A monoclonal antibody combination for HPV18 type E7 protein detection and its application

Abstract

The present invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody combination for detecting HPV18 E7 protein and its application. This combination consists of monoclonal antibodies 5F8 and 6G6, wherein the heavy and light chain variable regions of 5F8 and 6G6, respectively, contain specific complementarity-determining region sequences (as shown in SEQ ID NOs. 1 to 12), conferring high specificity for HPV18 E7 protein. Verification using a double-antibody sandwich ELISA and a biotin-avidin amplification system demonstrated that this antibody combination specifically recognizes HPV18 E7 protein, exhibits no cross-reactivity with other high-risk HPV oncoproteins, and exhibits a detection sensitivity of 1 ng / mL. This combination can be used to construct detection systems such as ELISA kits and immunochromatographic test strips, suitable for rapid screening of HPV18 E7 protein in cervical exfoliated cell samples.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection technology, and specifically relates to a monoclonal antibody combination and application for detecting HPV18 type E7 protein. Background Art

[0002] Human papillomavirus (HPV) is a DNA virus that infects epidermal cells through skin damage and can be transmitted between people. In addition to causing cervical cancer in women, HPV infection is also highly associated with anal and genital cancers in the general population.

[0003] The papillomavirus genome is divided into an early region (E), which contains six open reading frames (ORFs) (E1, E2, E4, E5, E6, and E7) expressed immediately after primary infection of host cells, and a late region (L), containing the major capsid protein L1 and the minor capsid protein L2. The HPV E7 protein is a key molecule in HPV carcinogenesis. E7 is composed of CR1, CR2, CR3, and a C-terminal domain, and is expressed as a dimer in cervical cancer. The HPV E7 protein is selectively expressed only in tumor cells and not in normal cells.

[0004] It is generally believed that approximately 13 types of HPV are persistently transmitted and are considered high-risk viruses. These include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68. They can cause precancerous lesions such as cervical intraepithelial neoplasia (CIN), which can then progress to invasive cancer. Studies have found that HPV infection is a necessary factor in the development of almost all cervical cancer cases, with high-risk HPV types 16 and 18 associated with over 80% of cervical cancers. When patients are persistently infected with high-risk HPV type 18, the E7 protein causes malignant transformation of cervical epithelial cells by inhibiting the activity of the tumor suppressor factor pRb, ultimately leading to cervical precancerous lesions and cervical cancer.

[0005] As cervical intraepithelial neoplasia progresses from CIN1 to CIN3, expression of the HPV18 E7 protein gradually increases and appears in cervical exfoliated cells. This information demonstrates the feasibility of detecting the HPV18 E7 protein in cervical exfoliated cells. Although HPV DNA testing is widely used for screening, E7 expression is absent or low in the early stages of HPV infection. Compared with DNA testing, E7 detection can more effectively reflect the active state of viral oncogenes and reduce interference caused by transient infection.

[0006] The E7 protein of high-risk HPV, an oncogenic protein produced after the virus integrates into the host cell genome, is a key factor in the development of high-grade precancerous lesions and ultimately cancer. Multiple prospective studies have demonstrated that testing for the HPV18 E7 protein is an effective means for early clinical detection of cervical cancer and can effectively assess the risk of lesions following HPV infection. Therefore, detecting the HPV18 E7 protein content in cervical epithelial cells can provide a basis for early screening and auxiliary diagnosis of cervical cancer and precancerous lesions, and provide an accurate and powerful reference for vaccination, clinical intervention, and efficacy evaluation. Summary of the Invention

[0007] This application uses prokaryotic expression technology to obtain an active HPV18 type E7 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 E7 protein with a sensitivity of 1 ng / ml. The double-antibody sandwich detection method based on this monoclonal antibody is specific for the HPV18 type E7 protein, i.e., an oncoprotein, 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.

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

[0009] The present invention provides a monoclonal antibody combination for detecting HPV18 type E7 protein, the monoclonal antibody combination comprising monoclonal antibody 5F8 and monoclonal antibody 6G6, the heavy chain variable region of monoclonal antibody 5F8 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;

[0010] The light chain variable region of monoclonal antibody 5F8 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO. 4 to SEQ ID NO. 6, respectively;

[0011] The heavy chain variable region of monoclonal antibody 6G6 includes three complementarity determining regions, the amino acid sequences of which are shown in SEQ ID NO.7 to SEQ ID NO.9, respectively;

[0012] The light chain variable region of monoclonal antibody 6G6 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.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5F8 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of monoclonal antibody 5F8 is shown in SEQ ID NO.14.

