Method for detecting treponema pallidum antibody based on chemiluminescence method and application thereof

Abstract

The invention provides a method for detecting a treponema pallidum antibody based on a chemiluminescence method and application thereof, and the method comprises the following steps: mixing a to-be-detected solution with a magnetic bead suspension mother solution coated with a TP recombinant antigen, incubating to form a TP recombinant antigen-anti-TP antibody compound, cleaning to remove free components, the to-be-detected solution being a cerebrospinal fluid sample or a calibrator; adding an alkaline phosphatase-labeled TP recombinant antigen into the TP recombinant antigen-anti-TP antibody compound, incubating to form a TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen compound, and cleaning to remove free components; adding a luminous substrate into the TP recombinant antigen-anti-TP antibody-alkaline phosphatase labeled TP recombinant antigen compound, and detecting the luminous value of the cerebrospinal fluid sample; and judging whether the cerebrospinal fluid sample contains the treponema pallidum antibody or not according to the ratio of the luminous value of the cerebrospinal fluid sample to the Cut-off value of the calibrator. The invention provides powerful technical support for clinical diagnosis and monitoring of syphilis.

Description

Technical Field

[0001] The present invention belongs to the technical field of Treponema pallidum antibody detection, and particularly relates to a method for detecting Treponema pallidum antibodies based on a chemiluminescence method and an application thereof. Background Art

[0002] Syphilis is a chronic infectious disease caused by Treponema pallidum that poses a serious threat to human health. Early diagnosis and treatment of syphilis are of great significance for controlling the spread of the disease and improving patient prognosis. Currently, commonly used clinical methods for syphilis diagnosis include serological testing and nucleic acid testing. However, existing detection methods have certain limitations when detecting Treponema pallidum antibodies in cerebrospinal fluid, such as insufficient sensitivity and specificity, complex operation, and long detection time. In particular, when testing cerebrospinal fluid samples, due to the low antibody content in cerebrospinal fluid, higher requirements are placed on the sensitivity and specificity of the detection method.

[0003] In recent years, chemiluminescence has gained widespread application in clinical diagnostics due to its high sensitivity, high specificity, and rapid detection. Chemiluminescence generates a light signal through a chemical reaction, and its signal intensity is proportional to the concentration of the analyte, enabling accurate detection of low concentrations of the target substance. However, currently, no chemiluminescence detection kit specifically for the detection of Treponema pallidum antibodies in cerebrospinal fluid is available, either domestically or internationally.

[0004] In view of this, the present invention proposes a method for detecting Treponema pallidum antibodies based on chemiluminescence and its application, which can achieve high-sensitivity and high-specificity detection of Treponema pallidum antibodies in cerebrospinal fluid. Summary of the Invention

[0005] The purpose of the present invention is to address the problems of insufficient sensitivity and specificity, complex operation, and long detection time in existing detection methods for detecting Treponema pallidum antibodies in cerebrospinal fluid, and to propose a method for detecting Treponema pallidum antibodies based on chemiluminescence and its application.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention provides a method for detecting Treponema pallidum antibodies based on chemiluminescence, the method comprising the following steps:

[0008] S1. Mixing the test solution with a stock solution of a magnetic bead suspension coated with TP recombinant antigen, incubating to form a TP recombinant antigen-anti-TP antibody complex, and washing to remove free components, wherein the test solution is a cerebrospinal fluid sample or a calibrator;

[0009] S2. adding alkaline phosphatase-labeled TP recombinant antigen to the TP recombinant antigen-anti-TP antibody complex, incubating to form a TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex, and washing to remove free components;

[0010] S3. Add a luminescent substrate to the TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex, catalyze the luminescent substrate to produce chemiluminescence by alkaline phosphatase, and detect the luminescence value of the cerebrospinal fluid sample;

[0011] S4. Determine whether the cerebrospinal fluid sample contains Treponema pallidum antibodies based on the ratio of the luminescence value of the cerebrospinal fluid sample to the cut-off value of the calibrator.

[0012] Preferably, in step S1, the method for preparing the mother solution of magnetic bead suspension coated with TP recombinant antigen comprises the following steps:

[0013] S11, dissolving the TP recombinant antigen in PBS solution and dialyzing it, adding 2-iminothiolane hydrochloride solution after dialysis, stirring for reaction, and then desalting through a G25 desalting column to obtain the thiolated TP recombinant antigen;

[0014] S12, taking tosyl magnetic beads with a particle size of 1.0 to 1.2 μm, adsorbing them using a magnetic separator, removing the supernatant, adding borate buffer at pH 9.0 to 10.0, and repeatedly washing the tosyl magnetic beads. The washed magnetic beads are diluted with borate buffer to obtain a magnetic bead suspension;

