Monoclonal antibody 4F6 for detecting HPV16 type E7 protein and application thereof

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody 4F6 for detecting HPV16 type E7 protein and application. The CDR sequences of the heavy chain and light chain variable regions of the antibody are respectively SEQ ID NO. 1-3 and SEQ ID NO. 4-6, and the antibody can specifically bind to HPV16 type E7 protein without cross reaction. A double antibody sandwich ELISA detection system based on the antibody and combined with biotin-avidin amplification technology has a sensitivity of 100 pg / mL, and is suitable for rapid detection of HPV16 type E7 protein in cervical exfoliated cell samples. The application also provides prokaryotic expression obtained HPV16 type E7 recombinant protein with immunocompetence, which is used for antibody screening and detection application. Compared with nucleic acid detection, the method has a lower false positive rate, can be used for early screening of cervical cancer and precancerous lesions, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a monoclonal antibody 4F6 for detecting HPV16 type E7 protein and its application. Background Art

[0002] Human papillomavirus (HPV) is a common double-stranded DNA virus that enters the body primarily through broken skin or mucous membranes, infects epidermal cells, and can be transmitted through direct contact. Epidemiological studies have shown that persistent infection with high-risk HPV is the main cause of cervical cancer and its precancerous lesions. In recent years, the incidence of cervical cancer in Chinese women has shown a significant upward trend, becoming the second most common female malignancy after breast cancer. Effective prevention and early detection of cervical cancer have become important issues in public health. Currently, systematic cervical cancer screening is considered a key measure to achieve early diagnosis and treatment and reduce mortality, and has been incorporated into my country's basic public health service programs.

[0003] The HPV genome consists of approximately 8,000 base pairs, divided into an early region (E region) and a late region (L region). The E7 gene, expressed early in viral infection, plays a central role in promoting abnormal cell proliferation and carcinogenesis. Studies have shown that the HPV16 E7 protein can immortalize normal epithelial cells, demonstrating significant carcinogenic potential.

[0004] As cervical lesions progress from mild to severe (i.e., from CIN1 to CIN3), E7 protein expression gradually extends to the outer cervical cells, with expression levels increasing continuously, eventually becoming detectable in cervical exfoliated cells. This suggests that E7 protein detection is clinically feasible and can serve as an important biomarker for identifying high-grade cervical lesions and early-stage cervical cancer.

[0005] Multiple studies have also confirmed that testing for the HPV16 E7 protein can help clinically identify individuals at risk for cervical cancer earlier. By specifically detecting E7 protein expression, the virus's activity and potential damage to cervical tissue can be better assessed. Therefore, detecting HPV16 E7 protein levels in cervical exfoliated cells not only provides a scientific basis for early screening and auxiliary diagnosis of cervical cancer and precancerous lesions, but also provides a reliable reference for evaluating vaccination effectiveness and monitoring treatment. Summary of the Invention

[0006] The present invention obtains active HPV16 type E7 recombinant protein, i.e., antigen, based on prokaryotic expression technology, and screens out highly binding monoclonal antibodies based on this, which can be used to detect HPV16 type E7 protein with a sensitivity of 100 pg / mL. The double-antibody sandwich detection method based on this monoclonal antibody is specific to HPV16 E protein, i.e., oncoprotein, and has no cross-reaction with oncoproteins of other HPVs. It can be used for the detection of cervical cancer in the future. Compared with exfoliative cytology, it is faster and more accurate, and can avoid the risks of false positives and misdiagnosis caused by the excessive sensitivity of nucleic acid detection, and has high application value.

[0007] In order to achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] The present invention provides a monoclonal antibody 4F6 for detecting HPV16 type E7 protein. The heavy chain variable region of the monoclonal antibody 4F6 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.

[0009] The light chain variable region of monoclonal antibody 4F6 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.

[0010] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.8.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.10.

[0012] In a second aspect, the present invention provides use of the monoclonal antibody 4F6 in the preparation of a tool for detecting HPV16 type E7 protein.

[0013] In some embodiments, monoclonal antibody 4F6 is used to construct an ELISA detection system, which is used to detect HPV16 E7 protein in biological samples. The ELISA detection system is not used for disease diagnosis.

