Anti-p504s protein monoclonal antibody, its preparation method and application

Abstract

The present application relates to a kind of monoclonal antibody that can identify human p504s antigen, its preparation method and its use in immunodetection.The present application provides a kind of anti-p504s protein rabbit monoclonal antibody, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.1;The amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO.2.The antibody has high specificity, sensitivity, and can specifically recognize the cell expressing p504s protein, is suitable for immunological detection, especially immunohistochemical detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical engineering, and particularly relates to an anti-p504s protein monoclonal antibody and a preparation method and application thereof. BACKGROUND

[0002] P504s (alpha-methylacyl-CoA-racemase, AMACR) is a high-sensitivity and high-specificity molecular marker for prostate cancer, a transmembrane protein composed of 382 amino acids, and its encoding gene is located at human chromosome 5p13.2-q11.1. P504s exists in multiple subtypes and mainly exists in mitochondria and peroxisomes. It is highly expressed in prostate cancer and little or undetectable in normal prostate.

[0003] Many studies have shown that the sensitivity of p504s for prostate cancer diagnosis reaches 82%-100%, and the specificity reaches 79%-100%, and high-sensitivity and high-specificity p504s is an important histological biomarker for diagnosing prostate cancer. P504s can also be positively expressed in many tumors, such as breast cancer, pancreatic islet tumor, renal papillary tumor, colorectal cancer, ovarian tumor, breast tumor, bladder tumor, lung tumor, lymphoma and melanoma, and the expression of colorectal cancer and prostate cancer is the highest. P504S for the diagnosis of prostate cancer is generally combined with PSA, HMW, CK, P63, etc., which can greatly improve the early detection rate of prostate cancer. SUMMARY

[0004] The present application provides an anti-p504s protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 2.

[0005] SEQ ID NO. 1:

[0006] METGLRWLLLVAVLKGVQCQSLEESGGRLVTPGTPLTLTCTASGFSLSSYYMTWVRQAPGKGLEYIGYIYAGSGSTYYASWAKGRFTISKTSTTVDLKMTSLTAADTATYFCARGYVSGMDIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

[0007] SEQ ID NO. 2:

[0008] MDTRAPTQLLGLLLLWLPGARCAYDMTQTPASVEVAVGGAVTIKCQASQSINNYLAWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQQGISGRDVENAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTW

[0009] EVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC

[0010] Further, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 4.

[0011] SEQ ID NO. 3:

[0012]

[0013] SEQ ID NO. 4:

[0014] ATGGACACGAGGGCCCCCACTCAGCTGCTGGGGCTCCTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCTATGATATGACCCAGACTCCAGCCTCTGTGGAGGTAGCTGTGGGAGGCGCAGTCACCATCAAGTGCCAGGCCAGTCAGAGCATTAATAACTACTTAGCCTGGTATCAGCAGAAACCAGGGCAGCCTCCCAAGCTCCTGATCTACAAGGCATCCACTCTGGCATCTGGGGTCTCATCGCGGTTCAAAGGCAGTGGATCTGGGACAGAGTTCACTCTCACCATCAGCGACCTGGAGTGTGCCGATGCTGCCACTTACTACTGTCAACAGGGTATTAGTGGTAGGGATGTTGAGAATGCTTTCGGCGGAGGGACCGAGGTGGTGGTCAAAGGTGATCCAGTTGCACCTACTGTCCTCATCTTCCCACCAGCTGCTGATCAGGTGGCAACTGGAACAGTCACCATCGTGTGTGTGGCGAATAAATACTTTCCCGATGTCACCGTCACCTGGGAGGTGGATGGCACCACCCAAACAACTGGCATCGAGAACAGTAAAACACCGCAGAATTCTGCAGATTGTACCTACAACCTCAGCAGCACTCTGACACTGACCAGCACACAGTACAACAGCCACAAAGAGTACACCTGCAAGGTGACCCAGGGCACGACCTCAGTCGTCCAGAGCTTCAATAGGGGTGACTGTTAG

[0015] Further, the monoclonal antibody specifically recognizes the p504s protein.

[0016] Further, the monoclonal antibody is a rabbit monoclonal antibody.

[0017] Further, the rabbit monoclonal antibody has clone number 2G5.

[0018] The present application also provides a method for preparing the anti-p504s protein monoclonal antibody, wherein the antigen for immunizing the rabbit is a recombinant protein expressed by E. coli.

[0019] Further, the recombinant protein comprises a GST protein tag, a p504s protein fragment and a HIS protein tag.

