An anti-pla2r antibody produced from a capture isolation method of reactive b cells

Abstract

The application belongs to the technical field of biology, and discloses a method for capturing and separating anti-PLA2R antibody-producing B cells. The method first constructs a double-target separation molecule with a targeting ability of CTLD1-CTLA4, captures B cells carrying anti-PLA2R antibodies in peripheral blood by using the CTLA4 domain of the double-target separation molecule, and realizes high-precision sorting by fluorescence resonance energy transfer principle after fluorescence labeling with the aid of specific recognition of the CTLD1 domain and cell surface antibodies. The single B cells sorted are further subjected to antibody gene cloning and recombinant expression, and finally, high-purity and high-affinity fully human anti-PLA2R antibodies are obtained. The method of the application not only realizes efficient capture of low-frequency self-reactive B cells in peripheral blood, but also successfully obtains fully human anti-PLA2R antibodies with high affinity and high specificity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for capturing and separating B cells that generate self-reactive anti-PLA2R antibodies. Background Technology

[0002] Primary membranous nephropathy (PMN) is a chronic kidney disease characterized by thickening of the glomerular basement membrane, primarily caused by an abnormal immune response. Phospholipase A2 receptor (PLA2R) and its antibodies play a crucial role in the pathogenesis of this disease. As the main autoantigen in PMN, PLA2R antibodies play a vital role in the deposition of glomerular immune complexes and activation of the complement system, thereby causing kidney damage and leading to proteinuria, hypoalbuminemia, and potentially progressing to chronic renal failure. Currently, due to the heterogeneity and widespread distribution of anti-PLA2R antibodies, existing antibody isolation and purification methods have certain limitations. Traditional isolation methods often fail to efficiently isolate specific anti-PLA2R antibodies, which significantly restricts in-depth research into the pathogenesis of PMN and the clinical application of anti-PLA2R antibodies.

[0003] As research deepens, more and more scientists are focusing on precisely enriching autoreactive B cells, especially those secreting anti-PLA2R antibodies, to further elucidate the pathogenesis of PMN. B cells play a crucial role in immune responses, especially in cases of immune system dysregulation. B cells mediate immune responses by producing autoantibodies, which often lead to glomerular damage. Huang et al. successfully sorted memory B cells from HIV-infected patients using cytokines and the memory B cell surface marker CD4, and screened for antibodies with highly efficient neutralizing capacity (DOI:10.1038 / nprot.2013.117). However, this method still has limitations, as overlap of B cell surface markers can lead to confusion between normal B cells and autoreactive B cells, further reducing the accuracy of sorting. Therefore, the efficient and specific isolation and enrichment of these autoreactive B cells will not only help elucidate the pathological mechanisms of PMN, but may also provide new means for the early diagnosis and targeted therapy of PMN.

[0004] In the development of B-cell sorting technology, various technical routes have been proposed and continuously improved. While traditional hybridoma technology is mature, it suffers from low fusion efficiency and long operation cycles. Single-B-cell screening technology, as a newly emerging method in recent years, can rapidly and accurately screen monoclonal antibodies with specific antigen-binding capabilities from immunized animals, offering advantages such as high efficiency, high specificity, and resource saving. In particular, the high-throughput single-B-cell screening method based on microfluidics developed by Zheng et al. (CN201310244248) combines cell phases and reagents, uses an immiscible oil phase to cut droplets, confirms fluorescence signals under a fluorescence microscope, and then applies an electric field to sort droplets with fluorescence intensity above a threshold, achieving high-throughput single-B-cell screening. Furthermore, Wang et al.'s B-cell screening method for small-molecule monoclonal antibody preparation (CN26752886) uses hapten-fluorescein labeling of specific B cells and rapid sorting using flow cytometry, pre-screening fused cells and removing B cells that recognize carrier proteins, significantly improving the positive rate of B cells recognizing small molecules. However, single B cell surface markers are often unable to effectively distinguish between autoreactive B cells and normal B cells, leading to cross-contamination.

[0005] To address this issue, researchers have gradually introduced the concept of dual-target separation molecules, using multiple targets to improve the specificity and accuracy of sorting. Dual-target separation molecules are those capable of simultaneously targeting two different target molecules; this molecular strategy can effectively identify and separate target cells. For example, Wang et al. proposed a method for obtaining autoreactive B cells (CN104232577A) using biotin-hinge dsDNA and streptavidin-based immunomagnetic beads to effectively sort high-purity autoreactive B cells. However, this method still faces challenges, particularly in the selection and construction of dual-target separation molecules; accurately selecting targets that can effectively bind and enhance specificity is a key issue.

