Monoclonal antibody against human tissue kallikrein 1 and application thereof

Abstract

The invention discloses an anti-human tissue kallikrein 1 monoclonal antibody and an application thereof. The anti-human tissue kallikrein 1 monoclonal antibody comprises any one of a monoclonal antibody CELL-2, a monoclonal antibody CELL-5 or a monoclonal antibody CELL-6 or a combination of at least two of the monoclonal antibody CELL-2, the monoclonal antibody CELL-5 and the monoclonal antibody CELL-6. The anti-human tissue kallikrein 1 monoclonal antibody provided by the invention is produced by immunizing natural tissue kallikrein, and can effectively recognize tissue kallikrein in human serum / urine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection technology, and relates to a monoclonal antibody against human tissue kallikrein 1 and application thereof, in particular to a monoclonal antibody combination for detecting human tissue kallikrein 1 by double antibody sandwich enzyme-linked immunosorbent assay (ELISA) and application thereof. BACKGROUND

[0002] Cardiovascular and cerebrovascular diseases are major chronic non-communicable diseases that seriously endanger human health, among which coronary heart disease and stroke are the most common causes of death in the world. Stroke is one of the major diseases that cause death and disability, ranking first among the causes of death in Chinese residents, and ischemic stroke is the most common type, accounting for 69.6%-70.8% of strokes in China.

[0003] Acute ischemic stroke, commonly known as acute cerebral infarction, is a class of diseases caused by various reasons leading to blood supply disorders of brain tissue, and thus ischemic and anoxic necrosis, thereby causing brain dysfunction. Basic research has proved that the kallikrein-kinin / bradykinin system (KKS) is closely related to the occurrence and development of ischemic stroke, and participates in the regulation of cell apoptosis, oxidative stress, inflammatory response and angiogenesis, etc.

[0004] The KKS system is an important physiological regulation system in mammalian organisms, which produces kinins by kallikrein cleavage of kininogen, and the KKS system includes the precursor of kinin (called kininogen), mainly tissue and plasma kallikrein, which releases kinins from high and low molecular weight kininogen, and then binds to kinin receptors in the body and exerts biological effects through multiple downstream signaling pathways. Among them, human tissue kallikrein 1 is a class of serine proteases that can promote the release of vasoactive peptides from low molecular weight kininogen to exert a series of biological activity effects.

[0005] The content of human tissue kallikrein 1 in human plasma / serum / urine samples can reflect the recurrence risk of the prognosis of stroke patients to some extent. Although there are ELISA kits for human tissue kallikrein 1 on the market, such as R&D (catalog: DY2337-05), the kit has been verified to be unable to identify the naturally occurring kallikrein 1 (KLK1) in plasma samples. It is speculated that the reason may be that the antibody is prepared by using recombinant KLK1, and thus it does not have specific recognition effect on natural KLK1.

[0006] Therefore, it is urgent to provide an antibody that specifically recognizes tissue kallikrein 1 to improve the sensitivity and specificity of the determination of the content of human tissue kallikrein 1. SUMMARY

[0007] In view of the deficiencies of the prior art and actual needs, the present application provides an anti-human tissue kallikrein 1 monoclonal antibody and application thereof, which can effectively recognize human tissue kallikrein 1 in human serum and human urine samples, and has high sensitivity and strong specificity.

[0008] To achieve the object of the present application, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides an anti-human tissue kallikrein 1 monoclonal antibody, which comprises any one of a monoclonal antibody CELL-2, a monoclonal antibody CELL-5 or a monoclonal antibody CELL-6 or a combination of at least two thereof.

[0010] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 1; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 2; and the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 3.

[0011] The amino acid sequence of the light chain CDR1 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 4; the amino acid sequence of the light chain CDR2 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 5; and the amino acid sequence of the light chain CDR3 of the monoclonal antibody CELL-2 comprises a sequence as shown in SEQ ID NO. 6.

[0012] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 7; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 8; and the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 9.

[0013] The amino acid sequence of the light chain CDR1 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 10; the amino acid sequence of the light chain CDR2 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 11; and the amino acid sequence of the light chain CDR3 of the monoclonal antibody CELL-5 comprises a sequence as shown in SEQ ID NO. 12.

[0014] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 13; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 15;

[0015] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 13; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-6 comprises a sequence as shown in SEQ ID NO. 15;

[0016] SEQ ID NO. 1: NYGMN.

[0017] SEQ ID NO. 2: WINTYTGEPIYADDFKG.

[0018] SEQ ID NO. 3: KRDYWSWFAY.

[0019] SEQ ID NO. 4: RSSTGAVTTSNYAN.

[0020] SEQ ID NO. 5: GINNRAP.

[0021] SEQ ID NO. 6: ALWYSHHWV.