[0014] In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0015] In some embodiments, the tool is used to detect HPV16 E7 protein in a biological sample in vitro, the biological sample is selected from cervical exfoliated cells, and the detection is not used for diagnosis of a disease.

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

[0017] In some embodiments, the kit comprises a double antibody sandwich ELISA kit.

[0018] In some embodiments, the kit is coated and labeled with the monoclonal antibody 4F6.

[0019] Beneficial effects:

[0020] The present invention provides a highly specific and sensitive monoclonal antibody 4F6 and its use in detecting HPV16 E7 protein. Monoclonal antibody 4F6 has well-defined heavy and light chain variable region structures: its heavy chain variable region comprises three complementarity determining regions (CDRs), as shown in SEQ ID NOs. 1 to 3, respectively; its light chain variable region also comprises three complementarity determining regions, as shown in SEQ ID NOs. 4 to 6, respectively. This antibody specifically recognizes HPV16 E7 protein, exhibits no cross-reactivity with other HPV subtypes (e.g., HPV16 E6 protein, HPV18 E6 protein, and HPV18 E7 protein), and exhibits excellent binding activity.

[0021] A double-antibody sandwich ELISA detection system constructed based on the monoclonal antibody 4F6, combined with a biotin-avidin amplification system, significantly improves detection sensitivity, reaching a minimum detectable concentration of 100 pg / mL. This system is suitable for the rapid and accurate detection of HPV16 E7 protein in cervical exfoliated cell samples. Compared to nucleic acid detection methods, this method effectively reduces the false positive rate and avoids the risk of clinical misdiagnosis due to the high sensitivity of nucleic acid. It has broad application prospects in early screening for cervical cancer and its precancerous lesions, evaluating vaccination effectiveness, and monitoring treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention 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 invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is the SDS-PAGE protein identification diagram;

[0024] Figure 2 This is the result of ELISA identification of HPV16 type E7 recombinant protein;

[0025] Figure 3 Figure 1 is a graph identifying the binding activity of paired monoclonal antibodies;

[0026] Figure 4 This is a diagram showing the sensitivity and specificity of biotin-avidin amplified ELISA. DETAILED DESCRIPTION

[0027] The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings and examples. The detailed description of the following embodiments and the accompanying drawings are intended to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention. The present invention can 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.

[0028] The present invention provides these embodiments to make the present invention thorough and complete, and to fully express the scope of the present invention 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.

[0029] All terms used herein have the same meaning as understood by one of ordinary skill in the art to which the present invention belongs, unless otherwise specifically defined. It should also be understood that terms defined in, for example, common dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and should not be interpreted in an idealized or highly formal sense, unless explicitly defined herein.

[0030] Example 1

[0031] 1. Preparation of recombinant antigens

[0032] The HPV16 E7 gene was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0033] Nucleotide sequence (SEQ ID NO.11): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACA GAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA.

[0034] Amino acid sequence (SEQ ID NO. 12): MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP*.

[0035] The “*” symbol at the end of the sequence represents the termination codon of the protein sequence.

[0036] The recombinant plasmid pET28a-HPV16 / E7 was transformed into BL21(DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) using conventional methods. Transformants were plated on LB agar plates (containing 50 μg / mL kanamycin) and cultured overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C, 220 rpm, and 1% of the total culture volume. The cells were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C, 220 rpm, for approximately 3 hours. The cells were then induced with IPTG (final concentration of 0.1 mM) at 30°C, 200 rpm, and harvested for 4 hours to obtain the HPV16 E7 recombinant protein.

[0037] In the present invention, HPV16 type E7 protein refers to the wild-type E7 protein naturally present in HPV16 virus-infected cells, which is directly encoded by the viral genome, expressed in host cells and participates in key carcinogenic processes such as regulating the cell cycle and inhibiting tumor suppressor proteins. It is a core pathogenic factor in the occurrence and development of cervical cancer. The HPV16 type E7 recombinant protein is an E7 protein artificially expressed in a prokaryotic expression system through genetic engineering technology, usually by cloning the HPV16 type E7 gene into an expression vector and inducing expression in the host. In the present invention, the HPV16 type E7 recombinant protein is used as an antigen for screening and identifying specific monoclonal antibodies against the E7 protein, which can effectively identify the HPV16 type E7 protein in its natural state, thereby realizing the detection and evaluation of HPV16 infection and potential lesions in clinical samples.

