CN105524166B

CN105524166B - Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof

Info

Publication number: CN105524166B
Application number: CN201410508253.XA
Authority: CN
Inventors: 常小迦; 施丽君; 时成龙; 韩凤丽
Original assignee: Attogen Biomedical Suzhou Inc ltd
Current assignee: Attogen Biomedical Suzhou Inc ltd
Priority date: 2014-09-28
Filing date: 2014-09-28
Publication date: 2020-07-10
Anticipated expiration: 2034-09-28
Also published as: CN105524166A

Abstract

The invention provides a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof, wherein the antibody can detect a cervical cancer biomarker HPV18E7 protein in tumor cells with high specificity, so that cancerous cervical epithelial cells and cervical abnormal or non-cancerous cervical epithelial cells can be distinguished, basis is brought for doctors to accurately diagnose cancers caused by HPV infection, and then treatment is timely taken to prevent cancer occurrence or cancer spread and relieve pain of patients in time.

Description

Monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof

Technical Field

The invention belongs to the field of biological diagnosis and medicine, and particularly relates to a monoclonal antibody for identifying HPV18 positive cervical epithelial cancer cells and application thereof.

Background

Cervical cancer is a common malignancy in the female reproductive system, second-place in female malignancies. In 1949, Sttaus first observed Human Papilloma Virus (HPV) particles in wart body leachate under an electron microscope. After Harald Zur Hausen suggested that HPV may be a sexually transmitted carcinogen in 1976, the study of the relationship between HPV infection and cervical cancer became a popular topic of research on the etiology of tumor viruses. A large number of researches find that the human papilloma virus HPV is an extremely powerful cause of cervical cancer and can also cause various other tumors including cancers of the reproductive tract, mammary gland, digestive tract and respiratory tract. In recent years, the spread of HPV in people in China is continuously reduced, and the research work of prevention and treatment of the cervical cancer is very important.

Most women will infect genital HPV in their lives, the infection rate of genital HPV is as high as 10.4% worldwide, most HPV infections will be cleared by the autoimmune system within 1-2 years, and persistent HPV infections will progress to high grade Cervical Intraepithelial Neoplasia (CIN) lesions, such as CIN2 and CIN3, and even further to cervical cancer, statistically, about 20% of low grade cervical lesions will turn to high grade lesions, and 30% will further turn to malignant tumors if not treated in time, therefore, early diagnosis and prevention of HPV infection are important breakthrough points to reduce mortality of related diseases such as cervical cancer and reduce the expense of cervical cancer treatment.

HPV is a species-specific epitheliotropic virus, a double-stranded closed-loop small DNA virus, comprising about 8000 base pairs. These included 8 early open reading frames (E1-E8), 2 late reading frames and 1 non-coding long control region. In the early open reading frame, the E6 and E7 genes were most important for cell growth stimulation. The research of Ziegent (2003) and the like has proved that HPV E6 and E7 genes have cell transformation function and are potential oncogenes, and the encoded HPV E6 and E7 proteins are oncoproteins, can transform mouse epithelial cells in vitro and can also enable human epithelial cells to be immortalized, and the continuous expression of HPV E6 and E7 proteins is necessary for maintaining the immortalization of in vitro cultured cells. Therefore, the early expression proteins E6 and E7 of high-risk HPV play an important role in the development of cervical cancer. During carcinogenesis, virus DNA is integrated into human cell genome, and E6 and E7 proteins are continuously expressed in epithelial cells of patients with high cervical dysplasia and cervical cancer along with the loss of expression control of E6 and E7 proteins. E7 is a tumor-origin protein, has strong antigenicity, can inactivate tumor cancer suppressor pRb, and finally causes uncontrolled cell growth, resulting in cell immortalization and canceration. This makes E7 a tumor marker for the detection of high grade cervical lesions and carcinoma.

Currently, HPV infection is detected clinically and immunohistochemically mainly by adopting HPV L1 and other auxiliary biomarkers, such as p16INK4A, Ki67, hTERT and the like (Valentina F, Renzo B, Serena B, and the like, Detection of HPV E7Oncoviral protein in central loss by a new antigen, 2013,21(4):341 and 350), clinical HPV Detection has no proper antibody mainly for three reasons, 1, HPV protein is low in expression amount in clinical tissues or cell samples and needs high-affinity antibody for Detection, 2, HPV virus cannot be cultured in a laboratory under the existing standard tissue culture technology, and 3, E7 protein has no immunosuppression, so that animals immunized by adopting E7 protein cannot obtain good immunoreaction, and in addition, prepared HPV cross-reaction antibody and other HPV protein often have specific antibody to E7.

Therefore, there is a need to provide a simple and objective method for detecting high-risk HPV-infected cancers (especially cervical cancer).

Disclosure of Invention

The invention aims to provide a monoclonal antibody capable of identifying HPV18 positive cervical epithelial cancer cells and application thereof.

In a first aspect of the present invention, there is provided a heavy chain variable region of an antibody, said heavy chain variable region comprising the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO. 4,

CDR2 shown in SEQ ID NO. 6, and

CDR3 shown in SEQ ID No. 8.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID No. 10.

In a second aspect of the invention, there is provided a heavy chain of an antibody, said heavy chain having a heavy chain variable region as described in the first aspect of the invention, an

A heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human or murine origin.

In a third aspect of the present invention, there is provided a light chain variable region of an antibody, said light chain variable region having complementarity determining regions CDRs selected from the group consisting of:

14, CDR1' shown in SEQ ID NO,

CDR2' as shown in SEQ ID NO. 16, and

18, CDR3' as set forth in SEQ ID no.

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID No. 20.

In a fourth aspect of the invention, there is provided a light chain of an antibody, said light chain having the light chain variable region as described in the third aspect of the invention, and

a light chain constant region.

In another preferred embodiment, the light chain constant region is of human or murine origin.

In a fifth aspect of the invention, there is provided an antibody having:

(1) a heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is an antibody specific to HPV; preferably, the antibody is an antibody specific against HPV 18; more preferably, the antibody is specific against HPV18E7 protein.

In another preferred embodiment, the antibody comprises: a single chain antibody, a diabody, a monoclonal antibody, a chimeric antibody (e.g., a human-murine chimeric antibody), a murine antibody, or a humanized antibody.

In another preferred embodiment, the antibody has the following properties:

(1) the affinity with HPV18E7 protein is less than or equal to 2.67 nM; and

(2) does not bind to HPV16E7 protein.

The HPV18E7 protein can be wild type HPV18E7 protein or derivative protein of wild type HPV18E7 protein. The HPV16E7 protein can be wild type HPV16E7 protein or derivative protein of wild type HPV16E7 protein.

In another preferred embodiment, the antibody is a monoclonal antibody capable of specifically distinguishing HPV18E7 protein from HPV16E7 protein.

In another preferred embodiment, the antibody does not bind to or has low affinity for other HPV subtypes.

In another preferred embodiment, the antibody further has the following properties:

(3) can specifically bind to protein containing HPV18E7 amino acids 44-58.

In another preferred embodiment, the antibody is an IgG 1-type antibody.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) optionally a tag sequence to facilitate expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein is specific against HPV; preferably, specific anti-HPV 18; more preferably, it is specific against HPV18E7 protein.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or

(2) A recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide has the sequence shown in SEQ ID No. 3, 5, 7, 9, 13, 15, 17, or 19.

In an eighth aspect of the invention, there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

According to a ninth aspect of the invention, there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the invention into which has been integrated a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention, there is provided an immunoconjugate comprising:

(a) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(b) a coupling moiety selected from the group consisting of: a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

In another preferred embodiment, the conjugate is selected from the group consisting of a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., I L-2, etc.), an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPH L)), a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, and the like.

In an eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) a heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention, a recombinant protein according to the sixth aspect of the invention, or an immunoconjugate according to the tenth aspect of the invention; and

(ii) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of injection.

In another preferred embodiment, the pharmaceutical composition is used for preparing a medicament for treating a tumor selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, adrenal gland tumor, or bladder tumor.

In a twelfth aspect of the invention, there is provided a use of the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or the immunoconjugate according to the tenth aspect of the invention for the manufacture of a medicament, a reagent, a test panel or a kit;

the reagent, assay plate or kit is for:

(1) detecting HPV18E7 protein in the sample; and/or

(2) Detecting endogenous HPV18E7 protein in the tumor cells; and/or

(3) Detecting tumor cells expressing HPV18E7 protein; and/or

(4) Identifying the type of HPV;

the medicament is used for treating or preventing tumors expressing HPV18E7 protein.