[0006] To further improve sorting accuracy, the performance of dual-target separation molecules in cell sorting is enhanced through the Fluorescence Resonance Energy Transfer (FRET) principle. Using flow cytometry combined with the FRET principle, dual-target separation molecules can accurately detect and sort self-reactive B cells at the cellular level. FRET technology relies on energy transfer between two fluorescent probes; when the distance between two fluorescent molecules is very close, energy is transferred from one molecule to the other. This characteristic allows for precise identification of molecular binding at the cellular level. Furthermore, the construction and optimization of dual-target separation molecules is currently a hot research topic. Studies have shown that strain-promoted azido-alkyne cycloaddition reactions (SPAAC) can link different functional domains together to construct separation molecules with dual-targeting capabilities. These dual-target separation molecules not only improve sorting efficiency but also enhance the recognition of target cells, providing a powerful tool for PMN specific antibody screening and B cell isolation. Summary of the Invention

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for capturing, separating, and characterizing functional antibodies against anti-PLA2R antibodies generated from self-reactive B cells. This method is characterized by its simple preparation process, high efficiency, and high specificity, and can effectively overcome the limitations of traditional separation methods.

[0008] The technical solution of the present invention: A method for capturing and separating self-reactive B cells generated by anti-PLA2R antibody, comprising the following steps: Step 1: Expression and purification of dual-target isolated monomeric proteins; The expression cells were Escherichia coli Rosetta (DE3) strain, the expression vector was PET28a(+), and the expressed proteins were cytotoxic T lymphocyte-associated protein 4 (CTLA4) and phospholipase A2 receptor domain C-type lectin-like domain 1 (CTLD1). Recombinant plasmids pET-28a-CTLA4 and pET-28a-CTLD1 were transformed into Escherichia coli Rosetta (DE3) strain using heat shock transformation. Expression was induced for 12 h at 16 °C and 200 rpm with 0.25 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The proteins CTLA4 and CTLD1 were obtained by affinity chromatography and Superdex 75 gel sieving. The CTLA4 protein specifically binds to the B7 molecule on the surface of B cells, and the CTLD1 protein specifically binds to the anti-PLA2R antibody on the surface of autoreactive B cells. Step 2: The primary amine groups on the surface of the protein are activated by amino groups, and a dual-target separation molecule is synthesized using the SPAAC reaction; CTLD1 protein was reacted with DBCO-PEG5-NHS ester, and CTLA4 protein with N3-PEG4-C2-NHS ester, respectively, at 25℃ and pH 7.0-9.0 for 45 min. The molar ratio of DBCO-PEG5-NHS ester to CTLD1 protein was controlled at 7.5:1, and the molar ratio of N3-PEG4-C2-NHS ester to CTLA4 protein was controlled at 2:1, so that each protein molecule was activated by an average of one primary amine group, thus obtaining DBCO-modified CTLD1 protein and N3-modified CTLA4 protein, respectively. Since the N3 group has no characteristic peak in the UV spectrum, the activation efficiency was quantitatively monitored using the characteristic UV absorption peak of the DBCO group at 309 nm. After the reaction, unreacted activating reagents were removed by ultrafiltration. DBCO-modified CTLD1 protein and N3-modified CTLA4 protein were mixed in a 1:1 molar ratio and reacted overnight at 4°C. The CTLD1-CTLA4 dual-target separation molecule was constructed by SPAAC reaction. The reaction product was purified by Strep-Tactin affinity chromatography to obtain the CTLD1-CTLA4 dual-target separation molecule with a purity ≥95%. Step 3: Flow cytometry is used to separate autoreactive B cells from peripheral blood; CTLD1 and CTLA4 proteins were fluorescently labeled with Cy3 NHS ester and Cy5 SE triethylamine salt, respectively, at a protein-to-fluorescent-dye molar ratio of 1:5. The reaction was carried out at room temperature for 1 h, and unreacted dye was removed by ultrafiltration to obtain Cy3-CTLD1 and Cy5-CTLA4, respectively. Cy3-CTLD1 and Cy5-CTLA4 were then mixed at a 1:1 molar ratio and reacted overnight at 4 °C. The Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecule was constructed by SPAAC reaction. Peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood using Ficoll density gradient centrifugation at 800×g for 20 min. The interfacial lymphomonocytes were washed 1-2 times with RPMI 1640 medium to obtain a PBMC suspension, which was then adjusted to a density of 1×10⁻⁶. 6 cells / mL, used for subsequent sorting experiments.

[0009] Flow cytometry was used to sort autoreactive B cells in peripheral blood. Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecules were incubated with PBMCs at 37°C and 5% CO2 for 4 h to allow the dual-target separation molecules to specifically bind to receptors and autoantibodies on the surface of autoreactive B cells. The BD FACSAria III flow cytometer was used for sorting, with 561 nm laser excitation and FRET signal detection at 670 nm wavelength to sort out FRET-positive autoreactive B cells.

