Syphilis monoclonal antibody with high binding capacity and screening process of syphilis monoclonal antibody
By developing engineered antibodies with high binding capacity, the problem of insufficient antibody binding capacity in syphilis detection is solved, and high sensitivity and low cost syphilis detection is achieved, which improves the accuracy and economic benefits of the detection.
Patent Information
- Application Number
- CN202510626350.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-15
AI Technical Summary
In the existing syphilis detection methods, the antibody binding capacity of traditional detection reagents is insufficient, resulting in low detection sensitivity and prone to false positives, and high cost, which limits the patient's need to test on time.
Develop an engineered antibody with high binding ability. By collecting syphilis antibodies in human serum for purification and sequencing screening, high-affinity secondary antibodies are prepared, and bind to antigens in the presence of syphilis antibodies to form a rapid aggregation network to improve detection sensitivity.
It significantly improves the sensitivity of syphilis detection, reaching 16 times that of traditional methods, reducing the detection cost, and improving the accuracy and economic benefits of the detection.
Smart Images
Figure CN120484113A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, and in particular to the screening and preparation of an antibody with high-efficiency binding ability and its application in judging syphilis detection results. Background Art
[0002] Laboratory diagnostic methods for syphilis primarily fall into two categories: etiological testing and serological testing. Serological testing is the most commonly used and most important clinical method. Syphilis serological tests are a general term for tests that detect antibodies to Treponema pallidum in a patient's blood. These antibodies can be divided into two categories: reagins and T. pallidum-specific antibodies. After infection with T. pallidum, the body rapidly mounts an immune response to damaged host cells and lipids released from the surface of T. pallidum cells, producing antibodies against lipid antigens, also known as reagins, within three to four weeks. Non-treponemal tests, such as the toluidine red unheated serum test (TRUST), use cardiolipin, phosphatidylcholine, and cholesterol as antigens in flocculent agglutination tests. Agglutination of varying intensity is positively correlated with antibody concentration, reflecting, to some extent, the activity of the syphilis infection. Treponema pallidum-specific antibodies, using extracts of Treponema pallidum or its recombinant protein as antigens, are highly sensitive and specific. Currently, they are primarily tested clinically using chemiluminescence immunoassays (CLIs), with other methods including the Treponema pallidum particle agglutination test (TPPA). It is generally believed that even with syphilis-specific antibody-positive treatment, the antibody remains positive for life due to immune memory. Therefore, these tests cannot be used to monitor treatment efficacy or determine recurrence or reinfection.
[0003] The non-treponemal test is a flocculent agglutination test using cardiolipin, phosphatidylcholine, and cholesterol as antigens. Reagin forms an antigen-antibody reaction with cardiolipin. Shaking and collision cause particles to adhere to each other, resulting in visible agglutination and precipitation, a positive reaction. However, many factors can influence the test. Phospholipids are widely distributed in the biological world, present on the surface of cell mitochondria. Non-syphilitic diseases can produce lipid antibodies that react with phospholipid antigens, resulting in a false-positive syphilis test.
[0004] The TPPA test is a flocculent agglutination test for syphilis-specific antibodies. Refined syphilis bacterial components are coated onto artificial carrier gelatin particles. These sensitized particles react with Treponema pallidum antibodies in the sample, agglutinating them and producing a particle agglutination reaction. This allows the detection of Treponema pallidum antibodies in serum and plasma and can be used to determine antibody titers. Both the TPPA and TRUST tests operate on the principle of agglutination, with the degree of agglutination determined visually. However, current test reagents have low agglutination rates, making visual assessment highly subjective. Furthermore, the reagents and consumables primarily used in hospital laboratory testing for syphilis testing are pharmaceuticals, which are expensive and subject to strict regulations. This increases the cost burden for medical institutions and limits patients' demand for on-demand testing. However, existing test reagents for both tests suffer from insufficient antibody binding capacity. This can lead to false-positive results in the TRUST test when used in conjunction with other diseases, such as rheumatoid arthritis. Therefore, developing new high-affinity binding antibodies and binding modalities is a promising approach to improving test accuracy.
[0005] This invention develops an engineered antibody with four-site binding capability based on the laboratory's high-throughput antibody screening and production platform. Based on the traditional antigen-antibody binding reaction, this four-site binding antibody can efficiently react with the antigen / antibody complex, rapidly aggregating small aggregated particles into large agglutination networks, and the detection sensitivity is increased by 16 times compared to traditional diagnostic reagents. Summary of the Invention
[0006] 1. First, syphilis antibodies are collected from human serum, purified, and sequenced for screening. These screened antibodies are then combined with Treponema pallidum as a whole antigen component, which is then used to immunize mice to produce the corresponding secondary antibody. Finally, high-affinity secondary antibodies are collected, purified, sequenced, and the target sequence is introduced into phage structures for mass production. The resulting monoclonal antibodies are conjugated with streptavidin and biotin, respectively. In the presence of syphilis antibodies, the antigen and antibody bind to form small aggregated particles. The streptavidin-modified secondary antibody then binds to complexes with other biotin sites, rapidly aggregating into large aggregated networks. BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Figure 1 It is a process for screening and preparing high-affinity binding antibodies.