[0020] Further, the p504s protein fragment is a fragment of 200-382 sites, which is an amino acid sequence shown in SEQ ID NO. 5.

[0021] SEQ ID NO. 5:

[0022] KLSLWEAPRGQNMLDGGAPFYTTYRTADGEFMAVGAIEPQFYELLIKGLGLKSDELPNQMSMDDWPEMKKKFADVFAEKTKAEWCQIFDGTDACVTPVLTFEEVVHHDHNKERGSFITSEEQDVSPRPAPLLLNTPAIPSFKRDPFIGEHTEEILEEFGFSREEIYQLNSDKIIESNKVKASL

[0023] Further, the plasmid vector selected in the recombinant process is pGEX-4T-AB1 or pET-28a-sumo.

[0024] The present application also provides a p504s protein immunological detection reagent, wherein the immunological detection reagent contains the above-mentioned anti-p504s protein monoclonal antibody as an effective component.

[0025] Compared with the prior art, the present application has the beneficial technical effects that the above-mentioned technical solution provides an anti-p504s protein rabbit monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is an amino acid sequence shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is an amino acid sequence shown in SEQ ID NO. 2. The antibody has high specificity and sensitivity, can specifically recognize the cells expressing the p504s protein, and is suitable for immunological detection, especially immunohistochemical detection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a gel map of the p504s protein purification result of the pET-28a-sumo-p504s vector recombinant p504s protein.

[0027] Figure 2 It is a gel map of the p504s protein purification result of the pGEX-4T-AB1-p504s vector recombinant p504s protein.

[0028] Figure 3 Comparison of immunohistochemical staining results for prostate cancer Figure 1 (Left: rabbit monoclonal p504s of the present application, right: commercially available p504s).

[0029] Figure 4 Comparison of immunohistochemical staining results for prostate cancer Figure 2 (Left: rabbit monoclonal p504s of the present application, right: commercially available p504s). DETAILED DESCRIPTION

[0030] In order to make the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects achieved by the present application clear, the following will be described in detail in combination with the specific embodiments listed and with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical schemes of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0031] In this text, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiments can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any way to form a corresponding implementable technical scheme.

[0032] Unless otherwise defined, the meanings of the technical terms used herein 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 text is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0033] 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 text generally represents that the associated objects before and after are a "or" logical relationship.

[0034] In the present application, terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0035] In the present application, the terms "comprise", "contain", "have" or other similar forms are intended to cover non-exclusive inclusions, and these terms do not exclude the presence of additional elements in the process, method or product comprising the stated elements, so that the process, method or product comprising a series of elements can not only include those defined elements, but also include other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0036] In the present application, the terms "greater than", "less than", "exceed" and the like are understood as not including the number itself; the terms "above", "below", "within" and the like are understood as including the number itself. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is two or more (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 specified.

[0037] Preparation of recombinant p504s protein fragment

[0038] I. Gene optimization and synthesis

[0039] According to the protein sequence with accession number NM_014324.6 in the NCBI database, the protein fragment at positions 200-382 of p504s was selected and directly optimized into a gene fragment suitable for expression in E. coli Rosetta (DE3), and then cloned into the pGEX-4T-AB1 vector or the pET-28a-sumo vector.

[0040] The expression vector was used to transform E. coli Rosetta (DE3) competent cells, 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.

[0041] Selecting different antigens for immunization can produce antibodies with different binding characteristics. The presence of multiple variants caused by variable splicing in the molecule ultimately leads to different recognition abilities and patterns of different antibodies to cells expressing antigens. According to the published sequence, the p504s molecule was analyzed based on structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids, and secondary structure, and a region suitable for soluble expression and good immunogenicity was selected for recombinant expression. The gene sequence at positions 200-382 of p504s was selected for codon optimization, and the molecular weight was about 40 kDa. The p504s protein was obtained by sequence optimization design using prokaryotic expression gene sequence. The recombinant immunogen is composed of the p504s protein fragment with antigenicity and the protein tag for purification of the recombinant protein, and the protein tag is GST and HIS.

[0042] II. Protein expression and purification

[0043] The single colony culture of the overnight bacteria was transferred to 100 mL LB medium at a ratio of 1:100, and antibiotics were added at a final concentration of 100 μg / mL (ampicillin for pGEX-4T-AB1 and kanamycin for pET-28a-sumo), and the bacteria were cultured at 37°C for 4 h with shaking. The bacteria were then collected and disrupted by ultrasonication. The recombinant protein with a histidine tag was subjected to affinity purification using a nickel column. Elution was performed using 150 mmol / L imidazole, and the purified protein was detected by SDS PAGE.

