CN105669859B - Human papilloma virus 18 type monoclonal antibody and application thereof - Google Patents
Human papilloma virus 18 type monoclonal antibody and application thereof Download PDFInfo
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Abstract
The present invention provides monoclonal antibodies specific for human papillomavirus type18 (HPV 18). The specific monoclonal antibody provided by the invention has no obvious cross reaction with other types of HPV, has the advantages of high specificity and high sensitivity when being used for detection, can accurately detect the content of HPV18 type bioactive vaccine in vaccine seeds, and can be widely applied in the clinical detection and vaccine production processes.
Description
Technical Field
The present invention relates to the fields of molecular virology and immunology, in particular, it relates to human papillomavirus type18 hybridoma cell lines and monoclonal antibodies produced thereby, as well as sequences encoding them, and their use for diagnosis, prevention and treatment.
Background
Human Papilloma Virus (HPV) is a double-stranded small DNA virus without an envelope, mainly invades human epithelial tissues and further induces various benign and malignant hyperplasia lesions. High risk HPV infections are associated with the development of multiple malignancies, and low risk HPV infections cause anogenital warts. The epidemic range of HPV infection is wide, the induced relative lethal malignant tumor and various sexually transmitted diseases have serious harmfulness to human health, and the development of safe and effective preventive or therapeutic vaccines has great significance.
The diameter of the HPV virus particle is 55-60 nm, the nucleocapsid is in 20-face symmetry and consists of pentamers of 72 main capsid proteins L1 and a minor capsid protein L2. A large number of studies have demonstrated that the HPV L1 protein is the major target protein for HPV vaccines. The HPV L1 protein expressed in various expression systems forms viroid-like particles (VLPs) with morphological structures similar to native Virus particles without the aid of the L2 protein. The recombinant HPV L1-VLP vaccine has been successfully marketed and used for preventing HPV infection and cervical cancer, condyloma acuminatum and other diseases caused by HPV infection, and fully proves that the L1-VLP has the same antigenicity and immunogenicity as wild homovirus, has a structure highly similar to that of natural HPV, retains most of neutralizing epitopes of natural virus, and can induce high-titer neutralizing antibodies.
The national food and drug administration (CFDA) points out in the technical guidelines for preclinical research of vaccines for prevention (CCK): "the products in each link and step of the production process should establish corresponding monitoring standard to ensure the quality of the products and the stability of the process in the subsequent process", and at the same time, the quality control in the semi-finished product and finished product stage at least includes identification test "
In the development process of HPV vaccines, four aspects of determination are required, namely type identification experiments, antigen content detection and in vitro potency determination. The method can adopt a double-antibody sandwich ELISA. Therefore, in vaccine research, monoclonal antibodies are important tools for vaccine antigen quality control, and especially antibodies with specificity and neutralization activity have irreplaceable roles in vaccine development.
At present, there are three main human papillomavirus vaccines which are marketed abroad, and the vaccines are respectively: the first vaccine was a tetravalent vaccine consisting of 16, 18, 6 and 11 human papillomavirus L1 recombinant proteins (Gardasil) marketed in 2006, the first bivalent vaccine consisting of 16 and 18 papillomavirus L1 recombinant proteins (Cervarix) marketed in 2007, and the first nine-valent vaccine consisting of 6, 11, 16, 18, 31, 33, 45, 52 and 58 human papillomavirus L1 recombinant proteins marketed in 12 months 2014 (Gardasil 9). The invention provides a monoclonal antibody which can aim at the specificity and the neutralization activity of HPV18 in a nine-valent range at most, and an ELISA detection kit prepared by utilizing two strains of the monoclonal antibody can be specifically used for quickly identifying and quantifying HPV 18L 1 protein, can be widely applied to clinical detection and quality detection in the process of vaccine production by current vaccine manufacturers, and has important significance on the health development and public health prevention and control of women.
Disclosure of Invention
It is a first object of the present invention to provide a monoclonal antibody capable of recognizing HPV18 and a hybridoma cell line producing the same.
The second purpose of the invention is to provide a double-antibody sandwich ELISA kit for detecting HPV 18.
The third object of the present invention is to provide a method for producing a monoclonal antibody.
The fourth purpose of the invention is to provide a preparation method of the HPV antigen detection kit.
The experimental aim of the invention is realized by the following technical scheme:
the monoclonal antibody provided by the invention is prepared by taking HPV 18L 1 pentamer protein as immunogen. Specifically, HPV 18L 1 pentamer protein is used as immunogen to immunize mice, hybridoma cell strains capable of continuously and stably secreting anti-HPV 18L 1 are obtained by cell fusion and screening through a hybridoma technology, and monoclonal antibodies are obtained by secretion of each cell strain.