[0022] SEQ ID NO. 7: SGYSWH.

[0023] SEQ ID NO. 8: YIHFSGRNIYNPSLKS.

[0024] SEQ ID NO. 9: TTVVKYYAMDY.

[0025] SEQ ID NO. 10: SASSSVSYMY.

[0026] SEQ ID NO. 11: LTSNLAS.

[0027] SEQ ID NO. 12: QQWSSNPYT.

[0028] SEQ ID NO. 13: RYAMS.

[0029] SEQ ID NO. 14: SFSSGGNTYYPDSVKG.

[0030] SEQ ID NO. 15: AGVRLPHWFFDV.

[0031] SEQ ID NO. 16: RASQSISDYLH.

[0032] SEQ ID NO. 17: YASQSIS.

[0033] SEQ ID NO. 18: QNGHSFPYT.

[0034] Heavy chain amino acid sequence of monoclonal antibody CELL-2 SEQ ID NO. 19:

[0035] MAWVWTLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPIYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKRDYWSWFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0036] Light chain amino acid sequence of monoclonal antibody CELL-2 SEQ ID NO. 20:

[0037] MAWISLILSLLALSSGAISQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGINNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSHHWVFGGGTKLTVLGQPKSSPSVTLFPPSSEELETNKATLVCTITDFYPGVVTVDWKVDGTPVTQGMETTQPSKQSNNKYMASSYLTLTARAWERHSSYSCQVTHEGHTVEKSLSRADCS.

[0038] Heavy chain amino acid sequence of monoclonal antibody CELL-5 SEQ ID NO. 21:

[0039] MRVLILLCLFTAFPGILSDVQLQESGPDLVKPSQSLSLTCTVTGYSITSGYSWHWIRQFPGNKLEWMGYIHFSGRNIYNPSLKSRISITRDTSKNQFFLQLNSVTTGDTATYYCAITTVVKYYAMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0040] Light chain amino acid sequence of monoclonal antibody CELL-5 SEQ ID NO. 22:

[0041] MDFQVQIFSFLLMSASVIMSRGQIVLTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0042] Heavy chain amino acid sequence of monoclonal antibody CELL-6 SEQ ID NO. 23:

[0043] MNFGFSLIFLVLVLKGVQCEVKLVESGGDLVKPGGSLKLSCAASGFTFSRYAMSWVRQTPEKRLEWVASFSSGGNTYYPDSVKGRFTISRDNARSILELQMSSLRSEDTAMYYCARAGVRLPHWFFDVWGAGTTVTVSSAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK.

[0044] Light chain amino acid sequence of monoclonal antibody CELL-6 SEQ ID NO. 24:

[0045] MVSTSQLGLLLFWTSASRCDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0046] In a second aspect, the present application provides an anti-human tissue kallikrein 1 monoclonal antibody pair, comprising a detection antibody and a capture antibody.

[0047] The detection antibody comprises the monoclonal antibody CELL-6 according to claim 1.

[0048] The capture antibody comprises the monoclonal antibody CELL-2 and / or the monoclonal antibody CELL-5 according to claim 1.

[0049] In a third aspect, the present application provides a nucleic acid molecule encoding the anti-human tissue kallikrein 1 monoclonal antibody according to the first aspect.

[0050] In a fourth aspect, the present application provides a recombinant vector comprising the nucleic acid molecule according to the third aspect.

[0051] In a fifth aspect, the present application provides an engineered immune cell expressing the anti-human tissue kallikrein 1 monoclonal antibody according to the first aspect.

[0052] In a sixth aspect, the present application provides the use of the anti-human tissue kallikrein 1 monoclonal antibody according to the first aspect or the anti-human tissue kallikrein 1 monoclonal antibody pair according to the second aspect in the preparation of a product for detecting the content of human kallikrein 1.

[0053] Preferably, the product comprises any one of a colloidal gold immunoassay kit, a chemiluminescence immunoassay kit, a radioimmunoassay kit, an enzyme-linked immunoassay kit or a fluorescent immunoassay kit.

[0054] In a seventh aspect, the present application provides an enzyme-linked immunoassay kit comprising an enzyme-labeled plate coated with the monoclonal antibody CELL-2 and / or the monoclonal antibody CELL-5 according to the first aspect, alkaline phosphatase-labeled streptavidin and biotin-labeled monoclonal antibody CELL-6 according to the first aspect.

[0055] Preferably, the enzyme-linked immunoassay kit further comprises a standard.