[0014] In some embodiments, the amino acid sequence of the heavy chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO.16.

[0015] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 5F8 is shown as SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 5F8 is shown as SEQ ID NO.18.

[0016] In some embodiments, the nucleotide sequence encoding the heavy chain variable region of monoclonal antibody 6G6 is shown as SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of monoclonal antibody 6G6 is shown as SEQ ID NO.20.

[0017] In a second aspect, the present invention provides use of the above-mentioned monoclonal antibody combination in the preparation of a tool for detecting HPV18 type E7 protein.

[0018] In some embodiments, the tool is used to detect HPV18 E7 protein in a biological sample in vitro, the biological sample is selected from cervical exfoliated cells, and the detection is not used for diagnosis of a disease.

[0019] In some embodiments, the tools include reagents, kits, test strips, and antibody chips.

[0020] 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 E7 protein in a biological sample. The ELISA detection system is not used for disease diagnosis.

[0021] In some embodiments, the ELISA detection system is a biotin-avidin amplified ELISA system.

[0022] Beneficial effects:

[0023] This application provides a monoclonal antibody combination for detecting HPV18 E7 protein, including monoclonal antibodies 5F8 and 6G6. Their heavy and light chain variable regions contain specific complementarity-determining region (CDR) sequences, respectively, resulting in high specificity and affinity. This antibody combination has been validated through double-antibody sandwich ELISA screening and biotin-avidin amplification, demonstrating its ability to specifically recognize HPV18 E7 protein with no cross-reactivity with other high-risk HPV oncoproteins. The detection sensitivity can reach 1 ng / mL, demonstrating promising application prospects. This monoclonal antibody combination not only provides an efficient and sensitive tool for in vitro detection of HPV18 E7 protein, but can also be used to construct detection systems such as ELISA kits and immunochromatographic test strips, suitable for rapid screening of HPV18 E7 protein in cervical exfoliated cell samples. Compared to existing HPV DNA detection methods, this application's detection based on E7 protein expression levels more accurately reflects viral oncogenic activity, avoids false-positive results caused by transient infection, and helps improve the specificity and clinical utility of early cervical cancer diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0025] Figure 1 This is the SDS-PAGE protein identification diagram;

[0026] Figure 2 Results of identifying HPV18 E7 protein with purchased antibodies;

[0027] Figure 3 Figure 1 is a graph identifying the binding activity of paired monoclonal antibodies;

[0028] Figure 4 To determine the sensitivity of biotin-avidin amplified ELISA. DETAILED DESCRIPTION

[0029] The following detailed description of the embodiments of the present application is provided in conjunction with the accompanying drawings and examples. The detailed description of the following examples and the accompanying drawings are intended to illustrate the principles of the present application, but are not intended to limit the scope of the present application. The present application may be implemented in many different forms and is not limited to the specific embodiments disclosed herein, but rather includes all technical solutions within the scope of the claims.

[0030] The present application provides these embodiments to make this application thorough and complete, and to fully express the scope of this application to those skilled in the art. It should be noted that: unless otherwise specifically stated, the relative arrangement of parts and steps, the composition of materials, numerical expressions and numerical values ​​set forth in these embodiments should be interpreted as merely exemplary, and not as limiting.

[0031] Example 1

[0032] 1. Preparation of recombinant antigens

[0033] 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. Transformants were plated 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 with shaking at 37°C, 220 rpm, and 1% of the total volume of the culture was inoculated into LB medium (containing 100 μg / mL ampicillin). The culture was shaken at 37°C, 220 rpm, and cultured for approximately 3 hours until the OD600 reached 0.6-0.9. The cells were then induced with 0.1 mM IPTG at 30°C, 200 rpm, and harvested.

[0034] The HPV18 E7 protein gene sequence used in this application 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 expression of the full-length E7 protein has been achieved.

[0035] In this application, HPV18 type E7 protein refers to the wild-type E7 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 E7 recombinant protein is an E7 protein artificially expressed in a prokaryotic expression system through genetic engineering technology. It is usually obtained by cloning the HPV18 type E7 gene into an expression vector and inducing expression in the host. In this application, HPV18 type E7 recombinant protein is used as an antigen for screening and identifying specific monoclonal antibodies against E7 protein, which can effectively identify HPV18 type E7 protein in its natural state, thereby realizing the detection and evaluation of HPV18 infection and potential lesions in clinical samples.