[0010] The beneficial effects of this invention are as follows: This invention effectively achieves the isolation of autoreactive B cells from PMN patients. This method allows for the rapid and accurate enrichment of autoreactive B cells with specific anti-PLA2R antibodies from patient samples, providing a reliable cell source for subsequent immunological analysis, antibody screening, and the development of potential treatment strategies. It also provides a solution for the enrichment of autoreactive B cells in future autoimmune diseases. Attached Figure Description

[0011] Figure 1 To analyze changes in the binding ability of dual-target separated molecules to the CTLD1 terminus using enzyme-linked immunosorbent assay (ELISA).

[0012] Figure 2 The results show the changes in the binding ability of the CTLA4 end of the dual-target separation molecule using biomembrane layer interferometry (BLI). In this study, A represents the binding kinetics of CTLA4 and anti-CTLA4 nanobodies at different concentrations, and B represents the kinetic analysis between CTLD1-CTLA4 and anti-CTLA4 nanobodies.

[0013] Figure 3 The effects of fluorescent labeling on the binding ability of CTLD1 and CTLA4 to antibodies are shown in Figure A, where A represents the effect of fluorescent labeling on the binding ability of CTLD1 to antibodies as analyzed by ELISA; and B represents the effect of fluorescent labeling on the binding ability of CTLA4 to antibodies as analyzed by ELISA.

[0014] Figure 4 To verify the construction of dual-target separation molecules based on the FRET principle, CTLD1-CTLA4 underwent a fluorescence transition after binding with the corresponding antibody, and reached the highest fluorescence intensity near 670 nm.

[0015] Figure 5To capture autoreactive B cells for CTLD1-CTLA4, the PE channel (561 nm) meets the excitation requirements of CY3 and can be detected at a wavelength of 582 nm; the excitation light of APC (633 nm) meets the excitation requirements of CY5 and can be detected at a wavelength of 660 nm; the FRET signal requires excitation at 561 nm and detection at 660 nm, and the excitation and emission wavelengths of the PE-Cy5 channel meet the screening conditions for FRET signals. Region Q1 represents a cell population labeled only with CY5-CTLA4; region Q2 represents a double-positive cell population labeled with both CY3-CTLD1 and CY5-CTLA4; region Q3 represents a cell population labeled only with CY3-CTLD1; and region Q4 represents a double-negative population.

[0016] Figure 6 This is a characterization of the capture rate of molecules separated by dual targets using ELISA.

[0017] Figure 7 To verify the BLI affinity of the anti-PLA2R antibody to the PLA2R single-domain CTLD1 protein. Detailed Implementation

[0018] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0019] Example 1: Expression, purification and characterization of recombinant CTLA4 and recombinant CTLD1 (1) The recombinant plasmids pET-28a-CTLA4 and pET-28a-CTLD1 were introduced into *Escherichia coli* Rosetta (DE3) strain via heat shock transformation. The plasmids were plated on LB agar plates containing kanamycin and cultured overnight at 37°C to screen for positive clones. Positive clones were picked and inoculated into 5 mL of LB liquid medium (containing kanamycin) and cultured at 37°C and 200 r / min for 12 h to activate the strain. The activated strain was then transferred to 100 mL of TB medium and cultured at 37°C and 200 r / min until OD was reached. 600=0.8-1.0, add IPTG to a final concentration of 0.25 mM, and induce expression at 16℃ for 12 h. Collect the induced bacteria by centrifugation at 3200×g, add 1×PBS containing 20 mM imidazole, homogenize by high pressure, centrifuge at 10000×g for 30 min, and collect the supernatant. CTLA4 protein was purified by nickel column affinity chromatography, and CTLD1 protein was purified by Strep-Tactin affinity chromatography. Further purification was performed by Superdex 75 gel sieving to obtain recombinant CTLA4 and recombinant CTLD1 proteins with a purity ≥95%. SDS-PAGE electrophoresis was used to detect the expression and purification status of the recombinant proteins. After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using wet transfer, blocked, and incubated with primary antibody. Horseradish peroxidase (HRP)-labeled secondary antibody was added, and protein band images were captured by chemiluminescence imaging. Semi-quantitative analysis of CTLA4 and CTLD1 protein expression levels was performed using standard proteins.

[0020] (2) The purity of recombinant CTLA4 and recombinant CTLD1 was characterized using an AdvanceBio SEC 130 Å column, a flow rate of 0.35 mL / min, and a detection wavelength of 280 nm.

[0021] (3) The recombinant CTLA4 protein and the recombinant PLA2R domain CTLD1 were diluted to 0.1 mg / mL with phosphate-buffered saline (PBS). The CD spectra of recombinant CTLA4 and recombinant CTLD1 were measured at 200-260 nm using a circular dichroism spectrometer. A quartz cuvette with a 1 mm optical path was used, the slit width was set to 2 nm, the scanning range was 200-260 nm, and the acquisition period was set to 1 s / point.