[0056] Preferably, the standard comprises human tissue kallikrein 1.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] (1) The anti-human tissue kallikrein 1 monoclonal antibody provided by the present application is prepared by immunizing animals with natural human tissue kallikrein 1 and combining specific screening techniques, and can efficiently recognize and detect natural tissue kallikrein 1 in human serum and human urine samples;

[0059] (2) The anti-human tissue kallikrein 1 monoclonal antibody provided by the present application is reverse screened using tissue kallikrein 2, and exhibits excellent selectivity, can specifically bind to natural tissue kallikrein 1, and has no cross-reaction with tissue kallikrein 2, and has high specificity;

[0060] (3) The present application screens a variety of antibody pairs by systematic screening, screens antibody pairs with high sensitivity, and optimizes the detection system, and finally develops an enzyme-linked immunoassay quantitative detection kit with excellent performance, and the sensitivity and other key indicators can meet the detection needs of the concentration of tissue kallikrein 1 in human serum and human urine;

[0061] (4) The anti-human tissue kallikrein 1 monoclonal antibody provided by the present application is applied to an enzyme-linked immunoassay quantitative detection kit, has a wide detection range, can detect not only recombinant human tissue kallikrein 1 but also purified human tissue kallikrein 1 in human urine, and has good clinical application value. BRIEF DESCRIPTION OF DRAWINGS

[0062] Figure 1 is a standard curve diagram corresponding to the antibody pair prepared according to the two antibodies in Example 1 of the present application;

[0063] Figure 2 is a standard curve diagram for detecting the concentration of human tissue kallikrein 1 in human serum samples in Example 2 of the present application;

[0064] Figure 3 is a standard curve diagram for detecting the concentration of human tissue kallikrein 1 in human urine samples in Example 3 of the present application;

[0065] Figure 4 is a standard curve diagram of a commercially available antibody pair in the present application;

[0066] Figure 5 is a standard curve diagram of the antibody pair CELL-2 (capture antibody) and CELL-6 (detection antibody) prepared by the present application;

[0067] Figure 6 Standard curve of CELL-5 (capture antibody) & CELL-6 (detection antibody) prepared for the present application;

[0068] Figure 7 Standard curve of CELL-2 (capture antibody) & CELL-6 (detection antibody) prepared for the present application for detecting human tissue kallikrein 1 in human tissue.

[0069] Figure 8 Standard curve of CELL-5 (capture antibody) & CELL-6 (detection antibody) prepared for the present application for detecting human tissue kallikrein 1 in human tissue. DETAILED DESCRIPTION

[0070] In order to further clarify the technical means adopted by the present application and its effects, the present application is further described below in conjunction with the embodiments and drawings. It can be understood that the specific embodiments described herein are merely used to explain the present application, but not to limit the present application.

[0071] The specific techniques or conditions not specified in the embodiments are carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be commercially available through regular channels.

[0072] Example 1

[0073] Preparation of human tissue kallikrein 1-Elisa kit.

[0074] 1. Preparation of human tissue kallikrein 1 monoclonal antibody

[0075] 1.1 Animal immunization

[0076] Balb / c female mice were immunized with human tissue kallikrein 1 extracted and purified from human urine with a SEC-HPLC purity higher than 98% according to the general immunization procedure, and the immunized mice with high immune response level (OD>1, titer≥1:8000) to the immunogen were selected.

[0077] 1.2 Cell fusion and counter screening

[0078] Cell fusion: the mouse spleen cells of the immunized mice with the highest serum titer were fused with mouse myeloma cells by electric fusion, and all the cells of each round of fusion were plated in 96-well plates.

[0079] Primary screening: the supernatant of the fused cells was screened by indirect Elisa method, and the supernatant positive to human tissue kallikrein 1 (target protein) was selected.

[0080] Counter-screening: All positive mother clone supernatants obtained in the primary screening stage were screened by indirect Elisa method to obtain specific clone cells that were positive for human tissue kallikrein 1 and negative for human tissue kallikrein 2 and 293 cell line WB (human cell lysate counter-screening). Three positive clone mother cells that specifically recognized human tissue kallikrein 1 and did not recognize human tissue kallikrein 2 were obtained by screening, and the specificity check results are shown in Table 1:

[0081] Table 1

[0082]

[0083] As can be seen from Table 1, the three positive clone mother cells CELL-2, CELL-5 and CELL-6 have high specificity for human tissue kallikrein 1. Further subcloning and expansion culture obtained three positive monoclonal cells.

[0084] 2. Antibody preparation of the best human tissue kallikrein 1

[0085] 2.1 Antibody preparation of the best human tissue kallikrein 1

[0086] Antibody preparation was performed on the three positive monoclonal cells CELL-2, CELL-5 and CELL-6 obtained by screening, antibody purification was performed by protein A / G affinity chromatography, and double antibody sandwich enzyme-linked immunosorbent assay was performed in a buffer matrix using the mode of anti-target protein antibody-target protein-biotin-labeled anti-target protein antibody for detection.

