Anti-MGMT protein monoclonal antibody as well as preparation method and application thereof

Abstract

The invention relates to a monoclonal antibody capable of recognizing a human MGMT antigen, a preparation method of the monoclonal antibody and application of the monoclonal antibody in immunodetection. According to the technical scheme, the full-length protein is selected as the antigen peptide, codon optimization is carried out, a gene segment suitable for being expressed in escherichia coli BL21 is formed, and the finally obtained recombinant protein contains an MGMT protein and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain secreting the anti-MGMT protein monoclonal antibody and the anti-MGMT protein monoclonal antibody secreted by the cell strain are obtained. The antibody obtained by the scheme has high specificity and sensitivity, can specifically recognize cells expressing MGMT protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, in particular to an anti-MGMT protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] 6-oxo-methylguanine-DNA methyltransferase (06-methylguanine-DNA methyltransferase MGMT) belongs to DNA repair proteins, which is widely present in prokaryotes and human cells. It is located at 10g26 of the chromosome, and its coding gene is about 170 kd in full length, which is composed of 5 exons and 4 introns. 6-oxo-methylguanine-DNA methyltransferase is a key enzyme for repairing alkylating agents in the body, which can quickly repair the DNA alkyl damage caused by alkylating agent drugs. After the administration of alkylating agent antitumor drugs, one or more active alkyl groups in the molecule will be transferred to the base guanine of the DNA molecule of the tumor cell, so that O6-guanine is methylated to form O6-MG. The presence of O6-MG hinders the replication process of tumor cell DNA, or affects the expression of genes, or causes DNA molecule to break, and ultimately achieves the purpose of killing tumor cells. And the DNA repair protein MGMT can transfer the methyl group from O6-MG to the 145th cysteine residue of itself, so that the cysteine residue becomes a new methyl acceptor, and the guanine on the DNA chain of the tumor cell is restored to O6-guanine, restoring the proliferation ability of the tumor cell. Therefore, the presence of MGMT is considered to be the main reason for the drug resistance of tumor cells to alkylating agent antitumor drugs.

[0003] Under normal circumstances, MGMT in cells can protect normal cell tissues from damage by alkylating agents, while in tumor tissues it shows resistance to alkylating agent drugs. The intracellular MGMT directly reflects the degree of DNA damage it can tolerate, and it is generally believed that tumor cells with negative or low-level expression of MGMT are effective for alkylating agent drugs, and vice versa, which means drug resistance. It is mainly used for the research of various malignant tumors such as colon cancer, glioma, breast cancer, etc. SUMMARY

[0004] The present application provides an anti-MGMT protein monoclonal antibody, the heavy chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1; the light chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0005] Further, the monoclonal antibody is a murine monoclonal antibody, and the clone number is MX114.

[0006] The inventors also provide a method for preparing the anti-MGMT protein monoclonal antibody, wherein the antigen for immunizing the mouse is a recombinant protein expressed by E. coli.

[0007] Further, the recombinant protein comprises the MGMT protein and a HIS protein tag.

[0008] Further, the MGMT protein is the amino acid sequence shown in SEQ ID NO. 3.

[0009] Further, the plasmid vector selected in the recombinant process is pET30a.

[0010] The inventors also provide an immunodetection reagent for MGMT protein, which comprises the above anti-MGMT protein monoclonal antibody as an effective component.

[0011] Further, the immunodetection comprises immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

[0012] Different from the prior art, the present application has the following beneficial technical effects: the above technical solution selects the MGMT full-length protein as the antigen peptide, performs codon optimization, becomes a gene fragment suitable for expression in E. coli BL21 (DE3), and finally obtains a recombinant protein comprising the MGMT protein and a histidine protein tag. The recombinant protein is used for immunizing a mouse, and through cell fusion, screening and subcloning, a mouse hybridoma cell strain MX114 secreting the anti-MGMT protein monoclonal antibody is obtained, and the anti-MGMT protein monoclonal antibody secreted by the cell strain. The antibody obtained by the present application has high specificity and sensitivity, can specifically recognize cells expressing the MGMT protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 FIG. 4 is a comparison chart of immunohistochemical staining results of colon adenocarcinoma, wherein the left is the antibody of the present application, and the right is a commercially available antibody.

