A monoclonal antibody combination for HPV18 type E6 protein detection and its application

Abstract

The present invention relates to the field of biological detection technology, and in particular to a monoclonal antibody combination and application for the detection of HPV18 type E6 protein. The provided combination consists of 5G3 and 2C7, and the amino acid sequences of the complementary determining regions of the heavy chain and light chain variable regions are clear, as shown in SEQ ID NO.1-12, respectively. The antibody combination has high specificity and sensitivity, with a minimum detection limit of up to 100 pg / ml, and does not cross-react with other HPV subtypes. The double-antibody sandwich ELISA and biotin-avidin amplification detection system constructed based on the combination significantly improves the signal intensity and detection accuracy, is suitable for the rapid detection of HPV18 type E6 protein in cervical exfoliated cell samples, and has application value in the early diagnosis of cervical cancer, risk stratification, vaccine development and efficacy evaluation.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a monoclonal antibody combination and application for detecting HPV18 type E6 protein. Background Art

[0002] Human papillomavirus (HPV) is a double-stranded DNA virus belonging to the family Papovaviridae. It is known to infect human skin and mucosal epithelial cells and is closely associated with the development of various benign warts and malignant tumors. Among them, persistent infection with high-risk HPV is considered the primary pathogenic factor for cervical cancer and its precancerous lesions.

[0003] The HPV genome primarily consists of an early region (E region) and a late region (L region). The early region encompasses multiple open reading frames, including E1 to E7. E6 and E7 are proven key oncogenic proteins. In HPV type 18, the E6 protein disrupts normal host cell regulatory pathways through multiple mechanisms, promoting abnormal cell proliferation and malignant transformation. Studies have shown that the HPV 18 E6 protein specifically binds to and mediates the ubiquitination and degradation of the tumor suppressor protein p53, thereby impairing the cell's response to DNA damage, reducing cell cycle control and apoptosis. Furthermore, E6 can act on other key regulatory proteins, such as NFX1-91, the E2F1 / DP1 complex, PDZ domain proteins, and MAGUK family proteins, further affecting telomerase activity, cell signaling, and polarity maintenance, driving cell transformation to a malignant phenotype.

[0004] During the development of cervical cancer, the expression level of HPV18 E6 protein increases significantly and is closely associated with the progression of cervical intraepithelial neoplasia (CIN). Its expression not only reflects the transcriptional activity of the viral genome but also positively correlates with the severity of the lesion. Therefore, detecting HPV18 E6 protein expression can serve as an important biomarker for assessing the risk of cervical precancerous lesions. Compared with traditional HPV DNA testing, E6 protein detection can better reflect whether the virus has entered a persistent infection state and has potential carcinogenic activity, helping to reduce false-positive results caused by transient infections and improving screening specificity and clinical utility.

[0005] Currently, cervical cancer screening relies primarily on cervical cytology (TCT) and HPV DNA testing (such as HC2). TCT, as the earliest method used for cervical cancer screening, has established a well-established screening system in many countries and regions. Studies have shown that large-scale, organized cytology testing can significantly reduce cervical cancer morbidity and mortality. However, in practice, this method is affected by sampling quality, slide preparation, and subjective judgment by pathologists, resulting in low sensitivity and poor reproducibility, limiting its widespread application in grassroots or low-resource settings where specialized imaging personnel are scarce. HPV DNA testing has high sensitivity and can effectively identify cervical intraepithelial neoplasia and higher lesions, but it also has limitations. Firstly, the testing process is susceptible to sample contamination. Secondly, because over 90% of HPV infections are transient and typically clear the virus within 2–3 years without progressing to precancerous lesions or cancer, relying solely on HPV DNA testing can easily lead to false-positive results, resulting in unnecessary psychological burden on the patient and excessive clinical interventions, such as colposcopy and biopsy. In addition, when the HPV genome integrates into the host cell genome, the L1 region is often lost, which may cause false negatives in detection methods designed based on the L1 region, resulting in missed diagnoses. Therefore, the development of a new detection method that can accurately reflect the persistent HPV infection status and its carcinogenic potential, especially a detection method for the high-risk HPV18 type E6 protein, is of great practical significance for improving the accuracy of cervical cancer screening, achieving precise stratification of lesion risk, optimizing clinical management strategies, and promoting vaccine development and efficacy evaluation. Summary of the Invention

