CN114935649B

CN114935649B - HPV virus detection kit

Info

Publication number: CN114935649B
Application number: CN202210685365.7A
Authority: CN
Inventors: 张倡埼; 朱高茂
Original assignee: Guangdong Cel Biotechnology Co ltd
Current assignee: Guangdong Cel Biotechnology Co ltd
Priority date: 2022-06-15
Filing date: 2022-06-15
Publication date: 2023-07-25
Anticipated expiration: 2042-06-15
Also published as: CN114935649A

Abstract

The invention relates to an HPV virus detection kit. According to the invention, the immune antigen peptide with HPV6/11 specificity is obtained through screening and optimization, the antigen peptide is used for immunizing mice to prepare the specific monoclonal antibody, the monoclonal antibody can specifically bind with HPV6/11 and has the effect of inhibiting HPV6/11 from infecting normal cells, and the monoclonal antibody can be effectively used for detecting HPV by preparing the kit, so that the monoclonal antibody has a good application prospect.

Description

HPV virus detection kit

Technical Field

The invention relates to the field of biological detection, in particular to an HPV virus detection kit.

Background

Cervical cancer is the second most common malignancy in women worldwide, with about 52.8 new cases and 27.5 death cases occurring annually, with 87% of the new cases occurring in developing countries. About 9.9 thousands of new cases and 3.1 dead cases occur in China each year. Numerous studies have shown that persistent infection with Human Papillomavirus (HPV) is closely related to the occurrence of cervical cancer, and that high-risk HPV infection is a major factor causing Cervical Intraepithelial Neoplasia (CIN) and cervical cancer. More than 200 HPV genotypes have been found, of which about 54 HPV genotypes can infect the genital mucosa, and further HPV subtypes are being identified. The criterion for a new HPV genotype is that the nucleotide sequence of the HPV genotype in the E6, E7 or L1 region differs by more than 10% from any other HPV of defined type. It can be classified into high-risk type HPV and low-risk type HPV according to their pathogenicity and the magnitude of the risk of pathogenicity. High-risk HPV infection can cause intraepithelial neoplasia or canceration of cervical, vaginal, anal, penile and oropharyngeal sites, and HPV types mainly include 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 and 82. Low-risk HPV infection can only cause benign lesions such as genital warts, and its HPV types mainly include 6, 11, 40, 42, 43, 44, 54, 61, 70, 72 and 81. The E6 and E7 proteins of low-risk HPVs interfere with the P53 and pRb genes relatively poorly compared to high-risk HPVs.

HPV detection technology is widely applied to screening of cervical cancer, epidemiological investigation and evaluation of effectiveness of cervical cancer vaccines. At present, detection methods for HPV at home and abroad are endless, and the advantages and disadvantages of different detection methods for HPV are mainly evaluated through two aspects: clinical value (i.e., clinical sensitivity) and detection limit (i.e., analytical sensitivity). Clinical sensitivity (also called functional sensitivity) refers to the proportion of patients whose test results are positive in HPV detection technology and the detection ability of HPV to CIN2 grade and above 2; assay sensitivity (also known as 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, about 150 different HPV detection experiments are being performed worldwide, and some are still underway. The detection methods adopted mainly comprise DNA detection, RNA detection, marker detection related to HPV and antigen detection.

Aptima HPV was obtained in 2012 as a batch, which was the first and only FDA-certified technique for HPVmRNA detection. The technology is based on transcription-mediated amplification (transcription-MediatedAmplification, TMA) technology to detect HPV E6/E7mRNA: namely, a T7 promoter is added at the 5' end of the upstream primer, RNA polymerase is introduced into a reaction system, target mRNA is reverse transcribed into cDNA, and target molecule amplification is realized in a transcription mode. The technical aim is to detect the mRNA of the HPV oncogene E6/E7, which can effectively avoid the interference of HPV primary infection on the detection result compared with HPV-DNA detection, but AptimaHPV detection cost is more expensive.

HPV antibody serological detection is of great importance in HPV vaccinology and epidemiological studies. The current rapid immunoassay kit for HPV is not enough, and needs to be further studied, and particularly, the detection reagents capable of simultaneously aiming at multiple HPV subtypes are not enough. Therefore, the development of kits for HPV multi-subtype detection has become particularly urgent.

