Detection method of treponema pallidum TPP17 specific antibody

Abstract

The invention belongs to the technical field of syphilis specific antibody detection, and particularly relates to a detection method of a treponema pallidum TPP17 specific antibody. The detection method comprises the following steps: uniformly mixing protein A / G modified magnetic beads with the concentration of 0.9 mg / mL to 1.2 mg / mL, TPP17-hGluc protein and a diluted serum sample to be detected, and incubating for 10 minutes to 20 minutes; wherein the volume ratio of the protein A / G modified magnetic beads to the TPP17-hGluc protein to the diluted serum sample is 1: (4-6): (3-5); and adding 5 [mu] L to 20 [mu] L of a coelenterazine substrate with the concentration of 180 [mu] M to 220 [mu] M into the magnetic bead particles containing the antibody-TPP17-hGluc compound, and carrying out measurement. According to the invention, a protein A / G immobilized magnetic particle is adopted to accurately capture an antibody in serum, and meanwhile, TPP17 antigen chimeric protein and humanized Gaussian luciferase are utilized to carry out specific labeling, so that high sensitivity and high specificity of detection are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of syphilis-specific antibody detection, and specifically relates to a method for detecting Treponema pallidum TPP17-specific antibodies. Background Art

[0002] Syphilis is a sexually transmitted disease caused by Treponema pallidum. Currently, in the process of diagnosing syphilis, in addition to a detailed analysis of the patient's epidemiological history and clinical characteristics, laboratory tests play a vital role. Given that Globus pallidum tapeworms cannot be cultured in artificial culture media, traditional diagnostic methods such as cerebrospinal fluid inoculation of rabbit testicles are time-consuming and complicated to operate. In addition, dark-field microscopy is effective but requires high-precision instruments and professional technical support. Histopathological biopsy and immunofluorescence staining are feasible, but are limited by their invasive operations and low sensitivity. Among them, Globus pallidum tapeworms are Treponema pallidum.

[0003] Serological testing is an indispensable part of diagnosing syphilis, and a variety of methods have been developed, mainly divided into non-treponemal tests and treponemal tests. NTT mainly detects antibodies against substances released by damaged host cells of syphilis, including STD research laboratory tests, rapid plasma reagin tests, and toluidine red apyretic serum tests. These screening methods are low-cost and suitable for wide screening, but their antigens: natural cardiolipin and bovine heart lecithin only target patient antibodies, so they have high sensitivity but relatively low specificity, and may produce cross-reactions with other treponemal-related diseases. Among them, the English abbreviation of non-treponemal tests is NTT. The English abbreviation of treponemal tests is TTs. The English abbreviation of STD research laboratories is VDRL. The English abbreviation of rapid plasma reagin is RPR. The English abbreviation of toluidine red apyretic serum test is TRUST.

[0004] TTs uses expensive treponemal antigens cultured from infected rabbits to detect specific treponemal antibodies, such as Treponema pallidum particle agglutination test, Treponema pallidum hemagglutination test, fluorescent treponemal antibody absorption method and chemiluminescent immunoassay. Among them, the English abbreviation of Treponema pallidum particle agglutination test is TPPA. The English abbreviation of Treponema pallidum hemagglutination test is TPHA. The English abbreviation of fluorescent treponemal antibody absorption method is FTA-ABS. The English abbreviation of chemiluminescent immunoassay is CIA.

[0005] All of the above detection methods have the problems of low specificity and sensitivity. Summary of the invention

[0006] In order to solve the problem of low specificity and sensitivity of the existing Treponema pallidum antibody detection methods in the prior art, the present invention provides a method for detecting Treponema pallidum TPP17-specific antibodies. To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for detecting Treponema pallidum TPP17-specific antibodies, comprising the following steps:

[0008] Protein A / G modified magnetic beads with a concentration of 0.9 mg / mL to 1.2 mg / mL, TPP17-hGluc protein and the diluted serum sample to be tested are mixed and incubated for 10 minutes to 20 minutes to obtain magnetic bead particles containing antibody-TPP17-hGluc complexes.

