Monoclonal antibody combination for HPV18 type E6 protein detection and application

Abstract

The invention relates to the technical field of biological detection, in particular to a monoclonal antibody combination for HPV18 type E6 protein detection and application. The provided combination is composed of 5G3 and 2C7, and the amino acid sequences of complementary determining regions of variable regions of a heavy chain and a light chain of the combination are clear and are respectively shown as SEQ ID NO.1-12. The antibody combination has high specificity and sensitivity, the lowest detection limit can reach 100 pg / ml, and cross reaction with other HPV subtypes is avoided. According to a double-antibody sandwich ELISA and biotin-avidin amplification detection system constructed based on the combination, the signal intensity and the detection accuracy are remarkably improved, and the combination is suitable for rapid detection of the HPV18 type E6 protein in a cervical exfoliated cell sample and has application value in early diagnosis of cervical cancer, risk stratification, vaccine research and development and curative effect evaluation.

Description

Technical Field

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

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

[0003] The HPV genome mainly includes an early region (E region) and a late region (L region). The early region includes multiple open reading frames such as E1 to E7, among which E6 and E7 are key oncogenic proteins that have been proven. In HPV type 18, the E6 protein interferes with the normal regulatory pathways of host cells through multiple mechanisms, promoting abnormal cell proliferation and malignant transformation. Research shows that the HPV 18 E6 protein can specifically bind to and mediate the ubiquitination and degradation of the tumor suppressor protein p53, thereby weakening the cell's response to DNA damage, reducing cell cycle regulation and apoptosis. In addition, E6 can also act on other key regulatory proteins, such as NFX1-91, E2F1 / DP1 complex, PDZ domain proteins, and MAGUK family proteins, further affecting telomerase activity, cell signal transduction, and polarity maintenance, and promoting the transformation of cells into a malignant phenotype.

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

[0005] Currently, cervical cancer screening mainly relies on cervical cytology (TCT) and HPV DNA testing (such as HC2). Among them, as the earliest method applied to cervical cancer screening, TCT has established a relatively complete screening system in many countries and regions. Studies have shown that large-scale and organized cytological examinations can significantly reduce the incidence and mortality of cervical cancer. However, in practical applications, this method is affected by sampling quality, slide preparation technology, and the subjective judgment of pathologists, and has problems such as low sensitivity and poor repeatability, which limit its popularization and application in grass-roots or low-resource areas lacking professional film readers. HPV DNA testing has high sensitivity and can effectively identify cervical intraepithelial neoplasia and above lesions. However, this method also has certain limitations. On the one hand, the detection process is easily affected by sample contamination. On the other hand, since more than 90% of HPV infections are transient, the body can usually clear the virus on its own within 2-3 years and will not progress to precancerous lesions or cancer. Therefore, relying solely on HPV DNA testing is likely to lead to false-positive results, which in turn cause unnecessary psychological burdens and excessive clinical interventions for subjects, such as colposcopy and biopsy. In addition, when the HPV genome integrates into the host cell genome, deletions in the L1 region often occur, making detection methods based on the L1 region likely to show false negatives and cause missed diagnoses. Therefore, developing a new detection method that can accurately reflect the persistent HPV infection status and its carcinogenic potential, especially a detection method for the E6 protein of high-risk HPV type 18, has important practical significance for improving the accuracy of cervical cancer screening, achieving precise stratification of lesion risks, optimizing clinical management strategies, and promoting vaccine research and development and efficacy evaluation. Summary of the Invention

[0006] In this application, an active recombinant E6 protein of HPV type 18, that is, an antigen, was obtained based on prokaryotic expression technology, and a monoclonal antibody with high binding was screened out, which can be used for the detection of the E6 protein of HPV type 18, with a sensitivity of 100 pg / ml. Based on this monoclonal antibody, a double-antibody sandwich detection method specifically targets the E6 oncoprotein of HPV18, that is, the E6 protein of HPV type 18, and has no cross-reaction with the oncoproteins of other HPVs. In the future, it can be applied to the detection of cervical cancer, which is faster and more accurate than exfoliative cytology examination, and at the same time can avoid the false-positive and misdiagnosis risks brought by the high sensitivity of nucleic acid detection, and has high application value.

