A monoclonal antibody 3C7 for the detection of BK virus VP1 protein and its application

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a monoclonal antibody 3C7 for detecting BK virus VP1 protein and its applications. The amino acid sequences of the heavy chain variable regions CDR1, CDR2, and CDR3 of the monoclonal antibody are SEQ ID NO. 1–3, and the amino acid sequences of the light chain variable regions CDR1, CDR2, and CDR3 are SEQ ID NO. 4–6, respectively. This antibody exhibits high specificity and can recognize the BK virus VP1 protein. The test strip includes a backing plate, a sample pad, a colloidal gold pad, a nitrocellulose membrane, and absorbent paper. The detection method provided by this invention is simple to operate, highly sensitive, and requires no instruments, enabling rapid and visual detection of BK virus VP1 in urine or recombinant protein samples. It is suitable for on-site screening and point-of-care testing and can supplement existing detection methods such as nucleic acid testing.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody 3C7 for detecting the VP1 protein of BK virus and its application. Background Technology

[0002] BK virus, or BK polyomavirus (BKV), belongs to the family Polyomaviridae 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 major protein on the viral surface. BKV can be classified into types I, II, III, and IV based on differences in the VP1 gene.

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

[0004] In kidney transplant patients, the long-term use of immunosuppressants to prevent rejection after surgery makes them susceptible to various pathogens, including BK virus. BK virus is common in the general population, but it is usually harmless to individuals with normal immune function. However, in immunosuppressed kidney transplant patients, BK virus can be activated, leading to a range of complications, including BK viruria, viremia, and the more serious BK virus nephropathy (BKVN). BKVN can severely affect the function of the transplanted kidney, potentially causing irreversible damage. In patients who have received hematopoietic stem cell transplants, BK virus infection is one of the leading causes of hemorrhagic cystitis.

[0005] Early reactivation of the Black-kine virus (BKV) is typically asymptomatic and can only be detected through laboratory tests of urine and blood. Without timely intervention, the virus can continuously damage the kidneys or bladder. Studies have shown that the progression from viruria to viremia and then to BKV nephropathy is rapid, potentially taking only weeks to months. Therefore, close monitoring and detection of BKV infection after transplantation, early screening and diagnosis of BKVuria patients, and identification of high-risk patients with BKV nephropathy or hemorrhagic cystitis, along with timely intervention, can effectively halt disease progression and improve transplant success rates.

[0006] Currently, the main methods for detecting BK virus are viral DNA testing, urine cytology, and kidney biopsy. Quantitative detection of BK virus DNA load in urine and blood can monitor viral activity, but it has drawbacks such as high cost and long processing time, and may face the risk of false positives due to nucleic acid contamination. Urine cytology, which searches for positive bait cells under a microscope, is often used as a screening method, but a negative result does not completely rule out BK virus infection. Kidney biopsy is an invasive procedure and is usually considered the gold standard for diagnosis, but it is highly invasive, has poor patient acceptance, and its results are greatly affected by the deviation between the puncture site and the lesion site, often leading to false negatives. Therefore, there is still an urgent need for non-invasive, efficient, and accurate detection methods to supplement current methods. Summary of the Invention

[0007] This invention utilizes hybridoma technology to screen and obtain monoclonal antibodies suitable for BK virus detection. These antibodies are then applied to colloidal gold test strips, which have been verified to have high detection sensitivity for BK virus VP1 recombinant protein and to detect BK virus in nucleic acid-positive urine samples. This enables rapid and immediate detection of BK virus and monitoring of BK virus infection status and viral activity in the population.

[0008] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0009] A monoclonal antibody 3C7 for detecting the VP1 protein of BK virus. The heavy 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.1-SEQ ID NO.3, respectively.

[0010] The light chain variable region of monoclonal antibody 3C7 includes three complementarity-determining regions, the amino acid sequences of which are shown in SEQ ID NO.4-SEQ ID NO.6, respectively.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.7.

[0012] In some embodiments, the amino acid sequence of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.8.

[0013] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.9.

[0014] In some embodiments, the nucleotide sequence encoding the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO. 10.

[0015] Secondly, this application is based on the application of the aforementioned monoclonal antibody 3C7 in the preparation of a tool for detecting the BK virus VP1 protein.

[0016] In some embodiments, the tools include reagents, kits, test strips, and antibody chips; the test strips include colloidal gold test strips.