[0009] Wherein, the volume ratio of the protein A / G modified magnetic beads, the TPP17-hGluc protein and the diluted serum sample is 1:4-6:3-5.

[0010] The obtained magnetic bead particles containing the antibody-TPP17-hGluc complex are then washed; the washed magnetic bead particles containing the antibody-TPP17-hGluc complex are released into the hGluc buffer to obtain the released magnetic bead particles containing the antibody-TPP17-hGluc complex.

[0011] Coelenterazine substrate was added to the released magnetic beads containing the antibody-TPP17-hGluc complex, and the bioluminescence intensity was measured.

[0012] The presence or absence of syphilis antibodies in the serum sample to be tested and the concentration thereof are determined by comparing the bioluminescence intensities measured in different wells in the second cell culture plate.

[0013] In the detection method of the above-mentioned Treponema pallidum TPP17-specific antibody provided by the present invention, protein A / G-immobilized magnetic particles are used to accurately capture antibodies in serum, and TPP17 antigen chimeric protein and humanized Gaussia luciferase are used for specific labeling, thereby ensuring high sensitivity and high specificity of the detection. That is, the detection method of the above-mentioned Treponema pallidum TPP17-specific antibody provided by the present invention solves the problem of low specificity and sensitivity of the existing Treponema pallidum antibody detection method in the prior art. Among them, A / G-immobilized magnetic particles are also called A / G modified magnetic beads.

[0014] Preferably, the protein A / G modified magnetic beads, the TPP17-hGluc protein and the diluted serum sample are mixed and then incubated using a nucleic acid extraction machine.

[0015] The program commands for the nucleic acid extractor during incubation are: combine for 8 to 12 minutes, rinse for 1.5 to 2.5 minutes, rinse for 1.5 to 2.5 minutes, rinse for 1.5 to 2.5 minutes, rinse for 1.5 to 2.5 minutes, rinse for 1.5 to 2.5 minutes, and discard the magnetic beads for 25 to 50 seconds. This process integrates washing and incubation, reduces operation time, and avoids the risk of personnel exposure.

[0016] Preferably, the incubation condition is 10 to 20 minutes at 35° C. to 40° C. The incubation time is reduced by dozens of times compared to the traditional luciferase immunoprecipitation system.

[0017] Preferably, the magnetic bead particles containing the antibody-TPP17-hGluc complex are washed by first washing with buffer A and then washing with hGluc buffer.

[0018] The preparation method of the buffer A is as follows: 2.2 g to 2.6 g of Tris, 28 g to 31 g of sodium chloride, and 9 mL to 11 mL of Triton X-100 are taken respectively, dissolved in water, the pH value is adjusted to 7.2 to 7.6, water is added to make the volume to 1 L, and then sterilized by filtering through a 0.22 μm filter membrane.

[0019] The hGluc buffer solution is prepared by taking 2.2g-2.6g Tris and 5.5g-6.0g sodium chloride, respectively, dissolving them in water, adjusting the pH value to 7.2-7.6, adding constant volume to 1L, and then filtering through a 0.22 μm filter membrane for sterilization.

[0020] Preferably, the diluted serum sample to be tested is obtained by diluting the serum sample to be tested by 20 to 80 times.

[0021] Preferably, the method for obtaining the serum sample to be tested is: first placing the blood at 36°C to 38°C for 1.5 hours to 2.5 hours, then placing it at a low temperature of 0°C to 4°C for 8 hours to 16 hours, then centrifuging the blood at a speed of 1800rpm to 3000rpm for 4 minutes to 6 minutes, and aspirating the upper serum to obtain the serum sample to be tested.

[0022] Preferably, the preparation method of the TPP17-hGluc protein is: expressing the TPP17-hGluc protein using a human renal epithelial cell line, and performing ultrafiltration concentration to obtain the TPP17-hGluc protein, and the protein structure expressed by mammalian cells has a spatial conformational epitope.

