Monoclonal antibody 3C7 for BK virus VP1 protein detection and application

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody 3C7 for BK virus VP1 protein detection and application. The amino acid sequences of heavy chain variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody are respectively SEQ ID NO.1-3, and the amino acid sequences of light chain variable regions CDR1, CDR2 and CDR3 of the monoclonal antibody are respectively SEQ ID NO.6-6. The antibody has high specificity and can recognize the BK virus VP1 protein. The test strip comprises a back plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane and absorbent paper. The detection method provided by the invention is simple and convenient to operate and high in sensitivity, does not need instruments, realizes rapid and visual detection of the BK virus VP1 in urine or a recombinant protein sample, is suitable for on-site screening and instant detection, and can be used as a supplement of existing detection means such as nucleic acid and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and particularly relates to a monoclonal antibody 3C7 for detecting BK virus VP1 protein and its application. Background Art

[0002] BK virus, also known as BK polyomavirus (BKV), belongs to the Polyomaviridae family and is a non-enveloped, double-stranded DNA virus. The BKV genome is approximately 5.3 kb long and encodes three structural proteins (VP1, VP2, and VP3), with VP1 being the predominant protein on the viral surface. BKV can be divided into types I, II, III, and IV based on differences in the VP1 gene.

[0003] The initial BK virus infection usually occurs in childhood and can be effectively controlled by the immune system. Therefore, BK virus hardly causes any disease in healthy people with normal immune function. However, in individuals with severely impaired immune function, such as high-risk groups who have undergone transplantation, especially kidney transplantation and hematopoietic stem cell transplantation (HSCT), BK virus may be reactivated and replicate in large quantities, leading to serious consequences.

[0004] In kidney transplant patients, long-term immunosuppressive therapy is often required to prevent rejection, making them susceptible to infection with various pathogens, including BK virus. BK virus is common in the general population but is generally harmless in immunocompetent individuals. However, in immunosuppressed kidney transplant recipients, BK virus can become activated, leading to a range of complications, including BK viruria, viremia, and the more severe BK virus nephropathy (BKVN). BKVN can severely affect the function of the transplanted kidney, potentially leading to irreversible damage. BK virus infection is one of the leading causes of hemorrhagic cystitis in patients undergoing hematopoietic stem cell transplantation.

[0005] The early stages of BK virus reactivation are usually asymptomatic and latent, and can only be detected through laboratory tests of urine and blood. Without timely intervention, the virus will continue to damage the kidneys or bladder. Studies have shown that the progression from viruria to viremia to BK virus nephropathy is rapid, perhaps taking only weeks to months. Therefore, close monitoring and detection of BK virus infection after transplantation, early screening and diagnosis of BK viruria patients, and identification of high-risk patients with BK virus nephropathy or hemorrhagic cystitis, and timely intervention measures can effectively block the progression of the disease and improve the success rate of transplantation.

[0006] Currently, BK virus testing primarily involves viral DNA testing, urine cytology, and renal biopsy. Quantitative detection of BK virus DNA in urine and blood can monitor viral activity, but this approach carries disadvantages such as high cost and time, and may also carry the risk of false positives due to nucleic acid contamination. Urine cytology, which uses microscopic examination to identify positive bait cells, is often used as a screening method, but a negative result cannot completely rule out BK virus infection. Renal biopsy is an invasive procedure, often used as the gold standard for diagnosis, but it is relatively invasive and has poor patient acceptance. Furthermore, its results are significantly affected by the discrepancy between the puncture site and the lesion site, often resulting in false negatives. Therefore, there is an urgent need for non-invasive, efficient, and accurate testing methods to complement current methods. Summary of the Invention

[0007] The present invention screened and obtained monoclonal antibodies that can be used for BK virus detection through hybridoma technology, and applied them to colloidal gold test strips. It has been verified that they have high detection sensitivity for BK virus VP1 recombinant protein and can detect BK virus in nucleic acid-positive urine samples, realizing rapid and instant detection of BK virus and monitoring of BK virus infection and virus activity in the population.

[0008] In order to achieve the above objectives, the main technical solutions adopted by the present invention include: A monoclonal antibody 3C7 for detecting BK virus VP1 protein, wherein the heavy chain variable region of the monoclonal antibody 3C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO. 1 to SEQ ID NO. 3, respectively; The light chain variable region of monoclonal antibody 3C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

[0009] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.7.