[0017] In some embodiments, the colloidal gold test strip uses monoclonal antibody 3C7 as the capture antibody and monoclonal antibody 3C7 as the labeling antibody.

[0018] In some embodiments, the colloidal gold test strip includes a nitrocellulose membrane, a colloidal gold pad, a sample pad, and absorbent paper sequentially connected to a backing plate.

[0019] In some embodiments, a detection line and a control line are provided on the nitrocellulose membrane; the detection line is coated with monoclonal antibody 3C7, the control line includes goat anti-mouse IgG, and the colloidal gold pad is coated with monoclonal antibody 3C7.

[0020] In some embodiments, the BK virus VP1 protein includes natural VP1 protein and recombinant VP1 protein, preferably recombinant protein prepared by a prokaryotic expression system, specifically including recombinant proteins of BKV-I / VP1, BKV-II / VP1, BKV-III / VP1 and BKV-IV / VP1 types.

[0021] In some embodiments, reagents, kits, test strips, and antibody chips are not used for the diagnosis of diseases.

[0022] Beneficial effects:

[0023] This invention clarifies the amino acid sequences of the complementarity-determining regions (CMRs) of the heavy chain and light chain variable regions of monoclonal antibody 3C7, as shown in SEQ ID NO.1–SEQ ID NO.3 and SEQ ID NO.4–SEQ ID NO.6, respectively. Monoclonal antibody 3C7 exhibits high specificity and strong binding ability against the BK virus VP1 protein, effectively recognizing the VP1 protein of BK virus types I, II, III, and IV.

[0024] Immunoassay tools (such as colloidal gold test strips) constructed based on this monoclonal antibody exhibit high sensitivity and rapid response characteristics, enabling visual detection of BK virus VP1 antigen in samples such as urine within minutes. They require no complex instruments, are easy to operate, and are suitable for point-of-care testing applications.

[0025] The monoclonal antibody and its detection application provided by this invention realize non-nucleic acid in vitro detection of BK virus antigen, which can serve as a powerful supplement to existing viral DNA detection, cytological examination and other technologies, improving detection efficiency and accessibility. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Image showing the results of SDS-PAGE protein identification;

[0028] Figure 2 The image shows the ELISA results for identifying the recombinant BK virus VP1 protein.

[0029] Figure 3 This is a schematic diagram of the assembly of colloidal gold test strips.

[0030] Figure 4 This is a graph showing the test results of the colloidal gold test strip on a urine sample.

[0031] Figure 5 The image shows the detection results of the colloidal gold test strip for recombinant protein, control protein, and dilution solution.

[0032] Figure 6 The graph shows the sensitivity test results of the colloidal gold test strip.

[0033] Figure 7 The image shows the results of the binding activity assay of monoclonal antibody 3C7 with recombinant BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 proteins. Detailed Implementation

[0034] The embodiments of this application will be further described in detail below with reference to the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are used to illustrate the principles of this application by way of example, but should not be used to limit the scope of this application. This application can be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

[0035] These embodiments are provided to make the application thorough and complete, and to fully express the scope of the application to those skilled in the art. It should be noted that, unless otherwise specifically stated, the relative arrangement of components and steps, material composition, numerical expressions, and values ​​illustrated in these embodiments should be interpreted as merely exemplary and not as limiting.

[0036] All reagents used in this application are commercially available products.

[0037] Example 1

[0038] 1. Preparation of BK virus VP1 recombinant antigen

[0039] The VP1 genes of BK viruses of types I, II, III, and IV were downloaded from NCBI, synthesized by Qingke Biotechnology, and cloned into the pET28a expression vector.

[0040] The nucleotide sequence of type I BK virus VP1 is shown in SEQ ID NO.11:

[0041]

[0042] The amino acid sequence of type I BK virus VP1 is shown in SEQ ID NO.12:

[0043] MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDENLRGFSLKLSVENDFSSDSPQRKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVQTEVIGITSMLNLHAGSQKVHEHGGGKPIQGSNFHFFAVGGDPLEMQGVLMNYRTKYPEGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTLTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRKRSVKNPYPISFLLSDLINRRTQRVDGQPMYGMESQVEEVRVFDGTEKLPGDPDMIRYIDKQGQLQTKML*.

[0044] The asterisk (*) indicates the stop codon, which is not shown in the sequence list.