[0023] Preferably, the human kidney epithelial cell line is 293F cells.

[0024] Preferably, the ultrafiltration concentration method is: using an 8 kDa to 12 kDa ultrafiltration tube for concentration, and the rotation speed during ultrafiltration concentration is 3750×g to 3800×g.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The present invention provides a method for detecting TPP17-specific antibodies of Treponema pallidum, which cleverly combines the advanced technologies of luciferase immuno-magnetic separation system and automatic magnetic processing machine. In this method, the present invention uses protein A / G immobilized magnetic particles to accurately capture antibodies in serum, and uses TPP17 antigen chimeric protein and humanized Gaussia luciferase for specific labeling, thereby ensuring high sensitivity and high specificity of the detection. Among them, the English abbreviation of luciferase immuno-magnetic separation system is LIMS. The English abbreviation of humanized Gaussia luciferase is hGluc. A / G immobilized magnetic particles are also called A / G modified magnetic beads.

[0027] The magnetic separation step is automatically performed on the nucleic acid extraction machine, which not only simplifies the operation process, but also greatly improves the detection efficiency. The present invention is equipped with a user-friendly custom program for this step, making the entire detection process more convenient and reliable. From sample processing to result output, the entire analysis process only takes 25 minutes, and up to 32 samples can be tested at a time, fully meeting the needs of large-scale screening and rapid diagnosis.

[0028] This automated testing platform has shown great commercial potential, especially in the detection of TPP17-specific antibodies in serum. Its high efficiency, accuracy and speed make it of great significance in controlling the spread of syphilis. By quickly and accurately detecting syphilis-specific antibodies, we can detect infected people earlier, so as to take timely and effective prevention and control measures, block the transmission chain of syphilis and protect public health safety.

[0029] 2. LIPS was originally proposed by Professor Burbelo for the detection of colon cancer, and now it has demonstrated its outstanding application value in many key areas. LIPS is a cutting-edge technology that efficiently measures antibodies in serum by fusion protein and luciferase labeling of antigens. With its rapid and sensitive characteristics, this technology has been widely used in the detection of antibodies for respiratory syncytial virus, human norovirus, autoimmune diseases, infectious diseases and SARS-CoV-2. In addition, the automation potential of LIPS has been highlighted, and the convenience of operation has been achieved by introducing magnetic particles instead of traditional resin particles. Among them, the full English name of LIPS is Luciferase Immunoprecipitation System, and its Chinese name is Luciferase Immunoprecipitation System.

[0030] The present invention is based on LIPS technology and combines protein A / G modified magnetic particles to construct a luciferase immunomagnetic separation system. This platform shows many significant advantages: first, the antigen in the solution can effectively bind to the antibody in its natural spatial conformation; second, due to the special properties of luciferase, the background noise is extremely low, which effectively improves the detection accuracy; third, low concentrations of TPP17-hGluc fusion protein can meet the needs of LIMS without cumbersome purification steps; finally, the introduction of magnetic particles makes the system easy to automate. Among them, the English abbreviation of luciferase immunomagnetic separation system is LIMS.

[0031] The established LIMS platform is not only fast and high-throughput, but also has a sensitivity that exceeds the traditional ELISA method. In clinical practice, the platform has been successfully applied to the detection of syphilis-specific antibodies. By combining magnetic particles with a simple magnetic particle processor, we have automated the detection process, greatly improving the convenience and efficiency of the operation. Therefore, this method has broad prospects for clinical application and provides strong technical support for the early diagnosis and prevention and control of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the procedure of LIMS detecting syphilis antibodies in the present invention.

[0033] Figure 2 Characterization of the recombinant TPP17-hGluc in the present invention; wherein, Figure 2 Figure A shows the rdb-hgluc protein model structure constructed using Alphafold2 (left), and the comparison of the TPP17 and TPP17-hgluc structures in PyMOL (right); Figure 2 Panel B in the figure is western-blot analysis of TPP17-hGluc Lane 1 marker (kDa); Lane 2, TPP17-hGluc binding to strong positive syphilis serum; Figure 2 Panel C in Figure 2 is a graph showing the relationship between hGluc bioluminescence and TPP17-hGluc concentration; the data shown are the average of three independent experiments. The errors are too small to be shown.