Disclosure of Invention

The invention overcomes the defects of the prior art, screens and obtains HPV6 and HPV11 high immunogenicity antigen peptide, and the sequence of the antigen peptide is shown as SEQ ID NO: 1.

In another aspect of the invention, corresponding monoclonal antibodies are prepared and obtained using HPV6 and HPV11 highly immunogenic antigenic peptides as immunogens.

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

Antibodies of the invention, fragments thereof or chains of VH chain VL antibodies include those antibodies, fragments or chains of VH chain VL antibodies described herein, which 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 sequences of antibodies may be different 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 which 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 anew by modification of whole antibodies or using recombinant DNA methods.

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

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

Conservative amino acid substitutions 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 to be 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 an 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 optic inspection, and laparoscopy). Specific, non-limiting examples of detectable labels include avidin, biotin, fluorophores, chemiluminescent agents, enzyme bonds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). In one embodiment, "labeling an antibody" refers to the incorporation of another molecule into the antibody. For example, the tag is a detectable label, such as incorporation of a radiolabeled amino acid or attachment of a biotin moiety to the polypeptide, which can be detected by a labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides are known in the art and may be used. Examples of polypeptide markers include, but are not limited to, the following: a radioisotope or radionuclide (e.g., 35S or 131 i), a fluorescent label (e.g., fluorescein Isothiocyanate (FITC), rhodamine, lanthanide phosphor), an enzyme label, a chemiluminescent label, a biotin-based, a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site of a secondary antibody, metal binding domain, epitope tag), or a magnetic agent, e.g., gadolinium chelate. In some embodiments, the labels are connected by spacer arms of different lengths to reduce potential steric hindrance. For example, sambrook et al discuss methods of using detectable labels and directing the selection of detectable labels suitable for various purposes.

Examples of suitable enzyme labels include malate hydrogenase, staphylococcal nuclease, 5-steroid isomerase, yeast alcohol dehydrogenase, glycerophosphate 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 tag, fluorescein tag, isothiocyanate tag, rhodamine tag, phycoerythrin tag, phycocyanin tag, allophycocyanin tag, phthaldehyde tag, example chemiluminescent substrates such as fluorescamine tag, include luminal substrates, isopipe substrates, aromatic acridinium ester substrates, imidazole substrates, acridinium salt substrates, oxalate tag, fluorescein substrates, luciferase tag, hirudin tag, and the like.

Antibodies, fragments thereof, and VH and VL chains of antibodies may be produced using genetic techniques including 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 (editorial), 1985, and Harlow et al, antibodies: laboratory Manual, cold spring harbor laboratory Press, second edition. Monoclonal antibodies can also be obtained by cloning immunoglobulin sequences directly 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 an antibody by binding a 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 a kit for carrying out the detection method of the invention. The kit comprises a container containing monoclonal antibodies produced by at least one hybridoma cell line of the invention and instructions for using the monoclonal antibodies 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 comprise a first container means comprising an antibody as described herein. The kit may also include other container means with solutions as needed or convenient for practicing the invention. The container means may be made of glass, plastic, and the kit may also contain written information, such as information for performing the process or analysis of the invention, such as the amount of reagent contained in the first container means. The container means may be in another container means (e.g. box, bag, etc.) together with the written information.

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

Further, the diagnostic kit of the present invention comprises instruments and reagents commonly used for immunological tests. When HPV antibodies are a component of a diagnostic kit based on an ELISA method, such a 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 suitable blocking substances, buffers for washing the plates, dilution of samples and reagents, substrates for enzymes, and solutions for inhibition of the enzyme reaction.

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

Advantageous effects

According to the invention, the immune antigen peptide with HPV6/11 specificity is obtained through screening and optimization, the antigen peptide is used for immunizing mice to prepare the specific monoclonal antibody, the monoclonal antibody can specifically bind with HPV6/11 and has the effect of inhibiting HPV6/11 from infecting normal cells, and the monoclonal antibody can be effectively used for detecting HPV by preparing the kit, so that the monoclonal antibody has a good application prospect.