[0014] Figure 2 FIG. 5 is a comparison chart of immunohistochemical staining results of normal hepatocyte surface epithelium (the left is the antibody of the present application, and the right is a commercially available antibody). DETAILED DESCRIPTION

[0015] In order to explain the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific embodiments listed and with the aid of the drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot be used to limit the protection scope of the present application.

[0016] The term "embodiment" is mentioned in this document means that the specific features, structures or properties described in connection with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit the independence or association between other embodiments. In principle, in this application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0017] Unless otherwise defined, the meanings of the technical terms used in this document are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms in this document is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0018] In the description of the present application, the phrase "and / or" is a description of the logical relationship between the objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this document generally represents that the associated objects before and after are a "or" logical relationship.

[0019] In this application, such as "first" and "second" language is only used to distinguish one entity or operation from another entity or operation, and does not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0020] In this application, without more limitation, the "includes", "contains", "has" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent in such process, method or product.

[0021] As the same understanding as in the "Guidelines for Examination", in this application, "greater than", "less than", "exceed" and other expressions are understood as not including the number; "above", "below", "within" and other expressions are understood as including the number. In addition, the meaning of "multiple" in the description of the embodiments of the present application is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0022] Example 1 Preparation of recombinant MGMT protein fragment

[0023] I. Gene optimization and synthesis

[0024] MGMT according to the protein sequence of accession number NP_002403.3 in NCBI database, the full-length protein was selected and directly optimized into a gene fragment suitable for expression in E. coli BL21 (DE3). BamH I and Xho I enzyme sites were added at the 5' and 3' ends of the gene during PCR.

[0025] The PCR product was recovered after agarose gel electrophoresis, and the recovered fusion protein gene and the plasmid vector pET30a for expression were subjected to BamH I and Xho I enzyme digestion, respectively, and then recovered by electrophoresis again, and ligated with T4 DNA ligase. The ligation product was transformed into competent E. coli cells BL21 (DE3), and the colonies on the plate were inoculated and subjected to bacterial liquid PCR identification. The colonies with positive PCR results were selected for sequencing analysis, and the colonies with completely correct sequences were used.

[0026] Different antigens may be selected for immunization to prepare antibodies with different binding properties. The molecule contains multiple variants caused by variable splicing, which ultimately leads to different recognition abilities and patterns of different antibodies to cells expressing the antigen. According to the published sequence, the MGMT molecule was analyzed, and based on its structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, a suitable region for soluble expression and good immunogenicity was selected for recombinant expression. The full-length MGMT protein was selected for codon optimization, and the molecular weight was about 22 kDa. The MGMT protein was obtained by sequence optimization design using prokaryotic expression gene sequence. The recombinant immunogen is composed of MGMT protein fragments with antigenicity and protein tags for purification of recombinant proteins. The protein tag is HIS.

[0027] II. Protein expression and purification

[0028] The single colony culture was transferred to 100 mL of LB medium at a ratio of 1:100, kanamycin was added at a final concentration of 10 μg / mL, and the culture was incubated at 37°C with shaking until the OD600 was 0.6-0.8. Then 0.5 mmol / L IPTG was added, and the culture was incubated at 16°C with shaking overnight. The bacteria were collected and sonicated. The recombinant protein has a histidine tag, and nickel column was used for affinity purification of the protein. Elution was performed with 500 mmol / L imidazole, and SDS PAGE separation was performed for detection. The concentration of the recombinant MGMT protein with a histidine tag was 0.5 mg / mL, which could be used for animal immunization and antibody screening and identification.

[0029] Example 2 Establishment of hybridoma cell lines

[0030] I. Immunization

[0031] The recombinant protein in Example 1 was emulsified with Freund's complete adjuvant (Sigma, F5881), and 4-6 week-old SPF female ICR mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were immunized by subcutaneous injection of 6 points per mouse at a dose of 20 μg per mouse. The mice were boosted every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (Sigma, F5506) at a dose of 20 μg per mouse. Seven days after the third booster, the anti-antigen polyclonal antibody titer in the serum of the mice was detected by indirect ELISA (wavelength 450 nm), and the mouse with the highest titer was given a boost by tail vein injection of the antigen mixed with normal saline at a dose of 20 μg per mouse.