[0036] 2. Purification and identification of HPV18 E7 recombinant protein

[0037] 2.1 Purification of HPV18 E7 recombinant protein

[0038] Because the expressed HPV18 E7 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 selected 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.

[0039] See also Figure 1 M: Protein Marker, 1: pET32a-HPV18 E7 recombinant protein. A distinct main band is visible between 33 and 43 kDa, with protein purity exceeding 85%. This differs from the estimated antigen size (29.7 kDa). This may be due to the significant negative charge of the HPV18 E7 recombinant protein, which results in an abnormal migration speed during SDS-PAGE electrophoresis, appearing larger than its actual molecular weight. During SDS-PAGE electrophoresis at different concentrations, the protein migrates within a certain range. Therefore, the purified protein can be used for further downstream experiments.

[0040] 2.2 Identification of recombinant proteins

[0041] The HPV18 E7 recombinant protein was identified by indirect ELISA.

[0042] The cells were coated with purified HPV18 E7 recombinant protein (1 μg / ml), and the reaction with the positive monoclonal antibody was identified by indirect ELISA. The positive monoclonal antibody was purchased commercially (Santa Cruz, F-7). First, the microplate was coated with HPV18 E7 recombinant protein (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of pure water) at a coating concentration of 1 μg / mL, 50 μl / well, and incubated at 4°C overnight. The plate was blocked with 1% BSA, 150 μl per well, and incubated at 37°C for 2 hours. The plate was washed once with washing buffer (PBST, PBS containing 0.05% Tween-20) and patted dry. The monoclonal antibody was diluted in PBS at a gradient of 1 μg / mL, 100 ng / mL, 10 ng / mL, and 1 ng / mL, and 50 μL was added to the antigen-coated microplate. At the same time, HPV16 E7 monoclonal antibody (Biodragon, BD-PA0179) was used as a negative control. The reaction was carried out 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. The result is as follows Figure 2 , Figure 2 F-7 denotes Santa Cruz, F-7, and Ctrl denotes the HPV18 E7 monoclonal antibody. HPV18 E7 recombinant protein, coated at a concentration of 1 μg / ml, showed a weak positive reaction with a commercially available HPV18 E7 monoclonal antibody (Santa Cruz, F-7) at a concentration of 1 ng / ml, demonstrating the activity of the purified HPV18 E7 recombinant protein. This experiment, using an indirect ELISA method, confirmed that the recombinantly expressed and purified HPV18 E7 recombinant protein was immunoreactive with the specific monoclonal antibody, particularly at low concentrations. This indicates that the protein has a correct structure, an intact epitope, and good immunoreactivity, making it suitable for further functional studies.

[0043] 3. Mouse immunization

[0044] Six-week-old female BALB / c mice were immunized with purified HPV18 E7 recombinant protein mixed with an equal volume of Freund's complete adjuvant (200 μl) via subcutaneous injection at multiple sites. Six-week-old female BALB / c mice were then immunized with the same dose of HPV18 E7 recombinant protein mixed with an equal volume of Freund's complete adjuvant (30 μg / mouse) at two and four weeks. At week 5, mouse sera were collected for antibody titer determination. Mice with the highest titer were selected for a booster immunization with 20 μg of HPV18 E7 recombinant protein via intraperitoneal bolus. Three days later, spleens were harvested for hybridoma cell production.

[0045] 4. Screening, preparation of hybridoma cell lines and antibody purification

[0046] 4.1 Screening of hybridoma cell lines

[0047] 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 height of the well bottom, clones positive for HPV18 E7 recombinant protein were screened using indirect ELISA. Because the immunogen is prokaryotically expressed and contains a His tag, background components must be screened to identify cell lines specific for HPV18 E7 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.

[0048] Positive clones were screened by indirect ELISA method, and hybridoma cell lines that could secrete recombinant protein that specifically recognized HPV18 type E7 were screened after cell fusion by indirect ELISA method.

[0049] Microplates were coated with HPV18 E7 recombinant protein and pET32a-derived HPV16 E6 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) at 4°C overnight. Plates were 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 hybridoma 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 E7 recombinant protein and not with the control antigen were selected for subsequent testing. The control antigen was HPV16 E6 recombinant protein (His tag) expressed in the pET32a vector. The screening process is shown in Table 1.