[0022] Example 2: Synthesis and purification of CTLD1-CTLA4 capture molecules (1) The primary amine groups on the surface of CTLD1 and CTLA4 proteins were modified at specific sites using NHS ester reagent. The molar ratio gradients of DBCO-PEG5-NHS ester to CTLD1 protein were set to 1:1, 2.5:1, 5:1, 7.5:1, 10:1, 15:1, 20:1, and 25:1, and the molar ratio gradients of N3-PEG4-C2-NHS ester to CTLA4 protein were set to 1:1, 2:1, 4:1, 6:1, 8:1, and 10:1. The reactions were carried out at 25℃ and pH 7.0-9.0 for 45 min, with the volume of DMSO controlled to not exceed 3% of the total reaction volume. The activation efficiency was quantitatively monitored using the characteristic UV absorption peak of the DBCO group at 309 nm. The UV absorbance A of each reaction system at 280 nm and 309 nm was measured using a microplate reader. 280 and A309 Calculate the number of activated primary amine groups using the formula: The molar extinction coefficient ε of the CTLD1 protein at 280 nm is shown in the figure. 280 The value was 44835 for CTLD1 and 26400 for CTLA4. The results indicate that the optimal molar ratio for CTLD1 to activate one primary amine group on average is 7.5:1, while the optimal molar ratio for CTLA4 to activate one primary amine group on average is 2:1.

[0023] (2) Weigh 1 mg of CTLD1 protein and 1 mg of CTLA4 protein respectively, and dissolve them in 2 mL of 1×PBS (pH 7.4). Add 0.44 μmol of DBCO-PEG5-NHS ester (molar ratio 7.5:1) to the CTLD1 protein solution and add 3.99 μmol of N3-PEG4-C2-NHS ester (molar ratio 2:1) to the CTLA4 protein solution, and react at 25℃ for 45 min. After the reaction is complete, perform three ultrafiltrations using an ultrafiltration tube to remove unreacted activating reagents, and obtain DBCO-modified CTLD1 protein and N3-modified CTLA4 protein respectively. Dilute the CTLD1 protein and CTLA4 protein solutions before and after amino modification with PBS to 0.1 mg / mL. Measure the circular dichroism using a circular dichroism spectrometer, using a quartz cuvette with a 1 mm path length, setting the slit width to 2 nm, the scanning range to 200-260 nm, the step size to 1 nm, and the acquisition period to 1 s / point.

[0024] (3) DBCO-modified CTLD1 protein and N3-modified CTLA4 protein were mixed at a 1:1 molar ratio and reacted overnight at 4°C. The CTLD1-CTLA4 dual-target separation molecule was constructed by SPAAC reaction. The reaction product was purified by Strep-Tactin affinity chromatography: a StrepTrap XT pre-packed column was used, equilibrated with equilibration buffer (50 mM sodium phosphate, 280 mM NaCl, 6 mM potassium chloride, pH 7.4), and eluted with 2.5 mM dethiobiotin. The elution peak was collected to obtain the CTLD1-CTLA4 dual-target separation molecule with a purity ≥95%. An AdvanceBio SEC 130 Å 2.7 μm column was used, with 1×PBS containing 0.02% sodium azide (NaN3) as the mobile phase, a flow rate of 0.35 mL / min, and a detection wavelength of 280 nm. The purified CTLD1-CTLA4 dual-target separated molecules were filtered through a 0.22 μm filter membrane and then injected to analyze the protein purity.

[0025] Example 3: Biological function testing of the dual-target isolated molecule CTLD1-CTLA4 (1) Dilute recombinant CTLD1 protein to 1 μg / mL with PBS, and dilute CTLD1-CTLA4 dual-target molecules to 2 μg / mL with PBS. Add 100 μL to each well of a 96-well ELISA plate and incubate at 25°C and 200 rpm for 2 h to fix the antigen on the plate surface. Wash the plate 5 times with 1×TBST, 300 μL per well each time, and incubate for 1 min. After washing, blot the plate dry. Add 100 μL of 5% skim milk powder to each well and incubate at 25°C and 200 rpm for 2 h to block non-specific binding sites. Wash the plate 5 times with 1×TBST, 300 μL per well each time, and incubate for 1 min. After washing, blot the plate dry.