[0032] II. Cell fusion

[0033] The spleen cell suspension of the immune qualified mice was prepared aseptically and mixed with mouse myeloma cells sp2 / 0 (ATCC Number CRL-8287) at a ratio of 5:1. After centrifugation at 1500 rpm for 5 min, the supernatant was discarded and the centrifuge tube was placed in a 37°C water bath. Within 1 min, 1 mL of PEG1500 (Roche) was slowly added and the cells were stirred. After standing in warm water for 1 min, 10 mL of serum-free IMDM (Sigma) was added, mixed well, and centrifuged at 1000 rpm for 5 min. After discarding the supernatant, 10 mL of serum (PAA) was carefully blown up, and 5 mL of 10x HAT (Sigma) mixed thymocytes were added, mixed well. Then 25 mL of semi-solid medium containing 2.1% nitrocellulose (Sigma) was added and mixed well, and then evenly poured into 20 cell culture dishes. The cell culture dishes were placed in a wet box and cultured in a 37°C 5% CO2 incubator.

[0034] III. Cloning and ELISA screening of positive hybridoma cells

[0035] Seven days after fusion, the size and density of the cloned cell clusters were moderate. Under a dissecting microscope, round, solid, and large cloned clusters were sucked into 96-well culture plates prepared with medium in advance and cultured in a 37°C 5% CO2 incubator. After 3 days, the cells occupied about 2 / 3 of the bottom area, and 100 μL of supernatant was taken for ELISA screening with the immunogen and synthetic polypeptide, respectively. The positive clones were completely changed, 200 μL of complete medium containing feeder cells and 1% HT (Sigma) was added. Two days later, the second ELISA screening was performed, and the positive clones were transferred to 24-well plates prepared with medium (containing feeder cells and HT) in advance. Five days later, 100 μL of supernatant was taken for the third ELISA screening, and the positive clones were successively transferred to 6-well plates and cell culture bottles for expansion and cryopreservation.

[0036] Example 3 Preparation of monoclonal antibody by ascites induction

[0037] I. Ascites preparation

[0038] The logarithmic growth phase cells were washed with serum-free medium and suspended, and counted to be about 5 x 10 5 , 1 mL. The suspended cells were injected intraperitoneally into mice previously sensitized with paraffin oil. Ascites was collected after 7 days. The removed ascites was centrifuged at 4000 rpm at 4°C for 10 min. The middle ascites was carefully sucked out and collected in a centrifuge tube, which was stored at 4°C or -20°C.

[0039] II. Purification of monoclonal antibody

[0040] The antibody was purified from ascites by HiTrap rProtein A FF (GE) affinity chromatography according to the instructions. The purity was identified by SDS-PAGE gel, and the concentration was determined by the Bradford method. The purified antibody was stored at -20°C.

[0041] Example 4 Identification of characteristics of monoclonal antibody

[0042] I. Subclass identification

[0043] Sheep anti-mouse IgG (Beijing Zhongshanjinqiao Biotechnology Co., Ltd.) was diluted to 0.5 µg / mL with 100 mM PBS (pH 7.4), 100 µL was added to each well, and incubated at 4°C overnight. The liquid was poured out, and washed 3 times with PBS containing 0.05% Tween (PBS-T), 200 µL of blocking solution (PBS containing 2% BSA and 3% sucrose) was added to each well, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 0.1 mL of HRP-labeled sheep anti-mouse (κ, λ) antibody diluted 1:1000 or HRP-labeled sheep anti-mouse (IgM, IgG1, IgG2a, IgG2b, IgG3, IgA) antibody (Southern Biotech) diluted 1:2000 was added to the appropriate wells, respectively, and incubated at 37°C for 1 h. The liquid was poured out, and washed 3 times with PBS-T. 50 µL of citric acid buffer (pH 4.0) containing 0.15% ABTS (Southern Biotech) and 0.03% H2O2 was added to each well for color development, and the OD value at 405 nm was determined within 10-20 min.

[0044] The results show that the monoclonal antibody of the application is an IgG2a type mouse monoclonal antibody.

[0045] II. Determination of affinity constant

[0046] The MGMT recombinant protein prepared in Example 3 was coated at a concentration of 2 μg / mL, 100 μL / well, and incubated overnight at 4°C, and washed with PBS-T for 3 times. 200 μL blocking solution was added to each well, and incubated at 37°C for 2 hours, and washed with PBS-T for 3 times. The monoclonal antibody purified in Example 3 was diluted in a gradient of 2 times from 1:200, and 1 well was left blank as a control, and incubated at 37°C for 1 hour, and washed with PBS-T for 3 times. HRP-labeled goat anti-mouse secondary antibody was diluted at 1:20,000, and 100 μL was added to each well, and incubated at 37°C for 1 hour, and washed with PBS-T for 3 times. 100 μL of citric acid-phosphoric acid buffer containing 0.1% TMB (Sigma) and 0.03% H2O2 was added to each well, and color development was performed for 10 minutes, and the reaction was terminated by adding 50 μL of 0.5 M sulfuric acid solution. The absorbance value at a wavelength of 450 nm was measured by an enzyme-labeled instrument. The OD value corresponding to the dilution multiple of the antibody was plotted, and the dilution multiple A corresponding to half of the maximum binding OD value was found, and the affinity constant of the antibody was calculated to be 1.92 x 10 9 .