[0008] Figure 2 The principle of syphilis detection is based on high affinity binding antibodies.
[0009] Figure 3 Diagnostic results of high-affinity secondary antibody TPPA.
[0010] Figure 4 This is the diagnostic result of the high-affinity secondary antibody TRUST. DETAILED DESCRIPTION
[0011] 1. Antibody Screening and Purification
[0012] First, a sample containing 2-3 mL of syphilis antibodies is collected from human serum. This step is fundamental to ensuring subsequent screening, as naturally produced antibodies are used as a starting point. The collected syphilis antibodies are then purified using affinity chromatography, a protein purification technique. The purpose of purification is to remove impurities and improve the purity and specificity of the antibodies. The purified antibodies are sequenced, and their genetic sequence information is obtained using high-throughput sequencing technology. Subsequently, based on this sequence information and expected antigen-binding ability, antibody sequences with specific properties are screened for use in subsequent experiments and production.
[0013] 2. Antigen Preparation
[0014] Based on the structure and properties of existing Treponema pallidum, an antigen capable of specifically binding to the screened antibody is designed and synthesized. This typically involves analyzing and modeling the surface proteins of Treponema pallidum to ensure the accuracy and effectiveness of the antigen. The designed antigen is synthesized using chemical synthesis or genetic engineering techniques. The synthesized antigen needs to be validated to ensure it has the expected immunogenicity and specificity. The screened antibody is conjugated to the synthesized antigen to form the overall antigen component. This step ensures that the antibody and antigen bind tightly, providing the necessary stimulation for the subsequent production of a secondary antibody.
[0015] III. Secondary Antibody Production and Purification
[0016] The conjugated whole antigen component is immunized into mice to stimulate the production of the corresponding secondary antibody. Parameters such as the immunization dose, number of immunizations, and intervals must be controlled to ensure sufficient secondary antibody production. Serum or ascites containing the secondary antibody is collected from the mice and purified to remove impurities, enhancing the purity and specificity of the secondary antibody. The purified secondary antibody is then used for subsequent testing and mass production.
Claims
1. A syphilis monoclonal antibody with high binding capacity and a screening process thereof, characterized in that: The antibody is a secondary antibody directed against the Treponema pallidum-antigen complex molecule.
2. An expression vector comprising the nucleotide sequence of claim 1.
3. A host cell, characterized in that The cell expresses the secondary antibody according to claim 1 or contains the expression vector according to claim 2.
4. Use of the secondary antibody according to claim 1 or the nucleotide sequence according to claim 2 in the preparation of a product for detecting Treponema pallidum antibodies.
5. Use of the secondary antibody according to claim 1 or the nucleotide sequence according to claim 2 in the preparation of a product for detecting diseases related to the secondary antibody.
6. The use according to claim 4 or 5, characterized in that: The diseases include syphilis caused by infection with Treponema pallidum.
7. The application according to claim 6, characterized in that The products include reagents, kits, test strips and probes.
8. A method for detecting Treponema pallidum antibodies in serum for non-diagnostic purposes, characterized in that: The following steps are involved: Bovine cardiolipin is used as an antigen to coat an ELISA plate, and the nanobody according to claim 1 is used as a secondary antibody for detection.
9. A method for screening a secondary antibody, comprising the following steps: 1) performing a first round of panning on the natural nanoantibody library to obtain rTpN15-17-47; 2) performing a second round of panning on the rTpN15-17-47 obtained in step 1) to obtain a phage solution; 3) mixing the phage solution obtained in step 2) with a Treponema pallidum antigen, infecting the mixture, and culturing the mixture to obtain a strain; 4) mixing the strain obtained in step 3) with a KM13 helper phage, infecting the mixture, and performing a first shaking culture and a first centrifugation on the obtained infection to obtain a first precipitate; resuspending the obtained first precipitate in a liquid culture medium, performing a second shaking culture, and performing a second centrifugation to obtain a second supernatant; The obtained second supernatant is mixed with the blocking solution, incubated and then indirectly detected; 5) plasmid extraction is performed on the strain reactive with the second antibody in step 4), and PCR amplification is performed using the plasmid as a template with a plasmid primer pair to obtain an antibody VHH fragment, and the antibody VHH fragment is connected to an expression vector to obtain a recombinant plasmid; 6) the recombinant plasmid obtained in step 5) is transformed into Escherichia coli to obtain an antibody expression strain, the antibody expression strain is induced, and the protein of the induced nanobody expression strain is extracted.
10. A method for preparing an engineered antibody, comprising the following steps: Using the antibody described in claim 9 as the basis for engineering modification, the screened secondary antibody is coupled with streptomycin to obtain SA-secondary antibody, and then biotin-labeled-secondary antibody is synthesized. The SA-secondary antibody and the biotin-labeled-secondary antibody are mixed in a ratio of 1:3 to obtain a tetravalent binding site-reactive antibody, which is used as a reaction reagent for subsequent detection of Treponema pallidum-antigen complex.