CN115028689A

CN115028689A - Bispecific detection kit for specifically detecting HPV6 and HPV11

Info

Publication number: CN115028689A
Application number: CN202210678942.XA
Authority: CN
Inventors: 张倡埼; 朱高茂
Original assignee: Beijing Reyan Biotechnology Co ltd
Current assignee: Zhejiang Orient Gene Biotech Co Ltd
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2022-09-09
Anticipated expiration: 2042-06-15
Also published as: CN115028689B

Abstract

The invention relates to a bispecific detection kit for specifically detecting HPV6 and HPV 11. According to the invention, the specific immune antigen peptide of HPV6/11 is obtained through screening optimization, the antigen peptide is used for immunizing a mouse to prepare a specific monoclonal antibody, the monoclonal antibody can be specifically combined with HPV6/11, and has the effect of inhibiting HPV6/11 from infecting normal cells, and the monoclonal antibody prepared into a kit can be effectively used for HPV detection and has a good application prospect.

Description

Bispecific detection kit for specifically detecting HPV6 and HPV11

Technical Field

The invention relates to the field of biological detection, in particular to a bispecific detection kit for specifically detecting HPV6 and HPV 11.

Background

Cervical cancer is the second most common malignancy in women worldwide. Numerous studies have shown that persistent infection with Human Papillomavirus (HPV) is closely associated with the development of cervical cancer, and high-risk HPV infection is a major factor in the development of Cervical Intraepithelial Neoplasia (CIN) and cervical cancer. More than 200 HPV genotypes have been found, of which about 54 can infect the genital mucosa and still more HPV subtypes are being identified. The new HPV genotype determination criteria means that the HPV genotype differs from any other HPV of the determined type by more than 10% in the nucleotide sequence of the E6, E7 or L1 region. According to the pathogenicity and the pathogenic risk, the HPV can be divided into high-risk HPV and low-risk HPV. High risk HPV infections can cause intraepithelial neoplasia or canceration in the cervical, vaginal, anal, penile and oropharyngeal regions, with HPV types mainly including 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. The low-risk type HPV infection can only cause benign lesions such as genital warts, and the HPV types mainly comprise 6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81. Compared with high-risk HPV, the E6 and E7 proteins of low-risk HPV interfere with the P53 gene and pRb gene to have weaker functions.

The HPV detection technology is widely applied to cervical cancer screening, epidemiological investigation and cervical cancer vaccine effectiveness evaluation. At present, the detection methods for HPV at home and abroad are infinite, and the advantages and disadvantages of different detection methods for HPV are mainly evaluated through two aspects: clinical utility value (i.e., clinical sensitivity) and detection limit (i.e., analytical sensitivity). The clinical sensitivity (also called functional sensitivity) refers to the proportion of patients with positive test results in the HPV detection technology and the detection capability of HPV on CIN2 grade and above 2; the assay sensitivity (also called the detection limit) refers to the lowest analyte concentration that can be detected. Good HPV detection technology needs to meet higher clinical sensitivity and lower analytical sensitivity. Currently, some 150 different HPV detection experiments are performed worldwide and still in progress. Detection methods that have been used mainly include DNA detection, RNA detection and HPV-associated marker detection as well as antigen detection.

Aptma HPV was approved in 2012, the first and only FDA-approved technique for HPVmRNA detection. The technology is based on transcription-mediated amplification (TMA) technology to detect HPV E6/E7 mRNA: namely, adding a T7 promoter at the 5' end of an upstream primer, introducing RNA polymerase into a reaction system, carrying out reverse transcription on target mRNA to form cDNA, and then realizing target molecule amplification in a transcription mode. The technology aims at detecting mRNA of HPV oncogene E6/E7, and compared with HPV-DNA detection, the technology can effectively avoid the interference of HPV transient infection on the detection result, but the cost of Aptima HPV detection is expensive.