[0015] S13, adding thiolated TP recombinant antigen to the magnetic bead suspension at a ratio of 10-25 μg antigen / mg magnetic beads, then adding 3 M ammonium sulfate solution, rotating and mixing for 16-24 hours to form a magnetic bead-TP recombinant antigen complex;

[0016] S14, after removing the supernatant of the reaction system in step S13, repeatedly washing the magnetic bead-TP recombinant antigen complex with TBST buffer solution at pH 7.0-8.0, adding TBST buffer solution containing 1% BSA, and rotating and mixing for 16-24 hours;

[0017] After removing the supernatant, the magnetic beads-TP recombinant antigen complex was repeatedly washed with TBST buffer solution at pH 7.0-8.0, and finally resuspended with TBST buffer solution containing 0.1% BSA to obtain a stock solution of magnetic beads suspension coated with TP recombinant antigen.

[0018] Further preferably, in step S11, the stirring reaction temperature is 25°C, and the stirring reaction time is 60 min; in step S12, the washed magnetic beads are diluted with borate buffer to a tosylated magnetic bead concentration of 10 mg / mL.

[0019] Further preferably, in step S14, after removing the supernatant of the reaction system in step S13, the magnetic bead-TP recombinant antigen complex is repeatedly washed multiple times with TBST buffer solution at pH 7.0-8.0, the supernatant is removed again, and TBST buffer solution containing 1% BSA is added, and the mixture is rotated and mixed at 37±2°C for 16-24 hours;

[0020] After removing the supernatant again, the magnetic beads-TP recombinant antigen complex was repeatedly washed several times with TBST buffer solution at pH 7.0-8.0, and the supernatant was removed again. Finally, the complex was resuspended with TBST buffer containing 0.1% BSA to obtain a 10 mg / mL stock solution of magnetic beads suspension coated with TP recombinant antigen.

[0021] Preferably, in step S2, the method for preparing the alkaline phosphatase-labeled TP recombinant antigen comprises the following steps:

[0022] S21, dialyzing alkaline phosphatase against a pH 7.2-8.0 buffer, adding a DMSO solution of succinimidyl-4-(N-maleimido)cyclohexane-1-carboxylate, and shaking for 0.5-2 hours. After the reaction, dialyzing against a pH 7.2-7.5 buffer to remove free succinimidyl-4-(N-maleimido)cyclohexane-1-carboxylate to obtain SMCC-activated alkaline phosphatase;

[0023] S22, dialyzing the TP recombinant antigen against a buffer solution at pH 7.0-9.5 containing 1-5 mM EDTA, adding 2-iminothiolane hydrochloride solution, and shaking for 1-2 hours. After the reaction, dialyzing against a buffer solution at pH 7.2-7.5 to remove free 2-iminothiolane hydrochloride solution to obtain 2-IT activated TP recombinant antigen;

[0024] S23. Mix SMCC-activated alkaline phosphatase and 2-IT-activated TP recombinant antigen, shake and react for 1 to 4 hours to form alkaline phosphatase-labeled TP recombinant antigen.

[0025] Further preferably, in step S23, SMCC-activated alkaline phosphatase and 2-IT-activated TP recombinant antigen are mixed at a mass ratio of 1:1 and shaken at room temperature for 1 to 4 hours.

[0026] Further preferably, in step S2, the method for preparing the alkaline phosphatase-labeled TP recombinant antigen further comprises the following steps:

[0027] S24, adding BSA to the alkaline phosphatase-labeled TP recombinant antigen obtained in step S23 to obtain a mixed solution, wherein the final concentration of BSA in the mixed solution is 0.1 mg / mL;

[0028] The mixed solution was dialyzed against 0.05 M Tris-HCl buffer, and an equal volume of glycerol was added to the dialyzed solution, and stored at -20°C for later use.

[0029] Preferably, the calibrator comprises a calibrator C0 and a calibrator C1, wherein the calibrator C0 is a 50 mM Tris-HCl buffer solution containing a rabbit anti-TP polyclonal antibody, and its S / CO value is calibrated to 0.5; the calibrator C1 is a 50 mM Tris-HCl buffer solution containing a rabbit anti-TP polyclonal antibody, and its S / CO value is calibrated to 3;

[0030] In step S4, the calculation formula of the cut-off value is:

[0031] Cut-off value = luminescence value of calibrator C0 × 0.8 + luminescence value of calibrator C1 × 0.2.

[0032] Preferably, the incubation time of step S1 and step S2 is 10 minutes, and the incubation temperature is 37±2°C.