In a preferred embodiment, the monoclonal antibody recognizing human papillomavirus type 18L 1 protein is produced by hybridoma cell line 1B1, having the accession number: CGMCC No. 11294; and (3) classification and naming: a hybridoma cell; the name of the depository: china general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 10 and 30 days in 2015.
In a preferred embodiment the present invention provides another monoclonal antibody to human papillomavirus type 18L 1 protein, characterized in that the monoclonal antibody is produced by hybridoma cell line 4C2 having the accession number: CGMCC No. 11297; and (3) classification and naming: a hybridoma cell; the name of the depository: china general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 10 and 30 days in 2015.
The monoclonal antibody 1B1 disclosed in the present invention, comprising at least 1 antibody heavy chain variable region and at least 1 antibody light chain variable region, and wherein the antibody light chain variable region has the CDR sequences CDRL1, CDRL2 or/and CDRL3, wherein:
CDRL1 includes SEQ ID NO: 1;
CDRL2 includes SEQ ID NO: 2;
CDRL3 includes SEQ ID NO: 3.
the antibody heavy chain variable region has a CDRH1, CDRH2 or/and CDRH3, wherein:
CDRH1 includes SEQ ID NO: 4;
CDRH2 includes SEQ ID NO: 5;
CDRH3 includes SEQ ID NO: 6.
in a preferred embodiment, the monoclonal antibody or antigen-binding fragment of the human papillomavirus type 18L 1 protein comprises at least 1 antibody light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 7 and at least 1 antibody heavy chain variable region comprising SEQ id no: 8.
in a preferred embodiment the invention provides an isolated nucleic acid encoding at least one light chain variable region of an antibody of the invention SEQ ID NO: 7 and heavy chain variable region SEQ ID NO: 8.
in a preferred embodiment the invention provides an expression vector for a nucleic acid which is operably linked to control sequences recognized by a host cell when the vector is transfected into the host cell.
In a preferred embodiment the invention provides a host cell for the expression of the vector.
In a preferred embodiment the present invention provides another monoclonal antibody to human papillomavirus type 18L 1 protein, characterized in that the monoclonal antibody is produced by hybridoma cell line 4C2 having the accession number: CGMCC No. 11297; and (3) classification and naming: a hybridoma cell; the name of the depository: china general microbiological culture Collection center; the address of the depository: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation date is as follows: 10 and 30 days in 2015.
A monoclonal antibody produced by hybridoma cell line 4C2, comprising at least 1 antibody heavy chain variable region and at least 1 antibody light chain variable region, and wherein the antibody light chain variable region has CDR sequences CDRL1, CDRL2 or/and CDRL3, wherein:
CDRL1 includes SEQ ID NO: 9;
CDRL2 includes SEQ ID NO: 10;
CDRL3 includes SEQ ID NO: 11.
the heavy chain variable region of a monoclonal antibody produced by hybridoma cell line 4C2 has a CDRH1, CDRH2 or/and CDRH3, wherein:
CDRH1 includes SEQ ID NO: 12;
CDRH2 includes SEQ ID NO: 13;
CDRH3 includes SEQ ID NO: 14.
in a preferred embodiment the present invention provides a monoclonal antibody or antigen-binding fragment recognizing human papillomavirus type 18L 1 protein characterized by a heavy chain variable region comprising at least 1 antibody light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and at least 1 antibody heavy chain variable region comprises SEQ ID NO: 16.
in a preferred embodiment the invention provides an isolated nucleic acid encoding at least one light chain variable region of an antibody of the invention SEQ ID NO: 15 and heavy chain variable region SEQ ID NO: 16.
in a preferred embodiment the invention provides an expression vector for a nucleic acid which is operably linked to control sequences recognized by a host cell when the vector is transfected into the host cell.
In a preferred embodiment the invention provides a host cell comprising an expression vector.
In another aspect, the invention provides a kit for detecting HPV 18L 1, comprising a monoclonal antibody or antigen-binding fragment disclosed herein.
The kit for detecting HPV 18L 1 disclosed by the invention further comprises detectable markers: a radioisotope, a fluorescent substance, a luminescent substance, a colored substance and/or an enzyme.
In another aspect, the present invention provides a composition for specifically detecting human papillomavirus type 18L 1 protein, the composition comprising the monoclonal antibody or antigen binding fragment of the present invention.