[0038] 2. Purification and identification of recombinant proteins

[0039] 2.1 Purification of recombinant protein

[0040] Because the expressed recombinant protein carries a histidine tag, it was purified using a protein purifier and HisTrapTM HP affinity chromatography column from Suzhou Taidu Biotechnology Co., Ltd. Buffer A is 50mM PB, 300mM NaCl, pH 8.0, and buffer B is 50mM PB, 300mM NaCl, 0.5M imidazole, pH 8.0. 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 disrupted 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. Finally, the column was gradient eluted with buffer B. The target protein peak was selected and dialyzed against 10mM phosphate pH 8.0 for exchange, and then subjected to ion exchange chromatography and gradient elution with different NaCl concentrations to collect the target protein peak. The purification was observed by SDS-PAGE protein gel electrophoresis. The electrophoresis results of the purified protein are as shown below. Figure 1 The protein concentration was determined using Thermo Nanodrop ultra-micro spectrophotometer and stored at -20°C.

[0041] Figure 1Middle M: ​​Protein Marker, 1: pET28a-HPV16 E7 recombinant protein, with a clear main band visible between 17-25 kDa, and protein purity of approximately 80% or more, but it differs from the estimated antigen size (15 kDa). This deviation may be related to the characteristics of the HPV16 E7 recombinant protein itself. The HPV16 E7 recombinant protein carries a large amount of negative charge, resulting in abnormal migration speed during SDS-PAGE electrophoresis, which appears to be larger than the actual molecular weight. It will migrate within a certain range during SDS-PAGE electrophoresis at different concentrations. This result is explainable, and the purified protein can be used for further downstream experiments.

[0042] 2.2 Identification of recombinant proteins

[0043] The HPV16 E7 recombinant protein was identified by indirect ELISA.

[0044] The purified HPV16 E7 recombinant protein was coated and the reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was a commercially available HPV16 E7 monoclonal antibody (Biodragon, BD-PA0179). First, the recombinant protein was coated in a microplate (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water) at a coating concentration of 1 μg / mL, 50 μL / well, and incubated at 4°C overnight; then the plate was blocked with 1% BSA, 150 μL per well, at 37°C for 2 hours, and washed once with washing buffer (PBST, PBS containing 0.05% Tween-20) and patted dry; the monoclonal antibody was diluted in PBS at a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / ml, and 50 μL was added to the antigen-coated microplate. At the same time, HPV18 type E7 (Santa Cruz, F-7) monoclonal antibody was used as a negative control, and the reaction was carried out 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. 450 nm value.

[0045] The purified HPV16 E7 recombinant protein was coated and its reaction with commercial monoclonal antibody was identified by indirect ELISA. Figure 2 , Figure 2BD-PA0179 represents the HPV16 type E7 monoclonal antibody, and Ctrl represents the HPV18 type E7 monoclonal antibody (F-7).

[0046] HPV16 E7 recombinant protein, coated at a concentration of 1 μg / ml, showed a weak positive reaction with a commercially available HPV16 E7 monoclonal antibody (Biodragon, BD-PA0179) at a concentration of 1 ng / ml, demonstrating the activity of the purified HPV16 E7 recombinant protein. This experiment, using an indirect ELISA method, confirmed that the recombinantly expressed and purified HPV16 E7 recombinant protein was capable of immunoreactivity with the specific monoclonal antibody, particularly at low concentrations. This demonstrates that the protein has a correct structure, an intact epitope, and good immunoreactivity, making it suitable for further functional studies.

[0047] 3. Mouse immunization

[0048] Six-week-old female BALB / c mice were immunized with purified HPV16 E7 recombinant protein mixed with an equal volume of Freund's complete adjuvant (200 μL) via subcutaneous injection at multiple sites. Six-week-old female mice were then immunized with the same dose of 30 μg per mouse mixed with an equal volume of MF59 adjuvant via intramuscular injection at two and four weeks. At five weeks, sera were collected from the mice for antibody titer determination. Mice with the highest titer were selected for a booster immunization with 20 μg of HPV16 E7 recombinant protein via intraperitoneal bolus. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0049] 4. Screening, preparation of hybridoma cell lines and antibody purification

[0050] 4.1. Screening of Hybridoma Cells

[0051] 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 HPV16 E7 recombinant protein were screened using indirect ELISA. Because the immunogen is prokaryotically expressed and contains a His tag, background components must be screened to identify cell lines specific for the HPV16 E7 recombinant protein, ensuring the specificity of the test results and avoiding false positives due to nonspecific binding. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were then expanded and cryopreserved.