[0033] In a second aspect, the present invention provides a Treponema pallidum antibody detection kit for implementing any of the methods described above, comprising: a stock solution of a magnetic bead suspension coated with a TP recombinant antigen, an alkaline phosphatase-labeled TP recombinant antigen, a calibrator, a luminescent substrate, and a concentrated cleaning solution;

[0034] The magnetic bead particle size in the stock solution of the magnetic bead suspension coated with the TP recombinant antigen is 1.0 to 1.2 μm, and the antigen coating amount is 10 to 25 μg / mg magnetic beads;

[0035] Alkaline phosphatase-labeled TP recombinant antigen is formed by coupling SMCC-activated alkaline phosphatase and 2-IT-activated TP recombinant antigen in a mass ratio of 1:1;

[0036] Calibrators: Rabbit anti-TP polyclonal antibody was prepared to the theoretical concentration using 50 mM Tris-HCl buffer, including calibrators C0 and C1. The S / CO value of calibrator C0 was calibrated to 0.5, and the S / CO value of calibrator C1 was calibrated to 3.

[0037] The luminescent substrate is 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[5.5.1.0 3,7 ]decane]-4-yl)phenyl phosphate disodium salt;

[0038] The concentrated cleaning solution contains 18-20% NaCl, 2-3% Tris, 0.4-0.6% MgCl2·6H2O and 0.5-1% preservative.

[0039] In summary, compared with the prior art, the method for detecting Treponema pallidum antibodies based on chemiluminescence and its application provided by the present invention have the following beneficial effects:

[0040] (1) Superior detection performance: Using the principle of double antigen sandwich method, the method utilizes magnetic particles coated with TP recombinant antigen and TP recombinant antigen labeled with alkaline phosphatase to specifically capture and detect Treponema pallidum antibodies, effectively reducing nonspecific binding and interference, and improving the accuracy and reliability of the test results. When testing cerebrospinal fluid samples, it demonstrates high sensitivity detection capability for low concentrations of antibodies, meeting the clinical demand for high sensitivity and high specificity of cerebrospinal fluid testing, and overcoming the limitations of existing detection methods in cerebrospinal fluid testing.

[0041] (2) Fast detection: The entire detection process, including incubation of the sample with the magnetic microparticle suspension, incubation with the alkaline phosphatase-labeled antigen, and chemiluminescence detection of the luminescent substrate, takes only 20 minutes, enabling rapid detection results. This is of great significance for early diagnosis of syphilis, timely treatment, and disease control, and significantly shortens the detection cycle compared to traditional detection methods.

[0042] (3) Simple and efficient operation: The detection process is automated, reducing manual operation steps and errors, and improving the repeatability and reliability of the test. It does not require complex sample pretreatment and tedious operation procedures, which reduces the technical requirements for operators and enables the test to be widely used in more laboratories and clinical scenarios.

[0043] (4) Wide range of applications: It can detect not only Treponema pallidum antibodies in serum and plasma, but also antibodies in cerebrospinal fluid. It provides a powerful tool for the diagnosis of neurosyphilis, makes up for the shortcomings of existing detection methods in cerebrospinal fluid detection, and provides more comprehensive support for clinicians in the diagnosis, treatment monitoring and prognosis assessment of syphilis patients, especially those suspected of neurosyphilis.

[0044] (5) The kit is well-composed: The components included, such as the stock solution of magnetic bead suspension coated with TP recombinant antigen, alkaline phosphatase-labeled TP recombinant antigen, calibrators, luminescent substrates, and concentrated cleaning solutions, have all been carefully designed and optimized. The synergistic effect of the components ensures the stability and accuracy of the detection method. For example, precise control of parameters such as the particle size of the magnetic beads, the amount of antigen coating, and the use of markers helps to improve the intensity and specificity of the detection signal; the setting of the calibrators provides an accurate reference for the determination of the test results.

[0045] (6) Good stability: Through specific preparation methods and storage conditions, such as multiple washing and blocking of the magnetic bead suspension stock solution coated with TP recombinant antigen, adding BSA to the alkaline phosphatase-labeled TP recombinant antigen and performing dialysis and glycerol storage, the stability and shelf life of the reagent are improved, ensuring the consistency of the results in different batches of tests.

[0046] (7) Objective and accurate result determination: By using the cut-off value calculation formula and combining it with the luminescence value of the calibrator, it is possible to objectively and accurately determine whether the cerebrospinal fluid sample contains Treponema pallidum antibodies. This determination method based on data and standard curves reduces the influence of human factors on the result determination and improves the credibility of the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Other features, objects and advantages of the present application will become more apparent upon reading the detailed description of non-limiting embodiments made with reference to the following drawings:

[0048] Figure 1 The present invention is a flow chart of a method for detecting Treponema pallidum antibodies based on chemiluminescence. DETAILED DESCRIPTION

[0049] The present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely for the purpose of explaining the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the relevant invention are shown in the accompanying drawings.