In another preferred embodiment, the sample contains HPV18E7 protein.

In another preferred embodiment, the tumor comprises: a tumor of the urogenital system, a small cell lung cancer, a melanoma or a head and neck tumor, a gastric cancer, a liver cancer, a leukemia, a kidney tumor, a lung cancer, a small intestine cancer, a bone cancer, a prostate cancer, a colorectal cancer, a breast cancer, a large intestine cancer, a prostate cancer, or an adrenal tumor.

In another preferred embodiment, the "tumor of urogenital system" includes: cervical cancer, bladder cancer, endometrial cancer, or penile cancer.

In another preferred embodiment, the reagent comprises a chip and immune microparticles coated with antibodies.

In a thirteenth aspect of the present invention, there is provided a method for detecting HPV E7 protein in a sample, the method comprising the steps of:

(1) contacting the sample with an antibody according to the fifth aspect of the invention;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of HPV E7 protein in the sample.

In another preferred example, the detection is performed by the ISA method of E L in step (2).

In another preferred embodiment, the HPV E7 protein comprises HPV18E7 protein.

In another preferred example, in step (1), the sample is contacted with two antibodies against HPV E7 protein, at least one of which is an antibody according to the fifth aspect of the present invention, and detected by the E L ISA method in step (2).

In another preferred embodiment, the "antigen-antibody complex" is a "first antibody-antigen-second antibody" ternary complex, wherein the first antibody is an antibody according to the fifth aspect of the invention and the binding epitope of the second antibody is different from the binding epitope of the first antibody.

In another preferred embodiment, the second antibody is a monoclonal antibody produced by hybridoma cell line CGMCC NO. 5714.

In another preferred embodiment, after the sample is contacted with the antibody according to the fifth aspect of the present invention in step (1), a third antibody against the first antibody is further added to the reaction system, and the formation of an "antigen-first antibody-third antibody" complex is detected in step (2).

In another preferred embodiment, the first antibody, the second antibody or the third antibody carries a detectable label.

In another preferred example, the detectable label is a biotin label, a colloidal gold label, a horseradish peroxidase label, a radionuclide label, or a fluorescein label.

In another preferred example, the sample comprises: human or animal tissue samples, tumor resection samples, exfoliated cell samples.

In another preferred embodiment, the method is used for non-diagnostic purposes.

In a fourteenth aspect of the present invention, there is provided a test plate comprising a substrate (support plate) and a test strip comprising an antibody according to the fifth aspect of the present invention or an immunoconjugate according to the sixth aspect of the present invention.

In another preferred embodiment, the test strip further comprises an antigen-sampling region.

In another preferred embodiment, the test strip is formed by overlapping the filter paper, the chromatographic material, the nitrocellulose membrane and the absorbent paper in sequence.

In a fifteenth aspect of the present invention, there is provided a kit comprising:

(1) a first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a secondary antibody against the antibody of the fifth aspect of the invention; and/or

(3) A third container comprising a cell lysis reagent;

alternatively, the first and second electrodes may be,

the kit comprises the detection plate according to the fourteenth aspect of the present invention.

In another preferred embodiment, the antibody in the first container is detectably labeled.

In another preferred embodiment, the antibody in the second container is detectably labeled.

In a sixteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression;

(b) isolating a recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the result of detection of HPV18E7 monoclonal antibody MAB-MAB-F2 antigen binding specificity E L ISA, and the details of the experiment are shown in the example 1, subsection 2.2, MAB-F2 can specifically bind to HPV18E7 recombinant protein but not to HPV16E7 recombinant protein.

FIG. 2 shows the result of ISA detection of HPV18E7 monoclonal antibody binding to epitope amino acid sequence region E L, and the details of the experiment are shown in the subsections of examples 1 and 2.3. FIG. 2A shows the schematic amino acid sequence of HPV18E7, FIG. 2B shows the result of ISA detection of E L. MAB-F2 can bind to His-HPV18E7 recombinant protein and polypeptides HPV18E7-1 and HPV18E7-2, while the polypeptide does not bind to His-HPV16E7 recombinant protein.

FIG. 3 is a diagram showing the detection results of HPV18E7 monoclonal antibody protein binding affinity analysis E L ISA, and the details of the experiment are shown in the subsections of example 1 and 2.4, the coating concentrations of His-HPV18E7 are respectively 8 mug/ml, 2 mug/ml, 0.5 mug/ml and 0.125 mug/ml, the incubation concentration of MAB-F2 is diluted from 8 mug/ml in a multiple ratio, seven gradients are obtained in total, and the seven gradients are substituted into K_aff＝(n-1)/2(n[Ab′]-[Ab]) Six K values were calculated and averaged to give MAB-F2 with 2.67nM protein level affinity.

FIG. 4 is a diagram showing the result of IP-WB detection of the HPV18E7 monoclonal antibody MAB-F2 on the endogenous HPV18E7 of Hela cells, and the details of the experiment are shown in the sections of example 1 and 2.5: mIgG as a negative control failed to bind HPV18E7 in the tumor cell lysate. mAb MAB-F2 was able to bind HPV18E7 in tumor cell lysates and therefore showed a clear band at 15 kDa. Of these, bands around 50kDa and 25kDa are the heavy and light chains of antibodies used in IP processes.

FIG. 5 is a diagram showing the result of HPV18E7 monoclonal antibody immunocytochemical staining detection, and details of the experiment are shown in example 1, section 2.6: a is the result of C-33A cell staining, B is the result of Hela cell staining, C is the result of CaSki cell staining, monoclonal antibody MAB-F2 has strong staining reaction with Hela cells, but has no staining with C-33A cells and CaSki cells.

FIG. 6 is a diagram showing the results of immunocytochemistry staining of cervical cancer cell lines C-33A and Hela cells fixed by different fixatives using HPV18E 7-specific monoclonal antibody MAB-F2, and the specific experimental details are shown in example 2. the C-33A cells have no immunoreaction with HPV18E 7-specific monoclonal antibody MAB-F2. after the two cells are mixed, the HPV18E 7-specific monoclonal antibody MAB-F2 makes the HeLa color (red arrow) 10% of the total number of the cells and the negative cells C-33A have no staining, the Hela cells fixed in suspension 100% have strong immunoreaction with HPV18E 7-specific monoclonal antibody MAB-F2. the 4% paraformaldehyde and L BC fixative have no influence on the specificity of HPV18E 7-specific monoclonal antibody MAB-F2 in ICC detection.

Detailed Description

The inventor unexpectedly obtains an anti-HPV E7 monoclonal antibody MAB-F2 through extensive and intensive research and a large number of screens, and experimental results show that the monoclonal antibody aiming at HPV E7 protein has high specificity and strong affinity, and can obviously distinguish HPV18E7 protein in cells. The monoclonal antibody can be specifically combined with HPV18E7 protein, but has no combination activity with HPV16E7 protein, so that the antibody not only can be used for detecting HPV18E7 protein, but also can be used for identifying different HPV types. The invention also provides a method for detecting and/or identifying the HPV18E7 protein, which has good stability and extremely high detection sensitivity. The invention also provides a kit containing the antibody and a detection plate.

Specifically, the recombinant GST-HPV18E7 fusion protein is adopted to immunize a mouse, and the other fusion protein His-HPV18E7 is used as a screening detection antigen, so that antibody IgG can be secreted and can be combined with the recombinant protein His-HPV18E7 when positive hybridoma clone strains are screened, and the antibody is specifically combined with the HPV E7 fusion protein in E L ISA detection.

In addition to the identification of the E L ISA binding recombinant protein antigen, the positive monoclonal antibody is further subjected to antigen binding epitope analysis, Affinity binding specificity, Western Blot, immunoprecipitation, Immunocytochemistry (ICC), and histological section Immunostaining (IHC).

In order to screen out an antibody reagent with clinical examination value, the monoclonal antibody is further used for a detection test of cervical cancer cell strains. Finally, a monoclonal antibody capable of specifically recognizing HPV18E7 positive cervical epithelial cells was obtained, and based on the specificity of the antibody, methods capable of distinguishing cervical epithelial cancer cells from normal cells were developed, which methods were sufficient in terms of sensitivity to identify those patients with early-stage cervical cancer, and in terms of specificity to distinguish between normal and malignant tumor cells.