[0050] Table 1: OD of different monoclonal antibodies against HPV18 E7 recombinant protein and control antigen 450 nm test results.

[0051]

[0052] 4.2 Preparation of Monoclonal Antibody Ascites

[0053] After the selected monoclonal cell lines were expanded and cultured, 0.2 ml (containing 2.5×10 6 Female BALB / c mice (100 cells) were pretreated with incomplete Freund's adjuvant. Approximately 10 days later, when the abdomen became noticeably swollen, ascites was collected using a sterile syringe needle. The collected ascites was centrifuged at 3000 rpm for 10 minutes, and the mid-layer was collected.

[0054] 4.3. Affinity chromatography purification of monoclonal antibodies (Protein G)

[0055] 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 purified monoclonal antibodies were stored in aliquots at -20°C.

[0056] 5. Screening of paired antibodies for double antibody sandwich ELISA

[0057] 5.1. HRP labeling of antibodies

[0058] The screened monoclonal antibodies were labeled according to the instructions of the G-Biosciences HOOK™ HRP PLUS Labeling Kit (Cat#:786-313). Finally, the labeled antigens were 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.

[0059] 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.

[0060] 5.2. Establishment of the Double Antibody Sandwich Method

[0061] The purified monoclonal antibody was coated at a concentration of 0.5 μg / mL, 1 μg / mL, and 2 μg / mL 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 next day, the coating solution was discarded 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 E7, and the control antigen, HPV16 type E6, 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 selected paired antibodies are then tested in a biotin amplification system. The screening process is shown in Table 2.

[0062] Table 2: OD of different monoclonal antibody combinations in double antibody sandwich ELISA 450 nm detection results and P / N value analysis.

[0063]

[0064] 1000* indicates a 1000-fold dilution. P / N = Positive signal (HPV18 E7) / Negative signal (HPV16 E6), used to evaluate the specificity of the antibody combination.

[0065] 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 E7 recombinant protein without cross-reactivity with other control antigens. Ultimately, the optimal antibody combination was selected: coating antibody 5F8 and labeling antibody 6G6.

[0066] 6. Identification of binding activity of paired monoclonal antibodies

[0067] To verify the binding ability of the monoclonal antibodies in this combination, an indirect ELISA method was used to identify their binding activity to the HPV18 E7 recombinant protein. This showed that the antibody still had a significant signal even at a concentration of 1 ng / mL, further demonstrating its high affinity and good detection performance. The details are as follows:

[0068] Based on the potential paired antibodies screened by double-antibody sandwich ELISA, the selected paired monoclonal antibodies and other unrelated mouse monoclonal antibodies were serially diluted (concentrations of 10 μg / mL, 1 μg / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, and 100 pg / mL) using the aforementioned indirect ELISA method to evaluate their binding activity to HPV18 E7 recombinant protein. An unrelated mouse monoclonal antibody, HPV16 E7 (Biodragon, BD-PA0179), was used as a negative control to exclude the influence of nonspecific binding.

[0069] By measuring the OD at each concentration 450 nm values, draw the binding curve (see Figure 3 ). Figure 3 Here, 5F8 represents monoclonal antibody 5F8, 6G6 represents monoclonal antibody 6G6, and Ctrl represents HPV16 type E7 monoclonal antibody.

[0070] The results showed that the selected monoclonal antibody could still react significantly with HPV18 E7 recombinant protein at a concentration as low as 10 ng / mL, indicating that it had high affinity and good binding titer.

[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 biotinylated 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. The final labeled antibody concentration was approximately 0.5 mg / mL.

[0075] The biotin-conjugated monoclonal antibody used in this experiment was the best monoclonal antibody 6G6 screened in the early stage. This antibody was labeled with biotin and used to construct a biotin-avidin signal amplification system.

[0076] 7.2 Establishment of Biotin-Avidin Amplified ELISA System

[0077] The above-screened monoclonal antibody combinations were used for coating and biotin coupling, 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.

[0078] Monoclonal antibodies were coated in microplates (coating buffer: carbonate buffer: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, diluted to 1 L of 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 plates were 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. After patting dry, the plates were dried and stored for later use. HPV18 E7 recombinant protein was diluted to a concentration of 100 ng / mL with PBS and 50 μL was added to the monoclonal antibody-coated microplate. At the same time, HPV16 E6 recombinant protein was diluted to 100 ng / mL as a negative control and reacted at 37°C for 30 min. 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 incubated 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 incubated 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. The OD was measured using a microplate reader. 450The coating concentration, biotin monoclonal antibody concentration, and HRP-labeled avidin dilution with the most obvious positive and negative differences were selected as the optimal reaction conditions.