[0045] The nucleotide sequence of type II BK virus VP1 is shown in SEQ ID NO.13:

[0046]

[0047] The amino acid sequence of type II BK virus VP1 is shown in SEQ ID NO.14:

[0048] MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDNDLRGYSLKLTAENAFDSDSPDKKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPVQGSNFHFFAVGGDPLEMQGVLMNYRTKYPQGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRKRSVKNPYPISFLLSDLINRRTQKVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0049] The asterisk (*) indicates the stop codon, which is not shown in the sequence list.

[0050] The nucleotide sequence of type III BK virus VP1 is shown in SEQ ID NO.15:

[0051]

[0052] The amino acid sequence of type III BK virus VP1 is shown in SEQ ID NO.16:

[0053] MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDDHLRGYSQHLTAENAFDSDSPDKKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPVQGSNFHFFAVGGDPLEMQGVLMNYRTKYPQGTITPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSKNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLARYFKIRLRSVKNPYPISFLLSDLINRRTQKVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0054] The asterisk (*) indicates the stop codon, which is not shown in the sequence list.

[0055] The nucleotide sequence of type IV BK virus VP1 is shown in SEQ ID NO.17:

[0056]

[0057] The amino acid sequence of type IV BK virus VP1 is shown in SEQ ID NO.18:

[0058] MAPTKRKGECPGAAPKKPKEPVQVPKLLIKGGVEVLEVKTGVDAITEVECFLNPEMGDPDNDLRGYSLRLTAETAFESDSPDRKMLPCYSTARIPLPNLNEDLTCGNLLMWEAVTVKTEVIGITSMLNLHAGSQKVHENGGGKPIQGSNFHFFAVGGDPLEMQGVLMNYRTKYPEGTVTPKN PTAQSQVMNTDHKAYLDKNNAYPVECWIPDPSRNENTRYFGTYTGGENVPPVLHVTNTATTVLLDEQGVGPLCKADSLYVSAADICGLFTNSSGTQQWRGLPRYFKIRLRSVKNPYPISFLLSDLINRRTQRVDGQPMYGMESQVEEVRVFDGTEQLPGDPDMIRYIDRQGQLQTKMV*.

[0059] The asterisk (*) indicates the stop codon, which is not shown in the sequence list.

[0060] The four 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 their contents using standard methods. The specific procedures were as follows: Transformed bacteria were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. Single colonies were picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours, until OD500 reached. 600 The concentration was 0.6-0.9, and IPTG was added to a final concentration of 0.1 mM. The cells were collected after induction at 30°C and 200 rpm for 4 hours.

[0061] 2. Purification and identification of recombinant proteins

[0062] 2.1 Purification of recombinant proteins

[0063] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purification instrument and HisTrap from Suzhou Taidu Biotechnology Co., Ltd. TMPurification was performed using an HP affinity chromatography column. Buffer A consisted of 50 mM BPB, 300 mM NaCl, pH 8.0, and buffer B consisted of 50 mM BPB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial culture was then centrifuged at 8000 rpm for 10 min. The precipitate was resuspended in buffer A and sonicated in ice water for 30 min, with 5-second intervals between sonications. The mixture was then centrifuged at 12000 rpm for 30 min. The supernatant was filtered through a 0.22 μm filter from JetBio, loaded onto the column, washed with buffer A, and finally eluted using a gradient of buffer B. The elution peak of the target protein was collected and dialyzed overnight at 4°C with buffer A. The purification process was observed by SDS-PAGE electrophoresis. The electrophoresis results of the purified protein are shown below. Figure 1 Protein concentrations were determined using a Thermo Nanodrop micro-volume spectrophotometer and stored at -20°C.

[0064] SDS-PAGE protein gel electrophoresis was used to observe the purification results. Figure 1 M: Protein Marker; numbers 1, 2, 3, and 4 represent the purified BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins, respectively. The results showed a clear and significant main 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 mainly concentrated in this molecular weight range, and the protein purity is high, consistent with the expected size. The purified proteins can be used for further downstream experiments.