[0006] This application uses prokaryotic expression technology to obtain an active HPV18 type E6 recombinant protein, i.e., an antigen, and uses this to screen out a highly binding monoclonal antibody, which can be used to detect the HPV18 type E6 protein with a sensitivity of 100 pg / ml. The double-antibody sandwich detection method based on this monoclonal antibody is specific to the HPV18 E6 oncoprotein, i.e., the HPV18 type E6 protein, and has no cross-reaction with other HPV oncoproteins. 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 risk of false positives and misdiagnosis caused by the excessive sensitivity of nucleic acid detection, and has high application value.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include: a monoclonal antibody combination for detecting HPV18 type E6 protein, the monoclonal antibody combination comprising monoclonal antibody 5G3 and monoclonal antibody 2C7, the heavy chain variable region of monoclonal antibody 5G3 comprising three complementarity determining regions, the amino acid sequences of the complementarity determining regions being shown as SEQ ID NO. 1 to SEQ ID NO. 3, respectively;

[0008] The light chain variable region of monoclonal antibody 5G3 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0009] The heavy chain variable region of monoclonal antibody 2C7 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 7 to SEQ ID NO. 9, respectively;

[0010] The light chain variable region of monoclonal antibody 2C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

[0011] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.14.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.16.

[0013] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G3 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G3 is shown as SEQ ID NO.18.

[0014] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2C7 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2C7 is shown as SEQ ID NO.20.

[0015] The present invention provides use of the above-mentioned monoclonal antibody combination in preparing a tool for detecting HPV18 type E6 protein.

[0016] In some embodiments, the tool is used to detect HPV18 type E6 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.

[0017] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0018] In some embodiments, the monoclonal antibody combination is used to construct an ELISA detection system, and the ELISA detection system is used to detect HPV18 type E6 protein in a sample. The ELISA detection system is not used for disease diagnosis.

[0019] In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0020] Beneficial effects:

[0021] The monoclonal antibody combination (5G3 and 2C7) for detecting HPV18 E6 protein provided by this invention exhibits high specificity and sensitivity, specifically recognizing the HPV18 E6 oncoprotein with a minimum detection limit of 100 pg / ml and exhibiting no cross-reactivity with other HPV subtypes (such as HPV16 E6, HPV16 E7, and HPV18 E7). This antibody combination, constructed based on well-defined complementary determining region (CDR) amino acid sequences, is suitable for use in double-antibody sandwich ELISAs and biotin-avidin amplification systems, significantly improving detection signal and accuracy. It can be used for the rapid detection of HPV18 E6 protein in cervical exfoliated cell samples. Compared to traditional HPV DNA detection methods, this antibody combination better reflects the persistent infection status and carcinogenic potential of the virus, helping to reduce false-positive results and improving the specificity and clinical utility of cervical cancer screening. It has significant application value and broad prospects for promotion in the risk assessment, early diagnosis, vaccine development, and efficacy evaluation of cervical precancerous lesions. 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 HPV18 type E6 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 meanings 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 meanings consistent with their meanings 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 HPV18 E6 gene sequence was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0033] The nucleotide sequence corresponding to the HPV18 type E6 gene is shown in SEQ ID NO.21:

[0034] ATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGACATTATTCAGACTCTGTGTATGGAGACACATTGGAAAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAACGACTCCAACGACGCAGAGAAACACAAGTATAA。

[0035] The amino acid sequence corresponding to the E6 gene of HPV type 18 is shown in SEQ ID NO. 22:

[0036] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV。

[0037] 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.