[0079] The experimental results showed that the optimal reaction conditions were: coated monoclonal antibody 5F8, concentration of 1ug / ml; biotin-coupled monoclonal antibody 6G6, concentration of 2ug / ml; HRP-polymer streptavidin 20,000-fold 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 dilute the HPV18 type E7 recombinant protein with PBS buffer solution in a gradient manner. The concentrations after dilution are 1μg / mL, 100ng / mL, 10ng / mL, 1ng / mL, 100pg / mL, and 10pg / mL, respectively. At the same time, take three recombinant proteins, including HPV16 type E6, HPV16 type E7, and HPV18 type E6, and add 50μL to each well at the same concentration for detection to determine the detection sensitivity and specificity of the detection system for recombinant proteins. The results are as follows Figure 4 , Figure 4 Herein, HPV16 E6, HPV16 E7, HPV18 E6, and HPV18 E7 represent HPV16 E6 recombinant protein, HPV16 E7 recombinant protein, HPV18 E6 recombinant protein, and HPV18 E7 recombinant protein, respectively.

[0082] The selected paired monoclonal antibodies were applied and optimized in the biotin-avidin amplified ELISA system. Under the optimal conditions of the reaction system, the results showed that the HPV18 E7 recombinant protein still showed a positive reaction when diluted to 1 ng / ml, and did not react with high-risk cancer proteins such as HPV16 E6, HPV16 E7, and HPV18 E6, demonstrating the good specificity and high sensitivity of the amplification system. Figure 4 This indicates that the detection method of this application has a high sensitivity, with a minimum detection limit of approximately 1 ng / ml. It can be used to detect HPV18 E7 oncoprotein in cervical exfoliated cells, and has important application prospects in the early screening and diagnosis of cervical cancer.

[0083] Among them, the preparation process of HPV16 E6, HPV16 E7, and HPV18 E6 recombinant proteins is as follows:

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

[0085] 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.

[0086] Nucleotide sequence (SEQ ID NO.21): ATGCATGGAGATACACCTACATTGCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACAGCTCAGAGGAGGAGGATGAAATAGATGGTCCAGCTGGACAAGCAGAACCGGACA GAGCCCATTACAATATTGTAACCTTTTGTTGCAAGTGTGACTCTACGCTTCGGTTGTGCGTACAAAGCACACACGTAGACATTCGTACTTTGGAAGACCTGTTAATGGGCACACTAGGAATTGTGTGCCCCATCTGTTCTCAGAAACCATAA.

[0087] Amino acid sequence (SEQ ID NO.22):

[0088] MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP.

[0089] 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 a final concentration of 0.1 mM IPTG at 30°C, 200 rpm, and harvested for 4 hours.

[0090] Preparation of HPV18 E6 recombinant protein: The HPV18 E6 gene sequence was downloaded from NCBI, synthesized by Anhui General Biotechnology Co., Ltd., and cloned into the pET28a expression vector.

[0091] Nucleotide sequence (SEQ ID NO.23):

[0092] ATGGCGCGCTTTGAGGATCCAACACGGCGACCCTACAAGCTACCTGATCTGTGCACGGAACTGAACACTTCACTGCAAGACATAGAAATAACCTGTGTATATTGCAAGACAGTATTGGAACTTACAGAGGTATTTGAATTTGCATTTAAAGATTTATTTGTGGTGTATAGAGACAGTATACCGCATGCTGCATGCCATAAATGTATAGATTTTTATTCTAGAATTAGAGAATTAAGACATTATTCAGACTCTGTGTATGGAGACACATTGGAAAAACTAACTAACACTGGGTTATACAATTTATTAATAAGGTGCCTGCGGTGCCAGAAACCGTTGAATCCAGCAGAAAAACTTAGACACCTTAATGAAAAACGACGATTTCACAACATAGCTGGGCACTATAGAGGCCAGTGCCATTCGTGCTGCAACCGAGCACGACAGGAACGACTCCAACGACGCAGAGAAACACAAGTATAA。

[0093] Amino acid sequence (SEQ ID NO.24):

[0094] MARFEDPTRRPYKLPDLCTELNTSLQDIEITCVYCKTVLELTEVFEFAFKDLFVVYRDSIPHAACHKCIDFYSRIRELRHYSDSVYGDTLEKLTNTGLYNLLIRCLRCQKPLNPAEKLRHLNEKRRFHNIAGHYRGQCHSCCNRARQERLQRRRETQV。

[0095] 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.