[0010] In some embodiments, the amino acid sequence of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.8.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.9.

[0012] In some embodiments, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.10.

[0013] In a second aspect, the present application is based on the use of the above-mentioned monoclonal antibody 3C7 in the preparation of a tool for detecting BK virus VP1 protein.

[0014] In some embodiments, the tools include reagents, kits, test strips, and antibody chips; the test strips include colloidal gold test strips.

[0015] In some embodiments, the colloidal gold test strip uses monoclonal antibody 3C7 as a capture antibody and monoclonal antibody 3C7 as a labeling antibody.

[0016] In some embodiments, the colloidal gold test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper sequentially connected to a back plate.

[0017] In some embodiments, a detection line and a quality control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 3C7, the quality control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 3C7.

[0018] In some embodiments, the BK virus VP1 protein includes natural VP1 protein and recombinant VP1 protein, preferably a recombinant protein prepared by a prokaryotic expression system, specifically including recombinant proteins of the BKV-I / VP1, BKV-II / VP1, BKV-III / VP1 and BKV-IV / VP1 types.

[0019] In some embodiments, the reagents, kits, test strips, and antibody chips are not used for diagnosis of diseases. Beneficial effects: The present invention defines the amino acid sequences of the complementarity-determining regions of the heavy and light chain variable regions of monoclonal antibody 3C7, as shown in SEQ ID NOs. 1 to 3 and 4 to 6, respectively. Monoclonal antibody 3C7 exhibits high specificity and strong binding to the BK virus VP1 protein, effectively recognizing the VP1 proteins of BK virus types I, II, III, and IV.

[0020] Immunoassay tools constructed based on this monoclonal antibody (such as colloidal gold test strips) exhibit high sensitivity and rapid response characteristics, and can achieve visual detection of BK virus VP1 antigen in samples such as urine within minutes. It does not require complex instruments, is easy to operate, and is suitable for instant detection scenarios.

[0021] The monoclonal antibodies and their detection applications provided by the present invention realize non-nucleic acid in vitro detection of BK virus antigens, and can serve as a powerful supplement to existing viral DNA detection, cytological examination and other technologies, thereby improving detection efficiency and accessibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0023] Figure 1 This is the result of SDS-PAGE protein identification; Figure 2 This is the ELISA identification result of BK virus VP1 recombinant protein; Figure 3 This is a schematic diagram of the assembly of the colloidal gold test strip; Figure 4 This is a diagram showing the test results of a urine sample using a colloidal gold test strip; Figure 5 The figure shows the test results of colloidal gold test strips on recombinant protein, control protein and diluent; Figure 6 This is the result of the sensitivity test of the colloidal gold test strip; Figure 7 This is a graph showing the results of identifying the binding activity of monoclonal antibody 3C7 with BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, and BKV-IV / VP1 recombinant protein. DETAILED DESCRIPTION

[0024] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0025] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0026] The reagents in this application are all commercially available products.

[0027] Example 1 1. Preparation of BK virus VP1 recombinant antigen The VP1 genes of BK virus types I, II, III, and IV were downloaded from NCBI, synthesized by Qingke Biotechnology, and cloned into the pET28a expression vector.

[0028] The nucleotide sequence of BK virus type I VP1 is shown in SEQ ID NO.11:

[0029] The amino acid sequence of BK virus type I VP1 is shown in SEQ ID NO.12: MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDENLRGFSLKLSVENDFSSSDSPQRKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVQTEVIGITSMLNLHAGSQKVHEHGGGKPIQGSNFHFFAVGGDPLEMQGVLMNYRTKYPEGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTLTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRKRSVKNPYPISFLLSDLINRRTQRVDGQPMYGMESQVEEVRVFDGTEKLPGDPDMIRYIDKQGQLQTKML*.

[0030] “*” indicates a stop codon, which is not shown in the sequence listing.