The monoclonal antibody of human papillomavirus type18 can be prepared by the following method:
1) the method comprises the steps of using a pentamer of human papilloma virus 18 type L1 protein as an immunogen, immunizing a Balb/c mouse after purification, collecting blood, detecting the serum titer by an indirect ELISA method, selecting the Balb/c mouse with high serum titer for boosting immunity, and preparing immune spleen cells from the mouse body;
2) preparing a myeloma cell (SP2/0) suspension, injecting a Balb/c mouse, preparing myeloma cells after the mouse grows solid tumors, fusing the myeloma cells with the immune spleen cells in the step 1), preparing thin and fragile hybridomas, detecting and screening hybridoma cell strains with high titer, and carrying out clone expansion culture;
3) carrying out expanded culture on the established hybridoma cells in the step 2), collecting supernatant, injecting the supernatant into a mouse through an abdominal cavity, collecting ascites of the mouse, purifying, and carrying out specificity identification and neutralization activity detection to obtain the monoclonal antibody of the human papilloma virus type 18.
The preparation method of the human papillomavirus double-antibody sandwich ELISA antigen detection kit comprises the following steps:
the monoclonal antibody 4C2 is used as a capture antibody to coat an enzyme label plate, the monoclonal antibody 1B1 is used as a detection antibody after being labeled by horseradish peroxidase, and the recombinant human papilloma virus-18 type VLP protein is used as a standard product to prepare a standard curve control. The kit also comprises a concentrated washing solution, a sample diluent dry powder, an enzyme-labeled antibody diluent dry powder, a substrate solution A, a substrate solution B, a stop solution and the like. The concentrated washing solution, the substrate solution A, the substrate solution B and the stop solution comprise the following components in percentage by weight:
concentrating the washing solution: 8.18g of NaCl, 3.58g of Na2HP04 & 12H2O 3.58, 0.2g of KCL and KH2PO40.25g, and adding double distilled water to 1000 mL;
substrate solution A: 200mg of 3, 3 ', 5', 5-tetramethyl diphenyldiamine, 100mL of absolute ethyl alcohol and 1000mL of double distilled water;
substrate solution B: Na2HPO414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, double distilled water is added to 1000mL, and the pH value is adjusted to 5.0-5.4;
stopping liquid: 1mol/L sulfuric acid solution.
In another aspect, the invention provides an application of the kit in preparing a detection composition for preventing or detecting human papilloma virus 18 type L1 infection.
In another aspect, the invention provides a use of a composition in preparing a detection composition for preventing or detecting human papillomavirus type 18L 1 infection.
The monoclonal antibody obtained by the invention has good specificity, experiments show that the monoclonal antibody has no cross reaction with other eight HPV types, and indirect ELISA shows that the monoclonal antibody has higher titer simultaneously in the currently marketed vaccine products, nine-valence is the product of the most types, so the monoclonal antibody obtained by the invention can be used for specific detection of HPV18 protein in bivalent, trivalent or tetravalent currently latest marketed nine-valence vaccines and vaccines or compositions comprising HPV18 types.
The invention adopts a double-antibody sandwich method, utilizes two monoclonal antibodies to carry out specific detection and quantification on HPV18, can detect pentamer of HPV18 and can detect HPV18 VLP, thereby, the invention provides a kit for specific detection and quantification of HPV18, the detection limit is as follows: 0.1ug/ml, linear range: 10-0.1ug/ml, has the advantages of high specificity and high sensitivity when used for detection, can accurately detect the level of HPV18 with biological activity in a sample, and can be widely applied to clinical detection and quality detection in the vaccine production process of vaccine manufacturers.
Drawings
FIG. 1: SDS-PAGE analysis detection results show that the purity of each purified subtype monoclonal antibody reaches more than 95 percent.
FIG. 2 is a drawing: the ELISA double antibody sandwich assay detects HPV 18L 1VLP standard curve with the ordinate being log of od450. and the abscissa being log of HPV 18L 1VLP concentration.
Detailed description of the invention
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents of which the sources are not indicated in the examples are all reagents which are conventional in the art or are commercially available.
Example 1 establishment of hybridoma cell lines
1. Animal immunization
1) Antigen preparation: a pentameric protein of L1 protein of HPV18 type was prepared using an E.coli expression system and observed by a transmission electron microscope (100,000 times), and it was revealed that a pentamer having a diameter of about 10nm was visible in the visual field, and the pentamer protein was diluted to 10. mu.g/ml.
2) Basic immunity: the antigen and Freund's complete adjuvant were mixed in equal volume and emulsified thoroughly, and injected subcutaneously in several portions, 10. mu.g per Balb/c mouse.
3) And (3) boosting immunity: the boosting immunity adopts emulsion of antigen and Freund's incomplete adjuvant. 3 days before cell fusion, a physiological saline solution containing 15ug of antigen was intraperitoneally injected.
