positive samples and 40 negative samples are shown in Table 1.

[0124] Experimental Example 2: Preparation and Detection of Negatively Charged Gelatin Particles Coated with Treponema pallidum Membrane Polypeptide 2

[0125] Replace Treponema pallidum membrane polypeptide 1 with Treponema pallidum membrane polypeptide 2. All other conditions were the same as in Experiment 1. The results of the Treponema pallidum antibody test are shown in Table 2.

[0126] Experimental Example 3: Preparation and Detection of Treponema pallidum Membrane Polypeptide-Coated Negatively Charged Gelatin Particles

[0127] The Treponema pallidum membrane polypeptide 1 was replaced with Treponema pallidum membrane polypeptide 3. All other conditions were the same as in Experiment 1. The results of the Treponema pallidum antibody test are shown in Table 3.

[0128] Experimental Example 4: Preparation and Detection of Negatively Charged Gelatin Particles Coated with Treponema pallidum Membrane Polyps

[0129] Replace Treponema pallidum membrane polypeptide 1 with Treponema pallidum membrane polypeptide 4. All other conditions were the same as in Experiment 1. The results of the Treponema pallidum antibody test are shown in Table 4.

[0130] Comparative Example 1

[0131] The Treponema pallidum membrane polypeptide 1 was replaced with a commercially available natural Treponema pallidum strain. All other conditions were the same as in Experiment 1. The results of the Treponema pallidum antibody test are shown in Table 5.

[0132] Table 1. Detection of the final feed ratio of Treponema pallidum membrane polypeptide coated with negatively charged gelatin particles

[0133]

[0134]

[0135] Table 2. Detection of the final feed ratio of Treponema pallidum membrane polypeptide-coated negatively charged gelatin particles

[0136]

[0137] Table 3. Detection of the final feed ratio of Treponema pallidum membrane polypeptide 3-coated negatively charged gelatin particles

[0138]

[0139] Table 4. Detection of the final feed ratio of Treponema pallidum membrane polypeptide 4-coated negatively charged gelatin particles

[0140]

[0141]

[0142] Table 5. Detection of final feed ratio of peptide-coated negatively charged gelatin particles from natural Treponema pallidum strains.

[0143]

[0144] Results Analysis:

[0145] Because there is a dissociation equilibrium between synthetic Treponema pallidum membrane peptides, natural Treponema pallidum strains, and negatively charged gelatin particles, and because the negatively charged gelatin particles cannot always maintain sufficient contact with the synthetic Treponema pallidum membrane peptides and natural Treponema pallidum strains, there will be uneven coating of synthetic Treponema pallidum membrane peptides and natural Treponema pallidum strains on the negatively charged gelatin particles, and the number of these substances will vary greatly. Therefore, the only standard to indicate the amount of antigen coated on the negatively charged gelatin particles is to calculate the average ratio of synthetic Treponema pallidum membrane peptides to negatively charged gelatin particles, or the ratio of natural Treponema pallidum strains to negatively charged gelatin particles after coating.

[0146] Based on a comprehensive assessment of sensitivity and specificity, the higher the sensitivity and the lower the specificity, the better the effect.

[0147] Table 1 shows that the final concentration ratios of Treponema pallidum membrane peptide 1 coated with negatively charged gelatin particles were 0.04 mg / mL, 0.08 mg / mL, and 0.1 mg / mL, with a sensitivity of 85%, a specificity of 90%, a false negative rate of 15%, and a false positive rate of 10%. The upper limit of the final concentration ratio of Treponema pallidum membrane peptide 1 coated with negatively charged gelatin particles was 0.1 mg / mL, and the lower limit was 0.04 mg / mL. Considering that the final concentration ratio of Treponema pallidum membrane peptide 1 coated with negatively charged gelatin particles may have a normal distribution relationship with the sensitivity and specificity results, it is speculated that a final concentration ratio of 0.08 mg / mL is the preferred ratio.

[0148] Table 2 shows that the final concentration ratios of Treponema pallidum membrane peptide 2 coated with negatively charged gelatin particles were 0.08 mg / mL, 0.1 mg / mL, and 0.15 mg / mL, with a sensitivity of 80%, a specificity of 92.5%, a false negative rate of 20%, and a false positive rate of 7.5%. The upper limit of the final concentration ratio of Treponema pallidum membrane peptide 2 coated with negatively charged gelatin particles was 0.15 mg / mL, and the lower limit was 0.08 mg / mL. Considering that the final concentration ratio of Treponema pallidum membrane peptide 2 coated with negatively charged gelatin particles may have a normal distribution relationship with the sensitivity and specificity results, it is speculated that a final concentration ratio of 0.1 mg / mL is the preferred ratio.