[0087] Materials:

[0088] Coating: capture antibody, 2.5 μg / mL;

[0089] Coating buffer: phosphate buffered saline, pH 7.4;

[0090] Antigen: human tissue kallikrein 10-100 ng / mL extracted and purified from human urine;

[0091] Sample matrix: phosphate buffered saline, pH 7.4;

[0092] Detection reagent: detection antibody, 0.25-1 μg / mL, labeled with biotin.

[0093] The results are shown in Tables 2 and 3:

[0094] Table 2

[0095]

[0096] Table 2 shows that the detection response OD of the two antibodies, CELL-2 (capture antibody) & CELL-6 (detection antibody) and CELL-5 (capture antibody) & CELL-6 (detection antibody), is... 450 The correlation between the two antibody pairs was high and showed good linearity, while the correlation between the two pairs was not linear. Therefore, the optimal concentrations of the two antibody pairs were further determined at gradient concentrations, and the results are shown in Table 3.

[0097] Table 3

[0098]

[0099] According to Table 3, the corresponding standard curves were prepared for the two antibody pairs CELL-2 (capture antibody 2.5 μg / mL) & CELL-6 (detection antibody 0.5 μg / mL) and CELL-5 (capture antibody 2.5 μg / mL) & CELL-6 (detection antibody 0.25 μg / mL), as shown in Table 3. Figure 1 As shown, the correlation coefficient R between the antibody and CELL-2 (capture antibody) & CELL-6 (detection antibody) is... 2 The correlation coefficient R between CELL-5 (capture antibody) and CELL-6 (detection antibody) is 0.99980. 2 The correlation coefficient R of the standard curves for the other two antibody pairs was 0.99804, while the correlation coefficient R of the other two antibody pairs was 0.99804. 2 All values ​​were below 0.99. Therefore, the following two pairs of paired antibodies were identified, and their sensitivities are shown in Table 4:

[0100] Table 4

[0101]

[0102]

[0103] The aforementioned antibody pairs involve a total of three antibodies, whose sequencing was performed by Genscript Biotech Inc. The first and second monoclonal antibody pairs were prepared by hybridoma cell culture amplification. The antibodies were purified using protein A / G affinity chromatography, and then preserved in phosphate-buffered saline (PBS) containing sodium azide preservative by dialysis. The protein concentration of each antibody was determined using the BCA method.

[0104] Example 2

[0105] Antibodies against CELL-2 (capture antibody) and CELL-6 (detection antibody) are used in an enzyme-linked immunosorbent assay (ELISA) kit to detect the concentration of human tissue kallikrein 1 in human serum samples.

[0106] 1. Reagent kit:

[0107] PBS buffer: precisely weigh 8 g of NaCl, 0.2 g of KCl, 3.63 g of Na2HPO4·12H2O, and 0.24 g of KH2PO4, dissolve in water, adjust the pH value to 7.4, and make up to 1 L, and store at room temperature for 1 month.

[0108] 1% BSA PBS solution: weigh about 1 g of BSA, dissolve in 100 mL of PBS buffer, mix well, and store in the refrigerator (2-8°C), with a shelf life of 7 days;

[0109] Preparation of coating human tissue kallikrein 1 capture antibody buffer: dilute the capture antibody CELL-2 to 2.5 μg / mL with 1% BSA PBS solution, and use immediately after preparation.

[0110] Preparation of biotin-labeled human kallikrein 1 detection antibody solution: dilute the biotin-labeled detection antibody CELL-6 (wherein the biotin-labeled detection antibody CELL-6 is labeled by commercially available Roche biotin protein labeling kit) to 0.5 μg / mL with 1% BSA PBS solution, mix well, and obtain.

[0111] Preparation of alkaline phosphatase-labeled streptavidin solution (SA-AP solution): dilute SA-AP (Hangzhou Jinghong Bioengineering) 5000 times with 1% BSA PBS solution.

[0112] Preparation of color developing solution: mix H2O2 and TMB color developing solution at a ratio of 1:1, and use immediately after preparation (Shanghai Biotech).

[0113] Preparation of washing solution: dilute the washing solution stock solution (25x) (Shanghai Biotech) 25 times with ultrapure water to prepare a washing solution containing 0.05% (V / V) Tween 20.

[0114] Termination solution: single-component reagent containing sulfuric acid (Shanghai Biotech).

[0115] Preparation of standard curve samples: the preparation process of the protein correction standard sample is shown in Table 5, and the preparation process of the quality control sample is shown in Table 6.

[0116] Table 5

[0117]

[0118] Table 6

[0119]

[0120]

[0121] Note: ULOQ is upper limit of quantification, HQC is high concentration quality control, MQC is medium concentration quality control, LQC is low concentration quality control, LLOQ is lower limit of quantification.