[0007] To achieve the above object, the main technical solutions adopted by the present invention include: a monoclonal antibody combination for detecting the E6 protein of HPV type 18, the monoclonal antibody combination includes monoclonal antibody 5G3 and monoclonal antibody 2C7, and the heavy chain variable region of monoclonal antibody 5G3 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are respectively shown as SEQ ID NO.1-SEQ ID NO.3; The light chain variable region of monoclonal antibody 5G3 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.4 - SEQ ID NO.6 respectively; The heavy chain variable region of monoclonal antibody 2C7 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.7 - SEQ ID NO.9 respectively; The light chain variable region of monoclonal antibody 2C7 includes three complementary determining regions, and the amino acid sequences of the complementary determining regions are shown in SEQ ID NO.10 - SEQ ID NO.12 respectively.

[0008] 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.

[0009] 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.

[0010] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.18.

[0011] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.20.

[0012] The present invention provides the application of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting HPV18 E6 protein.

[0013] In some embodiments, the tool is used for in vitro detection of HPV18 E6 protein in biological samples, the biological samples are selected from cervical exfoliated cells, and the detection is not used for the diagnosis of diseases.

[0014] In some embodiments, the tool includes reagents, reagent kits, test strips and antibody chips.

[0015] In some embodiments, the monoclonal antibody combination is used to construct an ELISA detection system, the ELISA detection system is used for detecting HPV18 E6 protein in samples, and the ELISA detection system is not used for the diagnosis of diseases.

[0016] In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0017] Beneficial effects: The monoclonal antibody combination (5G3 and 2C7) for detecting HPV18 E6 protein provided by the present invention has high specificity and high sensitivity, can specifically recognize the HPV18 E6 oncoprotein, has a minimum detection limit of up to 100 pg / ml, and has no cross-reaction with other HPV subtypes (such as HPV16 E6, HPV16 E7, HPV18 E7). This antibody combination is constructed based on the clear amino acid sequences of the complementarity-determining regions (CDRs), is applicable to the double-antibody sandwich ELISA and the biotin-avidin amplification system, significantly improves the detection signal and accuracy, and can be used for the rapid detection of HPV18 E6 protein in cervical exfoliated cell samples. Compared with the traditional HPV DNA detection method, this antibody combination can better reflect the persistent infection status and carcinogenic potential of the virus, helps to reduce false positive results, improves the specificity and clinical practicability of cervical cancer screening, and has important application value and broad popularization prospects in the risk assessment, early diagnosis, vaccine development and efficacy evaluation of cervical precancerous lesions. Brief description of the drawings

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is a SDS-PAGE protein identification diagram; Figure 2 It is an ELISA identification result diagram of the HPV18 E6 recombinant protein; Figure 3 It is a binding activity identification diagram of the paired monoclonal antibodies; Figure 4 It is a sensitivity and specificity identification diagram of the biotin-avidin amplified ELISA. Detailed implementation manners

[0020] The following will further describe in detail the implementation manners of the present invention in conjunction with the drawings and embodiments. The following detailed description of the embodiments and the drawings are used to exemplarily illustrate the principle of the present invention, but cannot be used to limit the scope of the present invention. The present invention can be implemented in many different forms, is not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

[0021] These embodiments of the present invention are provided to make the present invention thorough and complete, and to fully convey the scope of the present invention to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangements of components and steps, the compositions of materials, numerical expressions, and numerical values set forth in these embodiments should be construed as merely exemplary, rather than as limitations.

[0022] All terms used in the present invention have the same meanings as those understood by those of ordinary skill in the art to which the present invention pertains, unless otherwise specifically defined. It should also be understood that terms defined in common dictionaries should be construed as having meanings consistent with their meanings in the context of the relevant art, and should not be interpreted in an idealized or overly formal sense, unless specifically defined as such herein.

[0023] Example 1 1. Preparation of recombinant antigen Download the E6 gene sequence of HPV type 18 from NCBI, synthesized by Anhui General Biology, and cloned into the pET28a expression vector.

[0024] The nucleotide sequence corresponding to the E6 gene of HPV type 18 is shown in SEQ ID NO.21: ATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGACATTATTCAGACTCTGTGTATGGAGACACATTGGAAAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAACGACTCCAACGACGCAGAGAAACACAAGTATAA.

[0025] The amino acid sequence corresponding to the E6 gene of HPV type 18 is shown in SEQ ID NO.22: MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV。

[0026] The recombinant plasmid pET28a-HPV18 / E6 expressing HPV18 E6 was transformed into BL21(DE3) competent cells by the conventional method (Molecular Cloning, Third Edition, Science Press). The transformed bacteria were spread on an LB agar plate (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 overnight with shaking at 37°C and 220 rpm. It was inoculated into LB medium (containing 50 μg / mL kanamycin) at a ratio of 1% of the total volume of the medium, and cultured with shaking at 37°C and 220 rpm for about 3 hours until the OD600 reached 0.6 - 0.9. IPTG with a final concentration of 0.1 mM was added, and the cells were induced at 30°C and 200 rpm for 4 hours, and then the cells were collected.