[0149] Table 3 shows that the final concentration ratios of Treponema pallidum membrane peptide 3 coated with negatively charged gelatin particles were 0.08 mg / mL, 0.1 mg / mL, and 0.15 mg / mL, with a sensitivity of 82.5%, a specificity of 90%, a false negative rate of 17.5%, and a false positive rate of 10%. The upper limit of the final concentration ratio of Treponema pallidum membrane peptide 3 coated with negatively charged gelatin particles was 0.15 mg / mL, and the lower limit was 0.08 mg / mL. Considering that the final concentration ratio of Treponema pallidum membrane peptide 3 coated with negatively charged gelatin particles may have a normal distribution relationship with the sensitivity and specificity results, it is speculated that a final concentration ratio of 0.1 mg / mL is the preferred ratio.

[0150] Table 4 shows that the final concentration ratios of Treponema pallidum membrane peptide 4 coated with negatively charged gelatin particles were 0.04 mg / mL, 0.08 mg / mL, and 0.1 mg / mL, with a sensitivity of 82.5%, a specificity of 90%, a false negative rate of 17.5%, and a false positive rate of 10%. The upper limit of the final concentration ratio of Treponema pallidum membrane peptide 4 coated with negatively charged gelatin particles was 0.1 mg / mL, and the lower limit was 0.04 mg / mL. Considering that the final concentration ratio of Treponema pallidum membrane peptide 4 coated with negatively charged gelatin particles may have a normal distribution relationship with the sensitivity and specificity results, it is speculated that a final concentration ratio of 0.08 mg / mL is the preferred ratio.

[0151] Table 5 shows that the final concentration ratios of commercially available natural Treponema pallidum strains coated with negatively charged gelatin particles were 0.1 mg / mL, 0.15 mg / mL, and 0.2 mg / mL, with a sensitivity of 87.5%, a specificity of 92.5%, a false negative rate of 12.5%, and a false positive rate of 7.5%. The upper limit of the final concentration ratio of natural Treponema pallidum strains coated with negatively charged gelatin particles is 0.2 mg / mL, and the lower limit is 0.1 mg / mL. Considering that the final concentration ratio of natural Treponema pallidum strains coated with negatively charged gelatin particles may have a normal distribution relationship with the sensitivity and specificity results, it is speculated that a final concentration ratio of 0.15 mg / mL is the preferred ratio.

[0152] II. Detection of mixed-phase syphilis treponemal membrane polypeptides coated with negatively charged gelatin particles

[0153] Experimental Example 5

[0154] (1) The first reagent prepared in Example 1, consisting of Treponema pallidum membrane polypeptide 1 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL, the first reagent prepared in Example 2, consisting of Treponema pallidum membrane polypeptide 2 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL, and the first reagent prepared in Example 3, consisting of Treponema pallidum membrane polypeptide 3 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL, was mixed at a ratio of 1 mL:1 mL:1 mL, replacing the first reagent in Example 1. All other conditions were the same as in Example 1. The results of the Treponema pallidum antibody test are shown in Table 6.

[0155] (2) The first reagent prepared in Example 1 (Treponema pallidum membrane polypeptide 1 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL), the first reagent prepared in Example 3 (Treponema pallidum membrane polypeptide 3 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL), and the first reagent prepared in Example 4 (Treponema pallidum membrane polypeptide 4 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL) were mixed at a ratio of 1 mL:1 mL:1 mL, replacing the first reagent in Example 1. All other conditions were the same as in Example 1. The results of the Treponema pallidum antibody test are shown in Table 6.

[0156] (3) The first reagent prepared in Example 1, consisting of Treponema pallidum membrane polypeptide 1 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL, the first reagent prepared in Example 2, consisting of Treponema pallidum membrane polypeptide 2 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL, and the first reagent prepared in Example 4, consisting of Treponema pallidum membrane polypeptide 4 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL, was mixed at a ratio of 1 mL:1 mL:1 mL, replacing the first reagent in Example 1. All other conditions were the same as in Example 1. The results of the Treponema pallidum antibody test are shown in Table 6.

[0157] (4) The first reagent prepared in Example 2 (Treponema pallidum membrane polypeptide 2 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL), the first reagent prepared in Example 3 (Treponema pallidum membrane polypeptide 3 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL), and the first reagent prepared in Example 4 (Treponema pallidum membrane polypeptide 4 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL) were mixed at a ratio of 1 mL:1 mL:1 mL, replacing the first reagent in Example 1. All other conditions were the same as in Example 1. The results of the Treponema pallidum antibody test are shown in Table 6.