[0031] The nucleotide sequence of BK virus type II VP1 is shown in SEQ ID NO.13:

[0032] The amino acid sequence of BK virus VP1 type II is shown in SEQ ID NO.14: MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDNDLRGYSLKLTAENAFDSDSPDKKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPVQGSNFHFFAVGGDPLEMQGVLMNYRTKYPQGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRKRSVKNPYPISFLLSDLINRRTQKVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0033] “*” indicates a stop codon, which is not shown in the sequence listing.

[0034] The nucleotide sequence of BK virus VP1 type III is shown in SEQ ID NO.15:

[0035] The amino acid sequence of BK virus VP1 type III is shown in SEQ ID NO.16: MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDDHLRGYSQHLTAENAFDSDSPDKKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPVQGSNFHFFAVGGDPLEMQGVLMNYRTKYPQGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSKNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRSVKNPYPISFLLSDLINRRTQKVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0036] “*” indicates a stop codon, which is not shown in the sequence listing.

[0037] The nucleotide sequence of BK virus VP1 type IV is shown in SEQ ID NO.17:

[0038] The amino acid sequence of BK virus VP1 type IV is shown in SEQ ID NO.18: MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDNDLRGYSLRLTAETAFESDSPDRKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPIQGSNFHFFAVGGDPLEMQGVLMNYRTKYPEGTVTPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLPRYFKIRLRSVKNPYPISFLLSDLINRRTQRVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0039] “*” indicates a stop codon, which is not shown in the sequence listing.

[0040] The type 4 recombinant plasmids pET28a-BKV-I / VP1, pET28a-BKV-II / VP1, pET28a-BKV-III / VP1, and pET28a-BKV-IV / VP1 were transformed into BL21 (DE3) competent cells and induced to express according to conventional methods. The specific operations are as follows: the transformed bacteria were spread on LB agar plates (containing 50 μg / mL kanamycin) and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm overnight. 1% of the total volume of the culture medium was inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C and 220 rpm for about 3 hours until the OD 600 The pH value was 0.6-0.9, and IPTG was added at a final concentration of 0.1 mM. The cells were collected after induction at 30°C and 200 rpm for 4 hours.

[0041] 2. Purification and identification of recombinant proteins 2.1 Purification of recombinant protein Because the expressed recombinant protein has a histidine tag, the protein was purified using the protein purification instrument and HisTrap TMHP affinity chromatography column was used for purification. Buffer A was 50mMPB, 300mMNaCl, pH8.0, and buffer B was 50mMPB, 300mMNaCl, 0.5M imidazole, pH8.0. The chromatography column was balanced with buffer A, and then the fermented bacterial solution was centrifuged at 8000rpm for 10min. The precipitate was resuspended with liquid A and ultrasonically broken in ice water for 30min, ultrasonicated for 5 seconds at intervals of 5 seconds, and centrifuged at 12000rpm for 30min. The supernatant was filtered with a Jet Bio 0.22 micron filter, loaded, and the chromatography column was washed with buffer A, and finally gradient eluted with buffer B. The target protein elution peak was collected and dialyzed overnight with buffer A at 4°C. The purified protein was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are as follows. Figure 1 The protein concentration was determined using Thermo Nanodrop ultra-micro spectrophotometer and stored at -20°C.

[0042] The purification status was observed by SDS-PAGE protein gel electrophoresis. Figure 1 M: Protein Marker. Serial numbers 1, 2, 3, and 4 represent purified BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins, respectively. The results showed a clear and prominent major band around 43-55 kDa, indicating that the purified BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins are primarily concentrated around this molecular weight. The proteins are highly pure, consistent with the expected size, and can be used for further downstream experiments.