Drawings

FIG. 1A diagram of results of antigen reaction specificity identification

FIG. 2 shows the effect of monoclonal antibody on HPV infection of HaCaT cells

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

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

According to amino acid sequences of HPV6 and HPV11, adopting computer simulation, combining Kyte-Doolittle and Hopp-Woods amino acid hydrophilicity scheme, karplus-Schulz plasticity scheme, emini surface area probability scheme, jameson-Wolf antigen probability scheme and Kolaskar-Tongaonkar epitope index prediction method, and assisting in analyzing flexible region in protein secondary structure by Cohen method, garnier-Robson method and Chou-Fasman method, multiparameter predicting main epitope sequences of HPV6 and HPV11, and designing 1 best common epitope capable of simultaneously aiming at HPV6 and HPV11 after comprehensive comparison analysis of prediction results: vqqwaydndiceeseeiafey qrgdfdsnaraflunmmqakyv (SEQ ID NO: 1). And (3) entrusting the epitope peptide to be artificially synthesized for later use.

BALB/c mice were immunized with epitope peptides as immunogens. Mixing the primary immunization with Freund's complete adjuvant, wherein the immunization dose is 100 mug/dose, and subcutaneous multipoint injection is adopted; mixing with Freund's incomplete adjuvant, and enhancing immunity for 3 times, and performing intraperitoneal injection. 5d after the last 1 immunization, blood is collected through a rat tail, serum is separated, the immune effect is detected through indirect ELISA, and a mouse with the best immune effect is selected; and directly injecting the antigen epitope peptide into the spleen at the dosage of 200 mug/dose for enhancing the immune effect 3d before cell fusion. Taking immunized mouse spleen cells and syngeneic mouse myeloma cells SP2/0 to carry out cell fusion, respectively adopting a culture medium containing HAT and HT to carry out 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 capability of hybridoma cell culture supernatant, and screening and identifying 3 hybridoma clones with strongest positive, namely 3A2a,7C4c and 8D2b respectively.

Preparation and purification of monoclonal antibodies: at 1.5X10 ⁶ Three 3A2a,7C4c,8D2b hybridoma cells were intraperitoneally injected into paraffin oil-pretreated BALB/c mice, respectively, 10d later, ascites were extracted, centrifuged at 12000 Xg for 30min, the supernatant was collected, diluted with an equal volume of binding buffer (0.1 mol/LPBS, pH 8.0), filtered through a 0.45 μm microporous filter membrane, and the antibody was purified by biological affinity chromatography using the Fc segment of the antibody. Balancing rProteinA chromatographic column, loading, washing with binding buffer, and eluting with eluting buffer (0.1 mol/L citric acid-sodium citrate buffer, pH 3.0); immediately, 1mol/L Tris-HCl buffer at pH8.9 was added to the collected purified antibody for sufficient neutralization, and the buffer was replaced in PBS, sampled, and the concentration of the antibody was measured by BCA method. An indirect ELISA method is adopted, epitope peptide is used as a coating antigen, and the dilution of monoclonal antibody 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 was taken as antibody titer. The results are shown in Table 1 below.

TABLE 1 concentration and titers of purified monoclonal antibodies

Monoclonal antibody	Concentration (mg/mL)	Titers (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: an indirect ELISA method is adopted, and SEQ ID NO:1, and khkallikcppllvtsinid (SEQ ID NO: 2), and a BSA protein as coating antigens, and determining the antigen-antibody reactivity of the monoclonal antibody, and analyzing the antigen reaction specificity of the monoclonal antibody. The results are shown in FIG. 1.

The indirect ELISA results show that 3 monoclonal antibodies (3A 2a,7C4c,8D2 b) can react with the immune antigen peptide of the sequence 1 strongly, but do not react with the epitope peptide of the sequence 2 and do not react with human BSA, so that the 3 monoclonal antibodies are specific monoclonal antibodies aiming at HPV6/11, and the figure 1 shows.