[0026] Plasma from patients with high antibody titers (anti-PLA2R titer of 870 RU / mL), medium antibody titers (anti-PLA2R titer of 124 RU / mL), and no antibody titers (anti-PLA2R titer less than 20 RU / mL) was diluted 2000-fold with 5% skim milk powder, and 100 μL was added to each well. The plates were incubated overnight at 4°C. The plates were washed 5 times with 1×TBST, 300 μL per well each time, and incubated for 1 min. After washing, the plates were dried. HRP-labeled goat anti-human antibody was diluted 10000-fold with 5% skim milk powder, and 100 μL was added to each well. The plates were incubated at 25°C and 200 rpm for 2 h. The plates were washed 5 times with 1×TBST, 300 μL per well each time, and incubated for 1 min. After washing, the plates were dried. 100 μL of TMB single-component chromogenic solution was added to each well, and the plates were incubated at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well, and immediately read the absorbance at 450 nm and 630 nm using a microplate reader. Results are as follows: Figure 1 As shown, the results indicate that the binding affinity of the CTLD1-CTLA4 dual-target separated molecule to anti-PLA2R antibodies in patient plasma is 1.068 times that of the CTLD1 monomer, which is significantly higher than that of the CTLD1 monomer, indicating that the introduction of CTLA4 promotes the antibody affinity of CTLD1.

[0027] (2) Anti-CTLA4 nanobody immobilization: Anti-CTLA4 nanobody was immobilized on the Octet® AR2G sensor. The AR2G biosensor has a carboxylated surface, which can covalently immobilize amino-containing biomolecules. First, the sensor was immersed in EDC / NHS activation solution to activate the carboxyl groups on the sensor surface; then, the activated sensor was immersed in the Anti-CTLA4 nanobody solution, so that the amino groups of the Anti-CTLA4 nanobody formed covalent bonds with the activated carboxyl groups on the sensor surface, achieving stable protein immobilization; finally, the sensor was immersed in ethanolamine solution to block unreacted carboxyl groups and avoid non-specific binding.

[0028] The AR2G sensor immobilized with the Anti-CTLA4 nanobody was immersed in 1×PBS buffer to set a baseline. Then, the sensor was immersed in either a CTLD1-CTLA4 dual-target separation molecule solution or a CTLA4 monomeric protein solution, and the binding curve was recorded. After binding, the sensor was immersed in dissociation buffer, and the dissociation curve was recorded. Concentration gradients of CTLA4 monomeric protein were set at 83.2 nM, 166.3 nM, 332.5 nM, and 665.0 nM, and concentration gradients of CTLD1-CTLA4 dual-target separation molecules were set at 172.0 nM, 225.3 nM, 305.2 nM, and 652.4 nM, with parallel controls at each concentration.

[0029] The binding dissociation curves were fitted and analyzed using Octet Analysis Studio software, and the results are as follows: Figure 2 As shown, the results indicate that the affinity between the CTLD1-CTLA4 dual-target separation molecule and the Anti-CTLA4 nanobody is 2.219 × 10⁻⁶. - 9 M, higher than the affinity of CTLA4 monomer protein for Anti-CTLA4 nanobody (6.907×10). -8 M), indicating that the CTLA4 end of the dual-target separation molecule has a strong binding ability.

[0030] (3) CTLD1 protein was fluorescently labeled using Cy3 NHS ester, and CTLA4 protein was fluorescently labeled using Cy5 SE triethylamine salt. The molar ratio of protein to fluorescent dye was 1:5. The reaction was carried out at room temperature for 1 h. Unreacted dye was removed by ultrafiltration to obtain Cy3-CTLD1 and Cy5-CTLA4 fluorescently labeled proteins. The binding ability of the protein to the antibody before and after fluorescent labeling was compared by ELISA. The results are as follows: Figure 3 As shown, the results indicate that the binding ability of CTLD1 and CTLA4 proteins to the antibody was slightly lower after fluorescent labeling than that of unlabeled proteins, but the difference was statistically significant (P<0.05), and they still maintained a strong binding ability. This suggests that fluorescent labeling has little effect on protein binding function and can be used for subsequent cell sorting experiments.

[0031] (4) Cy3-labeled CTLD1 protein and Cy5-labeled CTLA4 protein were mixed at a 1:1 molar ratio and reacted overnight at 4°C. The resulting Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecule was constructed via SPAAC reaction. The reaction product was purified by Strep-Tactin affinity chromatography to obtain a fluorescently labeled dual-target separation molecule with a purity ≥95%. This fluorescently labeled dual-target separation molecule exhibited high specificity and stability in subsequent flow cytometry sorting and can be used for the precise sorting of autoreactive B cells.

[0032] (5) Anti-PLA2R antibody and anti-6×His antibody were pre-coated in polystyrene black well plates. 5% BSA blocking buffer was added, and the plates were blocked at room temperature for 30 min. After washing with PBST, Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecules were added, and the plates were incubated for 1 h. The emission signal was measured using a microplate reader at 550 nm excitation, and the fluorescence intensity at 670 nm was recorded as follows: Figure 4 As shown, the results indicate that Cy3-CTLD1-CTLA4-Cy5 undergoes a fluorescence transition after binding with the corresponding antibody, reaching the highest fluorescence intensity near 670 nm, and the FRET signal is significantly enhanced, proving that the dual-target separation molecule was successfully constructed and that both functional ends can bind to the corresponding target sites simultaneously.