[0065] 2.2 Indirect ELISA identification of BK virus VP1 recombinant protein

[0066] Purified BK virus VP1 recombinant protein was coated onto microplates, and its reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was a commercially available BK virus VP1 monoclonal antibody (Abnova, MAB3204-M01). First, BK virus VP1 recombinant protein was coated onto microplates (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, 50 μL / well, and incubated overnight at 4°C. The next day, the coating buffer was discarded, and the plates were blocked with 1% BSA, 150 μL per well, and incubated at 37°C for 2 hours. The plates were then washed once with PBST wash buffer (PBS containing 0.05% Tween-20) and patted dry. Commercially available BK virus VP1 monoclonal antibody was diluted with PBS in gradients of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL. 50 μL of each diluted antibody was added to each well of a microplate coated with the antigen. JC virus VP1 monoclonal antibody (abcam, ab34756) was used as a negative control. The reaction was carried out at 37°C for 30 min. The liquid in the wells was discarded, and the plates were washed four times with PBST. After drying, 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) was added to each well. The reaction was carried out at 37°C for 30 min, washed four more times, and dried. 50 μL of TMB chromogenic buffer was added to each well, and the plates were incubated at room temperature for 10 min. Finally, 50 μL of TMB stop solution (acidic, Beijing Meike Wande Biotechnology, 1001SA) was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. Results are as follows: Figure 2 MAB3204-M01 is a commercially available monoclonal antibody against BK virus VP1, and Ctrl is a monoclonal antibody against JC virus VP1. The purified recombinant BK virus VP1 protein was coated onto an ELISA plate at a concentration of 1 μg / mL. The plate was then serially diluted using the commercially available monoclonal antibody against BK virus VP1 for detection. 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 in the dilution range of 10 ng / mL to 1 μg / mL. This indicates that the purified BK virus VP1 recombinant protein has biological activity and can be used for further experiments.

[0067] 3. Mouse immunization

[0068] Purified BKV-I / VP1 recombinant protein was mixed with an equal volume of Freund's complete adjuvant (200 μL) and subcutaneously injected at multiple sites into 6-week-old female BALB / c mice at a dose of 30 μg / mouse. Then, at weeks 2, 4, and 6, the four antigens BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 were diluted to the same concentration, mixed in equal volumes, and finally injected intramuscularly with an equal volume of MF59 adjuvant at a dose of 20 μg / mouse. At week 7, mouse serum was collected to detect antibody titers. Mice to be fused were selected and immunized with a booster immunization of 20 μg of the four protein mixture via intraperitoneal pulse. Three days later, the spleens of these mice were harvested for hybridoma cell preparation.

[0069] 4. Screening, preparation, and antibody purification of hybridoma cell lines

[0070] 4.1 Screening of hybridoma cells

[0071] All spleen cells from immunized mice were fused with SP2 / 0 myeloma cells in logarithmic growth phase and then cultured in HAT medium for selection. When the fused cells reached halfway to the bottom of the well, clones positive for BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins were selected by indirect ELISA. Since the immunogen was of prokaryotic origin and contained a His tag, background components needed to be screened to select specific cell lines targeting the BKV virus VP1 recombinant protein. The positive cells were cloned to a monoclonal state by limiting dilution, and then the cell lines were expanded and cryopreserved.

[0072] 4.2 Screening of positive clones using indirect ELISA method

[0073] Recombinant proteins BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, BKV-IV / VP1, and other recombinant proteins of the pET28a vector (pET28a-HPV16 / E7, His tag) were coated in microplates (coating buffer: carbonate buffer: sodium carbonate 1.59 g, sodium bicarbonate 2.93 g, diluted to 1 L of pure water), with a coating concentration of 1 μg / mL, and incubated overnight at 4 °C; blocked with 1% BSA, 150 μL per well, at 37 °C for 2 hours, washed once with washing buffer, and patted dry; 50 μL of cell culture supernatant was added, and the reaction was carried out at 37 °C for 30 min. Discard the liquid from the wells, wash the plate four times with PBST, pat dry, and add 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS). Incubate at 37°C for 30 min, wash four more times, pat dry, and add 50 μL / well of TMB chromogenic buffer for incubation at room temperature for 10 min. Finally, add 50 μL of TMB stop solution (acidic, Beijing Mecowand Biotechnology, 1001SA) to stop the reaction. Measure the OD using a microplate reader. 450 nm value. Positive cell lines that reacted with all 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.

[0074] The preparation process of the HPV16 / E7 recombinant protein in this application is as follows:

[0075] The HPV16 E7 gene was downloaded from NCBI, synthesized by Anhui General Biotechnology, and cloned into the pET28a expression vector.