[0047] Affinity constant

[0048] III. Specificity of monoclonal antibody reaction and application effect

[0049] The MGMT recombinant protein prepared in Example 1 was used to detect the recognition specificity of the monoclonal antibody of the present application by immunoblotting, and 12% polyacrylamide gel electrophoresis was performed. The gel protein band was transferred to a PVDF membrane (Millipore) in a Bio-Rad electrotransfer system according to a conventional method. The membrane was placed in a TBS-T blocking solution containing 5% skim milk at 4°C overnight. The MGMT protein monoclonal antibody purified in Example 3 (diluted at 1:1000) was added and incubated at 4°C overnight. After washing the membrane with TBS-T, goat anti-mouse secondary antibody (Beijing Zhongshen Jinqiao Biotechnology Co., Ltd.) diluted at 1:5000 was added, and incubated at room temperature for 1 hour. The membrane was washed again with TBST, ECL ultra-sensitive color developing liquid (Beijing Puli Lei Gene Technology Co., Ltd.) was added, and ChemiDocMP multi-color fluorescence imaging system (Bio-Rad) was used to collect the chemiluminescence image data.

[0050] Example 5 Sequence determination of antibody

[0051] Fresh hybridoma cells were taken, and the supernatant was taken for antigen binding property verification, and it was confirmed that the cell strain used for cloning could indeed secrete the required antibody. After the results were confirmed, 10 6The above hybridoma cells. Trizol method was used to extract total RNA from hybridoma cells. 9 μL of total RNA, 2.5 μL oligo (dT) 12-18 primer (10 mM), and 5 μL dNTPs were mixed uniformly, and then incubated at 70°C for 5 min and placed on ice for 5 min, or denatured according to the reverse transcriptase used. Then 5 μL of RT buffer (5X), 2.5 μL of DTT (0.1 M), and 1 μL of reverse transcriptase were added, and the reaction was carried out at 42°C for 1 hour. The reaction was terminated by incubation at 70°C for 15 min, and the obtained cDNA was stored at -20°C. The obtained first-strand cDNA was subjected to PCR amplification. In a 50 μL reaction system, 25 pmol of primers were added, and the sequences of the primers for amplifying the heavy chain variable region and the light chain variable region were designed and synthesized according to the mouse monoclonal antibody primer sequence design and synthesis in the book "Recombinant Antibodies" (Science Press, published in 2005) edited by Shen Bei-fen.

[0052] The remaining dNTPs and buffers were added according to the conventional method, and finally 1 μL of cDNA template and 1 U of hot-start Taq DNA polymerase were added. The PCR amplification program was set as 94°C for 40 s, 52°C for 40 s, and 72°C for 40 s, and 20 to 25 cycles were performed, and finally 72°C was extended for 3 min. The product can be stored at 4°C or directly electrophoresed. 20 μL of the PCR product was subjected to electrophoresis analysis, separated on a 1.5% agarose gel, and the length of the light chain (κ light chain) was between 320-340 bp and the length of the heavy chain was between 340-370 bp. When specific products in this region were present, the gel was cut and recovered for cloning into a T vector or an expression vector for sequencing.

[0053] Example 6. Immunohistochemical tissue chip staining and identification

[0054] I. Chip preparation process

[0055] Each sample was first subjected to HE section staining to determine the tumor site. A full-automatic tissue chip instrument from 3DHISTECH was used to make the tissue chip. The prepared tissue chip wax block was placed into a wax block making mold, and then placed into a 68°C oven for 10 min to make the tissue chip and the wax of the receptor wax block into one body. Then the mold was gently taken out of the oven, and the semi-melted paraffin was cooled at room temperature for about 30 min. Then the tissue chip wax block was taken out of the mold, and then sliced or stored in a 4°C refrigerator for standby use. After trimming, continuous sections were made with a thickness of 3 μm. The continuous sections were floated in 40% alcohol to naturally expand, and then the separated sections were transferred to warm water at 50°C for 30 s. The sections were attached to glass slides treated with polylysine, and the prepared tissue chip was placed in a 68°C oven for 2 h for sectioning. Then the chip was taken out, cooled at room temperature, and stored in a -4°C refrigerator.