Serological detection of HPV antibodies is of great importance in HPV vaccinology and epidemiological studies. At present, the research on rapid immunoassay kits for HPV is insufficient, and the rapid immunoassay kits are ready for further research, particularly, the detection reagents capable of simultaneously targeting multiple HPV subtypes are insufficient. Therefore, the development of kits for HPV multi-subtype detection becomes particularly urgent.

Disclosure of Invention

The invention overcomes the defects of the prior art, and the HPV6 and HPV11 high-immunogenicity antigen peptides are screened and obtained, and the sequences of the antigen peptides are shown as SEQ ID NO: 1 is shown.

In another aspect of the invention, HPV6 and HPV11 high immunogenicity antigenic peptides are used as immunogens to prepare and obtain corresponding monoclonal antibodies.

In one aspect, the invention provides a monoclonal antibody specific for HPV6/11, characterized in that the light and heavy chain variable region sequences thereof are set forth in SEQ ID nos:3 and 4.

The chains of the antibodies, fragments thereof or VH chain VL antibodies of the invention include those antibodies, fragments or VH chain VL chains of the antibodies described herein that have sequence identity. For example, the invention includes sequences having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.8% sequence identity to the exemplified sequences described herein (e.g., SEQ ID NOS: 3-4). Thus, the heavy and/or light chain variable regions of the amino acid sequence of the antibody may differ from the sequences set forth herein and still be within the scope of the embodiments disclosed herein. For example, one or more Complementarity Determining Regions (CDRs) may be different from the heavy and/or light chain variable regions of the antibody amino acid sequences described herein. Alternatively, one or more Complementarity Determining Regions (CDRs) may be identical to the sequences described herein (e.g., SEQ ID NOS:3-4), but other portions of the heavy or light chain variable regions may be different. Such sequence variations of the exemplary sequences described herein (e.g., SEQ ID NOs:3-4) are to be considered embodiments within the scope of the invention disclosed herein.

Non-limiting examples of antibodies of the invention include, for example, intact immunoglobulins and variants and fragments thereof known in the art that retain antigen binding affinity. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab ', Fab ' -SH, F (ab ') 2; a diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments synthesized de novo by modifying whole antibodies or using recombinant DNA methods.

Antibodies of the invention include conservative variants: "conservative" amino acid substitutions are those that do not substantially affect or reduce the function of the protein, e.g., the ability of the protein to interact with a target protein. For example, FTC-specific antibodies comprise up to 1, 2, 3,4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions as compared to a reference antibody sequence and retain specific binding activity for FTC. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

In addition, one of ordinary skill will recognize that individual substitutions, altered deletions or additions, additions or deletions of a single amino acid or a small percentage (e.g., less than 5%, in some embodiments less than 1%) of amino acids in a coding sequence are conservative variations, wherein the variation results in the substitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitution representatives that provide functionally similar amino acids are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered conservative substitutions for one another:

1) alanine (a), serine (S), threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W).

The antibodies of the invention are also conjugated with a detectable label: a detectable molecule (also referred to as a label) conjugated directly or indirectly to the antibody to facilitate detection. For example, the detectable label can be detected by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (e.g., CT scanning, MRI, ultrasound, fiber optics and laparoscopy). Specific, non-limiting examples of detectable labels include avidin, biotin, fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). In one embodiment, "labeling an antibody" refers to incorporating another molecule into the antibody. For example, the tag is a detectable label, e.g., incorporating a radiolabeled amino acid or attachment to a polypeptide of a biotin moiety that is detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or an enzymatic activity that is detectable by optical or colorimetric methods). Various methods of labeling polypeptides are known in the art and can be used. Examples of polypeptide tags include, but are not limited to, the following: a radioisotope or radionuclide (e.g., 35S or 131i), a fluorescent label (e.g., Fluorescein Isothiocyanate (FITC), rhodamine, lanthanide fluorophor), an enzymatic label, a chemiluminescent label, a biotin group, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag), or a magnetic reagent, e.g., gadolinium chelate. In some embodiments, the tags are connected by spacer arms of different lengths to reduce potential space obstruction. For example, Sambrook et al discusses methods of using detectable labels and guidance in selecting detectable labels suitable for various purposes.

Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, 5-steroid isomerase, yeast alcohol dehydrogenase, glycerol phosphate dehydrogenase, trisaccharide phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase and the like. Examples of suitable radioisotope labels include: examples of suitable fluorescent labels for 3h, 125i, 131i, 32P, 35S, 14c, 51Cr, 57 to 58CO, 59Fe, 75 selenium, 152Eu, 90Y, 67 copper, 217CI, 211AT, 212Pb, 47SC, 109 include A152EU tags, fluorescein tags, isothiocyanate tags, rhodamine tags, phycoerythrin tags, phycocyanin tags, allophycocyanin tags, o-phthaldehyde tags, fluorescamine tags and other chemiluminescent substrates include luminal substrates, abluminal substrates, aromatic acridinium ester substrates, imidazole substrates, acridinium salt substrates, oxalate tags, fluorescein substrates, luciferase tags, hirudin tags and the like.

Antibodies, fragments thereof, and VH and VL chains of antibodies can be produced using genetic techniques that include expressing all or part of the coding sequence into a host cell. Antibodies can be produced using techniques including hybridoma technology, recombinant and phage display technology, or combinations thereof (see U.S. Pat. Nos. 4,902,614, 4,543,439 and 4,411,993; see also monoclonal antibodies, hybridomas: new dimensions in biological assays, Plenum Press, Kennett, McKearn and Bechtol (eds.), 1985, and Harlow et al, antibodies: laboratory manuals, Cold spring harbor laboratory presses, second edition monoclonal antibodies can also be obtained by directly cloning immunoglobulin sequences from animals, including mammals such as rabbits, primates or human subjects.

In further embodiments, the method of detecting HPV in a sample comprises contacting the sample with a capture antibody by binding the capture antibody, and then detecting the capture antibody with a detection antibody. The detection antibody may be directly labeled with an enzyme, a fluorophore, or the like, and thus may be directly detected. The detection antibody in the present assay may be labeled using any label known in the art.

The invention also provides kits for carrying out the detection methods of the invention. The kit comprises a container containing a monoclonal antibody produced by at least one hybridoma cell line of the invention and instructions for using the monoclonal antibody to bind HPV to form an immune complex, such that the presence or absence of the immune complex correlates with or indicates the presence or absence of HPV in the sample. The kit may include a first container means containing an antibody as described herein. The kit may also include other container means having solutions as needed or convenient for practicing the invention. The container means may be made of glass, plastic, and the kit may further comprise written information, e.g. for performing the process of the invention or analytical information, e.g. the amount of reagents contained in the first container means. The container device may be in another container device (e.g., a box, bag, etc.) with the written information.

The invention further provides application of the HPV monoclonal antibody in preparing a kit for detecting HPV6/11 subtype.

Further, the diagnostic kit of the present invention comprises instruments and reagents generally used for immunological tests. When HPV antibodies are a component of a diagnostic kit based on an ELISA method, such kit additionally comprises:

plates coated with mAbs (sandwich ELISA) or HPV antigens (Belisa, Celisa) of the invention, in which the non-specific binding sites are fully saturated with appropriate blocking substances, buffers for washing the plates, dilution of samples and reagents, substrates for enzymes, and solutions for inhibition of enzyme reactions.

Further, the kit is prepared by adopting a preparation method which is conventional in the field. The preparation method specifically comprises the following steps: coupling the anti-HPV monoclonal antibody with a gold label; preparing a detection antigen release pad; preparing a latex combined pad; preparing a sample treatment solution; diluting and fixing the anti-HPV monoclonal antibody; manufacturing a herpes simplex virus detection kit; the steps of diluting and fixing the anti-HPV monoclonal antibody specifically comprise: diluting the avidin to 1-5mg/mL by using a diluent, and diluting the monoclonal antibody to 1-5 mg/mL; spraying the anti-HPV monoclonal antibody solution onto a nitrocellulose membrane by using an automatic spot spraying machine as detection lines under the conditions that the temperature and the humidity are respectively 30-40 ℃ and 40-50%; spraying the avidin solution onto the nitrocellulose membrane as a quality control line; the dilution included 50nM PBS buffer, 2.5% sucrose by mass, and 80nM EDTA.