[0033] Example 4: Flow cytometry sorting of autoreactive B cells (1) Collect human peripheral blood and mix it with an equal volume of PBS buffer containing 2% fetal bovine serum. Carefully spread the mixture on the upper layer of Ficoll lymphocyte separation medium and centrifuge at 800×g for 20 min. After centrifugation, discard the upper plasma layer. The white cell layer is the isolated lymphomonocytes. Add 1 mL of RPMI 1640 medium to the lymphomonocytes and wash them by centrifugation at 1000×g for 10 min. Repeat the washing twice. Resuspend the cells in RPMI 1640 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution) and adjust the cell density to 1×10⁻⁶. 6 cells / mL, take 8×10 6Cells were divided into three groups on average: a blank group (no fluorescent labeling molecule incubation), a control group (incubated with 50 μg Cy3-CTLD1 and 50 μg Cy5-CTLA4 monomeric fluorescent proteins per million cells), and an experimental group (incubated with 50 μg Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecules per million cells). Cells in each group were incubated at 37℃ in a 5% CO2 incubator for 4 h in the dark. After staining, the cells were washed three times with RPMI 1640 complete medium, centrifuged at 1500 rpm for 3 min to remove unbound fluorescently labeled molecules, filtered through a 300-mesh filter to remove cell aggregates, and the cell suspension was transferred to flow cytometry tubes.

[0034] (2) Flow cytometer setup and sorting Cell sorting was performed using a BD FACSAria III flow cytometer. Cy3 labeling was performed using a 561 nm yellow laser excitation and detected at 582 nm; Cy5 labeling was performed using a 633 nm red laser excitation and detected at 660 nm; and FRET signals were performed using a 561 nm laser excitation and detected at 670 nm.

[0035] First, single-cell populations (P1 gate) were selected using forward scatter (FSC) and side scatter (SSC) scatter plots to exclude dead cells and cell aggregates. Then, Cy3 and Cy5 fluorescence signals were analyzed in the PE-A / APC-A scatter plot to exclude single-positive and double-negative cell populations. Finally, FRET signals were detected in the PE-Cy5-A histogram, and FRET-positive cells (P2 gate) were selected, representing autoreactive B cells that bind to the dual-target separation molecules. The selected cells were collected in 96-well plates containing 200 μL of RPMI 1640 complete medium, one cell per well. Figure 5 The results of self-reactive B cell sorting obtained by signal monitoring using flow cytometry were demonstrated, proving the effectiveness of this method in highly specific B cell sorting. Combining FRET signaling with flow cytometry improves sorting precision and reduces interference from non-specific cells, thereby enhancing the specificity and accuracy of sorting.

[0036] Example 5: Validation of autoreactive B cells (1) Cells sorted in Example 4 were subjected to ELISA to verify and evaluate the capture efficiency of the CTLD1-CTLA4 dual-target separation molecules. Cell lysis buffer (1 mL 2 M Tris-HCl pH 8.0, 0.85 mL RNase inhibitor, and DEPC water to a final volume of 66 mL) was prepared, and 60-80 μL of lysis buffer was added to each well of a 96-well plate to lyse single B cells. The ELISA plate was coated with the cell lysis buffer as the primary antibody and CTLD1 protein (1 μg / mL) as the capture antigen. HRP-labeled goat anti-human antibody was used as the secondary antibody. After TMB color development, OD was measured. 450 Value. Result as follows Figure 6 As shown, the capture rate of CTLD1-CTLA4 separated molecules is demonstrated, verifying the high efficiency and accuracy of dual-target separated molecules in the sorting of self-reactive B cells.

[0037] (2) Single-cell PCR was performed on the wells with the highest ELISA positive rate to clone the anti-PLA2R antibody gene. The RT-PCR reaction system was prepared as follows: 5 μL 5× buffer, 1 μL dNTP (400 μM), 0.75 μL each of mixed primers (heavy chain primer and light chain primer), 1 μL RT-PCR enzyme, 0.5 μL RNase inhibitor, 4 μL B cell lysis buffer, and DEPC water to a final volume of 25 μL. Reaction conditions: incubation at 50℃ for 30 min; treatment at 95℃ for 15 min; 40 cycles (denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 1 min).

[0038] After verification by agarose gel electrophoresis, the RT-PCR products were subjected to a second round of PCR to obtain more specific sequences. The reaction mixture consisted of: 19.75 μL DEPC water, 0.5 μL dNTPs, 0.5 μL each primer, 2.5 μL 10×Buffer, and 0.25 μL Taq enzyme. The PCR products were verified by agarose gel electrophoresis, yielding specific bands. The heavy chain variable region gene VH was approximately 350-400 bp in size, and the light chain variable region gene VL was approximately 300-350 bp in size. The PCR products were then sent to a sequencing company for DNA sequencing to obtain the sequence information of the cloned heavy chain variable region gene VH and light chain variable region gene VL.