[0027] In this application, the HPV18 E6 protein refers to the wild-type E6 protein naturally present in cells infected with the HPV18 virus, which is directly encoded by the viral genome, expressed in host cells, and participates in regulating key carcinogenic processes such as the cell cycle and inhibiting tumor suppressor proteins. It is a core pathogenic factor in the occurrence and development of cervical cancer. The recombinant HPV18 E6 protein, on the other hand, is an E6 protein artificially expressed in a prokaryotic expression system through genetic engineering techniques, usually obtained by cloning the HPV18 E6 gene into an expression vector and inducing its expression in a host. In this application, the recombinant HPV18 E6 protein is used as an antigen to screen and identify specific monoclonal antibodies against the E6 protein, which can effectively recognize the HPV18 E6 protein in its natural state, thereby enabling the detection and evaluation of HPV18 infection and potential lesions in clinical samples.

[0028] 2. Purification and Identification of Recombinant HPV18 E6 Protein 2.1 Purification of Recombinant HPV18 E6 Protein Since the expressed HPV18 E6 recombinant protein carries a histidine tag, it was purified using an AKTA Start and HisTrapTM HP affinity chromatography column from GE Healthcare. Buffer A was 50 mM PB, 300 mM NaCl, pH 8.0, and Buffer B was 50 mM PB, 300 mM NaCl, 0.5 M imidazole, pH 8.0. The chromatography column was equilibrated with Buffer A. Then, the fermented bacterial solution was centrifuged at 8000 rpm for 10 min. The pellet was resuspended in Buffer A, sonicated in ice water for 30 min with 5 s sonication intervals, centrifuged at 12000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter. The sample was loaded onto the column, and the column was washed with Buffer A and finally eluted with a gradient of Buffer B. The purification was observed by SDS-PAGE protein gel electrophoresis. The peak of the target protein was selected, dialyzed and exchanged to Buffer A, and the protein concentration was measured by Nanodrop. It was aliquoted into 1 ml / tube and stored at -20°C.

[0029] The purification was observed by SDS-PAGE protein gel electrophoresis as shown in Figure 1 . Figure 1 In which, M: protein Marker, 1: pET28a-HPV18 E6 recombinant protein. An obvious main band was visible between 17 - 25 kDa. The protein purity was approximately above 80%, consistent with the estimated antigen size (21.5 kDa). The purified protein can be used for further downstream experiments.

[0030] 2.2 Identification of HPV18 E6 recombinant protein by indirect ELISA Coated with purified HPV18 E6 recombinant protein, the reaction with positive monoclonal antibody was identified by indirect ELISA method. The positive monoclonal antibody was a commercially available monoclonal antibody purchased externally (Santa Cruz, G-7). First, coat the recombinant protein in a microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), the coating concentration was 1 μg / mL, 50 μl per well, overnight at 4 °C; block with 1% BSA, 100 μl per well, 2 hours at 37 °C, wash the plate once with washing solution (PBST, PBS containing 0.05% Tween-20), pat dry; dilute the commercially available monoclonal antibody purchased externally in gradients of 1 μg / ml, 100 ng / ml, 10 ng / ml, 1 ng / ml (PBS), add 50 μL to the microplate coated with antigen, and at the same time use other irrelevant HPV18 E7 monoclonal antibodies (Santa Cruz, F-7) as negative controls, react at 37 °C for 30 min. Discard the liquid in the wells, wash the plate 4 times with PBST washing solution, pat dry and then add 50 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS) per well, react at 37 °C for 30 min, wash the plate 4 times again, pat dry and then add 50 μL of TMB chromogenic solution per well for color development at room temperature for 10 min, finally add 0.5 M sulfuric acid to terminate the reaction, and measure the OD 450 nm value. The results are shown in Figure 2 , Figure 2 in which G-7 is the commercially available monoclonal antibody Santa Cruz G-7, and Ctrl is the HPV18 E7 monoclonal antibody (Santa Cruz, F-7). The purified HPV18 E6 recombinant protein was coated on the ELISA plate at a concentration of 1 μg / mL, and the commercially available monoclonal antibody Santa Cruz G-7 was used for gradient dilution detection. The results showed that significant positive signals were still presented in the dilution range of 10 ng / mL to 1 ng / mL. It indicated that the purified HPV18 E6 recombinant protein was active and could be used for the next experiment.