[0034] Figure 3 is the cutoff value for determining TPP17 antibody by LIMS method in the present invention; wherein, Figure 3 Figure A in the figure is a scatter plot of 70 Treponema pallidum antibody-positive and 90 Treponema pallidum antibody-negative serum samples; Figure 3 Figure B is the ROC curve showing that the sensitivity and specificity of this method are 100% and 100% respectively; Figure 3Figure C is a scatter plot of 29 pregnant women with positive Treponema pallidum antibodies and 29 pregnant women with negative Treponema pallidum antibodies; Figure 3 Figure D is the ROC curve showing that the sensitivity and specificity of this method are 96.5% and 100%, respectively.

[0035] Figure 4 The comparison of the detection sensitivity results of the LIMS method of the present invention and the commercial ELISA kit; wherein, Figure 4 Figure A in the figure is the result of the LIMS method; Figure 4 Panel B in Figure 1 shows the results of a commercial ELISA kit; the results are obtained by serially diluting positive and negative serum samples by 2-fold. Error bars represent the standard deviation of three replicates. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0037] Example 1

[0038] A method for detecting Treponema pallidum TPP17-specific antibodies comprises the following steps:

[0039] S1. Preparation of TPP17-hGluc protein:

[0040] HEK293F cells express TPP17-hGluc protein. The specific method is as follows: First, 80 μg pCAGGS-TPP17-hGluc was transfected into 80 mL HEK293F cells with PEI. TPP17-hGluc was secreted in the supernatant. After 96 hours, the cell supernatant was harvested, and the TPP17-hGluc protein was concentrated using a 10kDa Millipore ultrafilter, and the expressed protein was enriched by centrifugation at 3800×g. The enriched protein did not need further purification, supplemented with 50% volume percentage glycerol, diluted 1000 times for bioluminescence measurement, and compared with the stored hGluc standard curve. Subsequently, the TPP17-hGluc protein concentration was calculated and stored at -20°C.

[0041] The method for measuring bioluminescence is as follows: First, the diluted TPP17-hGluc is transferred to a black 96-well luminescent microplate to ensure uniform distribution in each well. Then, 5 μL of a 200 μM coelenterazine solution is accurately added to each well. After this step is completed, the bioluminescence signal in the microplate is accurately measured and recorded using a high-performance microplate reader.

[0042] Among them, the high-performance microplate reader: Bio-Tek Synergy H1, made in the United States.

[0043] The Chinese name of PEI is polyethyleneimine.

[0044] Millipore ultrafiltration machine: MA, USA.

[0045] hGluc standard curve Figure 2 As shown in Figure C.

[0046] The method and results for calculating the TPP17-hGluc protein concentration are as follows:

[0047] The sample to be tested was diluted 1000 times and then bioluminescence was measured. Subsequently, the obtained bioluminescence data was compared and analyzed with the pre-established and properly stored hGluc standard curve to accurately evaluate the activity or concentration level of hGluc in the sample. The obtained TPP17-hGluc concentration was 0.5 mg / mL.

[0048] S2. Acquisition of serum samples to be tested

[0049] The drawn blood was placed in a 37°C incubator for 2 hours and then in a 4°C refrigerator for 12 hours. The blood was centrifuged at 2000 rpm for 5 minutes, and the upper serum was aspirated to obtain the serum sample to be tested.

[0050] The serum sample to be tested was diluted 50 times with PBS buffer to obtain a diluted serum sample to be tested.

[0051] Among them, the blood came from Jiaozuo Maternal and Child Health Hospital and Yichang First People's Hospital.

[0052] The source of PBS buffer was Gibco, the concentration was 1X, and the pH was 7.4.