[0158] Experimental Example 6

[0159] The first reagent (prepared in Example 1 with Treponema pallidum membrane polypeptide 1 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL), the first reagent (prepared in Example 2 with Treponema pallidum membrane polypeptide 2 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL), the first reagent (prepared in Example 3 with Treponema pallidum membrane polypeptide 3 coated with negatively charged gelatin particles at a final concentration of 0.1 mg / mL), and the first reagent (prepared in Example 4 with Treponema pallidum membrane polypeptide 4 coated with negatively charged gelatin particles at a final concentration of 0.08 mg / mL) were mixed at a ratio of 1 mL:1 mL:1 mL:1 mL, replacing the first reagent in Example 1. All other conditions were the same as in Example 1. The results of the Treponema pallidum antibody test are shown in Table 6.

[0160] Table 6. Detection of mixed-phase negatively charged gelatin particles coated with synthetic Treponema pallidum membrane peptides.

[0161]

[0162] Results analysis:

[0163] As shown in Table 6, the Treponema pallidum antibody detection kit prepared by mixing Treponema pallidum membrane peptide 1 coated with negatively charged gelatin particles at a final concentration ratio of 0.08 mg / mL, Treponema pallidum membrane peptide 2 coated with negatively charged gelatin particles at a final concentration ratio of 0.1 mg / mL, Treponema pallidum membrane peptide 3 coated with negatively charged gelatin particles at a final concentration ratio of 0.1 mg / mL, and Treponema pallidum membrane peptide 4 coated with negatively charged gelatin particles at a final concentration ratio of 0.08 mg / mL in a 1 mL:1 mL:1 mL:1 mL ratio exhibits superior sensitivity and specificity compared to the Treponema pallidum antibody detection kit prepared by mixing commercially available natural Treponema pallidum strains coated with negatively charged gelatin particles at a final concentration ratio of 0.15 mg / mL. The false negative rate decreased from 12.5% ​​to 2.5%, a reduction of 80%; the false positive rate decreased from 7.5% to 2.5%, a reduction of 66.7%, demonstrating a significant improvement in both sensitivity and specificity. The inventors speculate that a mixture of Treponema pallidum membrane polypeptide 1, Treponema pallidum membrane polypeptide 2, Treponema pallidum membrane polypeptide 3, and Treponema pallidum membrane polypeptide 4, compared to natural Treponema pallidum strains, is more conducive to the exposure of antigen binding sites, thus improving both sensitivity and specificity.

[0164] III. Detection of final concentration and feed ratio of synthesized Treponema pallidum membrane polypeptide coated with negatively charged gelatin particles

[0165] Experimental Example 7

[0166] The following syphilis membrane peptides coated with negatively charged gelatin particles—Treponema pallidum membrane peptide 1, 2, 3, and 4—were mixed at the final concentration ratios shown in Table 7, with all other conditions identical to those in Experiment 1. The results of the Treponema pallidum antibody test are shown in Table 8.

[0167] Table 7. Final concentration and feed ratio of synthesized Treponema pallidum membrane polypeptide coated with negatively charged gelatin particles

[0168]

[0169] Table 8. Detection of final concentration and feed ratio of synthetic Treponema pallidum membrane polypeptide coated with negatively charged gelatin particles

[0170]

[0171] Results analysis:

[0172] Based on Tables 6 and 7, the Treponema pallidum anti-inflammatory drug prepared by mixing Treponema pallidum membrane peptide 1 coated with negatively charged gelatin particles at a final concentration ratio of 0.08 mg / mL, Treponema pallidum membrane peptide 2 coated with negatively charged gelatin particles at a final concentration ratio of 0.1 mg / mL, Treponema pallidum membrane peptide 3 coated with negatively charged gelatin particles at a final concentration ratio of 0.1 mg / mL, and Treponema pallidum membrane peptide 4 coated with negatively charged gelatin particles at a final concentration ratio of 0.08 mg / mL in a 1 mL:1 mL:1 mL:1 mL ratio The optimal combination of the antibody detection kits yields the highest sensitivity and specificity for Treponema pallidum antibody detection, with a sensitivity of 97.5%, a specificity of 97.5%, a false positive rate of 2.5%, and a false negative rate of 2.5%. Compared to commercially available natural Treponema pallidum strains coated with negatively charged gelatin particles at a final concentration of 0.15 mg / mL, which achieves a sensitivity of 87.5%, a specificity of 92.5%, a false negative rate of 12.5%, and a false positive rate of 7.5%, the kits demonstrate a significant reduction in false positives (66.7%) and false negatives (80%).