[0096] 8. Monoclonal antibody variable region gene cloning and sequencing

[0097] 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.

[0098] Monoclonal antibody 5F8:

[0099] Heavy chain:

[0100] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 5F8 is shown in SEQ ID NO.17:

[0101] GAGTTCCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCAGCTACCTGATGCACTGGGTGAAGCAGAGCCACGTGAAGAGCCTGGAGTGGATCGGCAGGATCAACCCCGTGCTGGAGGCCACCAACTAC AACCAGAACTTCAAGGACAAGGCCAGCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGCACAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAGGGACTACGGCCTGAGGGACTGGTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCC.

[0102] The amino acid sequence of the heavy chain variable region of monoclonal antibody 5F8 is shown in SEQ ID NO.13:

[0103] EFQLQQSGPELVKPGASVKISCKASGYSFTSYLMHWVKQSHVKSLEWIGRINPVLEEATNYNQNFKDKASLTVDKSSSTAYMELHSLTSEDSAVYYCARRDYGLRDWFAYWGQGTLVTVSA.

[0104] CDR region annotation:

[0105] The heavy chain variable region CDR-H1 sequence of 5F8 is shown in SEQ ID NO. 1: SYLMH;

[0106] The heavy chain variable region CDR-H2 sequence of 5F8 is shown in SEQ ID NO. 2: RINPVLEATNYNQNFKD;

[0107] The heavy chain variable region CDR-H2 sequence of 5F8 is shown in SEQ ID NO. 3: RDYGLRDWFAY.

[0108] Light chain:

[0109] The nucleotide sequence of the light chain variable region of monoclonal antibody 5F8 is as SEQ ID Shown in NO.18: GACATCCAGATGACCCAGACCACCCTGACCCTGAGCGTGACCATCGGCCAGCCCGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGGACAGCGACGGCGAGACCTTCCTGAACTGGCTGATCCAGAGGCCCAGCCAGAGCCCCAAGAGGCTGATCGTGCTGGTGA GCAGGCTGACCAGGGGCGTGCCCGACAGGTTCACCGGCAGCGGCAGCGGCACCGACTTCACCCTGAAGATCAGCAGGGTGGAGGCCGAGGACCTGGGCGTGTACTACTGCTGGGGCACCTTCCACCAGACCCAGCACTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0110] The amino acid sequence of the light chain variable region of monoclonal antibody 5F8 is shown in SEQ ID NO.14:

[0111] DIQMTQTTLTLSVTIGQPASISCKSSQSLLDSDGETFLNWLIQRPSQSPKRLIVLVSRLTRGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWGTFHQTQHFGGGTKLEIKRTV.

[0112] CDR region annotation:

[0113] The light chain variable region CDR-L1 sequence of 5F8 is shown in SEQ ID NO. 4: KSSQSLLDSDGETFLN;

[0114] The light chain variable region CDR-L2 sequence of 5F8 is shown in SEQ ID NO. 5: LVSRLTR;

[0115] The CDR-L3 sequence of the light chain variable region of 5F8 is shown in SEQ ID NO.6: WGTFHQTQH.

[0116] 6G6 monoclonal antibody variable region:

[0117] Heavy chain:

[0118] The nucleotide sequence of the heavy chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO.19:

[0119] CAGATGCAGCTGCAGCAGAGCGGCCCCGAGCTGGTGAAGCCCGGCGCCAGCGTGAAGATCAGCTGCAAGGCCAGCGGCTACAGCTTCACCAGGGTGCTGCACATCTGGGTGAAGCAGAGCCACGTGAAGAGCCTGGAGTGGATCGGCCAGAGCAACCCCTACTACGAGGCCAGCAACATC ATGGAGAACAAGAAGCTGAAGGCCAGCCTGACCGTGGACAAGAGCAGCAGCACCGCCTACATGGAGCTGCACAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGGAGGCAGTACGGCCTGAGCCACTGGGACGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGC.