[0031] 3. Mouse immunization Mix the purified recombinant protein with an equal volume of Freund's complete adjuvant (200 μl) and immunize 6-week-old female BALB / c mice by subcutaneous multi-point injection, with a dose of 30 μg per mouse. Then, at the 2nd and 4th weeks, mix with an equal volume of MF59 adjuvant according to the above dose and immunize again by intramuscular injection. At the 5th week, collect mouse serum to detect the antibody titer. Select the mouse with the highest titer and boost immunize it intraperitoneally with 20 μg of HPV18 E6 recombinant protein. Three days later, take the mouse spleen for the preparation of hybridoma cells.

[0032] 4. Screening, preparation and antibody purification of hybridoma cell lines 4.1 Screening of hybridoma cell lines After fusing all splenocytes from immunized mice with SP2 / 0 myeloma cells in the logarithmic growth phase, they were placed in HAT medium for screening culture. When the fused cells grew to half the bottom of the well, positive clones of HPV18 E6 recombinant protein were screened by indirect ELISA. Since the immunogen was from prokaryotic expression and contained a His tag, the background components needed to be re-screened to select specific cell lines targeting the HPV18 E6 recombinant protein, ensuring the specificity of the detection results and avoiding false positive results caused by non-specific binding. The positive cell clones were cloned to the monoclonal state by limiting dilution method, and then the cell lines were expanded and cryopreserved.

[0033] Screening of positive clones by indirect ELISA: By indirect ELISA, hybridoma cell lines that could secrete specific antibodies recognizing the HPV18 E6 recombinant protein were screened after cell fusion.

[0034] In microtiter plates, the HPV18 E6 recombinant protein and the HPV16 E7 recombinant protein (His tag) of the pET28a vector were respectively coated (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentration was 1 μg / mL, overnight at 4°C; blocked with 1% BSA, 150 μL per well, blocked at 37°C for 2 hours, washed the plate once with washing solution, and patted dry; added 50 μL of cell culture supernatant, reacted at 37°C for 30 min. Discarded the liquid in the wells, washed the plate 4 times with PBST washing solution, patted dry and then added 50 μL / well of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000 with PBS), reacted at 37°C for 30 min, washed the plate 4 times again, patted dry and then added 50 μL / well of TMB chromogenic solution, developed color at room temperature for 10 min, and finally added 0.5 M sulfuric acid to terminate the reaction, and measured the OD 450 nm value. Positive cell lines that reacted only with the HPV18 E6 recombinant protein and not with the control antigen were selected for subsequent experiments. The screening process is shown in Table 1.

[0035] Table 1: Indirect ELISA reaction results of hybridoma cell lines against HPV18 E6 recombinant protein and control antigen.

[0036]

[0037] 4.2 Preparation of monoclonal antibody ascites After expanding the screened monoclonal cell lines, 0.2 ml (containing 2.5×10 6Female BALB / c mice (about 10 mice) pretreated with Freund's incomplete adjuvant. Approximately 10 days later, when the abdomen of the mice was significantly swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 r / min for 10 minutes, and the middle layer was collected.

[0038] 4.3 Affinity Chromatography Purification of Monoclonal Antibodies (Protein G) The ascites was centrifuged at 12000 r / min for 5 minutes. The supernatant was diluted 10-fold with binding buffer (20 mM PBS, 150 mM NaCl, pH 7.4), filtered through a 0.22-μm filter, and the filtered sample was slowly pumped into a Protein G (Cytiva) purification column equilibrated with binding buffer using a peristaltic pump. The AKTA purifier was connected, and the column was washed with binding buffer for 5 - 10 column volumes until the UV absorption peak was washed flat, and then eluted with elution buffer (0.1 M glycine, pH 2.7). The elution peak was collected, and the collected sample was adjusted to neutral with 1 M Tris-HCl, pH 9, loaded into a dialysis bag (MW: 8000 - 14000), and dialyzed in 0.01 M PBS, pH 7.4 solution at 2 - 8 °C for 14 hours. The liquid in the dialysis bag was transferred to a centrifuge tube and centrifuged at 12000 r / min 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.

[0039] 5 Screening of Paired Antibodies for Double Antibody Sandwich ELISA 5.1 HRP Labeling of Antibodies 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 0.01 M PBS, pH 7.4 buffer, and glycerol was added in a 1:1 volume ratio, and stored in aliquots at -20 °C.