[0043] 2.2. Identification of BK virus VP1 recombinant protein by indirect ELISA Purified BK virus VP1 recombinant protein was coated on microtiter plates, and the reaction with a commercially available BK virus VP1 monoclonal antibody (Abnova, MAB3204-M01) was assessed by indirect ELISA. Microplates were first coated with BK virus VP1 recombinant protein (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate diluted to 1 L of pure water) at a concentration of 1 μg / mL, with 50 μL / well incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 1% BSA, with 150 μL per well incubated at 37°C for 2 hours. The plates were then washed once with PBST (PBS containing 0.05% Tween-20) and patted dry. The commercially purchased BK virus VP1 monoclonal antibody was diluted with PBS at a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / ml. 50 μL was added to the antigen-coated microplate. At the same time, JC virus VP1 monoclonal antibody (abcam, ab34756) was used as a negative control and reacted at 37°C for 30 minutes. The liquid in the wells was discarded, the plate was washed 4 times with PBST, and after patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 minutes, and then washed 4 times. After patting dry, 50 μL / well of TMB color development solution was added for color development at room temperature for 10 minutes. Finally, 50 μL of TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction and the OD was measured using a microplate reader. 450 nm value. The result is as follows Figure 2 MAB3204-M01 is a commercial monoclonal antibody against BK virus VP1, and Ctrl is a monoclonal antibody against JC virus VP1. The purified BK virus VP1 recombinant protein was coated on an ELISA plate at a concentration of 1 μg / mL, and a gradient dilution test was performed using the commercial monoclonal antibody against BK virus VP1. The results showed that BKV-I / VP1 ( Figure 2 A), BKV-II / VP1 ( Figure 2 B), BKV-III / VP1 ( Figure 2 C), BKV-IV / VP1 ( Figure 2 D) All four recombinant VP1 proteins showed significant positive signals within the dilution range of 10 ng / mL to 1 μg / mL, indicating that the purified BK virus VP1 recombinant proteins are biologically active and can be used for further experiments.

[0044] 3. Mouse immunization Purified BKV-I / VP1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (200 μL) and injected subcutaneously at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg per mouse. At weeks 2, 4, and 6, BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 were diluted to the same concentration and mixed again by equal volume. Finally, each antigen was mixed with an equal volume of MF59 adjuvant at a dose of 20 μg per mouse and injected intramuscularly for further immunization. At week 7, mouse sera were collected for antibody titer analysis. Mice selected for fusion were then boosted intraperitoneally with 20 μg of the four protein mixture. Three days later, spleens were harvested for hybridoma production.

[0045] 4. Screening, preparation of hybridoma cell lines and antibody purification 4.1. Screening of Hybridoma Cells All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in the logarithmic growth phase and then cultured in HAT medium for screening. When the fused cells reached half the bottom of the well, clones positive for BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins were screened by indirect ELISA. Because the immunogen is prokaryotically expressed and contains a His tag, background components must be screened to identify specific cell lines targeting the BK virus VP1 recombinant protein. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were then expanded and cryopreserved.

[0046] 4.2. Screening of positive clones by indirect ELISA BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, BKV-IV / VP1 recombinant proteins and other recombinant proteins of the pET28a vector (pET28a-HPV16 / E7, His tag) were coated in microwell plates (the coating buffer was carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water), the coating concentration was 1 μg / mL, and the plate was incubated at 4°C overnight; 1% BSA was blocked, 150 μL per well, and the plate was blocked at 37°C for 2 hours. The plate was washed once with washing solution and patted dry; 50 μL cell culture supernatant was added and the plate was reacted at 37°C for 30 minutes. The liquid in the wells was shaken out, and the plate was washed 4 times with PBST solution. After patting dry, 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added. The plate was reacted at 37°C for 30 min. The plate was washed 4 times again, and after patting dry, 50 μL / well of TMB color development solution was added to develop at room temperature for 10 min. Finally, 50 μL of TMB stop solution (acidic, Beijing Meikewande Biological, 1001SA) was added to terminate the reaction. The OD was measured using a microplate reader. 450The positive cell lines that reacted with the four recombinant proteins BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 but not with the control antigen were selected for subsequent experiments.

[0047] Preparation process of HPV16 / E7 recombinant protein in this application: The HPV16 E7 gene was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0048] Nucleotide sequence (SEQ ID NO.19): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACA GAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA.

[0049] Amino acid sequence (SEQ ID NO. 20): MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP*.

[0050] The “*” symbol at the end of the sequence represents the termination codon of the protein sequence and is not shown in the sequence listing.

[0051] The recombinant plasmid pET28a-HPV16 / E7 was transformed into BL21 (DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) according to conventional methods. The transformed bacteria were spread on LB agar plates (containing 50 μg / mL kanamycin) and cultured at 37°C overnight. A single colony was picked and inoculated into 5 mL LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm, and shaken overnight. 1% of the total volume of the culture medium was inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C, 220 rpm, and shaken for about 3 hours until the OD 600The concentration of the protein was 0.6-0.9, and IPTG was added to a final concentration of 0.1 mM. After induction at 30°C and 200 rpm for 4 hours, the bacteria were collected to obtain the HPV16 / E7 recombinant protein.

