Anti-laminin monoclonal antibody and application thereof
By developing the highly specific and sensitive anti-laminarin monoclonal antibody HK02TJ and its chemiluminescence reagent kit, the shortcomings of existing laminarin detection methods have been overcome, enabling high-precision disease diagnosis and treatment.
Patent Information
- Application Number
- CN202511340394.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-19
- Publication Date
- 2025-12-12
AI Technical Summary
Existing methods for detecting laminin lack sensitivity and specificity, are easily affected by non-disease factors, and single-indicator detection is difficult to accurately reflect the disease state. There is a lack of effective treatments that target laminin.
We developed a highly specific and sensitive anti-lamin monoclonal antibody, HK02TJ, and a matching chemiluminescence reagent kit for high-precision detection and treatment of diseases mediated by laminin abnormalities.
It achieves high specificity and high sensitivity in the detection of laminin, with a detection limit of less than 0.1 ng/mL, supporting early diagnosis and personalized treatment of the disease.
Smart Images
Figure CN121108331A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of antibodies, specifically to an anti-laminar protein monoclonal antibody and its applications. Background Technology
[0002] In the biomedical field, laminin (LN), as a core component of the extracellular matrix, plays a crucial role in the development and progression of diseases. Currently, laminin detection is widely used in clinical diagnosis: in liver diseases, serum laminin levels are an important indicator for assessing the progression of liver fibrosis and cirrhosis; in oncology, the ability of tumor cells to degrade laminin and the abnormal expression of laminin in tissues can be used to determine the risk of tumor invasion and metastasis and prognosis; furthermore, in kidney diseases, cardiovascular diseases, and genetic diseases, abnormal laminin structure or expression also provides important evidence for pathological diagnosis.
[0003] However, current technologies related to laminin still have significant limitations. At the detection level, existing methods such as ELISA and radioimmunoassay suffer from insufficient sensitivity and specificity, are easily affected by non-disease factors such as inflammation and pregnancy, leading to false positives. Furthermore, single-indicator detection cannot accurately reflect the disease state, and there is a lack of effective combined analytical models with other biomarkers, hindering early diagnosis and the development of personalized treatment plans. At the treatment level, there are currently no effective treatments targeting laminin for diseases mediated by laminin abnormalities, such as tumor invasion and liver fibrosis progression. Therefore, developing highly specific and high-affinity anti-laminin antibodies, and based on these antibodies, developing novel detection technologies and treatment regimens, is of great significance for improving the accuracy of disease diagnosis and opening up new directions for treatment. Summary of the Invention
[0004] To overcome the above problems, the present invention provides a monoclonal antibody against laminin, HK02TJ, which has high specificity and sensitivity, and is characterized as follows: A monoclonal antibody HK02TJ targeting laminin binding is characterized in that it comprises three antigen complementarity-determining regions (CDRs) of the heavy chain variable region, namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region, namely LCDR1, LCDR2, and LCDR3. The specific amino acid sequences of these six CDRs are as follows: HCDR1 sequence as shown in SEQ ID NO:3; HCDR2 sequence as shown in SEQ ID NO:4; HCDR3 sequence as shown in SEQ ID NO:5; LCDR1 sequence as shown in SEQ ID NO:6; LCDR2 sequence as shown in SEQ ID NO:7; LCDR3 sequence as shown in SEQ ID NO:8; the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:1; and the amino acid sequence of the light chain variable region as shown in SEQ ID NO:2.
[0005] In addition, this invention also introduces a chemiluminescent reagent kit that can specifically detect laminin.
[0006] This invention provides an anti-laminarin monoclonal antibody HK02TJ. The detection kit prepared using the antibody has excellent accuracy and the detection sensitivity can reach below 0.1 ng / mL. Attached Figure Description
[0007] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0008] Figure 1 The image shows the purification process of the anti-lamin monoclonal antibody HK02TJ. Figure 2 This is a standard curve for the detection of laminin standards in the kit. Detailed Implementation
[0009] The embodiments are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
[0010] Example 1: Preparation of monoclonal antibodies.
[0011] Materials and reagents preparation.
[0012] Balb / c mice aged 6-8 weeks were selected as experimental animals. Recombinant human laminin with a purity greater than 95% was used as the antigen, and SP2 / 0 myeloma cell line was used in the experiment. The experiment also required reagents such as Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol (PEG), HAT selective medium, HT medium, fetal bovine serum, and ELISA kits, as well as instruments such as centrifuges, cell culture incubators, clean benches, microplate readers, and inverted microscopes.
[0013] Immunization program.
[0014] First, recombinant C was emulsified with Freund's complete adjuvant in a 1:1 ratio and used for primary immunization of mice, with each mouse receiving an intraperitoneal injection of 100 μg of the emulsified antigen. On day 21 post-primary immunization, a first booster immunization was performed using recombinant human laminin emulsified with Freund's incomplete adjuvant in a 1:1 ratio. Subsequent booster immunizations were performed every 14 days for a total of 3-4 days. A final booster immunization was performed 3-4 days before cell fusion, via tail vein injection of 50 μg of recombinant human laminin.
[0015] Cell fusion.
[0016] Three to four days after the final booster immunization, the spleens of immunized mice were aseptically removed, and spleen cell suspensions were prepared. Simultaneously, SP2 / 0 myeloma cells in logarithmic growth phase were prepared. Spleen cells and myeloma cells were mixed at a ratio of 5:1 to 10:1, and fusion was induced using PEG. After terminating the PEG treatment, the cells were resuspended in HAT selective medium and seeded into 96-well plates for culture.