[0040] Specifically: Dilute the antibody to be labeled with coupling buffer (supplied with the kit) to a final concentration of 2 mg / mL. Subsequently, add the diluted antibody solution to the tube containing HRP (supplied with the kit), and pipette to mix well. Incubate at room temperature for 1 h, and mix well regularly during the incubation. Add 50 μL of termination solution, mix well for 15 min to terminate the labeling reaction, dialyze overnight in PBS buffer, and add an equal volume of glycerol for freezing storage.

[0041] 5.2 Establishment of Double Antibody Sandwich Method Purify the monoclonal antibody and coat it overnight at 4°C with 50 μL / well of coating buffer (1.59 g sodium carbonate, 2.93 g sodium bicarbonate, made up to 1 L of pure water, pH 9.6) at concentrations of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL. The next day, discard the coating solution, block with 1% BSA at 150 μL / well, incubate at 37°C for 2 h, discard the blocking solution, dilute the antigen to be tested (HPV18 E6 recombinant protein) and the control antigen (HPV16 E7 recombinant protein) with PBS to 100 ng / ml and add 50 μL / well to the ELISA plate, incubate at 37°C for 35 min, wash the plate 4 times with PBST washing solution, add the enzyme-labeled monoclonal antibody diluted 500, 1000, and 2000 times with PBS at 50 μL / well, incubate at 37°C for 35 min, wash the plate 4 times again, pat dry, add 50 μL / well of TMB chromogenic solution, develop color at room temperature for 10 min, finally add 50 μL / well of 0.5 M sulfuric acid to terminate the reaction, and measure the OD 450 nm value. Calculate the P / N value, and select the combination with the largest P / N value and strong positive reaction as the screened paired monoclonal antibody. The screening process is shown in Table 2.

[0042] Table 2: Comparison of the reactivity of different monoclonal antibody combinations to HPV18 E6 recombinant protein and control antigen in a double-antibody sandwich ELISA.

[0043]

[0044] In the table, "*" indicates the dilution factor.

[0045] In this experiment, to confirm the best antibody combination, the candidate monoclonal antibodies were used as the coating antibody and the HRP-labeled antibody for double-antibody sandwich ELISA pairing tests to screen for combinations that can specifically detect the HPV18 E6 recombinant protein without cross-reacting with other control antigens (such as HPV16 E7 recombinant protein). Finally, the coating antibody 5G3 and the labeled antibody 2C7 were selected as the best paired antibody combination.

[0046] 6. Identification of the binding activity of paired monoclonal antibodies To verify the binding ability of the monoclonal antibody in this combination, the indirect ELISA method was used again to identify its binding activity with the recombinant HPV18 E6 protein. It was shown that the antibody still had obvious signals even at a concentration of 1 ng / mL, further proving its high affinity and good detection performance. Specifically, based on the potential paired antibodies screened by the double-antibody sandwich ELISA, referring to the aforementioned indirect ELISA method, the screened paired monoclonal antibodies and other mouse-derived irrelevant monoclonal antibodies, the HPV18 E7 monoclonal antibody (Santa Cruz, F-7), were serially diluted (concentrations were 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL) to evaluate their binding activity with the recombinant HPV18 E6 protein. In the experiment, other mouse-derived irrelevant monoclonal antibodies were used as negative controls to exclude the influence of non-specific binding.

[0047] By measuring the OD 450 nm value at each concentration, a binding curve was plotted (see Figure 3 ). Figure 3 In it, 5G3 represents the monoclonal antibody 5G3, 2C7 represents the monoclonal antibody 2C7, and Ctrl represents the HPV18 E7 monoclonal antibody (Santa Cruz, F-7). Among them, the monoclonal antibodies 5G3 and 2C7 are the preferred coating antibody and labeled antibody in the double-antibody sandwich ELISA respectively. The indirect ELISA results of the binding activity detection of these two antibodies showed that the two monoclonal antibodies 5G3 and 2C7 still had a weak positive reaction with the antigen (recombinant HPV18 E6 protein) at a concentration of 1 ng / ml, and the control monoclonal antibody did not react with the recombinant HPV18 E6 protein, indicating that the two monoclonal antibodies had a high binding titer.

[0048] 7. Application of paired monoclonal antibodies in the biotin amplification system 7.1. Conjugation of biotin with antibodies Biotin can specifically bind to avidin or biotin monoclonal antibodies and has a multi-level amplification effect. The binding between the two has high stability and strong specificity. In practical applications, it can not only greatly improve the sensitivity of the detection method but also minimize the non-specific binding of reaction reagents. Therefore, using the biotin-avidin amplification system can effectively improve the detection performance of antibody pairs.