[0052] The screening results are shown in Table 1 below.

[0053] Table 1: Results of indirect ELISA screening of monoclonal antibodies.

[0054]

[0055] As shown in Table 1 , 30 monoclonal antibodies that reacted with BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins were screened using indirect ELISA, and none of them reacted with the control antigen. However, the reactivity with these four types of K virus P1 recombinant proteins varied in strength, and the above-mentioned BK virus VP1-specific cell lines were selected for subsequent experiments.

[0056] 4.3 Preparation of Monoclonal Antibody Ascites After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0057] 4.4. Affinity chromatography purification of monoclonal antibodies Ascites was centrifuged at 12,000 rpm for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4) and filtered through a 0.22 μm filter. The filtered sample was pumped at a low speed via a peristaltic pump onto a Protein L purification column equilibrated with binding buffer. The column was then connected to a protein purifier and washed with binding buffer for 5-10 column volumes until the UV absorption peak leveled out. The column was then eluted with elution buffer (0.1 M glycine, pH 2.7). The eluted peak was collected and adjusted to neutral with 1 M Tris-HCl, pH 9. The sample was placed in a dialysis bag (MW: 8,000-14,000) and dialyzed against 20 mM PBS, pH 7.4, at 2-8°C for 16 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12,000 rpm for 5 minutes. The supernatant was the purified monoclonal antibody. The concentration of the purified monoclonal antibody was measured using an ultra-micro spectrophotometer and then aliquoted for storage.

[0058] 5. Preparation of test strips Monoclonal antibodies reactive with the selected BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins were streaked onto nitrocellulose membranes of varying sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL in PBS, pH 7.4) were applied horizontally in a line pattern using a streaking device at a rate of 0.8 μL / cm to form the test line (T line). Goat anti-mouse IgG antibody (diluted to a concentration of 1 mg / mL in 0.01 M PBS, pH 7.4) was applied horizontally in a line pattern at 6 mm intervals at a rate of 0.8 μL / cm to form the control line (C line).

[0059] 6. Preparation of Antibody-Colloidal Gold Labeled Complex Antibody labeling: Prepare colloidal gold solution using the trisodium citrate reduction method. Specifically, take 100 mL of 0.01% chloroauric acid solution, bring to a boil, then quickly add 1 mL of 1% trisodium citrate solution until the solution turns wine red. Continue boiling for 5 minutes. Once the colloidal gold particles are stable, cool to room temperature and set aside. Place 1 mL of colloidal gold solution in a centrifuge tube and add 0.2 M potassium carbonate solution in a gradient of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to achieve a pH that allows efficient coupling of the antibody and colloidal gold. After mixing, add 5 μg of the monoclonal antibody to be labeled, mix quickly, and incubate at room temperature for 10 minutes. Subsequently, add 10 μL of 10% (w / v) bovine serum albumin (BSA) to block nonspecific binding sites, and incubate at room temperature for another 10 minutes. Add 10 μL of 10% (w / v) polyethylene glycol 20,000 (PEG20,000) to enhance labeling stability. Mix thoroughly and centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of resuspending solution (0.01 M phosphate buffer + 1% BSA + 2% sucrose, pH 7.4) to obtain the antibody-colloidal gold labeled complex. Store at 4°C in the dark until used.

[0060] 7. Screening of paired monoclonal antibodies Nitrocellulose membranes marked with different monoclonal antibodies were paired with colloidal gold-labeled monoclonal antibodies for a one-to-one reaction. The four recombinant proteins (BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1) were first mixed at a final concentration of 20 ng / mL to serve as the positive antigen. HPV16 / E7 recombinant protein was simultaneously diluted to 20 ng / mL and used as the negative antigen for detection. The assay screened for combinations that exhibited a strong color reaction with the mixed BK virus VP1 proteins and did not react with the control protein. Subsequently, the initial screening pairings were further tested for their effectiveness in detecting the VP1 proteins of the four BK virus types. BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 were diluted to 20 ng / mL and tested. The combination that exhibited a strong color reaction with all four VP1 types was selected as the optimal pair for further testing. The screening process is shown in Tables 2 to 5. The control HPV16 / E7 recombinant protein and blank dilution were both negative and not displayed.