[0038] In this application, HPV18 type E6 protein refers to the wild-type E6 protein naturally present in HPV18 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 HPV18 type E6 recombinant protein is an E6 protein artificially expressed in a prokaryotic expression system through genetic engineering technology. It is usually obtained by cloning the HPV18 type E6 gene into an expression vector and inducing expression in the host. In this application, HPV18 type E6 recombinant protein is used as an antigen for screening and identifying specific monoclonal antibodies against E6 protein, which can effectively identify HPV18 type E6 protein in its natural state, thereby realizing the detection and evaluation of HPV18 infection and potential lesions in clinical samples.

[0039] 2. Purification and identification of HPV18 E6 recombinant protein

[0040] 2.1 Purification of HPV18 E6 recombinant protein

[0041] Because the expressed HPV18 E6 recombinant protein carries a histidine tag, it was purified using GE AKTA Start HisTrap™ HP affinity chromatography columns. Buffer A consisted of 50 mM PB, 300 mM NaCl, pH 8.0, and buffer B consisted of 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The column was equilibrated with buffer A. The fermented bacterial broth was then centrifuged at 8000 rpm for 10 minutes. The pellet was resuspended in buffer A and disrupted in ice water by sonication for 30 minutes, followed by 5-second intervals and 5-second intervals. The pellet was then centrifuged at 12000 rpm for 30 minutes. The supernatant was filtered through a 0.22 μm filter and loaded onto the column. The column was washed with buffer A and then eluted with a gradient of buffer B. Purification was assessed by SDS-PAGE. The target protein peak was collected and dialyzed into buffer A. The protein concentration was determined using a Nanodrop assay. The pellet was aliquoted into 1 ml tubes and stored at -20°C.

[0042] The purification status was observed by SDS-PAGE protein gel electrophoresis. Figure 1 . Figure 1 M in the middle: protein marker, 1: pET28a-HPV18 type E6 recombinant protein, with a clear main band visible between 17-25 kDa, and the protein purity is approximately over 80%, which is consistent with the estimated antigen size (21.5 kDa). The purified protein can be used for further downstream experiments.

[0043] 2.2. Identification of HPV18 E6 recombinant protein by indirect ELISA

[0044] The purified HPV18 E6 recombinant protein was coated and the reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was purchased from a commercial manufacturer (Santa Cruz, G-7). 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; 1% BSA was blocked, 100 μl per well, 37°C for 2 hours, and the plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20) and patted dry; the purchased commercial monoclonal antibody was diluted (PBS) according to a gradient of 1ug / ml, 100ng / ml, 10ng / ml, and 1ng / ml, and 50 μL was added to the antigen-coated microplate. At the same time, other unrelated HPV18 E7 monoclonal antibodies (Santa Cruz, F-7) were used as negative controls and reacted at 37°C for 30 minutes. The liquid in the wells was shaken off, 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 again 4 times. After patting dry, 50 μL / well of TMB color development solution was added and color was developed at room temperature for 10 min. Finally, 0.5 M sulfuric acid was added to stop the reaction. The OD was measured using a microplate reader. 450 nm value. See the results Figure 2 , Figure 2 G-7 is the commercial monoclonal antibody Santa Cruz G-7, and Ctrl is the HPV18 E7 monoclonal antibody Santa Cruz, F-7. Purified HPV18 E6 recombinant protein was coated onto an ELISA plate at a concentration of 1 μg / mL. A serial dilution of the commercial monoclonal antibody Santa Cruz G-7 was used for detection. Results showed a significant positive signal across the dilution range of 10 ng / mL to 1 ng / mL, indicating that the purified HPV18 E6 recombinant protein is active and suitable for use in further experiments.

[0045] 3. Mouse immunization

[0046] The purified 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 / mouse. At weeks 2 and 4, the same dose was mixed with an equal volume of MF59 adjuvant and injected intramuscularly. At week 5, sera were collected from mice for antibody titer analysis. Mice with the highest titer were selected for a booster immunization with 20 μg of HPV18 E6 recombinant protein via intraperitoneal bolus. Three days later, spleens were harvested from the mice for hybridoma cell production.