2. Preparation of hybridoma cells
Spleen cells from mice were harvested conventionally and fused with SP2/0 cells at a 10:1 ratio with 500g/L PEG 4000. Selectively culturing with HAT culture solution, taking supernatant after 10-15 days of fusion, and screening hybridoma cell strains by adopting an indirect ELISA method. The obtained positive clones were subcloned by limiting dilution method. The indirect ELISA method was performed as follows: plates were packed with 200 ng/well of HPV18 VLP, immune mouse serum 1:2000 as a positive control, medium supernatant without clonal growth and normal mouse serum as negative controls, and 1:2000 HRP-goat anti-mouse IgG 100. mu.l was added to each well, and finally OD450nm was determined. If the OD450 value is more than 2 times larger than the negative control, the positive clone can be primarily determined.
3. Establishment of hybridoma cell lines
And (3) continuously cloning the positive clones obtained in the step (2), continuously culturing and passaging the hybridoma cell line in a DMEM culture medium containing 10% fetal calf serum, and after culturing for 10 generations, enabling the hybridoma cell line to still grow well and be passaged stably to obtain the hybridoma cell line capable of stably secreting the monoclonal antibody.
EXAMPLE 2 preparation of monoclonal antibody against HPV18
Selecting adult BALB/c mouse, intraperitoneally inoculating norphytane, 0.5ml of hybridoma cells after 7-10 days, 1 × 10 days6-2×106And (4) respectively. After 5 days, when the abdomen is obviously enlarged and the hand touches the abdomen, the skin is tense, and the ascites can be collected by using a 16-gauge needle.
The ascites fluid was centrifuged (13000 r/min for 30 minutes), the cell fraction and other precipitates were removed, and the supernatant was collected. Purifying with Protein G-Sepharose CL-4B, wherein the upper column liquid is 20mM PBS buffer solution, and the column chromatography eluent is: the monoclonal antibody against HPV18 was obtained at pH2.7 in 20mM glycine buffer. SDS-PAGE detection shows that the purity of the purified monoclonal antibody reaches more than 95 percent, and the specific figure is shown in figure 1.
Example 3: antibody subtype identification
The IgG subclasses of the antibodies produced by the above hybridoma cells were identified using antibodies against various IgG subclasses of mice by an indirect ELISA method.
The results showed that 10 clones, 3a2 clone was IgG2a,3D2 and 4H1 were IgG3, and the rest were IgG1, and the results are shown in table 1.
Table 1 identification of antibody subtypes by ELISA (OD 450 values.)
*: all values are averages of duplicate wells
Example 4: ELISA detection of antibody reactivity with HPV18 VLP
Whether the purified antibody can be conveniently and rapidly detected by the HPV18 is verified by indirect ELISA, and the affinity of the antibody can be preliminarily judged by using the antibodies with different concentrations.
HPV18 VLPs were coated in 96-well plates at 200 ng/well, then added to each well at antibody use concentrations of 1ug/ml, 0.2ug/ml, and 0.04ug/ml, and the binding strength of each monoclonal purified antibody to HPV18 VLPs was tested by ELISA. The results show that 10 antibodies, all at the concentrations used in this experiment, bind to HPV18 VLP, and that the signal intensity is greater than 1 at concentrations greater than 0.05ug/ml, the results are shown in Table 2.
TABLE 2 ELISA test antibody binding strength to HPV18 VLP
*: all values are averages of duplicate wells
Example 5: identification of the specificity of antibodies
The HPV-16L 1VLP, HPV-18L 1VLP and HPV-58L 1VLP are subjected to alkali denaturation and heat denaturation to break secondary or tertiary structure and preserve primary structure. Then reacting with the monoclonal antibody, and detecting the monoclonal antibody by adopting indirect ELISA. Through the experiment, the identification condition of the antibody on three VLPs of HPV-16L 1VLP, HPV-18L 1VLP and HPV-58L 1VLP can be identified, and whether the antibody is a conformation type identification antibody can also be identified. If the OD450 value of the protein after denaturation and the reaction with the monoclonal antibody is obviously reduced, the monoclonal antibody is proved to be a conformation type recognition antibody.
The experimental steps are as follows: VLP denatured protein treatment: 0.2M sodium carbonate, 0.01M DTT, pH 10.6 was incubated at room temperature for 30 minutes and then boiled for 5 minutes. Coating: the VLP protein was diluted to 2. mu.g/ml. Add 100. mu.l/well to 96-well microplate and coat overnight at 4 ℃. And (3) sealing: and (5) drying the coated ELISA plate. Add 300. mu.l/well blocking solution (2% BSA) to the plate and allow to stand at room temperature for 1-2 hours. Sample dilution: and (3) respectively diluting the antibody samples to 0.3 mu g/ml by using sample diluent, uniformly mixing, adding 100 mu l/hole into an enzyme label plate, and standing at room temperature for 1 h. Adding a secondary antibody: and (3) drying the enzyme label plate after sealing, adding 100 mul/hole of HRP-labeled goat-anti-mouse secondary antibody into the enzyme label plate at the concentration of 1:4000, and standing for 1 h at room temperature. Color development: washing the plate for 5 times with 300 mul per hole, and drying by spin. The bottom was wiped with toilet paper. The developing solution was added at 100. mu.l/well, and the mixture was developed in the dark at room temperature for 10 minutes. And (4) terminating: the reaction was terminated by adding 100. mu.l/well of a stop solution. Reading: the microplate was placed in the microplate reader and OD450 read and data analyzed.