[0061] Table 2: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein1.

[0062]

[0063] Table 3: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein2.

[0064]

[0065] Table 4: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein3.

[0066]

[0067] Table 5: Detection effects of different monoclonal antibody combinations on four types of BK virus VP1 recombinant proteins (BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, BKV-IV / VP1).

[0068]

[0069] - indicates negative, i.e. no color development; + / ++ / +++ indicates positive, i.e. a color reaction. The more + signs there are, the darker the color development, i.e. the stronger the positive reaction.

[0070] Thirty monoclonal antibodies reactive with all four BK virus VP1 proteins were stripped and gold-labeled, then paired one by one. The results in Table 5 show that three combinations produced the strongest color development: 3C7 stripping, 3C7 gold-labeling; 3F9 stripping, 6C11 gold-labeling; and 4E3 stripping, 1G5 gold-labeling. These three combinations demonstrated excellent detection of mixtures of BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1. Furthermore, we used four different types of BK virus VP1 proteins to test the ability of different monoclonal antibody combinations to recognize each protein, comparing the results for the different types of VP1 recombinant proteins. As shown in Table 3, the combination of 3C7 streaking and 3C7 gold labeling showed a deep color development for 20 ng / mL of the four types of BK virus VP1 recombinant protein, while the other two combinations showed weaker color development for some types. Therefore, the combination of 3C7 streaking and 3C7 gold labeling was the best pair for detecting BK virus VP1 recombinant protein.

[0071] 8. Preparation and assembly of colloidal gold test strips Preparation of gold-labeled pad: Use a 6mm x 300mm glass fiber membrane, treat it with PBS containing 1% BSA and 1% Tween-20, pH 7.4, and evenly add 1200ul of colloidal gold-labeled antibody to the glass fiber. After air drying, place it in a drying oven at 37°C for 2 hours.

[0072] See also Figure 3 , Figure 3 This is a schematic diagram of the colloidal gold test strip assembly. A 60mm x 300mm PVC backing plate is used as the support, with a sample pad, gold label pad, nitrocellulose membrane, and absorbent paper attached. The nitrocellulose membrane is coated with two lines and dried at 37°C for 12 hours before use. The nitrocellulose membrane is coated with a test line (monoclonal antibody 3C7 strip) and a quality control line (goat anti-mouse IgG). The assembled large plate is cut into 4.05mm bare strips using a strip cutter and wrapped with a colloidal gold plastic card holder, with the sample pad exposed in the sample well of the plastic card holder and the quality control line and test line exposed in the result viewing well. The colloidal gold test strip assembly is complete.

[0073] 9. Test strip specificity test Urine samples: 5 urine samples that tested negative for BK virus nucleic acid and 1 urine sample that tested positive for BK virus nucleic acid. The urine samples were quantitatively tested for BKV DNA copy number using a commercial BKV nucleic acid detection kit. The nucleic acid test results of the 5 negative samples were all less than 2000 copies / mL, and the nucleic acid test result of the 1 positive sample was 3.2*10 7Copies / mL, urine samples were diluted with sample diluent (0.01MPB+0.1%Tween20+1.5%NaCl+0.1%SDS, pH7.4) with equal volume before detection.

[0074] Recombinant protein samples: BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, BKV-IV / VP1 recombinant protein, and HPV16 / E7 recombinant protein were diluted to 1 μg / mL with sample diluent for detection.

[0075] Take 100uL of the diluted sample and add it to the sample well of the test paper card. At the same time, take another 100uL of the dilution solution and add it to a new test paper card as a blank control. Determine the result within 20 minutes. If both the T line and the C line show clear red strips, it is considered positive; if only the C line shows color, it is negative; if the C line does not show color, it is considered invalid. Figure 4 and Figure 5 As shown, Figure 4 In the table, (-) represents 5 urine samples that tested negative for BK virus nucleic acid, and (+) represents 5 urine samples that tested positive for BK virus nucleic acid. Figure 5 From left to right, the image shows the test results for BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, and BKV-IV / VP1 recombinant protein diluted to 1 μg / ml, respectively. The control protein, HPV16 / E7 recombinant protein, was diluted to 1 μg / ml. The blank diluent is a sample diluent without any additives. The test strip can effectively detect BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, and BKV-IV / VP1 recombinant protein, as well as nucleic acid-positive urine samples. There is no cross-reaction with HPV16 / E7 recombinant protein. The blank diluent and BKV-nucleic acid-negative urine samples also show no color development, demonstrating the test strip's excellent specificity.