Example 2 sequence analysis and affinity identification of 7C4c monoclonal antibody

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

Affinity determination of the 7C4C antibody and epitope was performed by the surface plasmon resonance SPR method. The measurement was performed with different concentrations of antibody as mobile phase. The result shows that the 7C4C antibody and the antigen epitope are recognized with high affinity, and the dissociation constant reaches Kd of 3.24X10 ^-9 M。

Designing light and heavy chain amplification primers aiming at the 5 'end and the 3' conserved sequences of the heavy chain and light chain variable region genes of the mouse antibody, amplifying by taking cDNA of a 7C4C hybridoma cell as a template, inserting the amplified product into pUC19 plasmid, and then sequencing and analyzing to obtain the light and heavy chain variable region sequences of the 7C4C antibody, wherein the sequences are respectively shown as follows.

Light chain variable region (SEQ ID NO: 3)

Heavy chain variable region (SEQ ID NO: 4)

Example 3 Effect of 7C4c monoclonal antibodies on in vitro infection of HaCaT cells with human papillomavirus 6/11 Virus particles

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

In a 24-well plate according to 1X 10 ⁵ Inoculating HaCaT cells in the wells; the next time the broth was discarded, 6 groups were diluted 1: 10. 1: 50. 1: 100. 1: 200. 1: 400. 1:1000 monoclonal antibodies (initial concentration 1 mg/mL) were added to HaCaT cells, respectively, and placed on ice for 1h, with the plates being gently shaken from time to time; discarding culture solution, adding appropriate amount of PBS, washing thoroughly, and washing with 10 ⁶ Infecting HaCaT cells in vitro by using copy/ml of HPV6/11 virus-like particles, using equal amount of PBS to replace monoclonal antibody and HPV6/11 virus-like particles as negative control, and adding only HPV6/11 virus-like particles without monoclonal antibody as positive control; taking infected HaCaT cell linesPCR detection; repeating the above process for 3 times; and (3) calculating: monoclonal antibody inhibition = (HaCaT virus copy number after 1-addition of monoclonal antibody/HaCaT virus copy number without addition of monoclonal antibody) ×100%.

FIG. 2 shows that the concentration of the monoclonal antibody pairs in 6 groups with different dilution factors is 10 ⁶ The inhibition rates measured after in vitro infection of HaCaT cells with HPV6/11 virus-like particles in copies/ml were different, and by comparison, it was found that with increasing concentration of monoclonal antibody, monoclonal antibody pair 10 ⁶ The inhibition rate of HPV6/11 virus-like particles in copies/ml to host cells increases with the difference being statistically significant (P < 0.05). Under the dilution condition of 1:10, the inhibition rate reaches (72.30+/-3.98)%, and the inhibition effect is good.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

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Claims

Use of bispecific monoclonal antibody 7C4C of HPV6 and HPV11 in the preparation of a kit for HPV6/11 detection, wherein the bispecific monoclonal antibody light chain variable region sequences of HPV6 and HPV11 are as set forth in SEQ ID NO:3, the heavy chain variable region sequences of the bispecific monoclonal antibodies of HPV6 and HPV11 are shown in SEQ ID NO: 4.
2. The use according to claim 1, wherein the monoclonal antibody is further conjugated with a detectable label.
3. Use according to claim 2, characterized in that the detectable label is an enzyme.
4. The method of claim 3, wherein the enzyme is catalase, glucose-6-phosphate dehydrogenase, glucoamylase, or acetylcholinesterase.
Use of bispecific monoclonal antibody 7C4C of HPV6 and HPV11 in the manufacture of a medicament for inhibiting HPV6/11 infection, wherein the bispecific monoclonal antibody light chain variable region sequences of HPV6 and HPV11 are as set forth in SEQ ID NO:3, the heavy chain variable region sequences of the bispecific monoclonal antibodies of HPV6 and HPV11 are shown in SEQ ID NO: 4.
6. The use according to claim 5, wherein the bispecific monoclonal antibodies to HPV6 and HPV11 are capable of inhibiting human papillomavirus 6/11 viral particle infection of HaCaT cells in vitro.
7. The use according to claim 6, wherein the medicament further comprises a pharmaceutically acceptable carrier.