The results of the specific detection of the antibody are shown in Table 3. The results showed that, except clone 7H8, which slightly cross-reacted with HPV16 and HPV58 VLPs, all were clones specific for HPV18 VLPs. These clones, except for 7H8, recognized non-denatured HPV18 VLPs with a value greater than 1, as a significant positive result, while recognized denatured HPV18 VLPs with a value less than 0.1, as a negative result. All the monoclonal antibodies are conformation recognition monoclonal antibodies. And 7H8 has certain cross reaction with denatured HPV18 VLP, which shows that the monoclonal antibody of the strain can also recognize linear epitopes.
Table 3 specific ELISA detection of antibodies (od450. values.)
All values are the average of duplicate wells L: undenatured protein D: denatured protein
Example 6: detection of neutralizing Activity of antibodies
The neutralizing activity of each antibody strain was tested by a pseudovirus-cell neutralization model.
Each subtype antibody was diluted to 200ug/ml with PBS, then the antibody was diluted in 4-fold gradient, 50ul of each concentration antibody was incubated with 50ul of HPV18 pseudovirus at the appropriate concentration in a 96-well plate at 4 ℃ for one hour, and the mixture of pseudovirus and PBS was used as a control. The mixtures were then added to 96-well plates previously plated with 293FT cells and incubated in a cell incubator for 72 hours. Thereafter, the cells were collected, and fluorescence was detected by flow cytometry, and the fluorescence inhibition rate was calculated as (1-experimental group/control group) × 100%. The fluorescence inhibition rate of more than 50% and 90% are respectively used as the neutralization titer of the monoclonal antibody to HPV18 pseudovirus. The results in Table 4 show that all clones have a certain inhibitory response, and that the monoclonal antibodies of the present invention have neutralizing activity.
TABLE 4 inhibitory Rate Titers of the respective subtype antibodies
Example 7: in vitro ELISA Competition assays for neutralizing sera with antibodies
The competitive ELISA test was used to determine whether the prepared antibody was competitive with the World Health Organization (WHO) Anti-HPV 18 neutralizing antibody international standard (1 st WHO Reference Reagent 2007, Anti-human papillomavir type18 serum, NIBSC 10/140). The WHO standard was a human-derived polyclonal antiserum purchased from National Institute for Biological Standards and controls, NIBSC. Through the experiment, the correlation between the epitope of each monoclonal antibody and the epitope of the international standard can be analyzed.
First, HPV18 VLP was diluted to 100ng/ml and 100ul/well added to 96-well plates and incubated overnight at 4 ℃. After washing the 96-well plate, the plate was blocked with a 2% BSA blocking solution at room temperature for 2 hours, and then 100ul of 0.5 IU/ml of an international standard and 100ul of 0.3ug/ml of an HRP-labeled antibody were added to the 96-well plate, and 100ul of PBS was used as a control for the international standard. After incubation for 1.5 hours at room temperature, the plates were washed, the reaction was stopped, developed and read with an microplate reader OD450. The inhibition rate was calculated as (1-experimental OD./control OD.) x 100%.
The results of the in vitro ELISA competition experiments for neutralizing serum with the antibody are shown in table 5, the international standard has obvious strong inhibition (inhibition rate > 30%) on the cloned antibodies 1B1, 3a4, 4C12, 4C2, 4D6 and 4H5, and the international standard has weak inhibition (inhibition rate < 20%) on the cloned antibody 4H 1. The results show that the clones have certain correlation with the epitope of the international standard.
TABLE 5 neutralizing sera of antibodies in vitro ELISA Competition experiments
Example 8: affinity identification of antibody 1B1 and antibody 4C2
Kinetics of antigen-antibody binding and dissociation were analyzed using a BIACORE3000 (GE) biosensor, and the binding constant K of monoclonal antibodies 1B1 and 4C2 to HPV18 VLPs was calculatedaDissociation constant KdAnd affinity KDTo reflect the strength of the binding of the antibody to HPV18 VLPs.
HPV18 VLPs were diluted to 40. mu.g/ml with pH5.5 acetate-sodium acetate buffer and HPV18 VLPs were coupled onto the chip according to the manufacturer's instructions (BIACORE 3000 biosensor, GE USA) to set the coupling level at 4000 RU.