[0053] S3. Preparation of protein A / G modified magnetic beads

[0054] Take 500 μL of commercial protein A / G magnetic beads, wash twice with 500 μL of PBS buffer, then resuspend with an equal volume of Buffer A and a 5% volume percentage skim milk powder mixture, and mix thoroughly to obtain protein A / G modified magnetic beads. The prepared protein A / G modified magnetic beads are used for subsequent experiments.

[0055] Among them, commercial proteinA / G magnetic beads were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0056] S4, add sample

[0057] Protein A / G modified magnetic beads at a concentration of 1 mg / mL, TPP17-hGluc protein and diluted serum samples to be tested are pre-added to the first cell culture plate; wherein the volume ratio of protein A / G modified magnetic beads, TPP17-hGluc protein and diluted serum samples is 1:5:4.

[0058] The first cell culture plate to which the protein A / G modified magnetic beads, TPP17-hGluc protein and diluted serum sample were pre-added was placed on a nucleic acid extraction machine and incubated according to the set operation program.

[0059] The set running program command for the nucleic acid extraction machine during incubation is: binding for 10 minutes, rinsing for 2 minutes, rinsing for 2 minutes, rinsing for 2 minutes, rinsing for 2 minutes, and discarding magnetic beads for 30 seconds.

[0060] It should be noted that the nucleic acid extraction machine was provided by GenFine Biotech, Beijing, China. The device was originally used for automatic nucleic acid extraction, but after being customized by us, it is suitable for the current experimental process. Start the device,

[0061] After the protein A / G modified magnetic beads, TPP17-hGluc protein and diluted serum samples were mixed, they were incubated for 15 minutes.

[0062] S4. After the incubation, the obtained magnetic beads containing the antibody-TPP17-hGluc complex are first thoroughly rinsed with buffer A to remove unbound impurities. Subsequently, they are rinsed once with hGluc buffer to optimize the detection environment.

[0063] The preparation method of buffer A is as follows: 2.4 g Tris, 29.0 g sodium chloride, 10 mL TritonX-100, add 900 mL ultrapure water to dissolve, adjust the pH value to 7.4, add ultrapure water to make up to 1 L, filter with a 0.22 μm filter membrane for sterilization, and store at room temperature for use.

[0064] The preparation method of hGluc buffer is as follows: 2.4g Tris and 5.8g sodium chloride are dissolved in 900mL ultrapure water, the pH value is adjusted to 7.4, the volume is fixed to 1L with ultrapure water, filtered through a 0.22μm filter membrane for sterilization, and stored at room temperature for later use.

[0065] S5. Release the washed magnetic beads containing the antibody-TPP17-hGluc complex into 180 μL of hGluc buffer to obtain the released magnetic beads containing the antibody-TPP17-hGluc complex for subsequent substrate luminescence detection.

[0066] The magnetic beads containing the antibody-TPP17-hGluc complex described later were released and transferred to a second black cell culture plate, and 5 μL of 200 μM coelenterazine substrate was added to each culture well; the bioluminescence intensity in the cell culture plate was then measured.

[0067] S6. Determine the presence or absence of syphilis antibodies in the serum sample to be tested and the concentration thereof by comparing the bioluminescence intensities measured in different wells in the second cell culture plate.

[0068] 1. Research on TPP17-hGluc fusion protein and TPP17 structure modeling by AlphaFold2

[0069] The recombinant TPP17-hGluc protein was simulated using Alphafold2 software version V2.2.0 to explore whether the structure of TPP17 and hGluc would change after recombination. To this end, we simulated two protein models, TPP17 and TPP17-hGluc. Subsequently, by running PyMOL and QuadBuffered3D Stereo software and using alignment technology, the structural differences between the two proteins and their homologous protein sequences were analyzed in detail.

[0070] The present invention aims to reveal the structural changes caused by the binding of TPP17 fragments fused with two repeated GGGGS linkers to hGluc protein. Through the precise modeling of AlphaFold2, the structures of TPP17-hGluc complex and independent TPP17 are clearly presented. Figure 2 In Figure A.

