Heparin binding protein immunodetection paired antibody and application thereof

Abstract

The invention provides a heparin-binding protein immunodetection paired antibody and application thereof. The heparin-binding protein immunodetection paired antibody comprises a first anti-heparin-binding protein monoclonal antibody and a second anti-heparin-binding protein monoclonal antibody, the sequences of CDR1, CDR2 and CDR3 of a heavy chain variable region of the first anti-heparin binding protein monoclonal antibody are respectively shown as SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, and the sequences of CDR1, CDR2 and CDR3 of a light chain variable region of the first anti-heparin binding protein monoclonal antibody are respectively shown as SEQ ID No.8, SEQ ID No.9 and SEQ ID No.10; the sequences of the CDR1, the CDR2 and the CDR3 of the heavy chain variable region of the second anti-heparin binding protein monoclonal antibody are respectively shown as SEQ ID No.13, SEQ ID No.14 and SEQ ID No.15, and the sequences of the CDR1, the CDR2 and the CDR3 of the light chain variable region of the second anti-heparin binding protein monoclonal antibody are respectively shown as SEQ ID No.18, SEQ ID No.19 and SEQ ID No.20.

Description

Technical Field

[0001] The invention belongs to the technical field of biological immunology, and in particular relates to a heparin binding protein immune detection paired antibody and application thereof. Background Art

[0002] Heparin-binding protein (HBP) is a granular glycoprotein secreted by mature neutrophils. It has a strong heparin-binding ability, hence its name. Mature HBP is composed of 222 amino acids, with a relative molecular weight of 37,000. It is positively charged and has bactericidal function, so it is also called CAP37 (cationic antimicrobial protein of 37,000). HBP is also known as Azurocidin.

[0003] HBP can act on vascular endothelial cells to increase their permeability, and can also activate monocytes / macrophages to expand inflammatory responses. The level of HBP in the blood of healthy people is very low, generally less than 10ng / mL. When an infection occurs, the level of HBP in the blood will increase, generally reaching 20-30ng / mL during infection; in severe infections, it may even exceed 100ng / mL.

[0004] HBP can be used as a biomarker for sepsis. Compared with other indicators such as PCT, CRP, and white blood cell count, it has higher specificity and sensitivity. Therefore, it has extremely high clinical application value.

[0005] HBP monoclonal antibody is the core raw material of HBP immunoassay reagents and determines the performance of HBP immunoassay reagents. Currently, most HBP monoclonal antibodies have the problems of narrow detection range and low sensitivity, and it is urgent to develop HBP monoclonal antibodies with excellent performance. Summary of the invention

[0006] The present invention provides a heparin binding protein immunoassay paired antibody and application thereof, which can effectively solve the above problems.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a heparin-binding protein immunoassay paired antibody, wherein the heparin-binding protein immunoassay paired antibody comprises a first anti-heparin-binding protein monoclonal antibody and a second anti-heparin-binding protein monoclonal antibody;

[0009] The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the first anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.3, SEQ.ID No.4, and SEQ.ID No.5, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the first anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.8, SEQ.ID No.9, and SEQ.ID No.10, respectively;

[0010] The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the second anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.13, SEQ.ID No.14, and SEQ.ID No.15, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the second anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.18, SEQ.ID No.19, and SEQ.ID No.20, respectively.

[0011] In a second aspect, the present invention provides an anti-heparin binding protein monoclonal antibody, which can specifically bind to heparin binding protein, and the sequences of CDR1, CDR2, and CDR3 in the heavy chain variable region are shown in SEQ.ID No.3, SEQ.ID No.4, and SEQ.IDNo.5, respectively, and the sequences of CDR1, CDR2, and CDR3 in the light chain variable region are shown in SEQ.ID No.8, SEQ.ID No.9, and SEQ.IDNo.10, respectively.

[0012] In a third aspect, the present invention provides an anti-heparin binding protein monoclonal antibody, which can specifically bind to heparin binding protein, and the sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ.ID No.13, SEQ.ID No.14, and SEQ.ID No.15, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region are shown in SEQ.ID No.18, SEQ.IDNo.19, and SEQ.ID No.20, respectively.