In a preferred embodiment of the present invention, the amino acid sequence of the HPV16E7 protein is as follows:

HGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP(SEQ ID NO.:1)。

in a preferred embodiment of the present invention, the amino acid sequence of the HPV18E7 protein is as follows:

MHGPKATLQDIVLHLEPQNEIPVDLLCHEQLSDSEEENDEIDGVNHQHLPARRAEPQRHTMLCMCC KCEARIELVVESSADDLRAFQQLFLNTLSFVCPWCASQQ(SEQ ID NO.:2)。

as used herein, the term "antibody" or "immunoglobulin" is an isotetrameric glycan protein of about 150000 daltons having the same structural characteristics, consisting of two identical light chains (L) each linked to a heavy chain by a covalent disulfide bond and two identical heavy chains (H) differing in the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which form the binding and specificity of each particular antibody for its particular antigen, however, the variability is not evenly distributed throughout the antibody variable region, it is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions.

The "light chains" of vertebrate antibodies (immunoglobulins) can be classified into one of two distinct classes (termed kappa and lambda) according to the amino acid sequence of their constant regions, immunoglobulins can be classified into different classes, there are mainly 5 classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2, the constant regions of the heavy chains corresponding to different classes of immunoglobulins are respectively termed α, gamma and mu, the subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known to those skilled in the art.

As used herein, the term "monoclonal antibody (mab)" refers to an antibody obtained from a substantially homogeneous population, i.e., the individual antibodies contained in the population are identical, except for a few naturally occurring mutations that may be present. Monoclonal antibodies are directed against a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody preparations (typically having different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The invention also comprises a monoclonal antibody with the corresponding amino acid sequence of the monoclonal antibody for resisting HPV18E7 protein, a monoclonal antibody with the variable region chain of the monoclonal antibody for resisting HPV18E7 protein, and other proteins or protein conjugates and fusion expression products with the chains. Specifically, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having light and heavy chains with hypervariable regions (complementarity determining regions, CDRs) so long as the hypervariable regions are the same as or at least 90% homologous, preferably at least 95% homologous to the hypervariable regions of the light and heavy chains of the invention.

As known to those skilled in the art, immunoconjugates and fusion expression products include: drugs, toxins, cytokines (cytokines), radionuclides, enzymes, and other diagnostic or therapeutic molecules are conjugated to the monoclonal antibody to HPV16E7 protein or a fragment thereof. The invention also comprises a cell surface marker or antigen combined with the anti-HPV18E7 protein monoclonal antibody or the fragment thereof.

The invention includes not only intact monoclonal antibodies, but also immunologically active antibody fragments, such as Fab or (Fab')₂A fragment; an antibody heavy chain; the light chain of the antibody.

As used herein, the terms "heavy chain variable region" and "V_H"may be used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDRs including:

CDR1, the amino acid sequence of which is GFTFNRYD (SEQ ID NO.:4), and the coding nucleotide sequence of which is ggattcactttcaatagatatgac (SEQ ID NO.: 3);

CDR2, the amino acid sequence of which is IRNDGSKT (SEQ ID NO.:6), and the coding nucleotide sequence of which is atcagaaatgatggtagtaagact (SEQ ID NO.: 5);

CDR3 with an amino acid sequence of TRDSYGTVFAY (SEQ ID NO.:8) and a coding nucleotide sequence of acaagggactcctatggtaccgtgtttgcttac (SEQ ID NO.: 7).

In another preferred embodiment, the amino acid sequence of the heavy chain variable region is:

LVQSGGGSVKPGGSLKLSCAASGFTFNRYDMSWFRQSPEKRLEWVAEIRNDGSKTHYSDTVTG RFTISRDNAKNTLYLEMSSLRSEDTAMYYCTRDSYGTVFAYWGQGTLVTVSA(SEQ ID NO.:10)；

the coding nucleotide sequence is as follows:

Ctggtgcagtcagggggaggctcagtgaagcctggagggtccctgaaactctcctgtgcagcctc tggattcactttcaatagatatgacatgtcttggtttcgccagtctccagagaagaggctggagtgggtcg cagagatcagaaatgatggtagtaagactcactattcagacactgtgacgggccgattcaccatctccaga gacaatgccaagaacaccctgtacctggaaatgagcagtctgaggtctgaggacacggccatgtattactg tacaagggactcctatggtaccgtgtttgcttactggggccaagggactctggtcactgtctctgcag(S EQ ID NO.:9)。

in a preferred embodiment of the invention, the heavy chain of the antibody comprises the above-described heavy chain variable region and a heavy chain constant region, which may be of murine or human origin.

As used herein, the terms "light chain variable region" and "V L" are used interchangeably.

In a preferred embodiment of the invention, the light chain variable region of the antibody according to the invention has complementarity determining regions CDRs selected from the group consisting of:

CDR1', the amino acid sequence of which is QS LL YSNGKTY (SEQ ID NO.:14), the coding nucleotide sequence of which is cagagcctcttatatagtaatggaaaaacctat (SEQ ID NO.: 13);

CDR2', the amino acid sequence of which is L VS (SEQ ID NO.:16), and the coding nucleotide sequence of which is ctggtgtct (SEQ ID NO.:15)

CDR3', having an amino acid sequence of VQGTHFPQT (SEQ ID NO.:18) and a coding nucleotide sequence of gtgcaaggtacacattttcctcagacg (SEQ ID NO.:17)

In another preferred embodiment, the amino acid sequence of the light chain variable region is:

VMTQTPLTLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPD RFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPQTFGGGTKLEIK(SEQ ID NO.:20)，

the coding nucleotide sequence is as follows:

gtcatgacccagactccactcactttgtcggttaccattggacaaccagcctctatctcttgcaagtcaagtc agagcctcttatatagtaatggaaaaacctatttgaattggttattacagaggccaggccagtctccaaagcgccta atctatctggtgtctaaactggactctggagtccctgacaggttcactggcagtggatcaggaacagattttacact gaaaatcagcagagtggaggctgaggatttgggagtttattactgcgtgcaaggtacacattttcctcagacgttcg gtggaggcaccaagctggaaatcaaac(SEQ ID NO.:19)。

in a preferred embodiment of the invention, the light chain of the antibody comprises the light chain variable region and a light chain constant region, which may be murine or human.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably and refer to an antibody that specifically binds HPV18E7 protein, such as a protein or polypeptide having a heavy chain variable region (e.g., the amino acid sequence of SEQ ID No.: 10) and/or a light chain variable region (e.g., the amino acid sequence of SEQ ID No.: 20). They may or may not contain the initial methionine.

In another preferred embodiment, the antibody is a murine or human murine chimeric monoclonal antibody directed against the HPV18E7 protein, and its heavy chain constant region and/or light chain constant region may be humanized heavy chain constant region or light chain constant region. More preferably, the humanized heavy or light chain constant region is that of human IgG1, IgG2, or the like.

The invention also provides other proteins or fusion expression products having an antibody of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having heavy and light chains with variable regions, provided that the variable regions are identical or at least 90% homologous, preferably at least 95% homologous, to the variable regions of the heavy and light chains of the antibody of the invention.

In general, the antigen binding properties of an antibody can be described by 3 specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs), which are separated by 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction.

The variable regions of the heavy and/or light chains of the antibodies of the invention are of particular interest, since at least some of them are involved in binding to an antigen. Thus, the invention includes those molecules having the light and heavy chain variable regions of a monoclonal antibody with CDRs that are more than 90% (preferably more than 95%, most preferably more than 98%) homologous to the CDRs identified herein.

The invention includes not only complete monoclonal antibodies, but also fragments of antibodies with immunological activity or fusion proteins of antibodies with other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which an mature polypeptide is fused to another compound, such as a compound that increases the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6 His-tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The antibody of the present invention refers to a polypeptide having HPV18E7 protein binding activity, comprising the CDR regions described above. The term also includes variant forms of the polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural variants, induced mutants, proteins encoded by DNA that hybridizes under high or low stringency conditions with DNA encoding an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising human antibodies or fragments thereof. In addition to almost full-length polypeptides, the invention also encompasses fragments of the antibodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, "conservative variations of the antibody of the present invention" means that at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced with amino acids having similar or analogous properties as compared with the amino acid sequence of the antibody of the present invention to form a polypeptide. These conservative variants are preferably produced by amino acid substitutions according to Table A.

TABLE A

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region as shown in SEQ ID NO. 3, 5, 7, 9, 13, 15, 17, 19 or may be a degenerate variant. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence which encodes a polypeptide having the same amino acid sequence as the polypeptide of the present invention, but differs from the sequence of the coding region as set forth in SEQ ID No. 3, 5, 7, 9, 13, 15, 17, 19.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, and may also include additional coding and/or non-coding sequences.