Advantageous effects

According to the invention, the specific immune antigen peptide of HPV6/11 is obtained through screening optimization, the antigen peptide is used for immunizing a mouse to prepare a specific monoclonal antibody, the monoclonal antibody can be specifically combined with HPV6/11, and has the effect of inhibiting HPV6/11 from infecting normal cells, and the monoclonal antibody prepared into a kit can be effectively used for HPV detection and has a good application prospect.

Drawings

FIG. 1 is a diagram showing the result of identifying the specificity of antigen reaction

FIG. 2 is a graph showing the effect of monoclonal antibody on HPV infection of HaCaT cells

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

EXAMPLE 1 preparation of monoclonal antibodies specific for HPV6 and HPV11 subtypes

According to the amino acid sequences of HPV6 and HPV11, by adopting computer simulation and combining an amino acid hydrophilicity scheme of Kyte-Doolittle and Hopp-Woods, a plasticity scheme of Karplus-Schulz, a surface region possibility scheme of Emini, an antigen possibility scheme of Jameson-Wolf and an antigen epitope index prediction method of Kolaskar-Tongaonkar, and analyzing a flexible region in a protein secondary structure by a Cohen method, a Garnier-Robson method and a Chou-Fasman method, the main antigen epitope sequences of HPV6 and HPV11 are predicted in a multiparameter way, and after comprehensive comparison and analysis of prediction results, 1 best common antigen epitope capable of simultaneously aiming at HPV6 and HPV11 is designed: vqwaydniceeseeiafeyaqrgdfdsnaraflnsnmqakyv (SEQ ID NO: 1). And (3) preparing the epitope peptide by manual synthesis.

BALB/c mice are immunized by taking the epitope peptide as an immunogen. Mixing the primary immunization with Freund's complete adjuvant, wherein the immunization dose is 100 mug/mouse, and subcutaneous multipoint injection is adopted; mixing with Freund's incomplete adjuvant, and performing intraperitoneal injection for 3 times. 5d after the last 1 immunization, blood is collected from the rat tail, serum is separated, the immune effect is detected by indirect ELISA, and one mouse with the best immune effect is selected; and the antigen epitope peptide is directly injected into the spleen 3 days before cell fusion to strengthen the immune effect at the dose of 200 mug/cell. Taking immune mouse spleen cells and homologous mouse myeloma cells SP2/0 for cell fusion, respectively adopting culture media containing HAT and HT for selective culture, cloning hybridoma cells by a limiting dilution method, taking epitope peptide as a detection antigen, adopting an indirect ELISA method to determine the antigen-antibody reaction capacity of hybridoma cell culture supernatant, and screening and identifying the hybridoma clone with the strongest positive strains, namely 3A2a, 7C4C and 8D2 b.

Preparing and purifying a monoclonal antibody: at 1.5X 10 ⁶ Three strains of 3A2a, 7C4℃ and 8D2b hybridoma cells are respectively injected into the abdominal cavity of BALB/C mice pretreated by paraffin oil, after 10 days, the ascites is extracted, the ascites is centrifuged at 4 ℃, 12000 Xg for 30min, the supernatant is collected, the supernatant is diluted by adding binding buffer solution (0.1mol/LPBS, pH8.0) with the same volume, and after the dilution is filtered by a 0.45 mu m microporous filter membrane, the antibody is purified by utilizing the biological characteristic affinity chromatography of the Fc segment of the antibody. Loading the balanced rProteinA chromatographic column, fully washing with a binding buffer solution, and eluting the target antibody with an elution buffer solution (0.1mol/L citric acid-sodium citrate buffer solution, pH3.0); immediately, 1mol/L Tris-HCl buffer solution (pH8.9) was added to the collected purified antibody to sufficiently neutralize it, and the buffer solution was replaced in PBS, and samples were taken to determine the concentration of the antibody by BCA method. Adopting indirect ELISA method, using antigen epitope peptide as coating antigen, and monoclonal antibody dilution degree is 10 ^－1 、10 ^－2 、10 ^－3 、10 ^－4 、10 ^－5 、10 ^－6 、10 ^－7 And 10-8, the highest dilution with an A450 value greater than 1.0 as the antibody titer. The results are shown in table 1 below.