[0076] 10. Test strip sensitivity test BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, and BKV-IV / VP1 recombinant protein were diluted to the same concentration, respectively, and then mixed in equal volumes to make the concentrations of the four antigens in the mixed protein the same. The mixed proteins were then diluted to concentrations of 50 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, and 1 ng / mL for testing. Figure 6The results showed that the colloidal gold test paper still showed weak color at the recombinant protein concentration of 2.5 ng / mL, while the blank diluent, i.e. the sample diluent (0.01MPB+0.1%Tween20+1.5%NaCl+0.1%SDS, pH7.4) (0 ng / mL) did not show any color, indicating that the minimum detection limit of the test paper card for BK virus VP1 recombinant protein was 2.5 ng / mL.

[0077] The present invention screened and isolated a monoclonal antibody, 3C7, through hybridoma technology that specifically recognizes the BK virus VP1 protein. This antibody can efficiently recognize the VP1 proteins of BK virus types I, II, III, and IV, with excellent specificity and sensitivity. The present invention applied this monoclonal antibody 3C7 to an immunoassay platform, constructing a rapid test strip or card based on colloidal gold immunochromatography. This test strip has high sensitivity to BK virus VP1 recombinant protein and can effectively detect BK virus antigens in urine samples, enabling rapid, convenient, and non-invasive detection of BK virus.

[0078] The colloidal gold test strip constructed based on this antibody adopts a double antibody sandwich method, with 3C7 serving as both a capture antibody and a labeling antibody to achieve rapid and visual detection of BK virus VP1 in urine or recombinant protein samples.

[0079] The monoclonal antibodies and their detection applications provided by the present invention can be used to prepare reagents, kits or test strips for detecting BK virus VP1 protein, and are suitable for qualitative or quantitative detection of BK virus antigens, especially for monitoring and screening BK virus activity in body fluid samples of patients after transplantation.

[0080] This technical solution is not used for the diagnosis or treatment of human diseases. Instead, as a supplementary detection tool for existing detection methods, it helps to improve the convenience and accessibility of BK virus detection and has important application value in clinical laboratories and POCT scenarios.

[0081] 11. Identification of paired monoclonal antibodies Referring to the aforementioned indirect ELISA method, the binding activity of the screened paired monoclonal antibodies was identified. The monoclonal antibody 3C7 and the control JC virus VP1 monoclonal antibody (abcam, ab34756) were serially diluted (concentrations were 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL and 100 pg / mL, respectively) to evaluate their binding activity to the BK virus VP1 recombinant protein. The OD values ​​at each concentration were measured. 450 nm values ​​and draw the binding curve. Figure 7The results of the binding activity evaluation of monoclonal antibody 3C7 with BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, and BKV-IV / VP1 recombinant protein are shown in the figure. The selected monoclonal antibodies bind to BKV-I / VP1 recombinant protein ( Figure 7 A), BKV-II / VP1 recombinant protein ( Figure 7 B), BKV-III / VP1 recombinant protein ( Figure 7 C), BKV-IV / VP1 recombinant protein ( Figure 7 D) Significant reactions occurred, with the antibody still showing positive reactions against BKV-I / VP1 recombinant proteins and BKV-IV / VP1 recombinant proteins at concentrations as low as 1 ng / mL, demonstrating high affinity and good binding titer. 3C7 represents the BK virus VP1 monoclonal antibody 3C7, and Ctrl represents the control, which is the JC virus VP1 monoclonal antibody.

[0082] 12. Paired monoclonal antibody sequences Total RNA from hybridoma cells was extracted using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using Random Primers. Universal primers for the mouse antibody variable regions were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the third-round PCR primers. The PCR products were gel-cleaved and purified, then ligated into the pUC19 vector and transformed into the TOP10 strain. After incubation at 37°C for 14 hours, single colonies were picked and sequenced to obtain the gene sequences of the monoclonal antibody light and heavy chains.