[0050] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in this application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0051] In the first aspect, the present invention provides a method for detecting Treponema pallidum antibodies based on chemiluminescence. Figure 1 : is a flow chart of a method for detecting Treponema pallidum antibodies based on chemiluminescence according to the present invention, such as Figure 1 As shown, the method includes the following steps:

[0052] S1. Mix the test fluid with the stock solution of magnetic bead suspension coated with TP recombinant antigen, incubate to form TP recombinant antigen-anti-TP antibody complex, and wash to remove free components. The test fluid is a cerebrospinal fluid sample or a calibrator. It should be understood that the test fluid can also be serum or plasma.

[0053] S2. Add alkaline phosphatase-labeled TP recombinant antigen to the TP recombinant antigen-anti-TP antibody complex, incubate to form a TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex, and wash to remove free components.

[0054] S3. Add a luminescent substrate to the TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex. Alkaline phosphatase catalyzes the luminescent substrate to produce chemiluminescence, and the luminescence value of the cerebrospinal fluid sample is detected.

[0055] S4. Determine whether the cerebrospinal fluid sample contains Treponema pallidum antibodies based on the ratio of the luminescence value of the cerebrospinal fluid sample to the cut-off value of the calibrator.

[0056] In a specific embodiment, the preparation process of the magnetic bead suspension coated with TP (Treponema pallidum) recombinant antigen is as follows:

[0057] (1) The labeled antigen needs to be dialyzed and thiolated before coating. The specific operation is as follows: add TP recombinant antigen and 0.01M PBS (Phosphate-Buffered Saline) solution into the dialysis bag, weigh the treated antigen, add 2-IT (2-iminothiolane) solution, mix well at room temperature for 60 minutes, and then desalt it with a G25 desalting column to obtain the thiolated TP recombinant antigen, which is collected for later use.

[0058] (2) Place tosylated magnetic beads with a particle size of 1.0-1.2 μm on a rotary mixer (speed 40-60 rpm) and mix thoroughly for more than half an hour. Pipette the required amount of magnetic beads (10 mg-25 mg) into a suitable reagent bottle, place the reagent bottle on a magnetic separator, wait until the magnetic beads are completely adsorbed, and remove the supernatant. Add pH 9.0-10.0 borate buffer solution and wash the magnetic beads repeatedly three times; add the above borate buffer solution to dilute the magnetic beads to 10 mg / mL to obtain a magnetic bead suspension.

[0059] (3) Add the required amount of TP recombinant antigen to the magnetic bead suspension at a coating concentration of 10-25 μg / mg, and then add the required amount (0.05-0.1 mL) of 3 M ammonium sulfate solution. Rotate and mix at 37 ± 2°C for 16-24 hours to form a magnetic bead-TP recombinant antigen complex.

[0060] (4) Remove the supernatant, add TBST buffer solution at pH 7.0-8.0, and wash the magnetic beads three times. After removing the supernatant, add TBST buffer solution containing 1% BSA (bovine serum albumin) and rotate and mix at 37±2°C for 16-24h. Finally, remove the supernatant, add TBST buffer solution at pH 7.0-8.0, and wash the magnetic beads three times. After removing the supernatant, add TBST buffer solution containing 0.1% BSA to obtain a 10 mg / mL stock solution of magnetic bead suspension coated with TP recombinant antigen, which is stored at 2-8°C for use.

[0061] In a specific embodiment, the preparation process of alkaline phosphatase-labeled TP recombinant antigen is as follows:

[0062] (1) The alkaline phosphatase is dialyzed in a buffer solution of pH 7.2 to 8 for 6 to 24 hours. Desalting columns or ultrafiltration can also be used instead of dialysis. Before the next reaction, a succinimidyl-4-(N-maleimide) cyclohexane-1-carboxylate (SMCC) solution is prepared, and the solvent used is DMSO. SMCC is added to the dialyzed alkaline phosphatase solution and shaken for 0.5 to 2 hours at room temperature. Subsequently, the reacted alkaline phosphatase is dialyzed again in a buffer solution of pH 7.2 to 7.5 for 6 to 24 hours. Similarly, desalting columns or ultrafiltration can be used to remove excess SMCC in the reaction system to obtain SMCC-activated alkaline phosphatase.

[0063] (2) The TP recombinant antigen is dialyzed in a buffer solution containing 1-5 mM EDTA at pH 7.0-9.5. The dialysis time is also 6-24 hours. Desalting columns or ultrafiltration are also feasible alternatives. Before use, prepare a 2-iminothiolane hydrochloride solution (2-IT·HCl). Then, add 2-IT·HCl to the TP recombinant antigen solution at a concentration of 1-2 mg / mL and shake at room temperature for 1-2 hours. After the reaction is completed, the excess 2-IT·HCl needs to be removed by desalting columns or ultrafiltration in a buffer solution at pH 7.2-7.5 to obtain the 2-IT activated TP recombinant antigen.