[0044] Figure 1 The gel image of the purified recombinant p504s protein from the pET-28a-sumo-p504s vector is shown in Figure 2. The concentration of the purified protein in the supernatant was 1 mg / mL, and the purity reached 80%, which met the requirements for animal immunization and antibody screening and identification.

[0045] Figure 2 The gel image of the purified recombinant p504s protein from the pGEX-4T-AB1-p504s vector is shown in Figure 3. The concentration of the purified protein in the supernatant was 4 mg / mL, and the purity reached 85%, which met the requirements for animal immunization and antibody screening and identification.

[0046] Example 2. Single B cell sorting for producing rabbit anti-human p504s monoclonal antibodies

[0047] I. Immunization and ELISA detection

[0048] The recombinant p504s protein in Example 1 was emulsified with Freund's complete adjuvant, and six rabbits were selected for immunization at a dose of 400 μg / rabbit. Booster immunization was performed once on days 7, 21, 42, 61, and 131, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 200 μg / rabbit. Shock immunization was performed on day 137, and the antigen was mixed with physiological saline at a dose of 400 μg / rabbit. Serum ELISA titer detection was performed after the third, fourth, and fifth immunizations, and the serum of four rabbits after the fourth immunization was subjected to IHC detection. According to the results (i.e., the positive control photograph corresponding to the antibody was used for detection, and immunohistochemical staining was observed at the corresponding detection site on the positive control photograph), one rabbit (number E15677) was selected for subsequent monoclonal antibody screening.

[0049] II. Spleen cell separation and B lymphocyte sorting

[0050] Single antibody preparation was performed on selected rabbits. Three days after the impact immunization, the spleen was taken out, the rabbit spleen was placed in RMPI basic medium containing 100 U / ml penicillin and 100 ug / ml streptomycin, cut into pieces with a scalpel, and then transferred to a 100 μm cell screen for grinding. The obtained cell suspension was filtered to remove large cell clumps and tissue envelopes, and the supernatant was removed after centrifugation at 400 g for 5 minutes to retain the spleen cell mass. The spleen cell mass was resuspended with a hypotonic solution and the red blood cells were lysed, and then centrifuged at 400 g for 5 minutes, and the spleen cells were retained. The spleen cells were resuspended with RMPI basic medium containing 100 U / ml penicillin and 100 μg / ml streptomycin, centrifuged at 400 g for 5 minutes, and the obtained spleen cells were resuspended with complete medium (RMPI basic medium containing 10% fetal bovine serum, 100 U / ml penicillin and 100 μg / ml streptomycin) for standby.

[0051] The specific steps of B lymphocyte sorting are described in Chinese Patent 201910125091.4 "Method for efficiently separating single antigen-specific B lymphocytes from spleen cells".

[0052] About 2000 single B cell clones were sorted and cultured, and positive clones specifically recognizing the recombinant p504s protein in Example 1 were screened by ELISA. According to the ELISA data, 22 supernatants from high to low were selected for IHC verification.

[0053] III. Detection of positive clone culture supernatant of B cells

[0054] Single B cell culture supernatant was verified by IHC using a multi-tumor tissue chip and a normal tissue chip containing p504s positive and negative. Among them, the IHC results of the culture supernatant of 13 clones were good, and the LEM supernatant was prepared for IHC verification to determine the clone (2G5) with excellent sensitivity and specificity.

[0055] The sensitivity and specificity screening criteria are: using the positive control photo corresponding to the antibody for detection, the corresponding detection site in the positive control photo can observe immunohistochemical staining, and no immunohistochemical staining is observed at the non-detection site. Compared with the control antibody, the staining intensity reaches or is even higher than that of the control antibody.

[0056] IV. Cloning of rabbit monoclonal antibody gene (2G5) and construction of rabbit monoclonal antibody expression plasmid

[0057] After cell collection and lysis of the positive clone, RNA was extracted and reverse transcribed into cDNA. Using PCR method, the naturally paired rabbit monoclonal antibody light and heavy chain variable region genes were amplified from the cDNA corresponding to the positive clone, and sequenced. The above naturally paired rabbit monoclonal antibody light and heavy chain variable region gene sequences were used to construct rabbit monoclonal antibody expression vector plasmids.