[0122] 2. Operation step (1) coating: add prepared coating human tissue kallikrein capture antibody CELL-2 buffer solution into plate holes, 100 μL / hole, and seal the plate with sealing film, and coat at 2-8°C overnight for 12-16 h.

[0123] (2) plate washing: discard the liquid in the plate, add 350 μL of washing solution per hole for 3 times, 60 s each time, and pat the residual washing solution on the blotting paper after the last washing.

[0124] (3) blocking: add 300 μL of 1% (W / V) BSA PBS solution per hole, seal the plate with sealing film, and incubate at 37±1°C for 1 h±5 min.

[0125] (4) plate washing: discard the liquid in the plate, add 350 μL of washing solution per hole for 3 times, 60 s each time, and pat the residual washing solution on the blotting paper after the last washing.

[0126] (5) sample addition: according to the experimental layout, add 100 μL of standard, quality control and sample to be tested into the corresponding plate holes respectively, each sample is set in duplicate, seal the plate with sealing film, and incubate at 37°C±1°C for 1 h±10 min.

[0127] (6) plate washing: discard the liquid in the plate, add 350 μL of washing solution per hole for 3 times, 60 s each time, and pat the residual washing solution on the blotting paper after the last washing.

[0128] (7) detection solution addition: add 100 μL of biotin-labeled anti-human tissue kallikrein 1 detection antibody CELL-6 solution per hole, and incubate at 37±1°C for 1 h±10 min.

[0129] (8) plate washing: discard the liquid in the plate, add 350 μL of washing solution per hole for 3 times, 60 s each time, and pat the residual washing solution on the blotting paper after the last washing.

[0130] (9) SA-AP solution addition: add 100 μL of alkaline phosphatase-labeled streptavidin solution (SA-AP solution) per hole, and incubate at 37±1°C for 30 min±5 min.

[0131] (10) plate washing: discard the liquid in the plate, add 350 μL of washing solution per hole for 3 times, 60 s each time, and pat the residual washing solution on the blotting paper after the last washing.

[0132] (11) color development: add 100 μL of color developing solution per hole, and incubate at 25±1°C for 15±2 min.

[0133] (12) Termination: Add 50 μL of termination solution to each well to terminate the reaction.

[0134] (13) Detection reading: Read the OD value of each well by using a microplate reader at a wavelength of 450 nm (within 10 min after the addition of the termination solution).

[0135] Standard curve and accuracy:

[0136] Take the theoretical concentration as the abscissa, and the difference between the measured OD value and the average OD value of the blank duplicate wells as the ordinate, and fit the standard curve by using a four-parameter regression model;

[0137] Four-parameter regression model: y = (A-D) / [1+(x / C) B ]+D, wherein A is the estimated value of the asymptote of the curve, D is the estimated value of the asymptote of the curve, B is the slope of the curve, and C is the concentration corresponding to half of the maximum binding. The determined standard curve is shown in Table 7 and Figure 2 , and the corresponding quality control determination results are shown in Table 8.

[0138] Table 7

[0139] Sample No. Labelled concentration (pg / mL) OD 450 ]] Calculated concentration (pg / mL) RE% STD1 1600 3.450 1550 -2.8 STD2 800 2.386 846 5.8 STD3 400 1.288 391 -2.2 STD4 200 0.678 195 -2.3 STD5 100 0.369 103 3.1 STD6 50 0.194 49.7 -0.7 STD7 25.0 0.117 25.0 -0.2

[0140] Table 8

[0141]

[0142] It can be found from the above data that the antibody has good linear correlation with the standard curve, the accuracy deviation of the quality control is ≤10%, the detection limit is 25 pg / mL, the standard curve is stable, the data is accurate, and the kit can be used for sample detection.

[0143] The content of human tissue kallikrein 1 in 10 normal human serum samples was detected by using the kit, two duplicate wells were prepared for each group, and the detection results are shown in Table 9.

[0144] Table 9

[0145]

[0146] As shown in Table 9, the content of human tissue kallikrein 1 in 10 normal human serum samples was detected by using the kit, and the coefficient of variation CV was <5%, which indicates that the antibody CELL-2 (capture antibody) and CELL-6 (detection antibody) have good repeatability for detecting the concentration of human tissue kallikrein 1 in human blood samples by using the enzyme-linked immunoassay quantitative detection kit.

[0147] Example 3

[0148] The antibody pair CELL-5 (capture antibody) & CELL-6 (detection antibody) is applied to the enzyme-linked immunoassay quantitative detection kit for detecting the concentration of human tissue kallikrein 1 in human urine samples.

[0149] 1. Kit:

[0150] Preparation of coating buffer for human tissue kallikrein 1 capture antibody: dilute the capture antibody CELL-5 to 2.5 μg / mL with 1% BSA in PBS solution, and prepare it immediately before use.