The present invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences, the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention, in which case "stringent conditions" mean (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 × SSC, 0.1% SDS,60 ℃, or (2) hybridization with denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃, etc., or (3) hybridization only if the identity between the two sequences is at least 90% or more, more preferably 95% or more, and the polypeptides encoded by the hybridizable polynucleotides have the same biological function and activity as the mature polypeptides of SEQ ID No. 10 and/or SEQ ID No. 20.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequences of interest, especially when the fragments are of short length. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the expanded host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form.

At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells capable of DNA uptake may be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptides in the above methods can be expressed intracellularly, or on the cell membrane, or secreted extracellularly, if desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties, which are well known to those skilled in the art.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Detectable labels for diagnostic purposes include, but are not limited to: a fluorescent or luminescent label, a radioactive label, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of producing a detectable product.

Therapeutic agents that can be conjugated or conjugated to the antibodies of the invention include, but are not limited to, 1. radionuclides (Koppe et al, 2005, Cancer metastasis review (Cancer metastasis) 24, 539), 2. biotoxin (Chaudhary et al, 1989, Nature 339, 394; Epel et al, 2002, Cancer Immunology and immunotherapy (Cancer Immunology) 51, 565), 3. cytokines such as I L-2 et al (Gillies et al, 1992, American national academy of sciences (PNAS)89, 1428; Card et al, 2004, Cancer and immunotherapy (Cancer Immunology) 53, 345; Halin et al, 2004, Cancer Research (Cancer Research)63, 3202), 4. gold nanoparticles/L, Immunotko et al, 2005, chemotherapeutic subunit (MRK) 53, 345, 2006, 2004, chemotherapeutic Research (Cancer Research)63, 3202), 4. gold nanoparticles/MRK et al (MRS), MRS, MR.

The invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-described antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these materials will be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, typically having a pH of from about 5 to about 8, preferably a pH of from about 6 to about 8, although the pH will vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical composition may be administered by conventional routes including, but not limited to: intratumoral, intraperitoneal, intravenous, or topical administration.

The pharmaceutical composition of the invention can be directly used for binding HPV16E7 protein molecules, thus being used for preventing and treating tumors. In addition, other therapeutic agents may also be used simultaneously.

The pharmaceutical composition of the present invention comprises a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the monoclonal antibody (or conjugate thereof) of the present invention as described above and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

In the case of pharmaceutical compositions, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms/kg body weight, and in most cases no more than about 8 mg/kg body weight, preferably the dose is from about 10 micrograms/kg body weight to about 1 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

Hybridoma cell strain

The invention also provides a hybridoma cell strain capable of producing the monoclonal antibody aiming at the HPV E7 protein; preferably, the present invention provides a hybridoma cell strain with high titer against HPV18E7 protein monoclonal antibody.

After obtaining the hybridoma producing the HPV E7 protein monoclonal antibody of the present invention, one skilled in the art can conveniently prepare the antibody using the hybridoma cell line. In addition, the structure of the antibody of the present invention (e.g., the heavy chain variable region and the light chain variable region of the antibody) can be easily known by those skilled in the art, and then the monoclonal antibody of the present invention can be prepared by recombinant methods.

Preparation of monoclonal antibodies

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, the antigens of the invention can be administered to an animal to induce the production of monoclonal antibodies. For Monoclonal antibodies, they can be prepared using hybridoma technology (see Kohler et al, Nature 256; 495, 1975; Kohler et al, Eur. J. Immunol.6:511,1976; Kohler et al, Eur. J. Immunol.6:292, 1976; Hammerling et al, In Monoclonal antibodies and T Cell hybrids, Elsevier, N.Y.,1981) or can be prepared using recombinant DNA methods (U.S. Pat. No. 4,816,567).

Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to medium (HAT medium matrix), including myeloma Cell lines, such as murine myeloma Cell lines, including myeloma Cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from Salk Institute Cell Distribution Center, san diego, california, usa), and SP-2, NZ0, or X63-Ag8-653 cells (available from American Type Culture Collection, rockwell, maryland, usa). Human myeloma and mouse-human hybrid myeloma cell lines have also been described for the production of human monoclonal antibodies [ Kozbor, j.immunol., 133: 3001 (1984); brodeur et al, Techniques for the Production and use of Monoclonal Antibodies (Monoclonal Antibodies Production Techniques and Applications), pp 51-63 (Marcel Dekker, Inc., New York, 1987).

The culture medium in which the hybridoma cells are grown is analyzed to detect the production of monoclonal antibodies of the desired specificity, e.g., by in vitro binding assays such as enzyme linked immunosorbent assay (E L ISA) or Radioimmunoassay (RIA). the location of antibody-expressing cells can be detected by FACS.then the hybridoma clones can be subcloned by limiting dilution procedures (subcloning) and grown by standard methods (Goding, monoclonal antibodies): Principles and Practice (Principles and Practice), Academic Press (1986) pp.59-103.) suitable culture media for this purpose include, e.g., DMEM or RPMI-1640 medium.

The monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

The invention provides a monoclonal antibody aiming at HPV E7 protein, in particular a monoclonal antibody aiming at HPV16E7 protein. In a preferred embodiment of the present invention, the monoclonal antibody is prepared by culturing hybridoma cells. Taking supernatant fluid of hybridoma cell culture, carrying out saturated ammonium sulfate precipitation to obtain IgG, and purifying the antibody obtained by crude extraction through an affinity chromatography column (Protein G-Sepharose).

In a preferred embodiment of the invention, the monoclonal antibody is prepared by a method for producing the monoclonal antibody by Balb/C mouse ascites. The hybridoma cells were inoculated into the abdominal cavity of the sensitized mice, and significant abdominal distension was observed within 2-4 weeks. Ascites is extracted, and after the crude extraction by saturated ammonium sulfate precipitation, the antibody of the crude extraction is purified by an affinity chromatography column (Protein G-Sepharose).

Labeled immunoglobulins

In a preferred embodiment of the invention, the immunoglobulin is provided with a detectable label. More preferably, the marker is selected from the group consisting of: a colloidal gold label, a colored label, or a fluorescent label.

The colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred embodiment of the present invention, the monoclonal antibody of HPV18E7 protein is labeled with colloidal gold to obtain a colloidal gold-labeled monoclonal antibody.

The HPV18E7 protein monoclonal antibody has good specificity and high titer.

Test plate and material therefor

The detection plate can be made of detection plate materials commonly used in the field by adopting a conventional detection plate preparation method.

The immunoassay plate for detecting HPV18E7 protein of the invention comprises a test strip and a support plate for supporting the test strip, for example, a PVC polyester rubber plate and the like can be adopted; the test strip is formed by sequentially overlapping sample filtering paper, chromatographic materials, a nitrocellulose membrane and absorbent paper, and the overlapped part can be fixedly connected by adopting a conventional method, such as an adhesive tape and the like; wherein: the chromatography material is pre-coated with a colloidal gold-labeled or color-labeled HPV E7 protein monoclonal antibody or polyclonal antibody, preferably a colloidal gold-labeled HPV E7 protein monoclonal antibody, and an adsorption detection line and a quality control line are arranged on a nitrocellulose membrane;

in a preferred embodiment: the HPV E7 protein monoclonal antibody pre-coated with the colloidal gold label on the chromatographic material is pre-coated by adopting the HPV E7 protein monoclonal antibody solution with the colloidal gold label at the concentration of 0.5-1.5mg/ml, and the coating amount is 50 mu l/cm²(ii) a The preferred concentration is 0.5 or 1.5mg/ml, 50. mu.l/cm²；

Detection method and result judgment

And (3) flatly placing the detection plate, dropping the sample on the filter sample paper, and observing the chromatographic result within 3-5 min after the sample is about 120 mu l. And judging the result according to the position of the appearing stripe.

Negative: the quality control area and the detection area both have obvious colored bands and are shown as negative;

positive: a clear color band appears only in the quality control area, and no color band appears in the detection area, which is shown as positive;

and (4) invalidation: the quality control area and the detection area have no color band or no color band appears in the quality control area and a color band appears in the detection area, which indicates that the detection method is wrong or the detection plate is deteriorated or invalid, and the detection plate is required to be replaced for detection.

Method and sample

The present invention relates to methods for detecting cervical cancer in cytolytic samples of cells and/or tissue. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving the sample in a medium; detecting the level of HPV oncoprotein in the lysed sample. The sample used in the method of the present invention may be any sample comprising cells present in a cell preservation solution, as used in liquid-based cytology methods.