[0071] Figure 2 Figure A in the figure clearly shows that the core structure of the TPP17 part (marked in green) in the TPP17-hGluc fusion protein maintains a high degree of structural similarity with the unfused TPP17 (marked in red).

[0072] Among them, TPP17-hGluc fusion protein is also called TPP17-hGluc protein.

[0073] The above findings provide important clues for understanding the structural stability of fusion proteins.

[0074] 2. Characterization of TPP17-hGluc fusion protein

[0075] In order to verify the functionality of the TPP17-hGluc fusion protein, we used western blot and protein gradient dilution to detect its fluorescence value for verification.

[0076] The fusion protein was successfully expressed in the supernatant of HEK293F cells and then effectively concentrated by 10kDa ultrafiltration, that is, the cell supernatant was centrifuged at 3800×g, the concentrate was retained, 50% by volume glycerol was added, and it was stored at -20°C for later use.

[0077] No additional purification steps were introduced in the above process, thus maintaining the native state of the protein.

[0078] Western blot analysis, that is, the SDS-PAGE method was used to separate the proteins in the supernatant. Subsequently, the proteins on the gel were electrophoretically transferred to a polyvinylidene fluoride membrane using a semi-dry transfer device. To evaluate the effect of electrophoretic transfer, we used pre-stained markers for detection. After the transfer was completed, the membrane was blocked for 2 hours with Tris-buffered saline containing 5% by volume skim milk and 0.05% by volume Tween-20. After that, the membrane was thoroughly rinsed with TBST and immersed in strong positive syphilis serum and placed overnight at 4°C. The next day, after rinsing the membrane three times with TBST, it was incubated with 10,000-fold diluted HRP-labeled rabbit anti-human IgG antibody at room temperature for 2 hours. After the incubation, the membrane was carefully washed with TBST, then stained in a color developing solution, and finally imaged using the GelDocTMEZ imaging system. Among them, the English name of polyvinylidene fluoride is PVDF. The English name of Tris-buffered saline is TBS.

[0079] Among them, the semi-dry transfer device was provided by Tanon, located in Shanghai, China.

[0080] Prestained markers were provided by Vazyme, Nanjing, China.

[0081] Rabbit anti-human IgG antibody was provided by Biodragon, Suzhou, China.

[0082] The colorimetric solution was provided by Yeasen, Shanghai, China.

[0083] The GelDocTM EZ imaging system is manufactured by Bio-Rad, Inc., located in the United States.

[0084] It not only confirmed the presence of TPP17-hGluc, but its band position accurately reflected the expected molecular weight of about 40 kDa, as shown in Figure 2 As shown in Figure B, this result verifies the integrity and expected size of the fusion protein.

[0085] 3. Relationship between hGluc bioluminescence and TPP17-hGluc concentration

[0086] The present invention evaluates the bioluminescent properties of the TPP17-hGluc fusion protein, which is conferred by the hGluc luciferase portion fused therein. The TPP17-hGluc sample is diluted continuously and its bioluminescent intensity is detected. Specifically, the purified TPP17-hGluc is diluted with a gradient of hGluc buffer, and coelenterazine is added to detect its luminescent value.

[0087] We found that the logarithmic value of luminescence showed a good linear relationship with the concentration of TPP17-hGluc, such as Figure 2 As shown in Figure C.

[0088] The above findings not only prove that hGluc maintains its inherent enzymatic activity in the TPP17-hGluc fusion protein, but also further emphasize the potential application value of this fusion strategy in the fields of biosensing and molecular diagnosis, namely, high-sensitivity and quantitative detection achieved by the TPP17-hGluc fusion protein.

[0089] 4. Determination of Cut-off Value

[0090] The present invention uses 70 syphilis antibody-positive sera and 90 negative controls, as well as 29 positive and 29 negative serum samples specifically for pregnant women, to draw the ROC curve through prism7.0, and accurately defines the optimal cutoff value for detecting anti-TPP17 antibodies by LIMS method.