[0151] Preparation of biotin-labeled human kallikrein 1 detection antibody solution: dilute the biotin-labeled detection antibody CELL-6 (wherein the biotin-labeled detection antibody CELL-6 is labeled by using a commercially available Roche biotin protein labeling kit) to 0.25 μg / mL with 1% BSA in PBS solution, and mix well to obtain the solution.

[0152] The preparation of the remaining solutions is the same as in Example 2.

[0153] Preparation of standard curve samples: the preparation process of the protein correction standard sample is shown in Table 10, and the preparation process of the quality control sample is shown in Table 11.

[0154] Table 10

[0155]

[0156] Table 11

[0157]

[0158] 2. Operation steps

[0159] Coating: add the prepared coating buffer for human tissue kallikrein 1 capture antibody CELL-5 to the plate wells, 100 μL / well, and seal the plate with a sealing film, and coat overnight at 2-8°C.

[0160] (2) Washing the plate: discard the liquid in the plate, add 350 μL of washing solution to each well for 3 times, 60 s each time, and after the last washing, pat the residual washing solution on the blotting paper.

[0161] (3) Blocking: add 300 μL of 1% (W / V) BSA in PBS solution to each well, seal the plate with a sealing film, and incubate at 25±1°C for 2 h±5 min.

[0162] (4) Washing the plate: discard the liquid in the plate, add 350 μL of washing solution to each well for 3 times, 60 s each time, and after the last washing, pat the residual washing solution on the blotting paper.

[0163] (5) Add sample: according to the experimental layout, 100 μL of standard, quality control and sample to be tested were added into the corresponding wells, each sample was set in duplicate, sealed with sealing film, incubated at 25±1℃ for 1h±10min.

[0164] (6) Wash plate: discard the liquid in the plate, add 250 μL of washing solution to each well for 3 times, 60s each time, after the last washing, pat the residual washing solution on the absorbent paper.

[0165] (7) Add detection solution: add 100 μL of biotin-labeled anti-human tissue kallikrein 1 detection antibody CELL-6 solution to each well, incubate at 25±1℃ for 1h±10min.

[0166] (8) Wash plate: discard the liquid in the plate, add 250 μL of washing solution to each well for 3 times, 60s each time, after the last washing, pat the residual washing solution on the absorbent paper.

[0167] (9) Add SA-AP solution: add 100 μL of alkaline phosphatase-labeled streptavidin solution (SA-AP solution) to each well, incubate at 25±1℃ for 30min±5min.

[0168] (10) Wash plate: discard the liquid in the plate, add 350 μL of washing solution to each well for 3 times, 60s each time, after the last washing, pat the residual washing solution on the absorbent paper.

[0169] (11) Color development: add 100 μL of color developing solution to each well, incubate at 25±1℃ for 15±2min.

[0170] (12) Termination: add 50 μL of termination solution to each well to terminate the reaction.

[0171] (13) Detection reading: the OD value of each well was read by the enzyme-labeled instrument, the wavelength was 450nm (measured within 10min after the addition of termination solution).

[0172] Standard curve and accuracy:

[0173] The theoretical concentration was taken as the abscissa, the difference between the measured OD value and the average OD value of the blank duplicate wells was taken as the ordinate, and the standard curve was fitted by four-parameter regression model;

[0174] Four-parameter regression model: y=(A-D) / [1+(x / C) B ]+D, wherein A is the estimated value of the asymptote of the curve, D is the estimated value of the asymptote of the curve, B is the slope of the curve, and C is the concentration corresponding to half of the maximum binding. The determined standard curve is shown in Table 12 and Figure 3 , and the corresponding quality control determination results are shown in Table 13.

[0175] Table 12

[0176] Sample No. Labelled concentration (pg / mL) OD 450 ]] Calculated concentration (pg / mL) RE% STD1 2000 3.715 1992 -0.4 STD2 1000 2.706 1012 1.2 STD3 500 1.608 486 -2.8 STD4 250 0.914 260 4.0 STD5 125 0.415 119 -4.8 STD6 62.5 0.221 64 2.4 STD7 31.25 0.111 30 -4.0

[0177] Table 13

[0178] Name Labelled concentration (pg / mL) OD 450 ]]> Calculated concentration (pg / mL) Accuracy (RE%) ULOQ 2000 3.690 1955 -2.2 HQC 750 2.228 746 -0.5 MQC 300 1.001 286 -4.7 LQC 90 0.301 87 -3.3 LLOQ 31.25 0.120 33 5.6

[0179] From the above data, it can be found that the antibody has good linear correlation with the standard curve, the accuracy deviation of the quality control product is ≤10%, the detection limit is 31.25 pg / mL, the standard curve is stable, the data is accurate, and the kit can be used for sample detection.