TABLE 1 concentration and potency of purified monoclonal antibodies

Monoclonal antibody	Concentration (mg/mL)	Potency (dilution)
			3A2a	4.6	2.2×10- ⁵
7C4c	5.2	4.3×10- ⁵
			8D2b	4.9	5.8×10- ⁵

Antigen reaction specificity identification of monoclonal antibodies: using an indirect ELISA method, the sequences shown in SEQ ID NOs: 1, epitope peptide of khkallikcppplvtsnid (SEQ ID NO: 2) and BSA protein as envelope antigens, measuring the antigen-antibody reaction capability of the monoclonal antibody, and analyzing the antigen reaction specificity of the monoclonal antibody. The results are shown in FIG. 1.

The indirect ELISA result shows that 3 monoclonal antibodies (3A2a, 7C4C, 8D2b) can have stronger antigen-antibody reaction with immune antigen peptide of sequence 1, but do not react with epitope peptide of sequence 2, and do not react with human BSA, which indicates that the 3 monoclonal antibodies are specific monoclonal antibodies aiming at HPV6/11, and the figure 1 shows.

Example 23A 2a monoclonal antibody sequence analysis and affinity identification

Surface plasmon resonance SPR antibody affinity assay: according to ProteOn XPR36(B10-RAD) instrument and corresponding chip instructions, antigens were attached to a GLC chip via amino coupling reagents EDAC, 100mM and Sulfo-NHS, 25mM) (ProteOn amino coupling kit), and then different concentrations (600, 300, 150, 75, 37.5, and 18.75nM in PBST, pH7.4) of the antibody solutions to be tested were injected into the instrument for detection.

The affinity of the 3A2a antibody and the epitope was measured by the surface plasmon resonance SPR method. The measurement was performed with different concentrations of antibody as mobile phase. The results showed that the 3A2a antibody and epitope were recognized with high affinity, with dissociation constants of 5.52X 10 Kd ^-9 M。

The light and heavy chain variable region sequences of 3A2a are obtained by designing light and heavy chain amplification primers aiming at conserved sequences of 5 'ends and 3' J regions of heavy chain and light chain variable region genes of a mouse antibody, amplifying by taking cDNA of 3A2a hybridoma cells as a template, inserting the amplified product into pUC19 plasmid, and then sequencing and analyzing.

Light chain variable region (SEQ ID NO: 3)

DIVITQRPALMAASPGEKVTITCGEPITLCKKTFIWYQQKSGISPKPWIYDQQSTCVGVPARFSGSG SGTSYSLTITSMEAEDAATYYCRESAAPVDDFGAGTKLELK

Heavy chain variable region (SEQ ID NO: 4)

EVQLEESATELARPGASVKLSCKASGYIFSKHMSDWIKQRPGQGLEWIGTQHRGTDSNTDVCCVVGK ATLTADKSSSTAYMQLSSLASEDSAVYYCAGALATHCHWGLGTTLAVSS。

Example 33 Effect of monoclonal antibody A2a on in vitro infection of human papillomavirus 6/11 virions with HaCaT cells