[0039] Example 6: Construction and expression of anti-PLA2R recombinant antibody (1) The heavy chain and light chain variable regions of the present invention are cloned into plasmids containing the heavy chain constant region of human IgG4 and the light chain constant region of κ. Different combinations of light and heavy chain plasmids can be transfected into cells to produce humanized antibodies.

[0040] (2) The heavy chain vector is designed as follows: signal peptide + heavy chain variable region sequence + human IgG constant region sequence.

[0041] (3) The light chain vector is designed as follows: signal peptide + light chain variable region sequence + human Kappa constant region sequence.

[0042] (4) Insert the above sequences into the pFUSE vector to construct the heavy chain expression plasmid pFUSE-HC and the light chain expression plasmid pFUSE-LC. Synthesize the expression vectors according to the above design, and after obtaining the vector plasmids, perform large-scale plasmid extraction. Co-transfect the heavy chain expression plasmid and the light chain expression plasmid into human embryonic kidney epithelial cells (293F cells) at a mass ratio of 1:1. Culture the 293F cells in serum-free medium until the density reaches 1.2 × 10⁶ cells / year. 6 The logarithmic growth phase (cells / mL) was used for cell transfection. 18 μg of humanized antibody light chain plasmid and 18 μg of humanized antibody heavy chain plasmid were dissolved in 200 μl of serum-free culture medium and mixed well. Then, 108 μl of polyethyleneimine (PEI, 1 mg / mL) was added and mixed well. After incubation for 15 min, the mixture was added to 30 mL of 293F cells. Cell culture conditions: 5% CO2, 37℃, 90 rpm / min. Cell viability and density were monitored daily during culture. After 5-7 days of culture, the cell density reached 7 × 10⁶ cells / mL. 6 Cells / mL, cell supernatant was collected. The supernatant was centrifuged at 4000×g for 20 min to remove cell debris, filtered through a 0.22 μm filter membrane, and the antibody was purified by Protein A affinity chromatography: the Protein A column was equilibrated with binding buffer (20 mM PBS, pH 7.0), loaded with the sample, washed to remove contaminating proteins, eluted with elution buffer (0.1 M glycine-hydrochloric acid, pH 3.0), and immediately neutralized with neutralization buffer (1 M Tris-HCl, pH 8.0) to obtain purified anti-PLA2R recombinant antibody.

[0043] The heavy and light chain variable region sequences of the anti-PLA2R antibody are as follows: The SEQ ID NO: 1 of hCHIgG is: QLVESGGGLVQPGGSLRLSVQPSGFTFSSYGMHWVRQAPGKGLEWVSVIYGDGSTIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYDSSGYYYCYYGMDVWGKGTTVTVSS hCK's SEQ ID NO: 2 is: LVALLGQPVSISCRSSQSLLHSNGGTYLSWYQQKPGKAPKLLIYGASNLASGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVEIK Example 7: Functional Verification of Recombinant Antibody (1) The purified anti-PLA2R recombinant antibody samples were separated by SDS-PAGE electrophoresis. The separating gel concentration was 10%, the stacking gel concentration was 5%, and the electrophoresis voltage was 120 V. After electrophoresis, the protein was transferred to a PVDF membrane using wet transfer and blocked with rapid blocking buffer for 20 min. Goat anti-human IgG Fc antibody (1:5000 dilution) was added and incubated at 4℃ for 12 h. After washing with TBST, HRP-labeled donkey anti-goat IgG (H+L) (1:10000 dilution) was added and incubated at room temperature for 2 h. After washing with TBST, the protein bands were captured using a chemiluminescent imaging system after development with a chemiluminescent substrate.

[0044] (2) The antigen-binding function of the anti-PLA2R recombinant antibody was characterized by ELISA. Recombinant PLA2R protein was diluted to 1 μg / mL with PBS and coated onto 96-well ELISA plates (100 μL per well) overnight at 4°C. The plates were washed three times with 1×TBST and blocked with 5% skim milk at 37°C for 2 h. Serially diluted anti-PLA2R recombinant antibody (starting concentration 10 μg / mL, 2-fold serial dilution) was added and incubated at 37°C for 1 h. The plates were washed three times with TBST, and HRP-labeled goat anti-human IgG Fc antibody (1:5000 dilution) was added and incubated at 37°C for 1 h. The plates were washed three times with TBST, and TMB chromogenic solution was added and incubated at 37°C in the dark for 15 min. Stop solution was added, and OD was measured. 450 value.