[0064] (3) After the activation reaction of the above two parts is completed, SMCC activated alkaline phosphatase and 2-IT·HCl activated TP recombinant antigen are mixed in a mass ratio of 1:1 and shaken at room temperature for 1 to 4 hours. After the reaction is completed, bovine serum albumin (BSA) is added to the mixture to adjust the final concentration of BSA in the mixture to 0.1 mg / mL. Subsequently, the mixture is placed in 0.05MTris-HCl (pH 7.0-8.0) for dialysis. Dialysis can also be replaced by desalting or ultrafiltration. Finally, an equal volume of glycerol is added to the alkaline phosphatase labeled TP recombinant antigen mixture, and the final product is stored at -20°C for use.

[0065] In a specific embodiment, the preparation process of the calibrator is as follows:

[0066] First, prepare the calibration concentration according to the S / CO value. The specific steps are as follows:

[0067] Prepare a calibrator with an S / CO ratio of 0.5: Dilute the rabbit anti-TP polyclonal antibody with calibrator buffer until the calibrator reaches the theoretical concentration requirement. The specific concentration should be determined based on the negative control and the national plate RLU. For example, if the antibody amount at this concentration is determined to be a specific value based on experimental requirements and relevant standards, dilute it according to the corresponding ratio.

[0068] Similarly, prepare a calibrator with S / CO=3: dilute the rabbit anti-TP polyclonal antibody using the calibrator buffer to obtain a calibrator concentration that meets the S / CO=3 requirement.

[0069] The formula of the calibrator buffer is as follows:

[0070] NaCl: 0.8-1.2%

[0071] Tris: 0.5-1%

[0072] HCl: 0.2-0.5%

[0073] BSA: 2-4%

[0074] ProClin300: 0.1-0.3%

[0075] Purified water: balance

[0076] Mix the above ingredients in proportion, stir evenly to fully dissolve and mix evenly, and prepare a calibration buffer solution for use.

[0077] The rabbit anti-TP polyclonal antibody was further prepared using the prepared calibrator buffer to obtain a calibration product. The specific steps are as follows:

[0078] Calculate the specific amount of rabbit anti-TP polyclonal antibody in the calibrator based on the theoretical concentration required for the experiment. Add the rabbit anti-TP polyclonal antibody to the calibrator buffer and mix thoroughly to evenly disperse the antigen. Aliquot the prepared calibrator mixture into prepared test tubes or other suitable containers, filling each tube with 0.5 mL. Store the aliquoted calibrators at 2°C-8°C for subsequent use.

[0079] Through the above steps, the preparation of calibrator concentration and calibration materials are completed. The prepared calibrators and calibration materials will be used in subsequent experimental tests and instrument calibration to ensure the accuracy and reliability of the test results.

[0080] In a second aspect, the present invention provides a Treponema pallidum antibody detection kit for implementing the above method, the kit comprising: a stock solution of a magnetic bead suspension coated with a TP recombinant antigen, an alkaline phosphatase-labeled TP recombinant antigen, a calibrator, a luminescent substrate, and a concentrated cleaning solution;

[0081] The magnetic bead particle size in the stock solution of the magnetic bead suspension coated with the TP recombinant antigen is 1.0 to 1.2 μm, and the antigen coating amount is 10 to 25 μg / mg magnetic beads;

[0082] Alkaline phosphatase-labeled TP recombinant antigen is formed by coupling SMCC-activated alkaline phosphatase and 2-IT-activated TP recombinant antigen in a mass ratio of 1:1;

[0083] Calibrators: Rabbit anti-TP polyclonal antibody was prepared to the theoretical concentration using 50 mM Tris-HCl buffer, including calibrators C0 and C1. The S / CO value of calibrator C0 was calibrated to 0.5, and the S / CO value of calibrator C1 was calibrated to 3.

[0084] The luminescent substrate is CSPD (3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[5.5.1.0 3,7 ]decane]-4-yl)phenyl phosphate disodium salt);

[0085] The concentrated cleaning solution contains 18-20% NaCl, 2-3% Tris, 0.4-0.6% MgCl2·6H2O and 0.5-1% preservative, wherein the preservative contains 0.5% Krovin 600 or 1% NaN3, 2-3% Triton X-100 and 0.95-1% HCL (35-40%).

[0086] Example 1: Chemiluminescence detection of anti-TP antibodies

[0087] In this example, the above-mentioned kit (comprising a stock solution of a magnetic bead suspension coated with TP recombinant antigen, an alkaline phosphatase-labeled TP recombinant antigen, a calibrator, a luminescent substrate, and a concentrated cleaning solution) was used to perform the test on a matching fully automatic instrument. The specific steps are as follows:

[0088] 1. Check and load consumables according to the instrument instructions to ensure they are sufficient.

[0089] 2. Reagent loading:

[0090] (1) When loading the test reagent for the first time, the magnetic bead chamber of the reagent pack should be gently rotated before opening the bottle to avoid generating bubbles until the magnetic particles are completely suspended. If the magnetic particles are still attached to the bottom of the reagent bottle after multiple rotations, the bottle of reagent cannot be used.