The antibodies were diluted to 0.3125, 0.625, 1.25, 2.5, 5, 10 and 20 nmol/L with PBS buffer, respectively. In the detection, the antibody is injected for 60 seconds, then combined for 60 seconds, dissociated for 500 seconds, and finally the chip is regenerated by 10 mM acetic acid-sodium acetate buffer solution with pH 5.0. Kinetic analysis of antigen-antibody binding was performed according to the manufacturer's instructions and the data were analyzed with Biacore3000 Evaluation software. The results of the affinity detection of the antibodies 1B1 and 4C2 with HPV18 VLPs are shown in table 6, and the results indicate that the antibodies 1B1 and 4C2 are specific, bind only to HPV18 VLPs, and do not bind to other 8 HPV VLPs.
TABLE 6 affinity identification of antibody 1B1 and antibody 4C2
a N/A: not Applicable, indicates no binding.
Example 9: determination of variable region sequences for clone 1B1 and clone 4C2
The obtained 1B1 and 4C2 monoclonal cells were separately extracted for mRNA, reverse transcribed into cDNA, subjected to high fidelity PCR amplification using variable region universal primers, the PCR product fragments were inserted into T-vectors for DNA sequencing, and the obtained sequences were translated into the amino acid sequence of proteins. The obtained sequences were aligned and showed no identical sequence, indicating that the obtained sequences were specific.
The sequence is shown in a sequence table.
Using the sequences identified above, various genetically engineered antibodies, such as chimeric antibodies, humanized antibodies, single chain antibodies, diabodies, and the like, can be prepared by known antibody engineering techniques, while retaining the biological properties of the monoclonal antibody from which it is derived.
Example 10: assembly of HPV18 detection kit
The monoclonal antibody 4C2 is used as a capture antibody to coat an enzyme label plate, the monoclonal antibody 1B1 is used as a detection antibody after being labeled by horseradish peroxidase, and the recombinant human papilloma virus-18 type VLP protein is used as a standard product to prepare a standard curve control.
The coated antibody 4℃ 2 is diluted to 10 mu g/mL by carbonate buffer solution with pH 9.60.05 mol/L, 100 mu L is added to each hole of an enzyme label plate, the enzyme label plate is coated overnight at 4 ℃, coating solution is poured out, PBST is used for washing for 2 times and dried by beating, then 200 mu L of 3% Bovine Serum Albumin (BSA) is added to each hole, the hole is put into a 37 ℃ incubator for sealing for 2 hours, PBS is used for washing for 1 time, 10% sucrose aqueous solution is added, room temperature protection is carried out for 1 hour, the dried hole is beaten dry, and aluminum foil bags are vacuumized and stored at 4 ℃.
The antibody 1B1 was labeled with horseradish peroxidase to give 1B1-HRP and stored. Adding 100 mu L/hole of VLP sample into the ELISA plate, incubating for 1.5 hours at 37 ℃, washing the plate, adding 100 mu L/hole of 1B1-HRP (0.5 ug/ml), incubating for 1 hour at 37 ℃, washing, drying, adding color developing agent for color development, incubating for 10min at 37 ℃, adding 50 mu L/hole of stop solution, and reading by an ELISA reader at the wavelength of 450 nm.
The kit also comprises a concentrated washing solution, a sample diluent dry powder, an enzyme-labeled antibody diluent dry powder, a substrate solution A, a substrate solution B, a stop solution and the like. The concentrated washing solution, the substrate solution A, the substrate solution B and the stop solution comprise the following components in percentage by weight:
concentrating the washing solution: 8.18g of NaCl, 3.58g of Na2HP04 & 12H2O 3.58, 0.2g of KCL and KH2PO40.25g, and adding double distilled water to 1000 mL;
substrate solution A: 200mg of 3, 3 ', 5', 5-tetramethyl diphenyldiamine, 100mL of absolute ethyl alcohol and 1000mL of double distilled water;
substrate solution B: Na2HPO414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea 6.4mL, double distilled water is added to 1000mL, and the pH value is adjusted to 5.0-5.4;
stopping liquid: 1mol/L sulfuric acid solution.
Example 11: linear and repetitive detection of HPV18 detection kit
An ELISA double-antibody sandwich method is applied, the clone 1B1 and the clone 4C2 antibody are used as a pairing experiment, the clone 4C2 is determined to be used as a coating antibody, the clone 1B1 marked by HRP is used as a detection antibody, an ELISA detection method is determined, and the detection linear range of the kit is shown in table 7. The figure is shown in FIG. 2, the linear range is 0.1ug/ml to 10 ug/ml.