[0049] The molar ratio of biotin (Thermo, EZ-Link NHS Biotin, 20217) conjugated with the antibody was 20:1. First, 2.0 mg of activated biotin was added to 360 μL of ultrapure water and dissolved to prepare a 10 mM biotin solution. 2 mg of the antibody and 26.6 μL of 10 mM biotin were shaken and reacted at room temperature for 3 h (the reaction system was controlled at about 2 mL). Then, the biotin-antibody mixture was dialyzed with 0.01 M PBS to remove the excess free biotin. After dialysis, the antibody concentration was measured, and an equal volume of glycerol was added, and it was stored at -20 °C.

[0050] The concentration of the finally obtained labeled antibody was about 0.5 mg / ml.

[0051] The biotin-conjugated monoclonal antibody used in this experiment was the best monoclonal antibody 2C7 screened in the previous stage. After being labeled with biotin, this antibody was used to construct a biotin-avidin signal amplification system.

[0052] 7.2. Establishment of the biotin-avidin amplified ELISA system The above-screened monoclonal antibody combinations were used for coating and biotin conjugation respectively. Then, by exploring the concentrations of the coating antibody, biotin antibody, and the dilution factor of HRP-labeled streptavidin, the most suitable reaction conditions for the amplification system were determined. Monoclonal antibodies were coated in the microplate (coating buffer carbonate buffer: 1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, made up to 1 L of pure water), and the coating concentration gradients were 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 50 μL / well, overnight at 4 °C; the plate was washed once with the washing solution (PBST, PBS containing 0.05% Tween-20), patted dry, blocked with 1% BSA, 150 μL per well, at 37 °C for 2 h, and stored dry after patting dry for later use; the 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 monoclonal antibodies. At the same time, the HPV16 type recombinant protein E6 was diluted to 100 ng / mL as a negative control, and reacted at 37 °C for 30 min. The liquid in the wells was flicked out, and the plate was washed 4 times with PBST washing solution and patted dry; the biotin-conjugated monoclonal antibody was diluted to concentrations of 1 μg / mL, 2 μg / mL, 4 μg / mL with PBS, and 50 μL / well was added to the microplate and reacted at 37 °C for 30 min. The liquid in the wells was flicked out, and the plate was washed 4 times with PBST washing solution and patted dry, then 50 μL / well of HRP-polymer streptavidin (BIOSYNTH, 65R-S105PHRP, diluted 10000, 20000, 40000 times with PBS) was added and reacted at 37 °C for 30 min, then washed 4 times again, patted dry, 50 μL / well of TMB chromogenic solution was added and developed color at room temperature for 10 min, and finally the termination solution was added to terminate the reaction, and the OD was measured with an enzyme-linked immunosorbent assay instrument. 450nm value. Select the coating concentration, biotin monoclonal antibody concentration, dilution of HRP-labeled avidin, etc. with the most obvious positive and negative differences as the optimal reaction conditions.

[0053] The experimental results showed that the optimal reaction conditions were: coating monoclonal antibody 5G3 at a concentration of 1 μg / ml; biotin-conjugated monoclonal antibody 2C7 at a dilution of 2 μg / ml; HRP-polymer streptavidin diluted 40,000 times.

[0054] 7.3 Sensitivity and specificity identification of biotin-avidin amplified ELISA Determine the optimal reaction conditions such as coating concentration, biotin monoclonal antibody concentration, HRP-avidin dilution, etc. Referring to the above detection steps, first gradient dilute the HPV18 E6 recombinant protein with PBS buffer solution, and the diluted concentrations 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 3 recombinant proteins, namely HPV16 E6, HPV16 E7, and HPV18 E7, and add 50 μL per well at the same concentration for detection to determine the detection sensitivity of the detection system to the recombinant proteins.