[0076] Nucleotide sequence (SEQ ID NO.19): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACA GAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA.

[0077] Amino acid sequence (SEQ ID NO. 20): MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP*.

[0078] The asterisk (*) at the end of the sequence represents the stop codon of the protein sequence, which is not shown in the sequence listing.

[0079] Recombinant plasmid pET28a-HPV16 / E7 was transformed into BL21(DE3) competent cells using standard methods (Molecular Cloning, 3rd Edition, Science Press). The transformed cells were plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and incubated overnight at 37°C with shaking at 220 rpm. Then, 1% of the total culture volume was inoculated into LB medium (containing 50 μg / mL kanamycin) and incubated at 37°C with shaking at 220 rpm for approximately 3 hours, until OD (dose eluent) was reached. 600 The concentration was 0.6-0.9, and IPTG was added to a final concentration of 0.1 mM. After induction at 30℃ and 200 rpm for 4 hours, the bacterial cells were collected to obtain the HPV16 / E7 recombinant protein.

[0080] The screening results are shown in Table 1 below.

[0081] Table 1: Results of indirect ELISA screening for monoclonal antibodies.

[0082]

[0083] 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. None of them reacted with the control antigen, but the reaction strength with the P1 recombinant proteins of these four types of K viruses varied. The above BK virus VP1-specific cell lines were selected for subsequent experiments.

[0084] 4.3 Preparation of Monoclonal Antibody Ascites

[0085] After the selected monoclonal cell lines were expanded and cultured, 0.2 mL (containing 2.5 × 10⁻⁶ cells) was injected intraperitoneally. 6 Female BALB / c mice (cells) were pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the mice's abdomens were significantly swollen, ascites fluid was collected using a sterile syringe needle. The collected ascites fluid was centrifuged at 3000 rpm for 10 minutes, and the intermediate layer was collected.

[0086] 4.4 Affinity chromatography purification of monoclonal antibodies

[0087] Centrifuge the ascites fluid at 12000 rpm for 5 minutes. Collect the supernatant and dilute it 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4). Filter the supernatant through a 0.22 μm filter. Pump the filtered sample slowly through a peristaltic pump into a Protein L purification column equilibrated with binding buffer. Connect the column to a protein purification instrument and wash with binding buffer for 5-10 column volumes until the UV absorption peak flattens. Then elute with elution buffer (0.1 M glycine, pH 2.7). Collect the elution peak and adjust the collected sample to neutral with 1 M Tris-HCl (pH 9). Transfer the solution to a dialysis bag (MW: 8000-14000) and dialyze for 16 hours at 2-8°C in 20 mM PBS (pH 7.4). Transfer the liquid from the dialysis bag to a centrifuge tube and centrifuge at 12000 rpm for 5 minutes. The supernatant is the purified monoclonal antibody. The purified monoclonal antibody was aliquoted and stored after concentration determination using an ultra-micro spectrophotometer.

[0088] 5. Preparation of test strips

[0089] The selected monoclonal antibodies that reacted with BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 recombinant proteins were scribed onto nitrocellulose membranes of different sizes (20 mm × 300 mm). Diluted monoclonal antibodies (diluted to 1.5 mg / mL with PBS at pH 7.4) were sprayed horizontally in a linear pattern using a scribing instrument, with a spray volume of 0.8 μL / cm per line, forming the detection line (T line). Goat anti-mouse IgG antibodies, diluted to 1 mg / mL with 0.01 M PBS at pH 7.4, were sprayed horizontally in a linear pattern at 6 mm intervals, coating the nitrocellulose membrane at a volume of 0.8 μL / cm, forming the control line (C line).