[0047] 4. Screening, preparation of hybridoma cell lines and antibody purification

[0048] 4.1 Screening of hybridoma cell lines

[0049] 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 HPV18 E6 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 HPV18 E6 recombinant protein, ensuring specificity and avoiding false-positive results due to nonspecific binding. Positive cells were cloned to a monoclonal state by limiting dilution, and the cell lines were then expanded and cryopreserved.

[0050] Screening of positive clones by indirect ELISA: After cell fusion, hybridoma cell lines that can secrete recombinant protein that specifically recognizes HPV18 type E6 are screened using the indirect ELISA method.

[0051] Microplates were coated with HPV18 E6 recombinant protein and pET28a-derived HPV16 E7 recombinant protein (HIS tag) at a concentration of 1 μg / mL (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water). The plates were incubated overnight at 4°C. Plates were then blocked with 150 μL of 1% BSA per well for 2 hours at 37°C. The plates were washed once with detergent and patted dry. 50 μL of cell culture supernatant was added and incubated at 37°C for 30 minutes. The wells were then discarded, washed four times with PBST, patted dry, and HRP-conjugated goat anti-mouse secondary antibody (diluted 1:5000 in PBS) was added at 50 μL / well. The plates were incubated at 37°C for 30 minutes. The plates were then washed four times, patted dry, and TMB color development solution (50 μL / well) was added for 10 minutes at room temperature. The reaction was terminated with 0.5 M sulfuric acid, and the OD was measured using a microplate reader. 450 nm value. Positive cell lines that reacted only with HPV18 E6 recombinant protein and not with the control antigen were selected for subsequent testing. The screening process is shown in Table 1.

[0052] Table 1: Indirect ELISA reaction results of hybridoma cell lines to HPV18 E6 recombinant protein and control antigen.

[0053]

[0054] 4.2 Preparation of Monoclonal Antibody Ascites

[0055] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6Female 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.

[0056] 4.3. Affinity chromatography purification of monoclonal antibodies (Protein G)

[0057] 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 an AKTA 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 0.01 M PBS, pH 7.4, at 2-8°C for 14 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 a micro-spectrophotometer and stored in aliquots.

[0058] 5. Screening of paired antibodies for double antibody sandwich ELISA

[0059] 5.1. HRP labeling of antibodies

[0060] The selected monoclonal antibodies were labeled according to the instructions of G-Biosciences' HOOK™ HRP PLUS Labeling Kit (Cat#: 786-313). Finally, the labeled antigen was dialyzed overnight in 0.01M PBS, pH 7.4 buffer, and glycerol was added at a volume ratio of 1:1. The cells were stored in aliquots at -20°C.

[0061] 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 tube containing HRP (provided with the kit) 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 the solution overnight against PBS buffer and add an equal volume of glycerol for storage.

[0062] 5.2. Establishment of the Double Antibody Sandwich Method

[0063] 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 to be tested (HPV18 type E6 recombinant protein) and the control antigen (HPV16 type E7 recombinant 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 times diluted enzyme-labeled monoclonal antibody, 50 μL / well, incubate at 37°C for 35 min, wash the plate 4 times, pat dry, add TMB color development solution 50 μL / well, develop at room temperature for 10 min, finally add 0.5 M sulfuric acid, 50 μL / well, stop the reaction, and measure OD with 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. The screening process is shown in Table 2.

[0064] Table 2: Comparison of the reactivity of different monoclonal antibody combinations against HPV18 E6 recombinant protein and control antigen in double antibody sandwich ELISA.

[0065]

[0066] “*” in the table indicates the dilution multiple.

[0067] To identify the optimal antibody combination, a double-antibody sandwich ELISA was performed using candidate monoclonal antibodies as coating antibodies and HRP-labeled antibodies, respectively. The goal was to identify a combination that specifically detected HPV18 E6 recombinant protein without cross-reactivity with other control antigens (such as HPV16 E7 recombinant protein). Ultimately, the optimal antibody combination was selected: coating antibody 5G3 and labeling antibody 2C7.