[0091] It should be noted that all test results for syphilis antibodies are based on data provided by standard commercial diagnostic kits used in hospitals.

[0092] Among them, 70 syphilis antibody-positive sera and 90 negative control sera were collected by Luo Jian and Luo Jun from the hospital.

[0093] Standard commercial diagnostic kits were purchased from Zhuhai Livzon Reagent Co., Ltd.

[0094] like Figure 3 Figure A and Figure 3 As shown in Figure C, the TPP17 antibody level in human serum measured by LIMS technology is intuitively expressed in the form of relative light units, which clearly shows the difference in antibody expression between different sample groups. The statistical analysis results show that there is an extremely significant difference in antibody levels between the positive group and the negative group, P<0.0001, which fully proves the high accuracy of LIMS in distinguishing syphilis infection status. Among them, the English abbreviation of relative light units is RLU.

[0095] 5. Determination of sensitivity and specificity of LIMS for syphilis antibody detection

[0096] ROC curve analysis set a clear cutoff value for the LIMS method: 88 in the general population, such as Figure 3 As shown in Figure B, in the pregnant women group, it is 254. Figure 3 As shown in Figure D in Figure 1. The setting of this differentiated cutoff value reflects the sensitivity of the LIMS method to the differences in antibody responses under different physiological states. ROC analysis also revealed that the LIMS method showed perfect sensitivity and specificity of 100% when testing non-pregnant serum. Even when testing pregnant serum, its sensitivity and specificity reached 96.5% and 100% respectively, proving the efficiency and reliability of the method.

[0097] 6. Comparison of sensitivity between LIMS and ELISA

[0098] When evaluating the sensitivity of the detection method, it is traditionally relied on standard samples containing known target antibody concentrations. However, such standard samples are often difficult to obtain, making this approach impractical. In view of this, the present invention adopts an innovative strategy, by directly comparing the performance of LIMS and ELISA methods when continuously diluting syphilis Treponema pallidum antibody positive serum samples, that is, continuously gradient diluting a positive serum and a negative serum, to detect the fluorescence value and OD450nm value of each gradient positive and negative serum, and compare them to evaluate their sensitivity.

[0099] like Figure 4 As shown in Figure A, the LIMS method can still clearly detect positive signals after diluting the positive serum sample up to 40,960 times, and its cutoff value is set at 8820.56. This result fully demonstrates the excellent ability of the LIMS method in detecting low-concentration antibodies. In contrast, the commercial ELISA kit can only maintain positive detection when the sample is diluted to 640 times under the same conditions, as shown in Figure 1. Figure 4 As shown in Figure B, it is obvious that the LIMS method shows significant advantages in terms of sensitivity.

[0100] In summary, the present invention provides a method for detecting TPP17-specific antibodies of Treponema pallidum. The present invention realizes a fully automated process from sample processing to result output, and the detection of 32 samples can be completed in only 25 minutes. It is particularly noteworthy that in the detection of clinical samples, we unexpectedly found that the antibody-negative serum samples of pregnant women showed higher bioluminescence than normal serum. The innovation of this method is that we have determined two clear cutoff values ​​for the first time. After ROC analysis and verification, the method achieved 100% sensitivity and specificity for the detection of normal serum, and for the detection of serum during pregnancy, its sensitivity and specificity were as high as 96.5% and 100%, respectively. In particular, the sensitivity of this method is 32 times that of the traditional ELISA method, showing a significant detection advantage. In addition, the automated platform shows potential commercial value and has far-reaching significance for controlling and preventing the spread of syphilis.

[0101] The present invention provides a method for detecting TPP17-specific antibodies of Treponema pallidum, which cleverly combines the advanced technologies of luciferase immuno-magnetic separation system and automatic magnetic processing machine. In this method, the present invention uses protein A / G-immobilized magnetic particles to accurately capture antibodies in serum, and uses TPP17 antigen chimeric protein and humanized Gaussia luciferase for specific labeling, thereby ensuring high sensitivity and high specificity of the detection.