[0090] 6. Preparation of antibody-colloidal gold labeled complexes

[0091] Antibody labeling: Colloidal gold solution was prepared using the trisodium citrate reduction method. The specific procedure was as follows: 100 mL of 0.01% chloroauric acid solution was heated to boiling, and then 1 mL of 1% trisodium citrate solution was quickly added until the solution turned wine-red. Boiling was continued for 5 minutes, and the colloidal gold particles were allowed to stabilize before cooling to room temperature. 1 mL of colloidal gold solution was placed in a centrifuge tube, and 0.2 M potassium carbonate solution was added in a gradient of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and 6 μL to obtain the optimal pH for efficient antibody-colloidal gold conjugation. After mixing, 5 μg of the monoclonal antibody to be labeled was added to each tube, and the mixture was quickly mixed and incubated at room temperature for 10 min. Then, 10 μL of 10% (w / v) bovine serum albumin (BSA) was added to block non-specific binding sites, and the mixture was incubated at room temperature for another 10 min. Add 10 μL of 10% (w / v) polyethylene glycol 20000 (PEG20000) to enhance labeling stability. After mixing, centrifuge at 12000 rpm for 10 min and discard the supernatant. Resuspend the lower precipitate in 1 / 10 volume of reconstitution 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 protected from light for later use.

[0092] 7. Screening of paired monoclonal antibodies

[0093] Nitrocellulose membranes coated with different monoclonal antibodies were individually paired with different colloidal gold-labeled monoclonal antibodies. First, four recombinant proteins—BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1—were mixed at a final concentration of 20 ng / mL to serve as positive antigens. Simultaneously, HPV16 / E7 recombinant protein was diluted to 20 ng / mL to serve as a negative antigen for detection. Combinations showing a strong color reaction to the mixed BKV VP1 protein and not reacting with the control protein were selected. Subsequently, the initial pairings were tested to assess their effectiveness in detecting the four BKV VP1 protein types. BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 were diluted to 20 ng / mL for detection. Combinations showing strong color reactions to all four VP1 protein types were selected as preferred pairings for further experiments. The screening process is shown in Tables 2-5. The control HPV16 / E7 recombinant protein and blank dilution were both negative and were not displayed.

[0094] Table 2: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein 1.

[0095]

[0096] Table 3: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein 2.

[0097]

[0098] Table 4: Results of screening paired monoclonal antibodies using BK virus VP1 recombinant protein 3.

[0099]

[0100] Table 5: Detection efficacy of different monoclonal antibody combinations against four types of BKV virus VP1 recombinant proteins (BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, BKV-IV / VP1).

[0101]

[0102] - indicates a negative result, meaning no color develops; + / ++ / +++ indicates a positive result, meaning a color reaction occurs. The more + signs there are, the deeper the color, and the stronger the positive reaction.

[0103] Thirty monoclonal antibodies that reacted with all four BKV VP1 types were scratched and labeled with gold, and then paired one-to-one. Table 5 shows that three paired combinations showed the deepest staining: 3C7 scratched and 3C7 labeled with gold; 3F9 scratched and 6C11 labeled with gold; and 4E3 scratched and 1G5 labeled with gold. These three combinations showed good detection performance for mixtures of BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 proteins. Furthermore, we used four different types of BKV VP1 proteins to test whether different monoclonal antibody combinations could recognize them, and compared the detection results for different types of recombinant VP1 proteins. As shown in Table 3, the 3C7 streak membrane and 3C7 gold labeling combination showed deep color development for 20 ng / mL of BK virus VP1 recombinant protein of all four types, while the other two combinations showed weaker color development for some types. Therefore, the 3C7 streak membrane and 3C7 gold labeling combination is the best pair for detecting BK virus VP1 recombinant protein.

[0104] 8. Preparation and assembly of colloidal gold test strips

[0105] Preparation of gold-labeled pads: A 6mm x 300mm glass fiber membrane was treated with PBS containing 1% BSA and 1% Tween-20 at pH 7.4. The prepared colloidal gold-labeled antibody was then uniformly added to the glass fiber at a rate of 1200ul / strip. After air drying, the membrane was dried at 37℃ for 2 hours before use.

[0106] See Figure 3 , Figure 3This is a schematic diagram of the colloidal gold test strip assembly. A 60mm x 300mm PVC backing plate is used as a support. Sample pads, gold-labeled pads, nitrocellulose membranes, and absorbent paper are attached to this backing plate. The nitrocellulose membrane is coated with two lines and dried at 37℃ for 12 hours before use. The nitrocellulose membrane is coated with a detection line (monoclonal antibody 3C7 streaking) and a control line (goat anti-mouse IgG). The assembled strip is cut into 4.05mm strips using a strip cutter and wrapped with a colloidal gold plastic casing. The sample pad is exposed at the sample application well of the plastic casing, while the control and detection lines are exposed at the result observation wells. The colloidal gold test strip assembly is now complete.