[0068] 6. Identification of binding activity of paired monoclonal antibodies

[0069] To validate the binding ability of the monoclonal antibodies in this combination, their binding activity to HPV18 E6 recombinant protein was again assessed using an indirect ELISA. This antibody demonstrated a significant signal even at a concentration of 1 ng / mL, further demonstrating its high affinity and excellent detection performance. Specifically, based on the potential matching antibodies screened using the double-antibody sandwich ELISA, a serial dilution of the selected matching monoclonal antibodies and another unrelated mouse monoclonal antibody, HPV18 E7 (Santa Cruz, F-7), was performed using the aforementioned indirect ELISA method (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 to HPV18 E6 recombinant protein. Other unrelated mouse monoclonal antibodies were used as negative controls to exclude the influence of nonspecific binding.

[0070] By measuring the OD at each concentration 450 nm values, draw the binding curve (see Figure 3 ). Figure 3 Here, 5G3 represents monoclonal antibody 5G3, 2C7 represents cloned antibody 2C7, and Ctrl represents HPV18 E7 monoclonal antibody (Santa Cruz, F-7). Monoclonal antibodies 5G3 and 2C7 serve as the preferred coating and labeling antibodies, respectively, for double-antibody sandwich ELISAs. Indirect ELISA assays testing the binding activity of these two antibodies revealed that both 5G3 and 2C7 exhibited weak positive reactions with the antigen (HPV18 E6 recombinant protein) at a concentration of 1 ng / ml. The control monoclonal antibody showed no reaction with the HPV18 E6 recombinant protein, indicating that both monoclonal antibodies possess high binding potency.

[0071] 7. Application of paired monoclonal antibodies in biotin amplification system

[0072] 7.1. Biotin-antibody conjugation

[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] 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.

[0075] 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 2C7 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 streptavidin. The monoclonal antibody 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) with 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 plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20), patted dry, and blocked with 1% BSA, 150 μL per well, at 37°C for 2 hours, patted dry, and stored dry for later use; HPV18 type E6 recombinant protein was diluted to a concentration of 100 ng / mL with PBS, and 50 μL was added to the microplate coated with the monoclonal antibody. At the same time, HPV16 type recombinant protein E6 was diluted to 100 ng / mL as a negative control, and reacted at 37°C for 30 minutes. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry. The biotin-conjugated monoclonal antibody was diluted with PBS to a concentration of 1μg / mL, 2μg / mL, and 4ug / mL, and 50μL / well was added to the microplate. The reaction was carried out at 37°C for 30 minutes. The liquid in the wells was shaken off, the plate was washed 4 times with PBST solution, and patted dry. HRP-polymer streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10,000, 20,000, and 40,000 times with PBS) was added at 50μL / well. The reaction was carried out at 37°C for 30 minutes. The plate was washed 4 times, patted dry, and TMB color development solution was added at 50μL / well. The color was developed at room temperature for 10 minutes. Finally, the stop solution was added to terminate the reaction, and the OD was measured using 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.

[0079] The experimental results showed that the optimal reaction conditions were: coated monoclonal antibody 5G3, concentration of 1ug / ml; biotin-conjugated monoclonal antibody 2C7, dilution of 2ug / ml; HRP-polymer streptavidin 40,000 times dilution.

[0080] 7.3 Sensitivity and Specificity Evaluation of Biotin-Avidin Amplified ELISA

[0081] Determine the optimal coating concentration, biotin monoclonal antibody concentration, HRP-avidin dilution and other reaction conditions. Referring to the above detection steps, first perform a gradient dilution of the HPV18 E6 recombinant protein with PBS buffer solution. The concentrations after dilution are 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 100 pg / mL, and 10 pg / mL, respectively. At the same time, take three recombinant proteins, including HPV16 E6, HPV16 E7, and HPV18 E7, and add 50 μL to each well at the same concentration for detection to determine the detection sensitivity of the detection system for recombinant proteins.