The invention can be used for detecting HPV oncoprotein in HPV infection-related cancers, wherein the cancers related to HPV infection comprise tumors of genitourinary systems such as cervical cancer, bladder cancer, endometrial cancer, penile cancer and the like, small cell lung cancer, melanoma, head and neck tumors and the prophase stages of the cancers.

According to the present invention, the use of molecular markers for HPV oncoproteins may support or even replace cytological and/or histological detection methods. In special cases, protein molecular markers can be used as diagnostic tools without the support of further cell-based morphological tests. Without support based on cellular information, diagnostic methods for cancer are limited to cases only at the protein molecule level, where the marker or the level of the marker should be specific for the situation to be detected. The biomarker used in the present invention is HPV oncoprotein, which is derived from a virus, and detection of HPV infection can be accomplished in a sample solution because the nature of the marker present in the tissue by the virus does not occur in uninfected human tissue.

Samples (specimens) used in the present invention include cells, tissue samples, and biopsy specimens. The term "biopsy" as used herein shall include all kinds of biopsies known to the person skilled in the art. Thus biopsies as used in the present invention may comprise e.g. resection samples of tumours, tissue samples prepared by endoscopic methods or needle biopsy of organs.

Samples for use in the present invention may include fixed or preserved cell or tissue samples. The cell or tissue sample may be, for example, preserved in a standard sample collection, storage or transport medium, such as those commercially available preservation media known to those skilled in the art (formalin, Cytyc "PreservCyt" or Tripath Imaging "Cytorich", etc.). Suitable cell preservation media may include one or more mixtures selected from alcohols, aldehydes, ketones, acids, metal ions or mercury, ethers, and the like, for preserving cellular components. The alcohol includes methanol, ethanol, (n-or iso-) propanol, (n-, iso-or tert-) butanol or highly branched or unbranched alcohols. Aldehydes include formaldehyde, acetaldehyde, glutaraldehyde, and the like. Ketones such as acetone may also be used. Acids used in standard sample media include organic acids (acetic, trichloroacetic, salicylic and picric) or inorganic acids such as chromic acid. Standard sample solutions may include metals such as silver, copper, chromium, mercury, osmium, and uranium. Salt solutions such as uranyl acetate, potassium dichromate, ammonium sulfate, and the like may be components of the preservation medium.

In addition, samples that are subjected to cell lysis immediately after they are obtained can be used in the methods disclosed herein. The sample is obtained and immediately lysed, during which morphological information is lost and protein molecular information of the sample is preserved. The sample may be transferred directly from the body of the individual into a solution containing suitable detergents and preservatives. Using appropriate reagents in the lysis medium, the molecular components of the starting material can be preserved without degradation. E.g., by using enzyme inhibitors but enzyme activity degradation is minimized. Thus, a solution of the test sample in the lysis medium may, upon dissolution, exhibit the protein molecular properties of the test sample.

According to the present invention, the sample may be dissolved in any suitable lysis medium, e.g., urea, formamide, aqueous solutions of detergents, e.g., anionic detergents (e.g., SDS, N-dodecanol creatine sodium, sodium deoxycholate, alkylaryl sulfonates, long chain (aliphatic) alcohol sulfates, olefin sulfates and sulfonates, α -olefin sulfates and sulfonates, sulfate glycerolates, sulfates ethers, thiosuccinates, alkane sulfonates, phosphate esters, alkyl isothionates, sucrose esters), cationic detergents (e.g., cetyl trimethylammonium chloride), nonionic detergents (e.g., Tween 20, Nonidet P-40, Triton X-100, NP-40, lgepal CA-630, N-octyl-glycoside) or amphoteric detergents (e.g., CHAPS, 3-dodecyl-dimethylamine-propane-1-sulfonic acid, dodecyl dimethylamine oxide) and/or hydroxide bases, e.g., sodium hydroxide or potassium hydroxide. generally any suitable liquid may be used as a lysis medium of the present invention and may be a 0.50% aqueous solution containing the subject matter as an organic solvent, e.g., sodium deoxycholate, sodium hydroxide, Tris, or a 0.40% aqueous solution containing the subject matter.

The lysis medium used to lyse the sample according to the present invention may further comprise one or more reagents that prevent degradation of components of the starting material, such as enzyme inhibitors, e.g. protease inhibitors, in embodiments of the invention the sample is lysed directly, protease inhibitors may e.g. comprise serpins, cystatins, aspartase inhibitors, metalloprotease inhibitors, acid protease inhibitors, alkaline protease inhibitors or neutral protease inhibitors in embodiments of the invention Pepstatin is used Pepstatin, L eupeptin (leupeptin), (aprotinin) and 100 μ g/ml PMSF (phenylmethylsulfonyl fluoride).

Reagent kit

The present invention also provides a kit comprising an antibody (or fragment thereof) of the invention or an assay plate of the invention, and in a preferred embodiment of the invention, the kit further comprises a container, instructions for use, a buffer, and the like.

The present invention further designs a detection kit for detecting the level of HPV oncoprotein, which comprises an antibody recognizing HPV oncoprotein, a lysis medium for solubilizing a sample, general reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The antibody is preferably an anti-HPV E7 antibody, more preferably an anti-HPV16E7 antibody. The test kit may be an in vitro diagnostic device.

The present invention further contemplates the development of a kit for diagnostic assessment of conditions associated with HPV infection from a sample solution, which can detect HPV oncoproteins present in the sample solution, wherein the cell preservation solution in which the sample is preserved can be, for example, a cell preservation solution in a liquid-based cytology procedure. The cells are lysed in a suitable lysis medium and used to develop a detection kit and an in vitro diagnostic device for the detection of HPV infection-associated tumours from lysed samples on a non-cellular assay basis.

One of the purposes of the invention is to provide a method for detecting the expression of HPV18E7 protein, and the method can be used for detecting HPV infection-related cancers, particularly cervical cancer.

The inventor of the invention prepares a monoclonal antibody MAB-F2 aiming at human papilloma virus HPV18E7 protein and studies the reactivity of the monoclonal antibody, wherein the antigen binding epitope peptide of the monoclonal antibody is between amino acid positions 44-58, and comprises an amino acid sequence VNHQH L P ARRAEPQR, and uses the monoclonal antibody MAB-F2 of the anti-HPV18E7 to carry out immunocytochemical staining on a cervical cancer cell strain C-33A not expressing HPV, a human cervical cancer cell line CaSki expressing HPV16E7 and a human cervical cancer cell strain Hela expressing HPV18E7, and the result shows that the MAB-F2 and the HPV18E7 positive cell Hela show strong staining reaction and react with the HPV16E7 positive cell CaSki and the C-33A cell not expressing HPV protein.

Furthermore, the present inventors have also found that the present inventors have carried out immunocytochemical staining of a cervical cancer cell line fixed with 4% paraformaldehyde or L BC stationary liquid for several days using the prepared anti-HPV18E7 monoclonal antibody MAB-F2, that is, the expression of HPV18E7 was not detected in a human cervical cancer cell line C-33A containing no HPV DNA, that the presence of positive tumor cells could be specifically detected by detecting HPV18E7 and MAB-F2 in tumor cells when the proportion of positive tumor cells was as low as 10% after Hela cells and C-33A cells were mixed at a ratio of (1:9), and that both the stationary liquids did not affect the judgment of positive and negative results.

That is, the method of the present invention is a method for detecting a tumor marker, characterized in that: comprising the step of detecting HPV18E7 in the sample.

In the method of the present invention, the test sample is preferably exfoliated cells collected from a specimen, or a culture of the tissue or a section of the tissue, or may be suspended cells prepared from a tissue collected from a specimen or a culture of the tissue. In addition, the cell is preferably a cervical exfoliated cell.

In the method of the present invention, the sample is preferably a patient who may suffer from cervical lesions, or a patient whose cervical lesions have already occurred.

Preferably, the HPV18E7 is an HPV18E7 protein or fragment thereof. In this case, the step of detecting HPV18E7 is preferably an immunocytochemical staining assay using HPV18E 7. The anti-HPV18E7 antibody used is preferably an anti-HPV18E7 monoclonal antibody.

The protein expressed by the oncogene from HPV18E7 used in the immunodetection of the method serves as a reliable indicator of HPV 18-associated malignancy or premalignancy. One of the most useful aspects of the invention is its use in the diagnosis of cervical cancer, squamous epithelial cells and adenocarcinomas and any epithelial abnormalities associated with oncogenic HPV18 infection, including hollow cell disease; hyperkeratosis; precancerous conditions including intraepithelial neoplasia or intraepithelial lesions; high dysplasia; and invasive or malignant cancers. In addition to cervical cancer, detection of HPV18E7 is useful for detection of genitourinary tumors such as bladder cancer, endometrial cancer, penile cancer, small cell lung cancer, melanoma, and head and neck tumors.