[0052] Screening of positive clones by indirect ELISA: HPV16 type E7 recombinant protein and other recombinant proteins of pET28a vector (pET28a-HPV18 / E6, His tag) were coated in microtiter plates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a coating concentration of 1 μg / mL, and incubated at 4°C overnight; 1% BSA was blocked, 150 μL per well, at 37°C for 2 hours, the plate was washed once with washing solution, and patted dry; 50 μL hybridoma cell culture supernatant was added and reacted at 37°C for 30 minutes. The liquid in the wells was discarded, and the plate was washed 4 times with PBST. 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. 450 nm value. Positive cell lines that reacted only with HPV16 E7 recombinant protein and not with the control antigen were selected for subsequent testing. The control antigen was another recombinant protein in the pET28a vector (pET28a-HPV18 / E6, His tag). The screening process is shown in Table 1.

[0053] Table 1. Reactivity of different monoclonal antibody clones to HPV16 E7 recombinant protein and HPV18 E6 recombinant protein detected by indirect ELISA.

[0054]

[0055] 4.2 Preparation of Monoclonal Antibody Ascites

[0056] 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] 4.3. Affinity chromatography purification of monoclonal antibodies

[0058] 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 G (Cytiva) 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 purified monoclonal antibodies were stored in aliquots at -20°C.

[0059] 5. Screening of paired antibodies for double antibody sandwich ELISA

[0060] 5.1. HRP labeling of antibodies

[0061] The monoclonal antibody was labeled according to the instructions of the G-Biosciences HOOK™ HRP conjugation kit (Cat#: 786-313). The labeled antigen was dialyzed overnight in 0.01M PBS, pH 7.4 buffer, and glycerol was added at a 1:1 volume ratio. The cells were stored in aliquots at -20°C.

[0062] Specifically, dilute the antibody to be labeled with coupling buffer (provided with the kit) to a final concentration of 2 mg / mL. Add the diluted antibody solution to the HRP tube and mix thoroughly by pipetting. Incubate at room temperature for 1 hour, mixing regularly during the incubation period. Add 50 μL of stop solution and mix for 15 minutes to terminate the labeling reaction. Dialyze overnight against PBS buffer and add an equal volume of glycerol for storage.

[0063] 5.2. Establishment of the Double Antibody Sandwich Method

[0064] The purified monoclonal antibodies were coated at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL respectively with coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water, pH 9.6) 50 μL / well for overnight coating at 4°C. The coating solution was discarded the next day and blocked with 1% BSA, 150 μL / well, incubated at 37°C for 2 h, and the blocking solution was discarded. The antigen HPV16 type E7 recombinant protein and the negative control antigen HPV18 type E6 protein were diluted with PBS at 100 ng / mL and added to the ELISA plate, 50 μL / well, incubated at 37°C for 35 min, and the plate was washed 4 times with PBST solution. 500, 1000, and 2000-fold diluted HRP-labeled monoclonal antibodies were added at 50 μL / well and incubated at 37°C for 35 min. The plate was then washed four times and patted dry before adding 50 μL / well of TMB color development solution. The color was developed 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. 450 nm value. Calculate the P / N value and select the combination with the highest P / N and the strongest positive reaction as the screening pair of monoclonal antibodies. Antibody combinations with high detection values ​​for HPV16 type E7 recombinant protein and no reaction with HPV18 type E6 recombinant protein were selected as the optimal pair for the double antibody sandwich. The screening process is shown in Table 2.

[0065] Table 2 shows the results of double antibody sandwich ELISA paired antibody screening.

[0066]

[0067] 1000* means 1000-fold dilution.