[0058] Example 2 was completed by Wuhan Aibotek Biotechnology Co., Ltd. on behalf of Fujian Maynew Biotechnology Development Co., Ltd., in which immunohistochemical screening was performed by Fujian Maynew Biotechnology Development Co., Ltd.

[0059] Example 3 p504s rabbit monoclonal antibody expression

[0060] I. Plasmid amplification and extraction

[0061] Take out a tube (100 μl) of competent bacteria (DH5a), insert into ice, ice bath for 5-10 min; add 5 μl of plasmid, gently shake and place on ice for 30 min; gently shake and place in a 42°C water bath for 90 s for heat shock, then quickly return to ice, stand for 5 min; add 800 μl of LB medium (note: without antibiotics) to the above-mentioned mixture in the clean bench, gently mix, and fix on a shaker at 37°C for 1 h; take 50-100 μl of the above-mentioned transformation mixture in the clean bench, add to the labeled solid LB plate containing Amp, and evenly spread with a glass spreader (sterilized); first stand in a 37°C incubator for 30 min, allowing the surface bacteria to completely penetrate the medium, then invert and place in a 37°C incubator for overnight culture. Use a gun head to pick a single colony into 4 ml of LB medium (containing 2 uL of 200 mg / ml Amp), and culture at 37°C on a shaker for 16 h at 220 rpm.

[0062] Add 1 ml of bacteria to 100 ml of LB medium (containing 50 μl of 200 mg / ml Amp), and culture at 37°C on a shaker for 16 h; use SanPrep endotoxin-free plasmid DNA extraction kit (Shenguo), and extract plasmid according to the instructions.

[0063] II. Transfection

[0064] Adjust the concentration of 293F cells to 2.5-3 x 10 6 viable cells / ml with Expi293FTM Expression Medium, and culture overnight.

[0065] 1) Count with a hemocytometer, and the viable cell concentration is about 4.5-5.5 x 10 6 viable cells / ml, and the number of viable cells needs to meet the requirements of the expression system;

[0066] 2) Dilute the cell concentration to 3 x 10 6 viable cells / ml with Expi293FTM Expression Medium;

[0067] 3) Add plasmid DNA to Opti-MEM™ I Reduced Serum Medium, gently flick, invert to mix;

[0068] 4) Invert ExpiFectamine TM 293 Reagent 4-5 times, mix ExpiFectamine TM 293 Reagent and Opti-MEM™ I Reduced Serum Medium, gently flick and invert 2-3 times, let stand at room temperature for 5 min;

[0069] 5) Mix solutions from steps 3) and 4), gently flick and invert 2-3 times, mix well;

[0070] 6) Let solution from step 5) stand at room temperature for 10-20 min;

[0071] 7) Slowly pipette mixture into cell culture, gently shake flask;

[0072] 8) Incubate at 37°C, 8% CO2, 5-7 days;

[0073] 9) After 18-22 hours, add ExpiFectamine TM 293 Transfection Enhancer 1 and ExpiFectamine TM 293 Transfection Enhancer 2 (Note: mix ahead of time), gently shake to mix, continue incubation.

[0074] III. Purification of Monoclonal Antibodies

[0075] Purify antibodies from supernatant using HiTrap rp50 Protein A FF affinity chromatography according to manufacturer's instructions. Determine purity by SDS-PAGE gel and concentration by Bradford method. Store purified antibodies at -20°C.

[0076] Example 4. Immunohistochemistry Tissue Chip Staining and Evaluation

[0077] I. Chip Preparation Procedure

[0078] HE staining was performed on each sample to determine the tumor site. The tumor target site was circled and prepared for punching. When making the blank recipient wax block, a plastic frame was placed on the mold, and melted wax (melting point at 55-58°C) was poured into the mold. After cooling to room temperature, the mold was placed in a -20°C refrigerator for 6 min, and the wax block was removed from the mold. On the tissue sample machine, a 1 mm diameter sample needle was selected to punch a hole in the recipient wax block, with a hole depth of 3-4 mm. Another 1 mm diameter punching needle was used to punch a hole in the marked site of the wax block to collect a tissue core, with a length of about 0.1 mm shallower than the hole depth of the recipient wax block. The collected tissue core was directly inserted or carefully clamped with tweezers into the empty hole of the recipient wax block. This was repeated until all sample points were prepared. Finally, a glass slide was used to flatten all the tissue cores, making the tissue core block flat and smooth. The prepared tissue core block was placed in the wax block making mold and placed in a 60°C oven for 15 min to fuse the tissue core with the wax of the recipient wax block. Then the mold was gently removed from the oven, and the semi-melted wax was allowed to cool at room temperature for about 30 min. The tissue core block was then placed in a -20°C refrigerator for 6 min, and then removed from the mold. The tissue core block was sectioned or stored in a 4°C refrigerator for future use. After trimming, continuous sections were cut with a thickness of 3 μm. The continuous sections were floated in cold water to naturally expand, and then transferred to warm water at 45°C for 30 seconds. The sections were mounted on slides treated with polylysine, and the prepared tissue core block was placed in a 65°C oven for 2 hours. The oven was removed and allowed to cool at room temperature, and then stored in a -4°C refrigerator.