[0180] The content of human tissue kallikrein 1 in 10 normal morning urine samples was detected by using the above kit, two duplicate wells were made for each group, and the detection results are shown in Table 14.

[0181] Table 14

[0182]

[0183]

[0184] Note: BDL represents below the detection limit, and NA represents not applicable.

[0185] As shown in Table 14, the content of human tissue kallikrein 1 in 10 normal morning urine samples was detected by using the above kit, the CV is <5%, which indicates that the antibody of the application has good repeatability for the application of CELL-5 (capture antibody) and CELL-6 (detection antibody) in the enzyme-linked immunoassay quantitative detection kit for detecting the concentration of human tissue kallikrein 1 in human urine samples.

[0186] Comparative Example

[0187] Comparison with commercially available kits (evaluation of antibody pairs).

[0188] Based on the purchased R&D human tissue kallikrein 1-Elisa kit (catalog: DY2337-05), the antibodies CELL-2 (capture antibody) and CELL-6 (detection antibody), CELL-5 (capture antibody) and CELL-6 (detection antibody) prepared by the application, and the antibody pair (commercially available antibody pair) provided in the kit were used to simultaneously detect the recombinant mouse human tissue kallikrein 1 standard (provided in the R&D human tissue kallikrein 1-Elisa kit) and the natural human tissue kallikrein 1 extracted and purified from human urine, and other reagents were all the reagents provided in the kit, and the operation was as follows:

[0189] (1) Antibody coating: coating the prepared capture antibodies CELL-2 (2.5 μg / mL), CELL-5 (2.5 μg / mL) and Capture antibody (R&D Human Kallikrein 1-Elisa Kit, 4 μg / mL) of the application, 100 μL / well, 2-8°C overnight coating;

[0190] (2) Washing: discard the liquid in the well, add 300 μL / well of washing solution, wash 3 times, pour off, and pat dry;

[0191] (3) Blocking: add 300 μL / well of blocking solution Superblock, cover the film, 37°C, block for 2 h ± 5 min;

[0192] (4) Washing: add 300 μL / well of washing solution, wash 3 times, pour off, and pat dry;

[0193] (5) Sample addition: dilute the recombinant mouse human kallikrein 1 standard and human urine-extracted and purified human kallikrein 1 with sample diluent to 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0 pg / mL, respectively, add 100 μL / well of the above concentration solution, cover the film, 37°C, 500 rpm incubation for 1 h ± 5 min;

[0194] (6) Washing: add 300 μL / well of washing solution, wash 3 times, pour off, and pat dry;

[0195] (7) Add detection antibody: dilute the detection antibody with 1% BSA PBS solution, add 100 μL / well of the prepared detection antibody CELL-6 (0.5 μg / mL), CELL-6 (0.25 μg / mL) and Detection antibody (commercial detection antibody, 0.5 μg / mL) after dilution and biotin labeling, cover the film, 37°C incubation for 1 h ± 5 min;

[0196] (8) Washing: add 300 μL / well of washing solution, wash 3 times, pour off, and pat dry;

[0197] (9) Add detection reagent: dilute the detection reagent Streptavidin-HRP (Jackson IR Inc) to a dilution of 1:30000 with 1% BSA PBS solution, cover the film, 37°C, 500 rpm incubation for 1 h ± 5 min;

[0198] (10) Washing: add 300 μL / well of washing solution, wash 3 times, pour off, and pat dry;

[0199] (11) Add TMB developing solution: 100 μL / well, cover the film, 37°C incubation for 20 min;

[0200] (12) Termination: add termination solution, 50 μL / well, terminate the reaction at room temperature;

[0201] (13) Measurement: measure OD value of each well in a microplate reader at 450 nm.

[0202] The recombinant mouse human tissue kallikrein 1 standard (purchased R&D human tissue kallikrein 1-Elisa kit) was set at a concentration of 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0 pg / mL, and the human urine extracted and purified human tissue kallikrein 1 standard was set at a concentration of 2000, 1000, 500, 250, 125, 62.5, 31.25 and 0 pg / mL, and the detection results are shown in Tables 15 and 16.

[0203] Table 15

[0204]

[0205] Table 16

[0206]

[0207] As can be seen from Tables 15 and 16, the two antibody pairs of the application can show detection trends for the original recombinant mouse human tissue kallikrein 1 standard in the commercially available kit.

[0208] The standard curves of the antibody pair (commercially available antibody pair) and the antibody pair CELL-2 (capture antibody) & CELL-6 (detection antibody) and CELL-5 (capture antibody) & CELL-6 (detection antibody) prepared by the application were drawn with the concentration of the recombinant mouse KLK1 standard as the abscissa and the detector response value OD 450 as the ordinate, respectively, as shown in Figures 1 and 2. Figure 4 - Figure 6 .