TABLE 7 kit Linear Range detection
The antigen is diluted according to 10ug/ml, 3ug/ml, 1ug/ml, 0.3ug/ml and 0.1ug/ml in sequence, and batch-to-batch repeatability experiments are carried out according to the ELASA experimental operation steps. The test was repeated 10 times for each sample in both the batch and the batch, and the standard deviation and the coefficient of variation were calculated. The average standard deviation is 0.234, and the average coefficient of variation is 5.558%, which shows that the double-antibody sandwich ELASA antigen detection method established by the invention has good repeatability.
Example 12: HPV18 detection kit specificity test
Sample detection of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 VLPs, VLP denatured protein treatment: 0.2M sodium carbonate, 0.01M DTT, pH 10.6 was incubated at room temperature for 30 minutes and then boiled for 5 minutes. The 96-well plate detects 100ul of 30ug/ml VLP per well. The results are shown in table 8 and show that the kit detects non-denatured HPV18 VLPs with good signal, does not recognize denatured HPV18 VLPs, and has no crossover with other types of HPV VLPs.
The results show that the kit can be used for specifically detecting the HPV18 VLP with biological activity, and therefore, the kit can be widely applied to development of HPV18 vaccines.
TABLE 8 evaluation results of HPV18 VLP specificity by ELISA method (OD 450)
All values are mean values of multiple wells
Example 13: procedure for determination of HPV18 detection kit
1. Preparing samples
1) Preparing a washing liquid: taking out the concentrated washing solution, adding distilled water, and metering to 1L, and mixing well for use;
2) preparing a sample diluent: diluting the sample diluted dry powder to 50ml by using the washing liquid obtained in the step 1, and fully and uniformly mixing for later use;
3) preparing an enzyme-labeled antibody diluent: diluting the enzyme diluted powder to 50ml by using the washing liquid obtained in the step 1, and fully and uniformly mixing for later use;
4) and diluting the standard substance by using the prepared sample diluent, and setting a dilution gradient. The concentration is 30ug/ml, 10ug/ml, 3ug/ml, 1ug/ml, 0.3ug/ml, 0.1ug/ml, and 0.03ug/ml, respectively, for use.
5) Diluting the enzyme-labeled antibody with prepared enzyme diluent, and taking a proper amount of 1300 times of the enzyme-labeled antibody for dilution for later use.
2. Measurement procedure
1) Sample adding: adding the prepared standard substance and the sample to be detected into a coating plate at 100ul/well, and simultaneously arranging a plurality of holes and a negative control hole. (negative control blank is sample diluent) cover the sealing plate membrane and put into 37 ℃ incubator to incubate for 45 min.
2) Adding an enzyme-labeled antibody: taking out the enzyme label plate from the 37 ℃ incubator, discarding the liquid in the hole, adding washing liquid of 300ul/well, and washing the plate for 5 times. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. Adding 100ul/well of the prepared enzyme-labeled antibody solution, covering a cover plate membrane, and incubating in a 37 ℃ incubator for 45 min.
3) Color development: taking out the enzyme label plate from the 37 ℃ incubator, discarding the liquid in the hole, adding washing liquid of 300ul/well, and washing the plate for 10 times. And finally, after the liquid in the holes is discarded for the last time, patting the paper on toilet paper to be dry. Taking the required volumes of chromogenic substrate solution A and chromogenic substrate solution B according to the ratio of 1: 1, adding 100ul/well into an enzyme label plate, and developing for 5 minutes at room temperature in a dark place.
4) And (3) terminating the reaction: the desired volume of stop solution was added to the microplate at 50 ul/well.
5) Reading value: the microplate was placed in the microplate reader and OD450nm read.
6) And (4) judging a result: the Cutoff value =2.1 × NC mean, and the result of sample detection higher than this is judged to be positive.
Example 14: application of HPV18 detection kit
The standard HPV18 VLP protein is taken for gradient dilution, and the diluted L110 mu g/ml pentamer antigens of the samples HPV18, HPV18 and HPV58 are detected by adopting the double-antibody sandwich ELISA antigen detection kit prepared by the invention, so that the primary application effect of the kit is evaluated. The result shows that the ELISA method established by the invention can detect the HPV18 pentamer protein and has specificity.
TABLE 9 evaluation result of HPV18 pentamer specificity by ELISA method (OD 450)
SEQUENCE LISTING
<110> Beijing Kangle guard Biotechnology Ltd
<120> human papilloma virus 18 type monoclonal antibody and application thereof
<130>2015
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Claims (15)
1. A monoclonal antibody for recognizing human papilloma virus 18 type L1 protein is characterized in that the monoclonal antibody is generated by hybridoma cell strain 1B1 with CGMCC collection number of 11294;
the antibody comprises at least 1 antibody heavy chain variable region and at least 1 antibody light chain variable region, and wherein the antibody light chain variable region has the CDR sequences CDRL1, CDRL2 or/and CDRL3, wherein:
CDRL1 includes SEQ ID NO: 1;
CDRL2 includes SEQ ID NO: 2;
CDRL3 includes SEQ ID NO: 3;
the antibody heavy chain variable region has a CDRH1, CDRH2 or/and CDRH3, wherein:
CDRH1 includes SEQ ID NO: 4;
CDRH2 includes SEQ ID NO: 5;
CDRH3 includes SEQ ID NO: 6.