[0056] II. IHC staining and analysis

[0057] Routine xylene dewaxing 3 times, 6 minutes each time, hydration in 100%, 100%, 95%, 85% gradient ethanol, 3 minutes each time, and finally tap water rinsing. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS 3x3 minutes. 3% H2O2 was added and incubated for 10 minutes, and then rinsed with PBS 3x3 minutes. The sections were spun dry, and the appropriate dilution of the primary antibody was added (the first dilution was designed according to the concentration of the antibody) and incubated at room temperature (25°C) for 1 hour, rinsed with PBS 3x3 minutes, the secondary antibody was added and incubated at room temperature for 15-30 minutes, rinsed with PBS 3x3 minutes, the PBS was spun off, and fresh DAB developing solution was added and developed for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and then PBS was added and blue-stained for 30 seconds. Sequential dehydration was performed according to the alcohol gradient of 85% (3 minutes)-95% (3 minutes)-100% (3 minutes)-100% (3 minutes), and finally xylene clearing was performed for 3 minutes, and neutral resin mounting was performed.

[0058] The results of immunohistochemical staining were divided into positive and negative. Positive expression must be at the site of cell- and tissue-specific antigens to be considered positive. In the case of clear tissue staining distribution and accurate cell localization, the staining results were further divided according to the difference in staining intensity, as follows:

[0059] 1. The sample is weakly positive, marked as "+";

[0060] 2. The sample is moderately positive, marked as "++";

[0061] 3. The sample is highly positive, marked as "+++".

[0062] 4. The sample is negative, marked as "-".

[0063] III. Data statistics

[0064] 1. Tumor tissue chip detection results:

[0065] The antibody MGMT (MX114) of the application and the commercially available antibody MGMT (MT3.1) were used to synchronously detect 28 cases of colon adenocarcinoma and compare the detection results.

[0066] The immunohistochemical results of MGMT were statistically analyzed. The entire test process adopted a double-blind design, and the statistical results are as follows:

[0067]

[0068] The results show that the anti-MGMT protein monoclonal antibody provided by the application has accurate staining positioning, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive rate is equivalent to that of the commercial antibody, but the positive intensity of one case is higher than that of the commercial antibody. It is indicated that the antibody has higher sensitivity and effectively avoids false negative results.

[0069] Figure 1 A comparison chart of immunohistochemical staining results of colon adenocarcinoma (left: antibody of the application, right: commercial antibody).

[0070] 2. Normal tissue chip test results:

[0071] The normal tissue chip includes 30 normal tissue samples, which are mainly selected from fresh and timely fixed surgical specimens; each tissue includes 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectum, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (infant), skin, bone marrow, peripheral nerve, lung, mesothelial cell.

[0072] The antibody (MX114) and the commercial antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, which indicates that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody.

[0073] Figure 2 A comparison chart of immunohistochemical staining results of normal liver cell surface epithelium (left: antibody of the application, right: commercial antibody).

[0074] Finally, it should be noted that although the above-mentioned embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc., are all included in the patent protection scope of the present application.

Claims

1. A monoclonal antibody against MGMT protein, characterized in that, The heavy chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1; the light chain amino acid sequence of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody is a mouse monoclonal antibody with clone number MX114.

3. A method for preparing a monoclonal antibody against MGMT protein, characterized in that, The antigen used to immunize mice is a recombinant protein, which is recombinantly expressed by Escherichia coli.

4. The preparation method according to claim 3, characterized in that, The recombinant protein contains MGMT protein and HIS protein tag.

5. The preparation method according to claim 3, characterized in that, The MGMT protein has the amino acid sequence shown in SEQ ID NO.

3.

6. The preparation method according to claim 3, characterized in that, The plasmid vector used in the recombination process was pET30a.

7. An MGMT protein immunoassay reagent, characterized in that, The immunoassay reagent contains the anti-MGMT protein monoclonal antibody as its active ingredient, as described in claim 1.

8. The immunoassay reagent according to claim 7, characterized in that, The immunoassays include immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assay (ELISA).

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