[0082] For the preparation of recombinant HPV16 E6 protein, refer to the preparation method disclosed in CN119350484A. For the preparation of recombinant HPV18 E7 protein, the HPV18 E7 gene sequence was downloaded from NCBI, synthesized by Anhui General Biotechnology, 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 volume of the culture medium 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, and harvest the cells for 4 hours. The HPV18 E7 protein gene sequence is from GenBank NC_001357.1, containing the complete coding sequence (CDS). A prokaryotic expression plasmid pET32a-HPV18 E7 recombinant protein was successfully constructed, expressing the full-length E7 protein. Preparation of HPV16 E7 recombinant protein: The HPV16 E7 gene was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector. The nucleotide sequence corresponding to the HPV16 E7 gene is shown in SEQ ID NO. 23: ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA. The amino acid sequence corresponding to the HPV16 E7 gene is shown in SEQ ID NO. 24: MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.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.

[0083] See also Figure 4 This combination detected HPV18 E6 recombinant protein in the biotin amplification system and still showed a positive reaction when diluted to 100pg / ml. It did not react with HPV16 E6 recombinant protein (HPV16 E6), HPV16 E7 recombinant protein (HPV16 E7), and HPV18 E7 recombinant protein (HPV18 E7), demonstrating the good specificity and high sensitivity of the amplification system. It can be used to detect HPV18 E6 oncoprotein in cervical exfoliated cells and has important application prospects in the early screening and diagnosis of cervical cancer.

[0084] 8. Monoclonal antibody variable region gene cloning and sequencing

[0085] 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.

[0086] Monoclonal antibody 5G3 monoclonal antibody variable region gene:

[0087] Heavy chain:

[0088] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO. 17: GAGGTGCAGCTGCAGGAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCCTGGTGAAGCTGAGCTGCAAGGTGAGCGGCTTCAACATCAAGGACGTGGTGATCCACTGGGTGAAGCAGAGGCCCGAGCAGGGCCTGGAGTGGATCGGCTGGATCGACCCCGAGAAGGTGGAGACCATCTACGACCCCAAGTTCAGCGGCAAGGCCAGGATCACCGTGGACACCAGCGCCAACATCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACGCCGCCGTGTACTACTGCAGCACCGGCAGGTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0089] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.13:

[0090] EVQLQESGAELVRPGALVKLSCKVSGFNIKDVVIHWVKQRPEQGLEWIGWIDPEKVETIYDPKFSGKARITVDTSANIAYLQLSSLTSEDAAVYYCSTGRYWGQGTSVTVSS.

[0091] The complementary determining region CDR-H1 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.1: GFNIKDVV; the complementary determining region CDR-H2 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.2: IDPEKVET; the complementary determining region CDR-H3 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.3: STGRY.

[0092] Light chain:

[0093] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.18:

[0094] GACATCCTGCTGACCCAGAGCCCCCTGACCCTGAGCGTGACCATCGGCCAGCCCGCCAGCATCAGCTGCAAGAGGAGCCAGAGCATCATCGACAGCGACGGCGAGACCGAGCTGAACTGGCTGCTGCAGAGGCCCGGCCAGAGCCCCAAGAGGCTGATCTACCTGGTGAGCCA GCTGCCCAGCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTTGGCAGGGCACCGAGTTCCCCCAGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0095] The amino acid sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.14:

[0096] DILLTQSPLTLSVTIGQPASISCKRSQSIIDSDGETELNWLLQRPGQSPKRLIYLVSQLPSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTEFPQTFGGGTKLEIKRTV.

[0097] CDR region annotation:

[0098] The complementary determining region CDR-L1 sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.4: KRSQSIIDSDGETELN; the complementary determining region CDR-L2 sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.5: LVSQLPS; the complementary determining region CDR-L3 sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.6: WQGTEFPQT.