[0091] (2) The instrument automatically obtains the parameters required for the test by scanning the barcode of the test kit.

[0092] 3. Calibration execution:

[0093] (1) Use the calibrator to perform the calibration test. Before the kit starts calibration, the instrument automatically obtains the parameters required for the test by scanning the calibrator barcode.

[0094] (2) All tests require effective calibration before testing. Recalibration is required if the following conditions occur:

[0095] a. The batch number of the substrate used in the kit or immunoassay system has been changed.

[0096] b. Calibration exceeds 28 days.

[0097] c. The measured value of the quality control material is still out of range after repeated measurement.

[0098] d. Specific repair procedures or maintenance completed.

[0099] 4. Quality control operations:

[0100] (1) Perform quality control using a quality control material containing at least two analyte levels (e.g., level 1: anti-TP antibody concentration 1.0 IU / mL; level 2: 20 IU / mL).

[0101] (2) Quality control is required in the following situations:

[0102] a. The kit has been used for more than 24 hours;

[0103] b. First needle test after recalibration;

[0104] c. The first operation of the instrument after shutdown and restart.

[0105] (3) Each laboratory can establish an appropriate quality control range and quality control cycle based on its own situation.

[0106] (4) The quality control value should fall within the specified range. If it exceeds the range, the user should check the detection system and recalibrate it if necessary.

[0107] 5. Standardized testing process:

[0108] (1) Place the sample container in the sample holder provided with the instrument, and keep the sample volume for each test above 200 μL.

[0109] (2) Load the sample rack and enter the sample information in the instrument software interface.

[0110] (3) Select "Run" to start the test, and the system will automatically perform the test operation:

[0111] a. The sample rack is transferred to the sample aspiration position.

[0112] b. Load the reaction cup into the sample loading position.

[0113] c. Add calibrators or cerebrospinal fluid samples to the reaction cups in sequence, with the sample volume being 50 μL per cup.

[0114] d. Add 50 μL of the stock solution of magnetic bead suspension coated with TP recombinant antigen to each cup.

[0115] e. Mix well and incubate for 10 minutes.

[0116] f. Wash with cleaning solution.

[0117] g. Add 100 μL of alkaline phosphatase-labeled TP recombinant antigen to each cup.

[0118] h. Mix well and incubate for 10 minutes.

[0119] i. Wash again with cleaning solution.

[0120] j. Add luminescent substrate for immunoassay system to each cup, mix well, and detect chemiluminescent signal.

[0121] 6. Calculation of results:

[0122] The instrument's automatic operating system automatically calculates the sample test results through the stored calibration curve and the luminescence intensity value obtained from the sample test, and reports the results in the form of negative or positive.

[0123] 7. Positive judgment value:

[0124] This kit was tested on 335 samples and the ROC curve method was used to determine the positive judgment value of this kit.

[0125] Cut-off value (CO) = Calibrator C0 RLU × 0.8 + Calibrator C1 RLU × 0.2

[0126] The sample was determined to be positive if RLU(S)≥Cut-off value (CO), i.e. S / CO≥1;

[0127] The sample was determined to be negative if RLU(S) < Cut-off value (CO), ie S / CO < 1.

[0128] 8. Interpretation of test results:

[0129] (1) For suspicious test results, it is recommended to repeat the test to confirm the results.

[0130] (2) The test results are reliable only when they meet the quality control indicators.

[0131] (3) For samples with positive test results (serum, plasma, cerebrospinal fluid), when used to diagnose syphilis or neurosyphilis, a comprehensive judgment should be made in combination with clinical symptoms and other indicators.

[0132] (4) A negative test result does not mean that the sample does not contain TP antibodies.

[0133] This kit demonstrated excellent performance when testing cerebrospinal fluid samples. Specific performance is as follows:

[0134] Negative reference product testing: Strict testing was performed using the company's negative reference products. Experimental data showed that the positive reaction was precisely controlled within a range of no more than 1 copy, and the compliance rate of the negative reference products stably reached or exceeded the standard requirements of 9 / 10, fully demonstrating the high accuracy of this kit in determining negative samples.

[0135] Positive reference product testing: The company's positive reference products were used to carry out testing experiments. The results showed that negative reactions were effectively limited to no more than 2 copies, and the positive reference product compliance rate was stably at a level of ≥8 / 10, effectively ensuring the reliability of the positive sample test results.

[0136] Minimum detection limit detection: With the help of the company's minimum detection limit reference material, detailed testing was carried out. L1 and L2 samples were both accurately detected as positive. L3 samples could also show positive or negative reactions during the test process, and L4 samples were accurately judged as negative, highlighting the excellent performance of this kit in detecting samples with different concentration gradients.