[0068] To identify the optimal antibody combination, this experiment used candidate monoclonal antibodies as coating antibodies and HRP-labeled antibodies, respectively, in a double-antibody sandwich ELISA test to evaluate their specific recognition of HPV16 E7 recombinant protein while excluding cross-reactivity with other control antigens. The results showed that using 4F6 as both the coating antibody and the labeling antibody produced the best detection signal and specificity, confirming it as the optimal antibody combination.

[0069] 6. Identification of binding activity of paired monoclonal antibodies

[0070] Referring to the aforementioned indirect ELISA method, the screened paired monoclonal antibodies and other unrelated mouse monoclonal antibodies were serially diluted at 10ug / ml, 1ug / ml, 100ng / ml, 10ng / ml, 1ng / ml, and 100pg / ml. The binding activity of the HPV16 E7 monoclonal antibody was determined using other unrelated mouse monoclonal antibodies HPV18 type E7 (Santa Cruz, F-7) as negative controls.

[0071] Based on the above double antibody sandwich ELISA pairing results, 4F6 was finally selected as the coating and labeling antibody, which had a good detection effect on the target protein. The binding activity of the screened monoclonal antibodies to the HPV16 type E7 recombinant protein was identified by indirect ELISA. The results are shown in Figure 3 . Figure 3 4F6 is the monoclonal antibody 4F6, and Ctrl is the HPV18 E7 (SantaCruz, F-7). Monoclonal antibody 4F6 still reacts with HPV16 E7 recombinant protein at a concentration of 1 ng / ml, indicating that this monoclonal antibody has a high binding titer.

[0072] 7. Application of paired monoclonal antibodies in biotin amplification system

[0073] Biotin can specifically bind to avidin or biotin monoclonal antibodies, resulting in a multi-stage amplification effect. The combination is highly stable and specific. In practical applications, it can not only greatly improve the sensitivity of the detection method, but also minimize non-specific binding of the reaction reagents. Therefore, the use of a biotin-avidin amplification system can effectively improve the detection performance of antibody pairs.

[0074] 7.1. Biotin-antibody conjugation

[0075] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) to antibody conjugation was 20:1. First, 2.0 mg of activated biotin was dissolved in 360 μL of ultrapure water to prepare a 10 mM biotin solution. Then, 2 mg of antibody was reacted with 26.6 μL of 10 mM biotin at room temperature with shaking for 3 hours (the reaction volume was kept around 2 mL). The biotin-antibody mixture was then dialyzed against 0.01 M PBS to remove excess free biotin. After dialysis, the antibody concentration was determined, and an equal volume of glycerol was added and stored at -20°C. The final labeled antibody concentration was approximately 0.5 mg / mL.

[0076] The biotin-conjugated monoclonal antibody used in this experiment was the best monoclonal antibody 4F6 screened in the early stage. This antibody was labeled with biotin and used to construct a biotin-avidin signal amplification system.

[0077] 7.2 Establishment of Biotin-Avidin Amplified ELISA System

[0078] The monoclonal antibody combination screened above was used for coating and biotin coupling respectively, and then the most suitable reaction conditions for the amplification system were determined by exploring the coating antibody, biotin antibody concentration and the dilution of HRP-labeled avidin.

[0079] Microplates were coated with monoclonal antibodies (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, fixed to 1 L pure water) at a coating concentration gradient of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL, 50 μL / well, and incubated at 4°C overnight. The plates were washed once with washing buffer (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 1% sucrose and 2% BSA, 150 μL per well, at 37°C for 2 hours. After patting dry, the plates were dried and stored for later use. HPV16 type E7 recombinant protein was diluted to a concentration of 10 ng / mL with PBS and 50 μL was added to the microplates coated with monoclonal antibodies. At the same time, HPV18 type E6 recombinant protein was diluted to 10 ng / mL as a negative control and reacted at 37°C for 30 min. Drain the liquid from the wells, wash the plate 4 times with PBST, and pat dry. Dilute the biotin-conjugated monoclonal antibody with PBS to a concentration of 1 μg / mL, 2 μg / mL, and 4 μg / mL, add 50 μL / well to the microplate, and react at 37°C for 30 minutes. Drain the liquid from the wells, wash the plate 4 times with PBST, pat dry, add 50 μL / well of HRP-polymer streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10,000, 20,000, and 40,000 times with PBS), react at 37°C for 30 minutes, wash the plate 4 more times, pat dry, add 50 μL / well of TMB colorimetric solution, develop at room temperature for 10 minutes, and finally add stop solution to terminate the reaction. Measure the OD value with a microplate reader. 450 The optimal reaction conditions were selected as the coating concentration, biotin monoclonal antibody concentration, and HRP-labeled avidin dilution with the most obvious positive and negative differences.