Another object of the present invention is to provide a detection kit by the method of the present invention. The kit may be a diagnostic kit or a research kit.

The kit of the present invention is a kit for detecting a tumor marker, and is characterized by having an anti-HPV18E7 monoclonal antibody. The kit of the present invention preferably further comprises general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, and the like. The antibody is preferably an anti-HPV18E7 antibody, more preferably an anti-HPV18E7 monoclonal antibody, and particularly preferably a mouse anti-HPV18E7 monoclonal antibody produced by hybridoma MAB-F2 or a monoclonal antibody having binding activity equivalent to that of the mouse anti-HPV18E7 monoclonal antibody.

The invention provides a method for distinguishing HPV18E7 infected tumor cells from tumor cells without HPV DNA by detecting HPV18E7 protein endogenous to the cells, which can accurately detect the positive cells when HPV18E7 positive tumor cells account for 10 percent of the total number of the cells and can also accurately detect the positive cells after the cells are clinically and widely adopted in L BC fixing solution, thereby diagnosing the cancer in the early stage of the evolution and providing a basis for timely treatment.

Furthermore, the invention also provides a detection kit formed by adopting the detection method.

The main advantages of the invention are:

(1) the antibody aiming at the HPV18E7 protein provided by the invention has high specificity and strong affinity, and can be prepared in large scale, and the quality of the monoclonal antibody is easy to control.

(2) The antibody aiming at the HPV18E7 protein can be specifically combined with the HPV18E7 protein, but not combined with the HPV16E7 protein, so that the antibody not only can be used for detecting the HPV E7 protein, but also can be used for identifying different HPV types.

(3) The antibody provided by the invention is used in the method for detecting the HPV E7 protein, the stability is good, the detection sensitivity is extremely high, and the binding affinity with the HPV18E7 protein reaches 2.67 nM.

(4) The monoclonal antibody and the detection method provided by the invention are suitable for early diagnosis of related cancers and large-scale patient screening, and can be used for monitoring relapsing patients.

The present invention is further illustrated below with reference to specific examples, which are intended to illustrate the invention only and not to limit the scope of the invention the experimental procedures, for which specific conditions are not indicated in the following examples, are generally performed according to conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: Cold Spring Harbor L laboratory Press,1989), or according to conditions set forth by the manufacturer.

Example 1

1. Preparation of human papilloma virus HPV18E7 monoclonal antibody

1.1 animal immunization

Taking female BA L B/C mice of 6-8 weeks, the immunogen is GST-HPV18E7 protein, the immunization program is shown in Table 1, the tail breaking blood sampling is carried out before each immunization, and Hi is adopted_sHPV18E7 is used as a detection antigen to coat an indirect E L ISA method for detecting the serum titer of the mice, and spleen cells are taken for fusion when the serum titer of the mice to be immunized reaches the maximum value and is not increased any more.

Table 1 mouse immunization procedure

1.2 cell fusion and culture

Collecting splenocytes from immunized mice and myeloma cells SP2/0 according to a conventional method, fusing the splenocytes with the fusion ratio of SP2/0 to 5:1, subpackaging the fused cells in a 96-hole culture plate, placing the culture plate in a constant-temperature culture box at 37 ℃ and 5% CO2 for selective culture, completely replacing the culture solution with HAT culture medium for 3 times after fusion, and detecting E L ISA when the hybridoma cells fully grow in a microscope field.

1.3 screening of Positive hybridomas

During detection, indirect E L ISA screening is adopted, antigen is His-HPV18E7 fusion protein, 2 microgram/ml plate wrapping is adopted, overnight at 4 ℃, PBST is washed and dried, 5% skimmed milk powder is added for sealing, the mixture is acted at 37 ℃ for 2 hours or overnight at 4 ℃, after PBST is washed and dried, hybridoma supernatant is added, positive (P), negative (N) and blank contrast (enzyme-labeled secondary antibody is directly added in blank), reaction is carried out for 1 hour at 37 ℃, goat anti-mouse-HRP secondary antibody (Sigma A2554) (1:10000) is added after PBST is washed and dried, reaction is carried out for 45-60 minutes at 37 ℃, and TMB coloration and 2M H are carried out after PBST is washed and dried₂SO₄And (6) terminating.

And E L ISA screening result takes the positive hole with OD value greater than 0.6 as positive hole, and rechecks the positive hole, and after two times of continuous detection, the positive hole cell is amplified, and is frozen and subcloned in time.

1.4 establishment of hybridoma cell lines

For the wells which are positive in2 times of screening, subcloning by using a limiting dilution method, taking a 96-well cell culture plate, and adding 150 mu l HAT culture solution into each well; the positive wells to be subcloned were gently pipetted into suspension, and 100. mu.l of cell suspension was pipetted into a 96-well cell plate and diluted in multiple ratios from the first well. Cell count approximately 100 cells in the well were selected and added to a well containing 6ml HAT medium, added to a feeder layer-plated cell plate at 100. mu.l/well, and the first three columns were added; then 3ml HAT culture medium is added, 100 mul/hole is added into a cell plate paved with a feeder layer, and three rows are added; then 5ml HAT medium was added, 100. mu.l/well was added to the feeder cells, followed by six columns. When the positive rate of each plate reaches 100%, stable cell strains can be obtained. Transfer to cell culture plates, expand culture and bulk cryopreservation.

And screening to obtain a positive hybridoma cell strain MAB-F2.

1.5 Mass production of monoclonal antibodies (ascites production)

Culturing hybridoma cells (MAB-F2), immunizing 8-10 week-old BA L B/C female mice with liquid paraffin, and injecting into abdominal cavity with 1x10⁶About 10 days after the abdomen of the mouse is obviously expanded to a certain degree, the ascites is extracted by a 9-gauge needle, the obtained ascites is coated by His-HPV18E7, the ascites titer is detected to be more than 1:10,000 by E L ISA, the ascites fluid is purified by Protein G affinity column chromatography after being removed of fibrin and salted out, Protein peak effluent is collected, the Protein peak effluent is dialyzed by Phosphate Buffer Solution (PBS), the concentration of antibody Protein is measured to be 0.7-1.5mg/ml by an ultraviolet spectrophotometer OD260,280, and the indirect E L ISA detection result shows that the titer of the purified monoclonal antibody is more than 1: 10000.

2. Identification of monoclonal antibodies

2.1 identification of monoclonal antibody Ig subclasses:

the purified monoclonal antibody is diluted by PBS 1:10000 and is operated according to the specification of a subtype identification kit of Sigma company, and the monoclonal antibody MAB-F2 is IgG 1.

2.2E L ISA detection anti-HPV18E7 monoclonal antibody specificity and cross reaction:

the antigen is selected from His-HPV18E7(HPV18E7 amino acid sequence SEQ ID NO.:2 MHGPKAT L QDIV L H L0 EPQNEIPVD L1L CHEQ L3L SDSEEENDEIDGVNHQH L4 PAR RAEPQRHTM L5 CMCCKCEARIE L VVESADD L RAFQQ L F L NT L SFVCPWCASQ Q) and His-HPV16E7(HPV16E7 amino acid sequence SEQ ID NO: 1 HGDTPT L1 HEYM L D L QPTD ETTD L YCYEQ L R L0L) fusion protein, 2 μ g/ml package plate, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, 37 ℃ for 2H or 4 ℃ overnight, PBST washing and drying, adding anti-I-HPV 18E L MAB-F L monoclonal antibody (1 μ g/ml), 37 ℃ for 1H reaction, 37 ℃ for 2H, 10000 ℃ for mouse (PBST washing and 60 ℃ for mouse) (Sigma A-10000A) (TMST 60-60 ℃ for TMST washing and TMST 60) for developing reaction, and adding anti-TMST 60B for mouse antibody after PBST washing and drying (PBST 37 ℃ for reaction)₂SO₄End, OD450 nm. The results are shown in FIG. 1: MAB-F2 was only able to specifically bind the HPV18E7 recombinant protein and not HPV16E 7.