[0209] As can be seen from Tables 15 and 16, the two antibody pairs of the application can show detection trends for the original recombinant mouse human tissue kallikrein 1 standard in the commercially available kit.

[0210] The standard curves of the antibody pair (commercially available antibody pair) and the antibody pair CELL-2 (capture antibody) & CELL-6 (detection antibody) and CELL-5 (capture antibody) & CELL-6 (detection antibody) prepared by the application were drawn with the concentration of the recombinant mouse KLK1 standard as the abscissa and the detector response value OD 450 as the ordinate, respectively, as shown in Figures 1 and 2. 450There is no obvious gradient trend, so the corresponding standard curve cannot be drawn, the antibody prepared by the application has a standard curve correlation coefficient R of 0.99829 and 0.99925 respectively for CELL-2 (capture antibody) & CELL-6 (detection antibody) and CELL-5 (capture antibody) & CELL-6 (detection antibody) 2 respectively, as shown in Figure 7 and Figure 8 .

[0211] It can be seen that the antibody prepared by the application can detect both recombinant mouse human tissue kallikrein 1 and purified human tissue kallikrein 1 extracted from human urine, while the commercially available kit cannot accurately detect the natural human tissue kallikrein 1 extracted and purified from human urine, which indicates that the antibody of the application has a wider detection range.

[0212] In summary, the monoclonal antibodies of the application are all immunized by natural tissue kallikrein 1 and can effectively recognize tissue kallikrein 1 in human serum / urine.

[0213] The applicant declares that the detailed method of the application is illustrated by the above examples, but the application is not limited to the above detailed method, that is, it does not mean that the application must rely on the above detailed method to be implemented. It should be understood by those skilled in the art that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the application.

Claims

1. A monoclonal antibody against human tissue kallikrein 1, characterized in that, The anti-human tissue kallikrein 1 monoclonal antibody includes any one or a combination of at least two of the monoclonal antibodies CELL-2, CELL-5, or CELL-6. The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.1; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.2; the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.

3. The amino acid sequence of the light chain CDR1 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.4; the amino acid sequence of the light chain CDR2 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.5; the amino acid sequence of the light chain CDR3 of the monoclonal antibody CELL-2 includes the sequence shown in SEQ ID NO.

6. The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.7; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.8; and the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.

9. The amino acid sequence of the light chain CDR1 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.10; the amino acid sequence of the light chain CDR2 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.11; and the amino acid sequence of the light chain CDR3 of the monoclonal antibody CELL-5 includes the sequence shown in SEQ ID NO.

12. The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.13; the amino acid sequence of the heavy chain CDR2 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.14; and the amino acid sequence of the heavy chain CDR3 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.

15. The amino acid sequence of the light chain CDR1 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.16; the amino acid sequence of the light chain CDR2 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.17; and the amino acid sequence of the light chain CDR3 of the monoclonal antibody CELL-6 includes the sequence shown in SEQ ID NO.

18.

2. A monoclonal antibody pair against human tissue kallikrein 1, characterized in that, The anti-human tissue kallikrein 1 monoclonal antibody pair includes a detection antibody and a capture antibody; The detection antibody includes the monoclonal antibody CELL-6 as described in claim 1; The capture antibody includes the monoclonal antibody CELL-2 and / or monoclonal antibody CELL-5 as described in claim 1.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human tissue kallikrein 1 monoclonal antibody as described in claim 1.

4. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 3.

5. An engineered immune cell, characterized in that, The immune cells express the anti-human tissue kallikrein 1 monoclonal antibody as described in claim 1.

6. The use of the anti-human tissue kallikrein 1 monoclonal antibody of claim 1 or the anti-human tissue kallikrein 1 monoclonal antibody of claim 2 in the preparation of products for detecting the content of human tissue kallikrein.

7. The application according to claim 6, characterized in that, The product includes any one of the following: colloidal gold immunoassay kit, chemiluminescence immunoassay kit, radioimmunoassay kit, enzyme-linked immunosorbent assay kit, or fluorescence immunoassay kit.

8. An enzyme-linked immunosorbent assay (ELISA) kit, characterized in that, The enzyme-linked immunosorbent assay (ELISA) kit contains an ELISA plate coated with the monoclonal antibody CELL-2 and / or monoclonal antibody CELL-5 as described in claim 1 or 2, alkaline phosphatase-labeled streptavidin, and biotin-labeled monoclonal antibody CELL-6 as described in claim 1.

9. The enzyme-linked immunosorbent assay kit according to claim 8, characterized in that, The enzyme-linked immunosorbent assay kit also includes standards.

10. The enzyme-linked immunosorbent assay (ELISA) kit according to claim 9, characterized in that, The standard includes human tissue kallikrein 1.

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