[0080] The experiment determined the optimal conditions for the reaction system, namely: the 4F6 antibody coating concentration was 1ug / ml, which was also used as a labeled antibody at a concentration of 2ug / ml, and the HRP-streptavidin dilution was 40,000 times.

[0081] 7.3 Sensitivity and Specificity Evaluation of Biotin-Avidin Amplified ELISA

[0082] To determine the optimal conditions of the reaction system, referring to the above detection steps, the HPV16 E7 recombinant protein was first serially diluted with PBS buffer solution to a concentration of 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. At the same time, three recombinant proteins, HPV16 E6, HPV18 E6, and HPV18 E7, were taken and tested at the same concentration, with 50 μL added to each well to determine the detection sensitivity of the detection system for the recombinant proteins.

[0083] The screened paired monoclonal antibodies were applied to the biotin-avidin amplified ELISA system, and the reaction system was optimized. Figure 4 As shown, Figure 4 HPV16 E6 is a recombinant HPV16 E6 protein, HPV16 E7 is a recombinant HPV16 E7 protein, HPV18 E6 is a recombinant HPV18 E6 protein, and HPV18 E7 is a recombinant HPV18 E7 protein. Under the optimized conditions described above, the HPV16 E7 recombinant protein remained positive even when diluted to 100 pg / ml. It also showed no reaction with high-risk oncoproteins, such as HPV16 E6, HPV18 E6, and HPV18 E7. This demonstrates the excellent specificity and high sensitivity of this amplification system, making it suitable for the detection of HPV16 E7 oncoprotein in cervical exfoliated cells and holds great promise for the early screening and diagnosis of cervical cancer.

[0084] For the preparation of HPV16 type E6 recombinant protein, please refer to the preparation method disclosed in CN119350484A.

[0085] Preparation of HPV18 E6 recombinant protein: The HPV18 E6 gene sequence was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0086] Nucleotide sequence (SEQ ID NO.13):

[0087] ATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGACA TTATTCAGACTCTGTGTATGGAGACACATTGGAAAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAACGACTCCAACGACGCAGAGAAACACAAGTATAA.

[0088] Amino acid sequence (SEQ ID NO.14):

[0089] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV.

[0090] The recombinant plasmid pET28a-HPV18 / E6 expressing HPV18 E6 was transformed into BL21(DE3) competent cells (Molecular Cloning, 3rd edition, Science Press) using conventional methods. Transformants were plated on LB agar plates (containing 50 μg / mL kanamycin) and cultured overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C, 220 rpm, and 1% of the total culture volume. The cells were inoculated into LB medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C, 220 rpm, for approximately 3 hours. The cells were then induced with a final concentration of 0.1 mM IPTG at 30°C, 200 rpm, and harvested for 4 hours.

[0091] Preparation of recombinant HPV18 E7 protein: The HPV18 E7 gene sequence was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET32a expression vector. The recombinant plasmid pET32a-HPV18 / E7 expressing HPV18 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 100 μg / mL ampicillin) and cultured overnight at 37°C. A single colony was picked and inoculated into 5 mL of LB medium (containing 100 μg / mL ampicillin) and cultured overnight at 37°C with shaking at 220 rpm. Inoculate 1% of the total culture volume into LB medium (containing 100 μg / mL ampicillin) and culture with shaking at 37°C, 220 rpm for approximately 3 hours until the OD600 reaches 0.6-0.9. Induce with 0.1 mM IPTG at 30°C, 200 rpm for 4 hours, and harvest the cells. The HPV18 E7 protein gene sequence is from GenBank NC_001357.1, which contains the complete coding sequence (CDS). A prokaryotic expression plasmid, pET32a-HPV18 E7 recombinant protein, has been successfully constructed, expressing the full-length E7 protein.