[0045] (3) The affinity between the anti-PLA2R recombinant antibody and CTLD1 was determined using BLI technology. The purified recombinant anti-PLA2R antibody was captured by its Fc fragment against Protein A immobilized on the surface of the Octet® Protein A biosensor. The antibody was then directionally immobilized on the sensor chip. After baseline stabilization, a series of concentrations of CTLD1 protein (256 nM, 512 nM, 1024 nM, 2048 nM) were sequentially passed through the antibody-immobilized sensor surface. The binding time was 180 s, and the dissociation time was 300 s. The instrument monitored the changes in the interference spectrum during the binding and dissociation processes in real time. The binding and dissociation curves were fitted using Octet Analysis Studio software, and the kinetic parameters were calculated. The results are as follows: Figure 7As shown in Table 1, the results indicate that the binding of the anti-PLA2R recombinant antibody to CTLD1 is a high-affinity binding, demonstrating that the recombinant antibody has specific and high-affinity binding characteristics to the target antigen.

[0046] Table 1. BLI results of anti-PLA2R recombinant antibody and CTLD1

Claims

1. A method for capturing and separating B cells generated by anti-PLA2R antibody self-reacting, characterized in that, The steps are as follows: Step 1: Expression and purification of dual-target isolated monomeric proteins; Step 2: The primary amine groups on the surface of the protein are activated by amino groups, and a dual-target separation molecule is synthesized using the SPAAC reaction; CTLD1 protein was reacted with DBCO-PEG5-NHS ester, and CTLA4 protein was reacted with N3-PEG4-C2-NHS ester at 25℃ and pH 7.0-9.0 for 45 min, respectively. The molar ratio of DBCO-PEG5-NHS ester to CTLD1 protein was controlled at 7.5:1, and the molar ratio of N3-PEG4-C2-NHS ester to CTLA4 protein was controlled at 2:1, so that each protein molecule was activated by an average of one primary amine group, thus obtaining DBCO-modified CTLD1 protein and N3-modified CTLA4 protein, respectively. The activation efficiency was quantitatively monitored using the characteristic UV absorption peak of the DBCO group at 309 nm. After the reaction was completed, the unreacted activating reagent was removed by ultrafiltration. DBCO-modified CTLD1 protein and N3-modified CTLA4 protein were mixed in a 1:1 molar ratio and reacted overnight at 4°C. The CTLD1-CTLA4 dual-target separation molecule was constructed by SPAAC reaction. The reaction product was purified by Strep-Tactin affinity chromatography to obtain the CTLD1-CTLA4 dual-target separation molecule with a purity ≥95%. Step 3: Flow cytometry is used to separate autoreactive B cells from peripheral blood; CTLD1 and CTLA4 proteins were fluorescently labeled with Cy3 NHS ester and Cy5 SE triethylamine salt, respectively, at a protein-to-fluorescent-dye molar ratio of 1:

5. The reaction was carried out at room temperature for 1 h, and unreacted dye was removed by ultrafiltration to obtain Cy3-CTLD1 and Cy5-CTLA4, respectively. Cy3-CTLD1 and Cy5-CTLA4 were then mixed at a 1:1 molar ratio and reacted overnight at 4 °C. The Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecule was constructed by SPAAC reaction. Peripheral blood mononuclear cells (PBMCs) were extracted from peripheral blood using Ficoll density gradient centrifugation at 800×g for 20 min. The interfacial lymphomonocytes were washed 1-2 times with RPMI 1640 medium to obtain a PBMC suspension, which was then adjusted to a density of 1×10⁻⁶. 6 cells / mL; Flow cytometry was used to sort autoreactive B cells in peripheral blood. Cy3-CTLD1-CTLA4-Cy5 fluorescently labeled dual-target separation molecules were incubated with PBMCs at 37°C and 5% CO2 for 4 h, allowing the CTLD1-CTLA4 dual-target separation molecules to specifically bind to receptors and autoantibodies on the surface of autoreactive B cells. The BD FACSAria III flow cytometer was used for sorting, with 561 nm laser excitation and FRET signal detection at 670 nm wavelength, to identify FRET-positive autoreactive B cells.

2. The method for capturing and separating B cells generated by anti-PLA2R antibody according to claim 1, characterized in that, The specific process of step 1 is as follows: The expression cells were Escherichia coli Rosetta (DE3) strain, the expression vector was PET28a(+), and the expressed proteins were cytotoxic T lymphocyte-associated protein 4, namely CTLA4, and phospholipase A2 receptor domain C-type lectin-like domain 1, namely CTLD1. Recombinant plasmids pET-28a-CTLA4 and pET-28a-CTLD1 were transformed into Escherichia coli Rosetta (DE3) strain using heat shock transformation. After expression was induced for 12 h with 0.25 mM isopropyl-β-D-thiogalactopyranoside at 16 °C and 200 rpm, the proteins CTLA4 and CTLD1 were purified by affinity chromatography and Superdex 75 gel sieving to obtain the proteins respectively. The CTLA4 protein specifically binds to B7 molecules on the surface of B cells, and the CTLD1 protein specifically binds to anti-PLA2R antibodies on the surface of autoreactive B cells.

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