[0017] Screening for positive hybridoma cells.
[0018] Seven to ten days after cell fusion, positive hybridoma cells secreting anti-laminin antibodies were screened using an indirect ELISA method. The initially screened positive hybridoma cells were then subjected to clonal culture to obtain pure clones of single cells. Commonly used clonal methods include limiting dilution and soft agar plate method; this example uses limiting dilution.
[0019] Large-scale production of monoclonal antibodies.
[0020] The selected positive hybridoma cells were prepared in large quantities using either in vivo induction or in vitro culture methods. In vivo induction involved inoculating positive hybridoma cells into the peritoneal cavity of syngeneic Balb / c mice, collecting the ascites fluid, and then purifying the antibody. In vitro culture involved expanding the positive hybridoma cells in cell culture flasks or bioreactors, collecting the supernatant, and then purifying the antibody.
[0021] Identification of monoclonal antibodies.
[0022] The titer of monoclonal antibodies was determined by indirect ELISA, and antibody specificity was identified by Western blotting and immunohistochemistry. Antibody subclasses were determined using a mouse monoclonal antibody subclass identification kit, and antibody affinity was determined by ELISA competition method.
[0023] Table 1. Results of titer assay for anti-laminarin monoclonal antibody HK02TJ.
[0024] Dilution factor 1:1000 1:3000 1:9000 1:27000 1:81000 1:243000 1:729000 Results (P / N) 11.19 9.45 8.12 5.37 3.01 1.91 1.02 A positive result is defined as P / N > 2 in the titer test. As shown in Table 1, the titer of the HK02TJ monoclonal antibody reached over 1:81000, indicating that the monoclonal antibody has good antigen affinity and can be used for subsequent experiments. The purified antibody SDS-PAGE electrophoresis image is shown in the attached instruction manual. Figure 1 As shown.
[0025] Example 2: Performance testing of the laminin detection kit.
[0026] 0.1 mL of 100 mg / mL magnetic microparticles (JSR, MS160 / Tosyl) with a particle size of 1.5 μm and 0.2 mg of monoclonal antibody HK02TJ were added to 1 mL of 0.5 M borate buffer, followed by 1 mL of 2 M ammonium sulfate buffer. After reacting at 37 °C for 24 hours, 0.1 mL of 10% BSA was added, and the reaction was continued at 37 °C for 12 hours. Finally, the mixture was magnetically separated and stored in 1 mL of 50 mM PBS buffer containing 10% BSA. This yielded the magnetic microparticles coated with monoclonal antibody HK02TJ.
[0027] The magnetic microparticles conjugated with the above-mentioned monoclonal antibody HK02TJ were diluted to 1 mg / mL and used as component 1. The purchased anti-laminar protein monoclonal antibody diluent conjugated with alkaline phosphatase was used as component 2. The recombinant laminar protein antigen was diluted to prepare the detection calibrator with a concentration gradient of 0 ng / mL, 1 ng / mL, 3 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. The antigen concentration was plotted on the x-axis and the corresponding luminescence value of each point of the calibrator was plotted on the y-axis using the four-parameter method. The standard curve is shown in Figure 2 of the instruction manual.
[0028] Minimum detection limit confirmed.
[0029] In this experimental method, a zero-concentration calibrator was selected as the sample for detection. Specifically, the sample was measured 20 times repeatedly, and the RLU value (relative luminescence value) was recorded after each measurement. Then, based on these 20 measurements, the mean (M) and standard deviation (SD) were calculated. The RLU value corresponding to M+2SD was then determined. Simultaneously, based on the concentration-luminescence value data between the zero-concentration calibrator and adjacent calibrators, a two-point regression was performed to derive a linear equation. Finally, the RLU value corresponding to M+2SD was substituted into this linear equation to solve for the corresponding concentration value. This concentration value is the limit of detection (LOD) for this experiment, which was calculated to be 0.082 ng / mL.
[0030] Electrophoresis of the purified antibody and performance analysis of the kit containing the magnetic microparticles coated with the monoclonal antibody HK02TJ showed that the monoclonal antibody described in this invention performs excellently in the detection of laminin and can be used as a target for further production and optimization.
Claims
1. A monoclonal antibody HK02TJ targeting and binding to laminin, characterized in that, The monoclonal antibody comprises three antigen complementarity-determining regions (CDRs) of the heavy chain variable region, namely HCDR1, HCDR2, and HCDR3, and three CDRs of the light chain variable region, namely LCDR1, LCDR2, and LCDR3; wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:3, the amino acid sequence of HCDR2 is shown in SEQ ID NO:4, the amino acid sequence of HCDR3 is shown in SEQ ID NO:5, the amino acid sequence of LCDR1 is shown in SEQ ID NO:6, the amino acid sequence of LCDR2 is shown in SEQ ID NO:7, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:
8.
2. The monoclonal antibody HK02TJ according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:
2.
3. A superparamagnetic bead conjugated with an antibody, characterized in that, The antibody conjugated to the superparamagnetic bead is the monoclonal antibody HK02TJ as described in claim 1.
4. A detection kit for specifically detecting laminin in human serum, characterized in that, The test kit contains the monoclonal antibody HK02TJ as described in claim 1.
5. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of laminin detection reagents.