2.3 antigen binding epitope analysis of Anti-HPV18E7 monoclonal antibody

Identification of monoclonal antibodiesThe antigen is selected from polypeptide or recombinant protein, wherein the polypeptide is HPV18E7-1 (amino acid sequence SEQ ID NO: 11SDSEEENDEIDGVNHQH L PARRAEPQRH), HPV18E7-2 (amino acid sequence SEQ ID NO: 12 IDGVHQH L PARRAEPQR), the recombinant protein is His-HPV18E7 and His-HPV16E7, protein antigen 4 mu g/ml coated plate, polypeptide antigen 2 mu g/ml coated plate, 4 ℃ overnight, PBST washing and drying, adding 5% skimmed milk powder for sealing, 37 ℃ for 2h or 4 ℃ overnight, PBST washing and drying, adding anti-HPV18E7 monoclonal antibody MAB-F2(1 mu g/ml), 37 ℃ for reaction for 1h, adding sheep anti-mouse-HRP (Sigma A2554) (1:10000) after PBST washing and drying, 60 min-60 min after PBST washing and M H developing color after PBST washing and drying₂SO₄The result is shown in figure 2, the binding epitope of MAB-F2 and HPV18E7 is initially deduced to be amino acids 41-55 because MAB-F2 can bind to the polypeptides HPV18E7-1 and HPV18E7-2, while the binding amino acid sequence of MAB-F2 and HPV18E7 is located between VNHQH L PARRAEPQR, i.e. between amino acids 44-58 of HPV18E7, because MAB-F2 can only specifically bind to HPV18E7 recombinant protein and not to HPV16E7 recombinant protein, and amino acids 41-43 are present in both recombinant proteins.

2.4 Anti-HPV18E7 monoclonal antibody protein binding affinity assay

Determination of the protein binding affinity constant of monoclonal antibodies reference the method established by Beatty et al (J. David Beatty, BarbaraG. Beatty and William G. Vlahos 1987. His-HPV18E7 protein was coated at a concentration of 8. mu.g/ml, 2. mu.g/ml, 0.5. mu.g/ml, 0.125. mu.g/ml, at a ratio of the four concentrations of 1:4:16:64, primary antibody MAB-F2 antibody was added, the concentration was diluted from 8. mu.g/ml by a double ratio, seven gradients were run for E L ISA detection_aff＝(n-1)/2(n[Ab′]-[Ab]) Calculation of affinity constant, [ Ab ] in the formula]And [ Ab']Respectively, the antibody concentrations that give half the absorbance when the antigen concentrations were Ag and Ag', i.e., the corresponding antibody concentrations at half the maximum OD value (i.e., OD 50%) of the curve were plotted. Wherein n is Ag/Ag'. Then comparing two by two, when n is 4, 3K values can be obtained, when n is 16, 2K values can be obtained, when n is 64, 1 can be obtainedK values, the average of 6K values was determined as the final result. The results are shown in FIG. 3: the binding affinity of MAB-F2 to His-HPV18E7 recombinant protein was calculated to be 2.67 nM.

2.5IP-WB detection of monoclonal antibody binding to tumor cell endogenous HPV18E 7:

take 5x10⁵0.5ml of cell lysate containing 1% Triton X-100,1mM EDTA, 50mM NaCl, 0.1(m/v) SDS, 1% Sodium deoxyholate, 1mM PMSF, 2 μ g/ml Aprotinin, 1 μ g/ml L euppeptin and 1 μ g/ml pepstatin is added into the HPV18E7 positive cervical cancer cell line Hela cells reported in the literature, wherein all protease inhibitors are added before use, the cells are placed on ice for lysis for 30min, and then are centrifuged at 12500rpm and 4 ℃ for 10min, and the supernatant is collected, namely the target protein extract.

Mu.g of monoclonal antibody MAB-F2 or mIgG was added to the cell lysis supernatant and incubated at 4 ℃ for two hours, followed by 15. mu.l of protein A/G beads and incubated overnight at 4 ℃. On the next day, after washing the beads twice with a cell lysate containing no protease inhibitor, 1xSDS loading buffer was added for immunoblotting detection, and the membrane was transferred for 35min with NC membrane at 100V. After blocking with 5% skimmed milk for 2h, MAB-F2 or mIgG protein diluted with 5% skimmed milk was added, the antibody was used at a concentration of 2. mu.g/ml, incubated overnight at 4 ℃, washed 4 times with TBS containing 0.1% Tween, and Anti-Mouse IgG (Fcspeccific) -peroxidase antibody (Sigma A2554) (1:1000) was added, incubated for 1h at room temperature, DAB (Wuhan Boehd SA2024) was added, and the mixture was allowed to develop, as shown in FIG. 4, at a molecular weight of 15kDa indicated by an arrow, and MAB-F2 antibody specifically recognized HPV16E7 protein endogenous to Hela cells.

2.6 immunocytochemical staining to detect expression of HPV18E7 in cervical cancer cell lines:

the immunocytochemical staining test of the cells of the cervical cancer cell line Hela expressing HPV18E7 protein, the cells of the cervical cancer cell line CaSki expressing HPV16E7 protein and the cells of the cervical cancer cell line C-33A not containing HPV DNA was carried out with the monoclonal antibody MAB-F2. The specific experimental method is as follows:

CaSki, Hela, C-33A cells were plated on poly-L-L ysine treated coverslips at 37 ℃ with 5% CO₂Culturing for 24h, and covering with glassTaking out the tablet, and fixing at room temperature for 30min by using 4% paraformaldehyde fixing solution; adding TBS lotion for washing for 5min, and spin-drying; adding TBS buffer solution containing 0.3% Triton X-100, standing at room temperature for 15min to allow cell membrane permeation; adding TBS lotion for washing for 5min, and spin-drying; addition of 1% H for inactivation of endogenous peroxidase₂O₂Treating TBS buffer solution at room temperature for 5min, spin-drying, adding TBS washing solution, washing for 5min, spin-drying, adding 10% FBS/TBS blocking solution, sealing overnight, spin-drying, adding mAbanti-HPV 18E7 MAB-F2(5 mu g/ml), incubating at 37 ℃ for 2h, adding TBS/0.1% Tween washing solution, washing for 5 times, each time for 5min, spin-drying, adding Anti-Mouse IgG (Fc specific) -peroxidase antibody (Sigma A2554) (1:1000) secondary antibody, incubating at 37 ℃ for 1h, adding TBS/0.1% Tween washing solution, washing for 5 times, spin-drying, adding DAB developing solution (Beijing China fir bridge Z L I-9017), reacting at room temperature for 10min, washing with distilled water, terminating reaction, observing under microscope, and recording.

The result is shown in FIG. 5, the examination under a microscope shows that the HPV18E7 monoclonal antibody MAB-F2 has strong immunochemical staining reaction only with the cervical cancer cell strain Hela expressing HPV18E7 protein, but has no staining reaction with CaSki cells expressing HPV16E7 and the cervical cancer cell strain C-33A not expressing HPV protein, and the result is consistent with the previous detection result of E L ISA, namely, the MAB-F2 has strong binding capacity with the HPV18E7 endogenous in cells, namely, the MAB-F2 can specifically recognize the HPV18E7 protein endogenous in the cells.

Example 2

Immunocytochemical staining detects the specificity of HPV18E7 monoclonal antibody MAB-F2 in suspension-immobilized cervical cancer cell lines:

the cervical cancer cell strain Hela cells expressing HPV18E7 protein and the cervical cancer cell strain C-33A cells without HPV DNA are fixed by different cell fixing solutions, and then the monoclonal antibody MAB-F2 is adopted to carry out immunocyte chemical staining test. The specific experimental method is as follows:

collecting C-33A cells and Hela cells, fixing with 4% paraformaldehyde or L BC stationary liquid for several days, collecting 50000 cells, wherein the cervical cancer Hela cells and negative control C-33A cells are mixed at a certain ratio, and the ratio of Hela cells to C-33A cells is 1:9,1:19,1:49,1:99,1:499, 1:999Air drying on poly-L-L ysine treated glass cover slip at room temperature to fix cells on the glass cover slip, adding TBS lotion, washing for 5min, spin-drying, adding 0.3% Triton X-100 TBS buffer, standing at room temperature for 15min to allow cell membrane permeation, adding TBS lotion, washing for 5min, spin-drying, adding 1% H for inactivating endogenous peroxidase, and drying₂O₂Treating TBS buffer solution at room temperature for 5min, spin-drying, adding TBS washing solution, washing for 5min, spin-drying, adding 10% FBS/TBS blocking solution, sealing overnight, spin-drying, adding mAbanti-HPV 16E7 MAb-F2(2 μ g/ml), incubating at 37 ℃ for 2h, adding TBS/0.1% Tween washing solution, washing for 5 times, 5min each time, spin-drying, adding Anti-Mouse IgG (Fc specific) -peroxidase antibody (Sigma A2554) (1:1000) secondary antibody, incubating at 37 ℃ for 1h, adding TBS/0.1% Tween washing solution, washing for 5 times, spin-drying for 5min each time, adding DAB developing solution (Cunninghamia Z L I-9017), reacting at room temperature for 10min, washing with distilled water to terminate the reaction, adding hematoxylin staining solution (Biyunyan day C0107), staining for 2min, soaking in tap water to remove excess dyeing solution, washing for about 10min, washing with distilled water again (several seconds), observing and recording the result under a microscope.