[0079] II. IHC Staining and Analysis

[0080] Routine xylene de-waxing was performed 3 times for 6 minutes each, with hydration in 100%, 100%, 95%, and 85% ethanol for 3 minutes each, and finally rinsed with tap water. Antigen retrieval was performed, and then the sections were placed in a wet box and rinsed with PBS for 3 x 3 minutes. 3% H2O2 was added and incubated for 10 minutes, and then rinsed with PBS for 3 x 3 minutes. The sections were spun dry, and the appropriate dilution of primary antibody was added (the first dilution was designed according to the concentration of the antibody). The sections were incubated at room temperature (25°C) for 1 hour, rinsed with PBS for 3 x 3 minutes, and then the secondary antibody was added and incubated at room temperature for 15-30 minutes. The sections were rinsed with PBS for 3 x 3 minutes, spun dry, and then developed with freshly prepared DAB developing solution for 3-10 minutes. Hematoxylin counterstaining was performed for 25 seconds, and then rinsed with PBS for 30 seconds. The sections were dehydrated in an alcohol gradient of 85% (3 minutes), 95% (3 minutes), 100% (3 minutes), and 100% (3 minutes), and finally cleared with xylene for 3 minutes, and then mounted with neutral resin.

[0081] The immunohistochemical staining results are classified as positive and negative. Positive expression must be at the site of cell and tissue specific antigen to be considered positive. In the case of clear distribution of tissue staining and accurate cell localization, the staining results are further classified according to the difference in staining intensity, as follows:

[0082] 1. The sample is weakly positive, marked as "+".

[0083] 2. The sample is moderately positive, marked as "++".

[0084] 3. The sample is highly positive, marked as "+++".

[0085] 4. The sample is negative, marked as "-".

[0086] III. Data Statistics

[0087] 1. Tumor tissue chip detection results:

[0088] The antibody p504s (2G5) of the present application and commercially available antibody p504s (rabbit polyclonal antibody) were synchronously detected in 29 cases of prostate cancer and the detection results were compared. The immunohistochemical results of p504s were statistically analyzed. The whole test process adopted a double-blind design, and the statistical results are as follows:

[0089]

[0090] The results show that the rabbit monoclonal antibody p504s (2G5) has accurate staining localization, clear staining and no non-specific staining, and clean background. In the immunohistochemical detection, the positive rate is comparable to that of the commercially available antibody, and the staining of 3 cases of p504s rabbit monoclonal antibody 2G5 prepared by the present application is higher than that of the control antibody in positive intensity.

[0091] Figure 3 Comparison of immunohistochemical staining results of prostate cancer Figure 1 (left: rabbit monoclonal antibody p504s of the present application, right: commercially available p504s).

[0092] Figure 4 Comparison of immunohistochemical staining results of prostate cancer Figure 2 (left: rabbit monoclonal antibody p504s of the present application, right: commercially available p504s).

[0093] 2. Normal tissue chip detection results:

[0094] 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.

[0095] The rabbit monoclonal p504s (2G5) and the commercial p504s antibody are synchronously detected on the normal tissue chip, and the positive and negative detection results are consistent, indicating that the specificity of the antibody in the normal tissue is equivalent to that of the commercial antibody.

[0096] Finally, it should be noted that although the above 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. An anti-p504s protein monoclonal antibody, characterized by, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that, The coding DNA sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO. 3, and the coding DNA sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

4.

3. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody specifically recognizes the p504s protein.

4. The monoclonal antibody of claim 1, wherein, The monoclonal antibody is a rabbit monoclonal antibody.

5. An immunoassay reagent for p504s protein, characterized by, The immunodetection reagent contains the anti-p504s protein monoclonal antibody of claim 1 as an effective component.

6. The immunoassay test reagent of claim 5, wherein, The immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

Images