[0107] 9. Test strip specificity test

[0108] Urine samples: Five urine samples were negative for BKV nucleic acid, and one urine sample was positive for BKV nucleic acid. The copy number of BKV DNA in the urine samples was quantitatively detected using a commercially available BKV nucleic acid detection kit. The nucleic acid detection results of the five negative samples were all less than 2000 copies / mL, and the nucleic acid detection result of the one positive sample was 3.2*10. 7 The urine sample was diluted with an equal volume of sample diluent (0.01 MPB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) before testing.

[0109] 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 1ug / mL with sample dilution buffer for detection.

[0110] Take 100 μL of the diluted sample and add it to the sample well of the test strip. Simultaneously, add another 100 μL of the diluent to a new test strip as a blank control. Determine the results within 20 minutes. If both the T and C lines show clear red bands, the result is positive; if only the C line shows color, the result is negative; if the C line does not show color, the result is invalid. Results are as follows: Figure 4 and Figure 5 As shown, Figure 4 In the table, (—) represents urine samples that tested negative for BK virus nucleic acid, totaling 5 samples, and (+) represents urine samples that tested positive for BK virus nucleic acid. Figure 5From left to right, the images show the detection results of 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. The control protein is HPV16 / E7 recombinant protein, diluted to 1 μg / ml. The blank diluent is the sample diluent without any additives. This test strip can effectively detect BKV-I / VP1 recombinant protein, BKV-II / VP1 recombinant protein, BKV-III / VP1 recombinant protein, BKV-IV / VP1 recombinant protein, and nucleic acid-positive urine samples. It shows no cross-reactivity with HPV16 / E7 recombinant protein. The blank diluent and BKV nucleic acid-negative urine samples also show no color development, indicating that the test strip has good specificity.

[0111] 10. Sensitivity test of test strips

[0112] 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, then mixed in equal volumes to ensure that the four antigens in the mixed protein had the same concentration. The mixture was then diluted to concentrations of 50 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, and 1 ng / mL before detection. Figure 6 The results showed that the colloidal gold test strip still showed weak color development at a recombinant protein concentration of 2.5 ng / mL, while the blank dilution, i.e. the sample dilution (0.01 MPB + 0.1% Tween 20 + 1.5% NaCl + 0.1% SDS, pH 7.4) (0 ng / mL), did not show color development, indicating that the limit of detection for BK virus VP1 recombinant protein was 2.5 ng / mL.

[0113] This invention utilizes hybridoma technology to screen and obtain a monoclonal antibody, 3C7, that specifically recognizes the VP1 protein of the BK virus. This antibody can efficiently recognize the VP1 protein of BK virus types I, II, III, and IV, exhibiting good specificity and sensitivity. This invention applies the monoclonal antibody 3C7 to an immunoassay platform, constructing a rapid test strip or test card based on colloidal gold immunochromatography. This test strip has high sensitivity to the recombinant VP1 protein of the BK virus and can effectively detect the BK virus antigen in urine samples, achieving rapid, convenient, and non-invasive detection of the BK virus.

[0114] The colloidal gold test strip constructed based on this antibody uses a double antibody sandwich method, with 3C7 serving as both the capture antibody and the labeling antibody, to achieve rapid and visual detection of BK virus VP1 in urine or recombinant protein samples.

[0115] The monoclonal antibody and its detection application provided by this 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 antigen, especially for monitoring and screening of BK virus activity in body fluid samples of transplant patients.

[0116] This technical solution is not intended for the diagnosis or treatment of human diseases, but rather serves as a supplementary testing tool to existing methods. It helps improve the convenience and accessibility of BK virus testing and has significant application value in clinical laboratories and point-of-care testing (POCT) scenarios.

[0117] 11. Identification of paired monoclonal antibodies

[0118] 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 (to concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL, respectively) to assess their binding activity with the BK virus VP1 recombinant protein. The OD at each concentration was measured. 450 Use nm values ​​to plot binding curves. Figure 7 The figure shows the binding activity of monoclonal antibody 3C7 with recombinant BKV-I / VP1, BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1 proteins. The figure indicates that the selected monoclonal antibodies bound to BKV-I / VP1 recombinant protein (BKV-II / VP1, BKV-III / VP1, and BKV-IV / VP1) were effective at concentrations as low as 10 ng / mL. 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) A significant response was observed, with positive reactions to BKV-I / VP1 recombinant protein and BKV-IV / VP1 recombinant protein even at concentrations as low as 1 ng / mL, indicating high affinity and good binding titer. 3C7 represents BKV virus VP1 monoclonal antibody 3C7, and Ctrl represents the control, i.e., JC virus VP1 monoclonal antibody.