[0173] The inventors hypothesize that the four Treponema pallidum membrane peptides (1, 2, 3, and 4) selected in this application each contain different antigenic determinants. The binding rate constants of the antigenic determinants of the four antigens and their corresponding antibodies are constant but different. The concentration level signal value of a single antigen is positively correlated with the signal value level. The immunoassay methodology determines the positive or negative result by comparing the signal value with a pre-set threshold. The four antigens have a complementary relationship in antibody detection in the population. The final feed ratio when the four antigens are used simultaneously is particularly important. Only when the signal value levels of the mixed antigens are adjusted to be similar, and the positive determination values ​​of the three antigens are close, will the sensitivity and specificity be highest. If the concentration of a certain antigen is relatively low, the signal value of the sample that can only be detected by that antigen will be below the threshold, resulting in a false negative and a decrease in sensitivity. Similarly, if the concentration of a certain antigen is relatively high, the overall signal value of the sample will be increased, the threshold will shift upward, and a false positive will be detected, resulting in a decrease in specificity.

[0174] This invention utilizes a mixture of negatively charged gelatin particles coated with Treponema pallidum membrane peptide 1 at a final concentration of 0.08 mg / mL, Treponema pallidum membrane peptide 2 at a final concentration of 0.1 mg / mL, Treponema pallidum membrane peptide 3 at a final concentration of 0.1 mg / mL, and Treponema pallidum membrane peptide 4 at a final concentration of 0.08 mg / mL, in a ratio of 1 mL:1 mL:1 mL:1 mL. This mixture replaces commercially available natural Treponema pallidum strains coated with negatively charged gelatin particles, further improving the sensitivity and specificity of Treponema pallidum antibody detection in the serum of different patients.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Treponema pallidum antibody detection kit, characterized in that, include: The first reagent comprises a mixture of gelatin particle complexes; The second reagent includes negatively charged gelatin particles; The gelatin particle complex mixture includes: Gelatin particle complex 1, obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 1 (amino acid sequence SEQ ID No. 1); gelatin particle complex 2, obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 2 (amino acid sequence SEQ ID No. 2); gelatin particle complex 3, obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 3 (amino acid sequence SEQ ID No. 3); and gelatin particle complex 4, obtained by coating negatively charged gelatin particles with Treponema pallidum membrane polypeptide 4 (amino acid sequence SEQ ID No. 4).

2. The reagent kit according to claim 1, characterized in that, In the gelatin particle complex 1, the gelatin particle complex 2, the gelatin particle complex 3, and the gelatin particle complex 4, the final feed ratios of the Treponema pallidum membrane polypeptide to the coated negatively charged gelatin particles are 0.08 mg / mL, 0.1 mg / mL, 0.1 mg / mL, and 0.08 mg / mL, respectively.

3. The reagent kit according to claim 2, characterized in that, The negatively charged gelatin particles are charged through activation with citric acid.

4. The reagent kit according to claim 2, characterized in that, The negatively charged gelatin particles in the second reagent are lyophilizing agents.

5. The reagent kit according to claim 2, characterized in that, The second reagent also includes a dissolving solution, which is a first phosphate buffer with a pH of 7.0 and a concentration of 0.01 mol / L, the first phosphate buffer containing 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300 and 0.005 mg / mL sodium deoxycholate.

6. The reagent kit according to claim 5, characterized in that, The negatively charged gelatin particles have a volume percentage of 5% in the solution.

7. The kit according to claim 1, characterized in that, In the gelatin particle complex mixture, the volume ratio of gelatin particle complex 1, gelatin particle complex 2, gelatin particle complex 3 and gelatin particle complex 4 is 1:1:1:

1.

8. The reagent kit according to claim 7, characterized in that, The first reagent is a lyophilized agent; the Treponema pallidum antibody detection kit also includes a dissolving solution; the dissolving solution is a first phosphate buffer with pH 7.0 and a concentration of 0.01 mol / L, the first phosphate buffer containing 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300 and 0.005 mg / mL sodium deoxycholate.

9. The reagent kit according to claim 7, characterized in that, The first reagent also includes a dissolving solution, which is a first phosphate buffer with a pH of 7.0 and a concentration of 0.01 mol / L, comprising 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300, and 0.005 mg / mL sodium deoxycholate; the gelatin particle complex mixture has a volume percentage of 5% in the dissolving solution.

10. The kit according to claim 1, characterized in that, It also includes a sample diluent; the sample diluent is a second phosphate buffer with a pH of 7.0 and a concentration of 0.01 mol / L, the second phosphate buffer comprising 0.05 mg / mL trehalose, 0.05 mg / mL sodium chloride, 0.005 mg / mL Proclin 300 and a non-pathogenic Treponema pallidum solution with a volume percentage of 0.002%.

11. The use of the kit according to any one of claims 1-10 in the in vitro detection of Treponema pallidum antibodies for non-disease diagnostic purposes.

12. The application according to claim 11, characterized in that, The kit is used for the quantitative detection of Treponema pallidum antibodies in vitro.

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