[0083] Antibody variable region genes: Monoclonal antibody 3C7: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.9: GAGTTCCAGCTGCAGCAGTCTGGAGCTGGGCTGGTGAAACCCGGGGCATCAGTGAAGCTGTCCTGCAAGGCTTCTGGGTACACCTTCACTGAGTATATTATACATTGGGTAAAGCAGAGTTCTGGACAGGGTCTTGAGTGGATTGGGTGGTTTTACCCTGGAAGTGGTACTATAAAGTACAATGAGAAATT CAAGGACAAGGCCACATTGACTGCGGACAAATCCTCCAGCACAGCCTATATGGAGCTGAGTAGATTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACACGAAGAGATGTTCCATTACTACGATAGTAATTATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0084] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.7: EFQLQQSGAGLVKPGASVKLSCKASGYTFTEYIIHWVKQSSGQGLEWIGWFYPGSGTIKYNEKFKDKATLTADKSSSTAYMELSRLTSEDSAVYFCARHEEMFHYYDSNYYYAMDYWGQGTSVTVSS.

[0085] CDR region annotation: The amino acid sequences of the heavy chain variable region of monoclonal antibody 3C7, including the complementarity determining region CDR-H1, are shown in SEQ ID NO. 1: EYIIH; The amino acid sequences of the heavy chain variable region of monoclonal antibody 3C7, including the complementarity determining region CDR-H2, are shown in SEQ ID NO. 2: WFYPGSGTIKYNEKFKD; The amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7, including the complementarity determining region CDR-H3, is shown in SEQ ID NO. 3: HEEMFHYYDSNYYYAMDY.

[0086] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.10: GACATCCAGATGAACCAGTCTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTTCTACACATCAAAATTACAC TCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGACATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCCTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0087] The amino acid sequence of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.8: DIQMNQSPSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGTVKLLIFYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTTFGGGTKLEIKRTV.

[0088] CDR region annotation: The amino acid sequences of the complementarity determining region CDR-L1 of the light chain variable region of monoclonal antibody 3C7 are shown in SEQ ID NO. 4: RASQDISNYLA; The amino acid sequences of the complementary determining region CDR-L2 of the light chain variable region of monoclonal antibody 3C7 are shown in SEQ ID NO. 5: YTSKLHS; The amino acid sequence of the complementary determining region CDR-L3 of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO. 6: QQGNTLPWT.

[0089] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0090] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody 3C7 for detecting BK virus VP1 protein, characterized in that: The heavy chain variable region of the monoclonal antibody 3C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3 respectively; The light chain variable region of the monoclonal antibody 3C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

2. The monoclonal antibody 3C7 for detecting BK virus VP1 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 3C7 is shown in SEQ ID NO.

7.

3. The monoclonal antibody 3C7 for detecting BK virus VP1 protein according to claim 1, characterized in that: The amino acid sequence of the light chain variable region of the monoclonal antibody 3C7 is shown in SEQ ID NO.

8.

4. The monoclonal antibody 3C7 for detecting BK virus VP1 protein according to claim 2, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 3C7 is shown in SEQ ID NO.

9.

5. The monoclonal antibody 3C7 for detecting BK virus VP1 protein according to claim 3, characterized in that: The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 3C7 is shown in SEQ ID NO.

10.

6. Use of the monoclonal antibody 3C7 according to claim 1 in the preparation of a tool for detecting BK virus VP1 protein.

7. The use according to claim 6, characterized in that The tools include reagents, test kits, test strips and antibody chips; the test strips include colloidal gold detection test strips.

8. The use according to claim 7, characterized in that The colloidal gold test strip uses monoclonal antibody 3C7 as a capture antibody and monoclonal antibody 3C7 as a labeling antibody.

9. The use according to claim 8, characterized in that The colloidal gold test strip comprises a nitrocellulose membrane, a colloidal gold pad, a sample pad and absorbent paper which are sequentially connected to a back plate.

10. The use according to claim 9, characterized in that A detection line and a quality control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 3C7, the quality control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 3C7.

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