[0119] 12. Sequences of paired monoclonal antibodies

[0120] Total RNA was extracted from hybridoma cells using the RNeasy Mini Kit (Cat. No. 74104), and cDNA was synthesized by reverse transcription using RandomPrimers. Universal primers for the variable region of mouse antibodies were designed, and the VH and VL genes were amplified by two rounds of PCR. Age1 and Bsiw1 restriction sites were introduced into the primers for the third round of PCR. The PCR products were purified by gel extraction and ligated into the pUC19 vector, transformed into TOP10 strain, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the light and heavy chains of the monoclonal antibody.

[0121] Antibody variable region genes:

[0122] Monoclonal antibody 3C7:

[0123] Heavy chain:

[0124] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.9:

[0125] GAGTTCCAGCTGCAGCAGTCTGGAGCTGGGCTGGTGAAACCCGGGGCATCAGTGAAGCTGTCCTGCAAGGCTTCTGGGTACACCTTCACTGAGTATATTATACATTGGGTAAAGCAGAGTTCTGGACAGGGTCTTGAGTGGATTGGGTGGTTTTACCCTGGAAGTGGTACTATAAAGTACAATGAGAAATT CAAGGACAAGGCCACATTGACTGCGGACAAATCCTCCAGCACAGCCTATATGGAGCTGAGTAGATTGACATCTGAAGACTCTGCGGTCTATTTCTGTGCAAGACACGAAGAGATGTTCCATTACTACGATAGTAATTATTACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA.

[0126] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.7:

[0127] EFQLQQSGAGLVKPGASVKLSCKASGYTFTEYIIHWVKQSSGQGLEWIGWFYPGSGTIKYNEKFKDKATLTADKSSSTAYMELSRLTSEDSAVYFCARHEEMFHYYDSNYYYAMDYWGQGTSVTVSS.

[0128] CDR area annotation:

[0129] The amino acid sequence of the heavy chain variable region of monoclonal antibody 3C7, including the complementarity-determining region CDR-H1, is shown in SEQ ID NO. 1: EYIIH;

[0130] 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;

[0131] 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.

[0132] Light chain:

[0133] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.10:

[0134] GACATCCAGATGAACCAGTCTCCATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGCAATTATTTAGCCTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTTCTACACATCAAAATTACAC TCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGACATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCCTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGTACGGTG.

[0135] The amino acid sequence of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO. 8:

[0136] DIQMNQSPSSLSASLGDRVTISCRASQDISNYLAWYQQKPDGTVKLLIFYTSKLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTTFGGGTKLEIKRTV.

[0137] CDR area annotation:

[0138] The amino acid sequence of the complementarity-determining region (CDR-L1) of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO.4: RASQDISNYLA;

[0139] The amino acid sequence of the complementarity-determining region (CDR-L2) of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO. 5: YTSKLHS;

[0140] The amino acid sequence of the complementarity-determining region CDR-L3 of the light chain variable region of monoclonal antibody 3C7 is shown in SEQ ID NO. 6: QQGNTLPWT.

[0141] The embodiments of this application have now been described in detail. To avoid obscuring the concept of this application, some details known in the art have not been described. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0142] While specific embodiments of this application have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art should understand that modifications can be made to the above embodiments or equivalent substitutions can be made to some technical features without departing from the scope and spirit of this application.

Claims

1. A monoclonal antibody 3C7 for detecting the VP1 protein of BK virus, characterized in that, The heavy chain variable region of the monoclonal antibody 3C7 includes three complementarity-determining regions, CDR-H1, CDR-H2, and CDR-H3. The amino acid sequence of CDR-H1 is shown in SEQ ID NO.1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR-H3 is shown in SEQ ID NO.

3. The light chain variable region of the monoclonal antibody 3C7 includes three complementarity-determining regions, CDR-L1, CDR-L2, and CDR-L3. The amino acid sequence of CDR-L1 is shown in SEQ ID NO.4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO.5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO.

6.

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

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

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

9. The application 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 backing plate.

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

Images