[0055] The preparation of the HPV16 E6 recombinant protein refers to the preparation method disclosed in CN119350484A. Preparation of the HPV18 E7 recombinant protein: Download the HPV18 E7 gene sequence from NCBI, synthesize it by Anhui General Biosciences, and clone it into the pET32a expression vector. The recombinant plasmid pET32a-HPV18 / E7 expressing HPV18 E7 was transformed into BL21(DE3) competent cells by conventional methods (Molecular Cloning, Third Edition, Science Press). The transformed bacteria were spread on an LB agar plate (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. It was inoculated into LB medium (containing 100 μg / mL ampicillin) at a ratio of 1% of the total volume of the medium, and cultured with shaking at 37°C and 220 rpm for about 3 hours until the OD600 was 0.6 - 0.9. IPTG with a final concentration of 0.1 mM was added, and the cells were induced at 30°C and 200 rpm for 4 hours, and then the cells were collected. The HPV18 E7 protein gene sequence is from GenBank NC_001357.1, which is a complete coding sequence (CDS). The prokaryotic expression plasmid pET32a-HPV18 E7 recombinant protein has been successfully constructed and the full-length E7 protein expression has been achieved. Preparation of the HPV16 E7 recombinant protein: Download the HPV16 E7 gene from NCBI, synthesize it by Anhui General Biosciences, and clone it into the pET28a expression vector. The nucleotide sequence corresponding to the HPV16 E7 gene is shown as SEQ ID NO.23: ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACAGAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA. The amino acid sequence corresponding to the HPV16 E7 gene is shown as SEQ ID NO.24: MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.The recombinant plasmid pET28a-HPV16 / E7 was transformed into BL21(DE3) competent cells by the conventional method (Molecular Cloning, Third Edition, Science Press). The transformed bacteria were spread on an LB agar plate (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 overnight with shaking at 37°C and 220 rpm. It was inoculated into LB medium (containing 50 μg / mL kanamycin) at a ratio of 1% of the total volume of the medium, and cultured with shaking at 37°C and 220 rpm for about 3 hours until the OD600 was 0.6 - 0.9. IPTG with a final concentration of 0.1 mM was added, and the cells were induced at 30°C and 200 rpm for 4 hours, and then the cells were collected to obtain the recombinant protein E7 of HPV16 type.

[0056] See Figure 4 , this combination detected the recombinant protein E6 of HPV18 type in the biotin amplification system, and it was still positive when diluted to 100 pg / ml, and did not react with the recombinant protein E6 of HPV16 type (HPV16 E6), the recombinant protein E7 of HPV16 type (HPV16 E7), and the recombinant protein E7 of HPV18 type (HPV18 E7), showing the good specificity and high sensitivity of this amplification system, which can be used for the detection of the oncoprotein E6 of HPV18 in cervical exfoliated cells and has important application prospects in the early screening and diagnosis of cervical cancer. 8. Cloning and sequencing of variable region genes of monoclonal antibodies The total RNA of hybridoma cells was extracted using the RNeasy Mini Kit kit (Cat. No. 74104), and cDNA was reverse-transcribed using RandomPrimers; universal primers for the variable regions of mouse antibodies were designed, and the VH and VL genes were amplified by 2 rounds of PCR. The Age1 and Bsiw1 restriction enzyme sites were introduced into the primers of the 3rd round of PCR. After the PCR products were purified by gel cutting, they were ligated to the pUC19 vector, transformed into TOP10 strains, and single colonies were picked and sequenced after culturing at 37°C for 14 h to obtain the gene sequences of the heavy and light chains of the monoclonal antibody.

[0057] Variable region gene of monoclonal antibody 5G3 monoclonal antibody: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.17: GAGGTGCAGCTGCAGGAGAGCGGCGCCGAGCTGGTGAGGCCCGGCGCCCTGGTGAAGCTGAGCTGCAAGGTGAGCGGCTTCAACATCAAGGACGTGGTGATCCACTGGGTGAAGCAGAGGCCCGAGCAGGGCCTGGAGTGGATCGGCTGGATCGACCCCGAGAAGGTGGAGACCATCTACGACCCCAAGTTCAGCGGCAAGGCCAGGATCACCGTGGACACCAGCGCCAACATCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACGCCGCCGTGTACTACTGCAGCACCGGCAGGTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC。

[0058] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.13: EVQLQESGAELVRPGALVKLSCKVSGFNIKDVVIHWVKQRPEQGLEWIGWIDPEKVETIYDPKFSGKARITVDTSANIAYLQLSSLTSEDAAVYYCSTGRYWGQGTSVTVSS。

[0059] The complementarity-determining region CDR-H1 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.1: GFNIKDVV; The complementarity-determining region CDR-H2 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.2: IDPEKVET; The complementarity-determining region CDR-H3 sequence of the heavy chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.3: STGRY。

[0060] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.18: GACATCCTGCTGACCCAGAGCCCCCTGACCCTGAGCGTGACCATCGGCCAGCCCGCCAGCATCAGCTGCAAGAGGAGCCAGAGCATCATCGACAGCGACGGCGAGACCGAGCTGAACTGGCTGCTGCAGAGGCCCGGCCAGAGCCCCAAGAGGCTGATCTACCTGGTGAGCCAGCTGCCCAGCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTGGCAGGGCACCGAGTTCCCCCAGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG。

[0061] The amino acid sequence of the light chain variable region of monoclonal antibody 5G3 is shown in SEQ ID NO.14: DILLTQSPLTLSVTIGQPASISCKRSQSIIDSDGETELNWLLQRPGQSPKRLIYLVSQLPSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTEFPQTFGGGTKLEIKRTV。

[0062] CDR region annotation: 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.