[0120] The amino acid sequence of the heavy chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO. 15: QMQLQQSGPELVKPGASVKISCKASGYSFTRVLHIWVKQSHVKSLEWIGQSNPYYEASNIMENKKLKASLTVDKSSSTAYMELHSLTSEDSAVYYCARRQYGLSHWDAYWGQGTLVTVSS.

[0121] CDR region annotation:

[0122] The heavy chain variable region CDR-H1 sequence of 6G6 is shown in SEQ ID NO. 7: RVLHI;

[0123] The heavy chain variable region CDR-H2 sequence of 6G6 is shown in SEQ ID NO. 8: QSNPYYEASNIMENKKL;

[0124] The heavy chain variable region CDR-H2 sequence of 6G6 is shown in SEQ ID NO.9: RQYGLSHWDAY.

[0125] Light chain:

[0126] The nucleotide sequence of the light chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO.20:

[0127] GACATCGTGATGAGCCAGAGCCCCGCCCTGATGGCCGCCAGCCCCGGCGAGAAGGTGACCATCACCTGCAGCGTGGGCAGCAGCATCAGCGGCAGCTACCTGCACTGGTACCAGCAGAAGAGCGAGACCAGCCCCAAGCCCTGGATCTACAGCACCAGCATGCTGGC CAGCGGCGTGCCCGTGAGGTTCAGCGGCAGCGGCAGCGGCACCAGCTACAGCCTGACATCAGCAGCATGGAGGCCGAGGACGCCGCCACCTACTGCCACAGGTGGAACAGCTACCCCCTGACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGAGGACCGTG.

[0128] The amino acid sequence of the light chain variable region of monoclonal antibody 6G6 is shown in SEQ ID NO.16: DIVMSQSPALMAASPGEKVTITCSVGSSISGSYLHWYQQKSETSPKPWIYSTSMLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCHRWNSYPLTFGGGTKLEIKRTV.

[0129] CDR region annotation:

[0130] The light chain variable region CDR-L1 sequence of 6G6 is shown in SEQ ID NO. 10: SVGSSISGSYLH;

[0131] The light chain variable region CDR-L2 sequence of 6G6 is shown in SEQ ID NO. 11: STSMLAS;

[0132] The CDR-L3 sequence of the light chain variable region of 6G6 is shown in SEQ ID NO.12: HRWNSYPLT.

[0133] So far, the various embodiments of the present application have been described in detail. To avoid obscuring the concept of the present application, some details well known in the art have not been described. Based on the above description, those skilled in the art can fully understand how to implement the technical solutions disclosed herein.

[0134] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art will understand that the above examples are for illustration only and are not intended to limit the scope of the present application. Those skilled in the art will understand that the above embodiments may be modified or some technical features may be replaced with equivalents without departing from the scope and spirit of the present application.

Claims

1. A monoclonal antibody combination for detecting HPV18 type E7 protein, characterized in that: The monoclonal antibody combination includes monoclonal antibody 5F8 and monoclonal antibody 6G6. The heavy chain variable region of the monoclonal antibody 5F8 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 5F8 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 6G6 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 6G6 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 E7 protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 5F8 is shown in SEQ ID NO.13; the amino acid sequence of the light chain variable region of the monoclonal antibody 5F8 is shown in SEQ ID NO.

14.

3. The monoclonal antibody combination for detecting HPV18 type E7 protein according to claim 2, characterized in that: The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6G6 is shown in SEQ ID NO.15; the amino acid sequence of the light chain variable region of the monoclonal antibody 6G6 is shown in SEQ ID NO.

16.

4. The monoclonal antibody combination for detecting HPV18 type E7 protein according to claim 3, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 5F8 is shown in SEQ ID NO.17; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 5F8 is shown in SEQ ID NO.

18.

5. The monoclonal antibody combination for detecting HPV18 type E7 protein according to claim 4, characterized in that: The nucleotide sequence encoding the heavy chain variable region of the monoclonal antibody 6G6 is shown in SEQ ID NO.19; the nucleotide sequence encoding the light chain variable region of the monoclonal antibody 6G6 is shown in SEQ ID NO.

20.

6. Use of the monoclonal antibody combination according to claim 1 in preparing a tool for detecting HPV18 type E7 protein.

7. The use according to claim 6, characterized in that The tool is used for in vitro detection of HPV18 type E7 protein in a biological sample, wherein the biological sample is selected from cervical exfoliated cells, and the detection is not used for diagnosis of a disease.

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

9. The 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 E7 protein in biological samples. The ELISA detection system is not used for disease diagnosis.

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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