[0099] Monoclonal antibody 2C7 variable region gene:

[0100] Heavy chain:

[0101] The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO. 19: GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAGGCCCGGCGCCCTGGTGAAGCTGAGCTGCAAGGTGAGCGGCTTCAACATCAAGGACGTGGGCATCCACTGGGTGAAGCAGAGGCCCGAGCAGGGCCTGGAGTGGATCGGCTGGAGCCAGGAGAAGGTGTACTACATCGAGCCCGAGTTCAGCGGCAAGGCCAGGATCACCGTGGACACCAGCGCCAACATCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACGCCGCCGTGTACTACTGCAGCACCAAGAGGAGGGTGATCTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC.

[0102] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.15:

[0103] EVQLQQSGPELVRPGALVKLSCKVSGFNIKDVGIHWVKQRPEQGLEWIGWSQEKVYYIEPEFSGKARITVDTSANIAYLQLSSLTSEDAAVYYCSTKRRVIYWGQGTSVTVSS.

[0104] CDR region annotation:

[0105] The complementary determining region CDR-H1 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.7: DVGIH; the complementary determining region CDR-H2 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.8: WSQEKVYYIEPEFSG; the complementary determining region CDR-H3 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.9: KRRVIY.

[0106] Light chain:

[0107] The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.20:

[0108] GACATCCAGATGAACCAGAGCCCCCTGACCCTGAGCGTGGCCATCGGCCAGCCCGCCAGCATCAGCTGCAAAAATAGTAGGCAAAGTGACATTAGTATTGACGAGGGCGAGCTGACATGGCTGCTGCAGAGGCCCGGCCAGAGCCCCAAGAGGCTGATCTACCTGAGTGTGGT GCTGCAACCCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCCAGCAGTGGACCGAGGGCTTCCCCCTGTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG.

[0109] The amino acid sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO. 16: DIQMNQSPLTLSVAIGQPASISCKNSRQSDISIDEGELTWLLQRPGQSPKRLIYLSVVLQPGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCQQWTEGFPLFGGGTKLELKRTV.

[0110] CDR region annotation:

[0111] The complementary determining region CDR-L1 sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.10: KNSRQSDISIDEGELT; the complementary determining region CDR-L2 sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.11: LSVVLQP; the complementary determining region CDR-L3 sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.12: QQWTEGFPL.

[0112] 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.

[0113] 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 combination for detecting HPV18 type E6 protein, characterized in that: The monoclonal antibody combination includes monoclonal antibody 5G3 and monoclonal antibody 2C7. The heavy chain variable region of the monoclonal antibody 5G3 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively. The light chain variable region of the monoclonal antibody 5G3 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; The heavy chain variable region of the monoclonal antibody 2C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively; The light chain variable region of the monoclonal antibody 2C7 includes three complementarity determining regions, and the amino acid sequences of the complementarity determining regions are shown in SEQ ID NO.10 to SEQ ID NO.12, respectively.

2. The monoclonal antibody combination for detecting HPV18 type E6 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5G3 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5G3 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting HPV18 type E6 protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 2C7 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 2C7 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting HPV18 type E6 protein according to claim 3, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5G3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5G3 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting HPV18 type E6 protein according to claim 4, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 2C7 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 2C7 is shown in SEQ ID NO.

20.

6. Use of the monoclonal antibody combination according to claim 1 in the preparation of a tool for detecting HPV18 type E6 protein.

7. The use according to claim 6, characterized in that The tool is used for in vitro detection of HPV18 type E6 protein in a biological sample, wherein the biological sample is selected from cervical exfoliated cells, and the detection is not used for diagnosis of the disease.

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

9. The use according to claim 8, characterized in that The monoclonal antibody combination is used to construct an ELISA detection system, which is used to detect HPV18 type E6 protein in a sample. The ELISA detection system is not used for diagnosis of diseases.

10. The use according to claim 9, characterized in that The ELISA detection system is a biotin-avidin amplified ELISA system.

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