[0102] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0103] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for detecting Treponema pallidum TPP17-specific antibodies, characterized in that: The steps include: Mixing protein A / G modified magnetic beads with a concentration of 0.9 mg / mL to 1.2 mg / mL, TPP17-hGluc protein and a diluted serum sample to be tested, and incubating for 10 minutes to 20 minutes to obtain magnetic bead particles containing an antibody-TPP17-hGluc complex; wherein the volume ratio of the protein A / G modified magnetic beads, the TPP17-hGluc protein and the diluted serum sample is 1:4-6:3-5; Then, the magnetic bead particles containing the antibody-TPP17-hGluc complex are washed; the washed magnetic bead particles containing the antibody-TPP17-hGluc complex are released into the hGluc buffer to obtain the released magnetic bead particles containing the antibody-TPP17-hGluc complex; Then, coelenterazine substrate was added to the released magnetic beads containing the antibody-TPP17-hGluc complex, and the bioluminescence intensity was measured; By comparing the measured bioluminescence intensities, the presence or absence of syphilis antibodies in the serum sample to be tested and the concentration thereof are determined.

2. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 1, characterized in that: The protein A / G modified magnetic beads, the TPP17-hGluc protein and the diluted serum sample are mixed and then incubated using a nucleic acid extraction machine; The set running program commands for the nucleic acid extractor during incubation are: binding for 8 minutes to 12 minutes, rinsing for 1.5 minutes to 2.5 minutes, rinsing for 1.5 minutes to 2.5 minutes, rinsing for 1.5 minutes to 2.5 minutes, rinsing for 1.5 minutes to 2.5 minutes, rinsing for 1.5 minutes to 2.5 minutes, and discarding magnetic beads for 25 seconds to 50 seconds.

3. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 1, characterized in that: The incubation conditions are 10 to 20 minutes at 35° C. to 40° C.

4. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 1, characterized in that: The method for washing the magnetic bead particles containing the antibody-TPP17-hGluc complex is: first washing with buffer A, and then washing with hGluc buffer; The preparation method of the buffer A is as follows: take 2.2g-2.6g Tris, 28g-31g sodium chloride, and 9mL-11mL TritonX-100, respectively, dissolve them in water, adjust the pH value to 7.2-7.6, add water to make the volume to 1L, and then filter and sterilize through a 0.22μm filter membrane to obtain; The hGluc buffer solution is prepared by taking 2.2g-2.6g Tris and 5.5g-6.0g sodium chloride, respectively, dissolving them in water, adjusting the pH value to 7.2-7.6, adding constant volume to 1L, and then filtering through a 0.22 μm filter membrane for sterilization.

5. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 1, characterized in that: The diluted serum sample to be tested is obtained by diluting the serum sample to be tested by 20 to 80 times.

6. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 5, characterized in that: The method for obtaining the serum sample to be tested is as follows: the blood is first placed at 36°C to 38°C for 1.5 hours to 2.5 hours, then placed at a low temperature of 0°C to 4°C for 8 hours to 16 hours, and then the blood is centrifuged at a speed of 1800rpm to 3000rpm for 4 minutes to 6 minutes, and the upper serum is aspirated to obtain the serum sample to be tested.

7. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 1, characterized in that: The preparation method of the TPP17-hGluc protein comprises: expressing the TPP17-hGluc protein using a human renal epithelial cell line, and performing ultrafiltration concentration to obtain the TPP17-hGluc protein.

8. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 7, characterized in that: The human kidney epithelial cell line is 293F cells.

9. The method for detecting Treponema pallidum TPP17-specific antibodies according to claim 7, characterized in that: The ultrafiltration concentration method is: using an 8kDa-12kDa ultrafiltration tube for concentration, and the rotation speed during ultrafiltration concentration is 3750×g-3850×g.

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