2. a monoclonal antibody or antigen-binding fragment recognizing human papillomavirus type 18L 1 protein, wherein the heavy chain variable region comprising at least 1 antibody comprises the amino acid sequence set forth in SEQ ID NO: 7 and at least 1 antibody heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 8.
3. an isolated nucleic acid encoding at least one light chain variable region of an antibody of the invention seq id NO: 7 and at least one heavy chain variable region of SEQ ID NO: 8.
4. an expression vector comprising the nucleic acid of claim 3, wherein said nucleic acid is operably linked to control sequences recognized by a host cell when the vector is transfected into the host cell.
5. A host cell comprising the expression vector of claim 4.
6. A monoclonal antibody for recognizing human papilloma virus 18 type L1 protein is characterized in that the monoclonal antibody is generated by a hybridoma cell strain 4C2, and the CGMCC (China general microbiological culture collection center) is 11297;
the antibody comprises at least 1 antibody heavy chain variable region and at least 1 antibody light chain variable region, and wherein the antibody light chain variable region has the CDR sequences CDRL1, CDRL2 or/and CDRL3, wherein:
CDRL1 includes SEQ ID NO: 9;
CDRL2 includes SEQ ID NO: 10;
CDRL3 includes SEQ ID NO: 11;
the antibody heavy chain variable region has a CDRH1, CDRH2 or/and CDRH3, wherein:
CDRH1 includes SEQ ID NO: 12;
CDRH2 includes SEQ ID NO: 13;
CDRH3 includes SEQ ID NO: 14.
7. a monoclonal antibody or antigen-binding fragment recognizing human papillomavirus type 18L 1 protein, wherein the heavy chain variable region comprising at least 1 antibody comprises the amino acid sequence set forth in SEQ ID NO: 15 and at least 1 antibody heavy chain variable region comprises SEQ id no: 16.
8. an isolated nucleic acid encoding at least one light chain variable region of an antibody of the invention seq id NO: 15 and at least one heavy chain variable region SEQ ID NO: 16.
9. an expression vector comprising the nucleic acid of claim 8, wherein said nucleic acid is operably linked to control sequences recognized by a host cell when the vector is transfected into the host cell.
10. A host cell comprising the expression vector of claim 9.
11. A kit for detecting HPV 18L 1, comprising the monoclonal antibody or antigen-binding fragment of any one of claims 1-2 and/or any one of claims 6-7.
12. The kit for detecting HPV 18L 1 of claim 11, further comprising a detectable label: a radioisotope, a luminescent material, a colored material, and/or an enzyme.
13. A composition for specifically detecting human papillomavirus type 18L 1 protein, characterized in that the composition comprises the monoclonal antibody or antigen binding fragment according to any one of claims 1-2 and/or any one of claims 6-7.
14. Use of a kit according to claim 11 or 12 for the preparation of a composition for the prevention or detection of human papillomavirus type18 infection.
15. Use of a composition according to claim 13 for the preparation of a composition for the prevention or detection of human papillomavirus type18 infection.
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| CN109021097A (en) * | 2017-06-08 | 2018-12-18 | 艾托金生物医药（苏州）有限公司 | A kind of monoclonal antibody and its application identifying HPV18 and/or HPV45 |
| CN108276491B (en) * | 2018-02-05 | 2021-01-12 | 南京薇熙生物医药科技有限公司 | Monoclonal antibody capable of specifically recognizing HPV18L1 protein and application thereof |
| CN112125972B (en) * | 2019-06-25 | 2022-07-29 | 神州细胞工程有限公司 | Monoclonal antibody for resisting HPV 16L 1 protein and detection method using same |
| CN111662378B (en) * | 2019-12-30 | 2022-05-31 | 中生方政生物技术股份有限公司 | Monoclonal neutralizing antibody of HPV18L1 and application thereof |
| CN111205364B (en) * | 2020-02-26 | 2022-05-17 | 中生方政生物技术股份有限公司 | Monoclonal neutralizing antibody against HPV31L1 and application thereof |
| CN113512109B (en) * | 2021-03-09 | 2022-03-25 | 北京康乐卫士生物技术股份有限公司 | Monoclonal neutralizing antibody against human papillomavirus 31 and application thereof |
| CN116199772B (en) * | 2022-11-16 | 2023-10-17 | 怡道生物科技(苏州)有限公司 | HPV31 type capsid protein L1 monoclonal antibody, preparation method and application |
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