Detection under a microscope shows that the HPV18E7 specific monoclonal antibody MAB-F2 only has strong immunochemical staining reaction with cervical cancer cell strain Hela expressing HPV18E7 protein, but has no immunological staining reaction with cervical cancer cell strain C-33A without HPV DNA, when the number of the Hela cells and the number of the C-33A cells are 1:9, the MAB-F2 can be specifically combined with the Hela cells and is not combined with the C-33A cells, namely, when positive tumor cells account for 10 percent of the total number of the cells, the MAB-F2 can still accurately distinguish negative cells from positive cells, and in addition, the judgment of negative and positive staining in ICC detection is not influenced by adopting 4 percent paraformaldehyde or L BC stationary liquid fixed cells.

Therefore, the result shows that after the tumor cells are fixed in advance by 4% paraformaldehyde or L BC fixing solution for several days, the HPV18E7 monoclonal antibody MAB-F2 can still specifically detect HPV18E7 protein in the tumor cells, namely, after the fixing solution treats the cells for several days, the chemical staining result of immunocytes is not influenced by the chemical staining result of the immunocytes, such as reduced staining or enhanced background, in addition, when the proportion of the HPV18E7 cells is as low as 10%, the MAB-F2 antibody can still accurately detect target cells, which indicates that the detection method has good specificity and sensitivity.

Discussion:

the inventor adopts the method to prepare the HPV18E7 monoclonal antibody, screens more than 1000 strains of cells, screens 8 strains of cells with better specificity and no cross reaction with HPV16E7 protein, turns 5 strains to negative after subcloning, and still turns 3 strains to positive. Finally, only the monoclonal antibody MAB-F2 can be used for IP-WB detection and immunocytochemistry detection, and has great value in clinical detection application.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it will be appreciated that various changes or modifications may be made by those skilled in the art after reading the above teachings of the invention, and such equivalents will fall within the scope of the invention as defined in the appended claims.

Claims

1. An antibody having a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following three complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO. 4,

CDR2 shown in SEQ ID NO. 6, and

CDR3 shown in SEQ ID No. 8;

and, the light chain variable region comprises the following three complementarity determining regions CDRs:

14, CDR1' shown in SEQ ID NO,

CDR2' as shown in SEQ ID NO. 16, and

18, CDR3' as set forth in SEQ ID no.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region is as set forth in SEQ id No. 10.

3. The antibody of claim 1, wherein the light chain variable region has the amino acid sequence of SEQ id No. 20.

4. The antibody of claim 1, wherein said antibody has a heavy chain and a light chain, and wherein said heavy chain has said heavy chain variable region and said heavy chain constant region.

5. The antibody of claim 4, wherein said light chain has said light chain variable region and a light chain constant region.

6. The antibody of claim 1, wherein said antibody is an antibody specific for HPV 18.

7. The antibody of claim 1, wherein said antibody is an antibody specific for anti-HPV18E7 protein.

8. The antibody of claim 1, wherein said antibody comprises: a single chain antibody, a diabody, a monoclonal antibody, a chimeric antibody, a murine antibody, or a humanized antibody.

9. The antibody of claim 1, wherein said antibody has the following properties:

(1) the affinity with HPV18E7 protein is less than or equal to 2.67 nM; and

(2) does not bind to HPV16E7 protein;

the HPV18E7 protein is wild-type HPV18E7 protein, and the HPV16E7 protein is wild-type HPV16E7 protein.

10. The antibody of claim 1, wherein the antibody is a monoclonal antibody capable of specifically distinguishing between the HPV18E7 protein and the HPV16E7 protein.

11. The antibody of claim 1, wherein said antibody does not bind to or has low affinity for other HPV subtypes.

12. The antibody of claim 1, wherein said antibody further has the following properties:

(3) can specifically bind to protein containing HPV18E7 amino acids 44-58.

13. The antibody of claim 1, wherein said antibody is an IgG 1-type antibody.

14. A polynucleotide encoding the antibody of claim 1.

15. The polynucleotide of claim 14, wherein the polynucleotide is set forth in SEQ ID No. 3, 5, 7, 9, 13, 15, 17, or 19.

16. A vector comprising the polynucleotide of claim 14.

17. The vector of claim 16, wherein the vector comprises: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or a retroviral vector.

18. A genetically engineered host cell comprising the vector or genome of claim 16 having the polynucleotide of claim 14 integrated therein.

19. An immunoconjugate, comprising:

(a) the antibody of claim 1; and

20. A pharmaceutical composition comprising:

(i) the antibody of claim 1 or the immunoconjugate of claim 19; and

(ii) a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is in an injectable dosage form.

22. The pharmaceutical composition of claim 20, wherein the pharmaceutical composition is used for the preparation of a medicament for the treatment of a tumor.

23. Use of the antibody of claim 1 or the immunoconjugate of claim 19 for the preparation of a medicament, a reagent, a test plate or a kit;

the reagent, assay plate or kit is for:

(1) detecting HPV18E7 protein in the sample; and/or

(2) Detecting endogenous HPV18E7 protein in tumor cells; and/or

(3) Detecting tumor cells expressing HPV18E7 protein; and/or

(4) Identifying the type of HPV;

24. The use of claim 23, wherein the sample comprises HPV18E7 protein.

25. The use of claim 23, wherein the tumor comprises: a tumor of the urogenital system, a small cell lung cancer, a melanoma or a head and neck tumor, a gastric cancer, a liver cancer, a leukemia, a kidney tumor, a lung cancer, a small intestine cancer, a bone cancer, a colorectal cancer, a breast cancer, a large intestine cancer, a prostate cancer, or an adrenal tumor.

26. The use of claim 25, wherein the neoplasm of the urogenital system comprises: cervical cancer, bladder cancer, endometrial cancer, or penile cancer.

27. The use of claim 23, wherein the reagent comprises a chip, antibody-coated immunoparticles.

28. A method for detecting HPV18E7 protein in a sample for non-diagnostic purposes, the method comprising the steps of:

(1) contacting a sample with the antibody of claim 1;

(2) detecting the formation of an antigen-antibody complex, wherein the formation of the complex indicates the presence of HPV18E7 protein in the sample.

29. The method of claim 28, wherein the detection in step (2) is performed by the E L ISA method.

30. The method of claim 28, wherein in step (1), the sample is contacted with two antibodies against HPV E7 protein, at least one of which is the antibody of claim 1, and in step (2) is detected by the E L ISA method.

31. The method of claim 28, wherein the antigen-antibody complex is a first antibody-antigen-second antibody ternary complex, wherein the first antibody is the antibody of claim 1 and the binding epitope of the second antibody is different from the binding epitope of the first antibody.

32. The method of claim 31, wherein the second antibody is a monoclonal antibody produced by hybridoma cell line cgmccno. 5714.

33. The method according to claim 28, wherein in step (1), after the sample is contacted with the antibody according to claim 1, a third antibody against the first antibody is further added to the reaction system, wherein the first antibody is the antibody according to claim 1, and the formation of an antigen-first antibody-third antibody complex is detected in step (2).

34. The method of claim 31, wherein said first antibody and said second antibody are detectably labeled.

35. The method of claim 33, wherein the first antibody and the third antibody are detectably labeled.

36. The method of claim 34 or 35, wherein the detectable label is a biotin label, a colloidal gold label, a horseradish peroxidase label, a radionuclide label, or a fluorescein label.

37. An assay plate comprising a substrate and a test strip comprising the antibody of claim 1 or the immunoconjugate of claim 19.

38. A kit, comprising:

(1) a first container comprising the antibody of claim 1; and

(2) a second container comprising a secondary antibody against the antibody of claim 1; and/or

(3) A third container comprising a cell lysis reagent;

alternatively, the first and second electrodes may be,

the kit comprising the assay plate of claim 37.

39. A method of producing an antibody, the method comprising:

(a) culturing the host cell of claim 18 under conditions suitable for expression;

(b) isolating an antibody from the culture, wherein the antibody is according to claim 1.