[0137] Precision testing: Repeatability testing was performed using the company's precision reference material. After 10 rigorous repeated tests, all results were positive without exception. The key coefficient of variation (CV) indicator was always strictly controlled within the range of less than 15%, which strongly confirmed the high precision and low variability of the test results of this kit.

[0138] Inter-batch difference detection: We continued to use the company's precision reference materials to test and compare three different batches of test kits. The experimental results showed that the inter-batch coefficient of variation (CV) was precisely controlled within a range of no more than 15%. This fully demonstrates that the performance of different batches of test kits maintains a high degree of consistency and stability, providing a solid guarantee for large-scale testing applications.

[0139] Interference test: The results of the interference test specifically conducted on hemoglobin and penicillin, two common potential interfering substances, showed that neither of them caused any interference with the test results, indicating that this kit has excellent anti-interference ability and can ensure detection accuracy in complex sample environments.

[0140] Cross-reaction detection: Through clinical testing of possible cross-reaction substances such as cerebrospinal fluid albumin, cerebrospinal fluid IgA, and cerebrospinal fluid IgG, the experimental results clearly showed that these substances did not interfere with the test results of the reagent, further confirming the excellent performance of this kit in specifically identifying the target antigen and effectively avoiding the occurrence of false positive results.

[0141] Hook Effect Assessment: Given that this product utilizes a two-step detection reaction model, theoretically and practically, there is no evidence of a hook effect. During actual testing, validation was conducted on a TP sample with a titer of approximately 1:2000, and the results also showed no observed hook effect. This provides strong support for accurate detection of high-concentration samples and broadens the dynamic range of the kit.

[0142] The above description is merely a preferred embodiment of the present application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in this application is not limited to the technical solutions formed by the specific combination of the above-mentioned technical features, but also encompasses other technical solutions formed by any combination of the above-mentioned technical features or their equivalents without departing from the above-mentioned inventive concept. For example, a technical solution formed by replacing the above-mentioned features with (but not limited to) technical features with similar functions disclosed in this application.

Claims

1. A method for detecting Treponema pallidum antibodies based on chemiluminescence, characterized in that: The method comprises the following steps: S1. Mixing a test solution with a stock solution of a magnetic bead suspension coated with a TP recombinant antigen, incubating to form a TP recombinant antigen-anti-TP antibody complex, and washing to remove free components, wherein the test solution is a cerebrospinal fluid sample or a calibrator; S2, adding alkaline phosphatase-labeled TP recombinant antigen to the TP recombinant antigen-anti-TP antibody complex, incubating to form a TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex, and washing to remove free components; S3, adding a luminescent substrate to the TP recombinant antigen-anti-TP antibody-alkaline phosphatase-labeled TP recombinant antigen complex, catalyzing the luminescent substrate with alkaline phosphatase to produce chemiluminescence, and detecting the luminescence value of the cerebrospinal fluid sample; S4. Determine whether the cerebrospinal fluid sample contains Treponema pallidum antibodies based on the ratio of the luminescence value of the cerebrospinal fluid sample to the cut-off value of the calibrator.

2. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 1, characterized in that: In step S1, the method for preparing the mother solution of magnetic bead suspension coated with TP recombinant antigen comprises the following steps: S11, dissolving the TP recombinant antigen in PBS solution and dialyzing it, adding 2-iminothiolane hydrochloride solution after dialysis, stirring for reaction, and then desalting through a G25 desalting column to obtain the thiolated TP recombinant antigen; S12, taking tosyl magnetic beads with a particle size of 1.0 to 1.2 μm, adsorbing them using a magnetic separator, removing the supernatant, adding a borate buffer having a pH of 9.0 to 10.0, and repeatedly washing the tosyl magnetic beads, diluting the washed magnetic beads with the borate buffer to obtain a magnetic bead suspension; S13, adding the thiolated TP recombinant antigen to the magnetic bead suspension at a ratio of 10 to 25 μg antigen / mg magnetic beads, then adding 3 M ammonium sulfate solution, and rotating and mixing for 16 to 24 hours to form a magnetic bead-TP recombinant antigen complex; S14, after removing the supernatant of the reaction system in step S13, repeatedly washing the magnetic bead-TP recombinant antigen complex with TBST buffer solution at pH 7.0-8.0, adding TBST buffer solution containing 1% BSA, and rotating and mixing for 16-24 hours; After removing the supernatant, the magnetic bead-TP recombinant antigen complex was repeatedly washed several times with TBST buffer solution at pH 7.0-8.0, and finally resuspended with TBST buffer solution containing 0.1% BSA to obtain a stock solution of magnetic bead suspension coated with TP recombinant antigen.

3. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 2, characterized in that: In step S11, the stirring reaction temperature is 25°C, and the stirring reaction time is 60 minutes. In step S12, the washed magnetic beads are diluted with the borate buffer to a concentration of tosylated magnetic beads of 10 mg / mL.

4. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 3, characterized in that: In step S14, after removing the supernatant of the reaction system in step S13, the magnetic bead-TP recombinant antigen complex was repeatedly washed several times with TBST buffer solution at pH 7.0-8.0, and then the supernatant was removed again. TBST buffer solution containing 1% BSA was added and the mixture was rotated and mixed at 37±2°C for 16-24 hours; After removing the supernatant again, the magnetic bead-TP recombinant antigen complex was repeatedly washed several times with TBST buffer solution at pH 7.0-8.0, and the supernatant was removed again. Finally, the complex was resuspended with TBST buffer containing 0.1% BSA to obtain a 10 mg / mL stock solution of magnetic bead suspension coated with TP recombinant antigen.

5. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 1, characterized in that: In step S2, the method for preparing the alkaline phosphatase-labeled TP recombinant antigen comprises the following steps: S21, dialyzing alkaline phosphatase against a pH 7.2-8.0 buffer, adding a DMSO solution of succinimidyl-4-(N-maleimido)cyclohexane-1-carboxylate, and shaking for 0.5-2 hours. After the reaction, dialyzing against a pH 7.2-7.5 buffer to remove free succinimidyl-4-(N-maleimido)cyclohexane-1-carboxylate to obtain SMCC-activated alkaline phosphatase; S22, dialyzing the TP recombinant antigen against a buffer solution at pH 7.0-9.5 containing 1-5 mM EDTA, adding 2-iminothiolane hydrochloride solution, and shaking for 1-2 hours. After the reaction, dialyzing against a buffer solution at pH 7.2-7.5 to remove free 2-iminothiolane hydrochloride solution to obtain 2-IT activated TP recombinant antigen; S23, mixing the SMCC-activated alkaline phosphatase and the 2-IT-activated TP recombinant antigen, shaking and reacting for 1 to 4 hours to form alkaline phosphatase-labeled TP recombinant antigen.

6. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 5, characterized in that: In step S23, the SMCC-activated alkaline phosphatase and the 2-IT-activated TP recombinant antigen are mixed at a mass ratio of 1:1 and shaken at room temperature for 1 to 4 hours.

7. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 5, characterized in that: In step S2, the method for preparing the alkaline phosphatase-labeled TP recombinant antigen further comprises the following steps: S24, adding BSA to the alkaline phosphatase-labeled TP recombinant antigen obtained in step S23 to obtain a mixed solution, wherein the final concentration of BSA in the mixed solution is 0.1 mg / mL; The mixed solution was dialyzed against 0.05 M Tris-HCl buffer, and an equal volume of glycerol was added to the dialyzed solution, and the solution was stored at -20°C for later use.

8. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 1, characterized in that: The calibrator includes calibrator C0 and calibrator C1, wherein the calibrator C0 is a 50mM Tris-HCl buffer containing rabbit anti-TP polyclonal antibody, and its S / CO value is calibrated to 0.5; the calibrator C1 is a 50mM Tris-HCl buffer containing rabbit anti-TP polyclonal antibody, and its S / CO value is calibrated to 3; In step S4, the calculation formula of the cut-off value is: Cut-off value = luminescence value of calibrator C0 × 0.8 + luminescence value of calibrator C1 × 0.

2.

9. The method for detecting Treponema pallidum antibodies based on chemiluminescence according to claim 1, characterized in that: The incubation time of step S1 and step S2 is both 10 minutes, and the incubation temperature is both 37±2°C.

10. A Treponema pallidum antibody detection kit for implementing the method according to any one of claims 1 to 9, characterized in that: include: TP recombinant antigen-coated magnetic bead suspension stock, alkaline phosphatase-labeled TP recombinant antigen, calibrator, luminescent substrate, and concentrated washing solution; The magnetic bead particle size in the mother solution of the magnetic bead suspension coated with the TP recombinant antigen is 1.0 to 1.2 μm, and the antigen coating amount is 10 to 25 μg / mg magnetic beads; The alkaline phosphatase-labeled TP recombinant antigen is formed by coupling SMCC-activated alkaline phosphatase and 2-IT-activated TP recombinant antigen in a mass ratio of 1:1; The calibrator is prepared by using 50mM Tris-HCl buffer to prepare rabbit anti-TP polyclonal antibody to a theoretical concentration, including calibrator C0 and calibrator C1, the S / CO value of calibrator C0 is calibrated to 0.5, and the S / CO value of calibrator C1 is calibrated to 3; The luminescent substrate is 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[5.5.1.0 3 , 7 ]decane]-4-yl)phenyl phosphate disodium salt; The concentrated cleaning solution contains 18-20% NaCl, 2-3% Tris, 0.4-0.6% MgCl2·6H2O and 0.5-1% preservative.

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