[0063] Variable region gene of monoclonal antibody 2C7: Heavy chain: The nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.19: GAGGTGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAGGCCCGGCGCCCTGGTGAAGCTGAGCTGCAAGGTGAGCGGCTTCAACATCAAGGACGTGGGCATCCACTGGGTGAAGCAGAGGCCCGAGCAGGGCCTGGAGTGGATCGGCTGGAGCCAGGAGAAGGTGTACTACATCGAGCCCGAGTTCAGCGGCAAGGCCAGGATCACCGTGGACACCAGCGCCAACATCGCCTACCTGCAGCTGAGCAGCCTGACCAGCGAGGACGCCGCCGTGTACTACTGCAGCACCAAGAGGAGGGTGATCTACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC。

[0064] The amino acid sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.15: EVQLQQSGPELVRPGALVKLSCKVSGFNIKDVGIHWVKQRPEQGLEWIGWSQEKVYYIEPEFSGKARITVDTSANIAYLQLSSLTSEDAAVYYCSTKRRVIYWGQGTSVTVSS。

[0065] CDR region annotation: The complementarity determining region CDR-H1 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.7: DVGIH; The complementarity determining region CDR-H2 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.8: WSQEKVYYIEPEFSG; The complementarity determining region CDR-H3 sequence of the heavy chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.9: KRRVIY。

[0066] Light chain: The nucleotide sequence encoding the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.20: GACATCCAGATGAACCAGAGCCCCCTGACCCTGAGCGTGGCCATCGGCCAGCCCGCCAGCATCAGCTGCAAAAATAGTAGGCAAAGTGACATTAGTATTGACGAGGGCGAGCTGACATGGCTGCTGCAGAGGCCCGGCCAGAGCCCCAAGAGGCTGATCTACCTGAGTGTGGTGCTGCAACCCGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCCAGCAGTGGACCGAGGGCTTCCCCCTGTTCGGCGGCGGCACCAAGCTGGAGCTGAAGAGGACCGTG。

[0067] The amino acid sequence of the light chain variable region of monoclonal antibody 2C7 is shown in SEQ ID NO.16: DIQMNQSPLTLSVAIGQPASISCKNSRQSDISIDEGELTWLLQRPGQSPKRLIYLSVVLQPGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCQQWTEGFPLFGGGTKLELKRTV。

[0068] CDR region annotation: 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。

[0069] Thus far, the 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. Those skilled in the art can fully understand how to implement the technical solutions disclosed herein based on the above description.

[0070] Although some specific embodiments of the present invention 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 not for limiting the scope of the present invention. Those skilled in the art should understand that the above embodiments can be modified or partial technical features can be equivalently replaced without departing from the scope and spirit of the present invention.

Claims

1. A monoclonal antibody combination for detecting HPV18 E6 protein, characterized in that, The monoclonal antibody combination includes monoclonal antibody 5G3 and monoclonal antibody 2C7. The heavy chain variable region of monoclonal antibody 5G3 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; The light chain variable region of 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 - SEQ ID NO.6 respectively; The heavy 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.7 - SEQ ID NO.9 respectively; 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 - SEQ ID NO.12 respectively.

2. The monoclonal antibody combination for detecting HPV18 E6 protein according to claim 1, characterized in that 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.

3. The monoclonal antibody combination for detecting HPV18 E6 protein according to claim 2, wherein 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.

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

18.

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

7. The application according to claim 6, wherein The tool is used for in vitro detection of HPV18 E6 protein in biological samples. The biological samples are selected from cervical exfoliated cells, and the detection is not used for the diagnosis of diseases.

8. The application according to claim 7, characterized in that, The tool includes reagents, kits, test strips and antibody chips.

9. The application according to claim 8, wherein The monoclonal antibody combination is used to construct an ELISA detection system for detecting HPV18 E6 protein in samples. The ELISA detection system is not used for the diagnosis of diseases.

10. The application according to claim 9, wherein The ELISA detection system is a biotin-avidin amplified ELISA system.

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