[0092] 8. Monoclonal antibody variable region gene cloning and sequencing

[0093] 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.

[0094] Monoclonal antibody 4F6:

[0095] Heavy chain:

[0096] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.9:

[0097] CAGGCCTACCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACACCTTCAGCTTCAGGTGGAACAACTGGGTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCAGGATCTTCCCCAGGGACGGCGAGATCAACTACAA CGAGAACTTCAGCGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGTGGACAGCGCCGTGTACTTCTGCAGGTACTACTACCAGAGCATCAGCGTGGTGGTGATGGACTACTGGGTCAGGGCACCAGCGTGACCGTGAGCAGC.

[0098] The amino acid sequence of the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.7:

[0099] QAYLQQSGPELVKPGASVKISCKASGYTFSFRWNNWVKQRPGQGLEWIGRIFPRDGEINYNENFSGKATLTADKSSSTAYMQLSSLTSVDSAVYFCRYYYQSISVVVMDYWVQGTSVTVSS.

[0100] CDR region annotation:

[0101] The sequence of the complementarity determining region CDR-H1 of the heavy chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO. 1: FRWNN;

[0102] The sequence of the complementary determining region CDR-H2 of the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO. 2: RIFPRDGEINYNENFSG; the sequence of the complementary determining region CDR-H3 of the heavy chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO. 3: YYQSISVVVMDY.

[0103] Light chain:

[0104] The nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO.10:

[0105] GAGATCGTGCTGACCCAGAGCCCCGAGAGCCTGGCCGTGAGCCTGGGCCAGAGGGCCACCATCAGCTGCCAGGCCAGCGACAGCGAGGACAGCAGGGGCAAGAGGTTCATGCACTGGTTCCAGCAGAAGCCCGGCCAGCCCCCCCAAGCTGCTGATCTACCTGGCCAGCATG ATCGAGAGCGGCGTGCCCGCCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCGACCCCGTGGAGGCCGACGACGTGGCCACCTACTACTGCCAGCAGTGGAACGAGCCCCTGGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0106] The amino acid sequence of the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.8: EIVLTQSPESLAVSLGQRATISCQASDSEDSRGKRFMHWFQQKPGQPPKLLIYLASMIESGVPARFSGSGSGTDFTLTIDPVEADDVATYYCQQWNEPPWTFGGGTKLEIKRTV.

[0107] CDR region annotation;

[0108] The complementary determining region CDR-L1 sequence of the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.4: QASDSEDSRGKRFMH; the complementary determining region CDR-L2 sequence of the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.5: LASMIES; the complementary determining region CDR-L3 sequence of the light chain variable region of monoclonal antibody 4F6 is shown in SEQ ID NO.6: QQWNEPPWT.

[0109] Thus far, various embodiments of the present invention have been described in detail. To avoid obscuring the concept of the present invention, 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.

[0110] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art will appreciate that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate 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 invention.

Claims

1. A monoclonal antibody 4F6 for detecting HPV16 type E7 protein, characterized in that: The heavy chain variable region of the monoclonal antibody 4F6 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 4F6 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 4F6 according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO.

8.

3. The monoclonal antibody 4F6 according to claim 2, characterized in that The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO.9; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 4F6 is shown in SEQ ID NO.

10.

4. Use of the monoclonal antibody 4F6 according to claim 1 in the preparation of a tool for detecting HPV16 type E7 protein.

5. The use according to claim 4, characterized in that The monoclonal antibody 4F6 is used to construct an ELISA detection system, which is used to detect HPV16 type E7 protein in biological samples. The ELISA detection system is not used for disease diagnosis.

6. The use according to claim 5, characterized in that The ELISA detection system is a biotin-avidin amplified ELISA system.

7. The use according to claim 6, characterized in that The tool is used for in vitro detection of HPV16 type E7 protein in a biological sample, wherein the biological sample is selected from cervical exfoliated cells, and the detection is not used for diagnosis of a disease.

8. The use according to claim 7, characterized in that The tools include reagents, test kits, test strips and antibody chips.

9. The application according to claim 8, characterized in that: The kit includes a double antibody sandwich ELISA kit.

10. The use according to claim 9, characterized in that: The kit utilizes the monoclonal antibody 4F6 for coating and labeling.

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