Taking HPV6/11 positive infected condyloma acuminatum tissue, putting the tissue in a plate, and fully washing and shearing the tissue with sterilized PBS; placing the cut tissue in a mortar, adding an extraction buffer solution, fully and uniformly grinding, and centrifuging at 12000r/min and 4 ℃ for 10 min; adding equal volume of polyethylene glycol into the supernatant, standing at 4 deg.C for 1h, centrifuging at 8000r/min and 4 deg.C for 10 min; discarding the supernatant, adding a proper amount of PBS buffer solution into the precipitate, and standing overnight at 4 ℃; centrifuging at 4 deg.C for 1 hr at 10000r/min the next day, collecting supernatant, filtering with 0.22 μm microporous membrane for sterilization, subpackaging, and standing at-70 deg.C; taking the virus suspension to be detected to carry out PCR detection, wherein the DNA carrying capacity of the prepared virus-like particle suspension is 5 multiplied by 10 ⁶ copy/mL.

HaCaT cells were seeded at 1X 105/well in 24-well plates; the culture solution was discarded the next day, and 6 groups were diluted by a factor of 1: 10. 1: 50. 1: 100. 1: 200. 1: 400. 1:1000 monoclonal antibodies (initial concentration of 1mg/mL) were added to HaCaT cells, respectively, and placed on ice for 1h with occasional light shaking of the plates; discarding the culture solution, adding appropriate amount of PBS, washing thoroughly, and using 10 ⁶ Infecting HaCaT cells in vitro by using copied/ml HPV6/11 virus-like particles, replacing monoclonal antibodies and HPV6/11 virus-like particles by using PBS with equal amount as negative control, and adding HPV6/11 virus-like particles without adding monoclonal antibodies as positive control; carrying out PCR detection on the infected HaCaT cells; the above processes are repeated for 3 times; and (3) calculating: the inhibition rate of the monoclonal antibody is (1-HaCaT virus copy number after adding the monoclonal antibody/HaCaT virus copy number without adding the monoclonal antibody) multiplied by 100%.

FIG. 2 shows that 6 groups of mAbs with different dilution times have a concentration of 10 ⁶ The inhibition rates measured after HaCaT cells are infected in vitro by copy/ml HPV6/11 virus-like particles are different, and comparison shows that the monoclonal antibody pairs 10 are increased along with the increase of the concentration of the monoclonal antibody ⁶ The inhibition rate of HPV6/11 virus-like particles adhering to host cells increased at copies/ml, and the difference was statistically significant (P < 0.05). Under the dilution condition of 1:10, the inhibition rate reaches (68.41 +/-2.03)%, and the inhibition effect is better.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

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Claims

Use of the bispecific monoclonal antibody 3A2a of HPV6 and HPV11 in preparation of a kit for HPV6/11 detection, wherein the light chain variable region sequences of the bispecific monoclonal antibody of HPV6 and HPV11 are shown as SEQ ID NO:3, the sequences of the heavy chain variable regions of the bispecific monoclonal antibodies of HPV6 and HPV11 are shown as SEQ ID NO: 4, respectively.
2. The use according to claim 1, wherein the monoclonal antibody is further conjugated to a detectable label.
3. Use according to claim 2, characterized in that the detectable label is an enzyme.
4. Use according to claim 3, characterized in that the enzyme is catalase, glucose-6-phosphate dehydrogenase, glucoamylase or acetylcholine ester.
The use of the bispecific monoclonal antibody 3A2a of HPV6 and HPV11 in the preparation of medicaments for inhibiting HPV6/11 infection, wherein the light chain variable region sequence of the bispecific monoclonal antibody of HPV6 and HPV11 is shown as SEQ ID NO:3, the sequences of the heavy chain variable regions of the bispecific monoclonal antibodies of HPV6 and HPV11 are shown as SEQ ID NO: 4, respectively.
6. The use according to claim 5, characterized in that the bispecific monoclonal antibody against HPV6 and HPV11 is capable of inhibiting the in vitro infection of HaCaT cells with human papillomavirus 6/11 virus particles.
7. The use according to claim 6, wherein said medicament further comprises a pharmaceutically acceptable carrier.