[0013] In a fourth aspect, the present invention provides a heparin-binding protein immunoassay kit, comprising the heparin-binding protein immunoassay paired antibody described in the first aspect.

[0014] In a fifth aspect, the present invention provides a heparin-binding protein immunoassay kit, comprising the anti-heparin-binding protein monoclonal antibody described in the second aspect or the anti-heparin-binding protein monoclonal antibody described in the third aspect.

[0015] The beneficial effects of the present invention are:

[0016] The invention provides a heparin binding protein immunoassay paired antibody, comprising a first anti-heparin binding protein monoclonal antibody and a second anti-heparin binding protein monoclonal antibody. The first anti-heparin binding protein monoclonal antibody and the second anti-heparin binding protein monoclonal antibody are used in a paired manner and have high detection activity.

[0017] The present invention provides a heparin-binding protein immunoassay kit, comprising the heparin-binding protein immunoassay paired antibody, wherein the first anti-heparin-binding protein monoclonal antibody is a coating antibody, and the second anti-heparin-binding protein monoclonal antibody is a detection antibody. The detection sensitivity can reach 0.1 ng / mL, and the linear detection range can reach 0.1-723.1 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the SDS-PAGE protein gel electrophoresis diagram of the HBP antigen expressed by 293F cells in the example of the present invention.

[0020] Figure 2 It is a linear graph of the ELISA detection activity of the paired antibodies Ab1 and Ab2 according to the example of the present invention.

[0021] Figure 3 This is a linear graph of the heparin binding protein immunoassay kit according to an embodiment of the present invention detecting clinical samples. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Sources of reagents in the embodiments of the present invention:

[0024] 293F cells were from Xiamen University;

[0025] The feed medium was OPM293 ProFeed medium, purchased from Shanghai Aopuma Biotechnology Co., Ltd.;

[0026] Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from Sigma;

[0027] The HBP protein assay kit was purchased from Zhonghan Shengtai Biotechnology Co., Ltd.;

[0028] 6-8 week old SPF grade Balb / c mice were purchased from Fuzhou Wu Animal Experiment Center;

[0029] RPMI 1640 basal culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.;

[0030] HAT culture medium was purchased from Sigma;

[0031] DMEM serum-free culture medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.;

[0032] Mouse myeloma cells Sp2 / 0 were from Xiamen University;

[0033] Fetal bovine serum was purchased from Shanghai Xiaopeng Biotechnology Co., Ltd.;

[0034] Goat anti-mouse antibody was purchased from Sigma.

[0035] Example 1

[0036] Preparation of monoclonal antibody against HBP

[0037] 1. Preparation of HBP recombinant antigen

[0038] The amino acid sequence encoding the mature protein of HBP is derived from NCBI: NP_001691. A His tag is added to the C-terminus, and the resulting amino acid sequence is shown in SEQ.ID No.21.

[0039] SEQ.ID No.21:

[0040] IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVSTVVLGAYDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLPLQN ATVEAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNVCTGVLTRRGGICNGDGGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGPHHHHHHH

[0041] The nucleotide sequence was optimized according to the human gene codon preference, and the obtained gene sequence is shown in SEQ.ID No. 22. Suzhou Jinweizhi was commissioned to synthesize the sequence into the pTT5 vector.

[0042] SEQ.ID No.22:

[0043] ATTGTGGGAGGAAGAAAAGCTAGACCCAGACAGTTCCCGTTCCTGGCATCTATTCAGAACCAGGGGCGACACTTCTGCGGGGGCGCCTTGATCCATGCAAGGTTCGTGATGACGGCGGCCTCATGTTTCCAATCCCAGAACCCCGGTGTGAGCACAGTCGTTCTTGGAGCA TACGATCTGCGCCGCCGGGAACGGCAAAGCAGGCAGACATTCAGCATCAGCTCCATGTCCGAGAACGGTTACGACCCCCAACAGAATCTCAACGATCTGATGCTGCTGCAACTGGACCGGGAAGCTAACCTGACCTCCTCAGTGACTATCCTCCCGCTTCCCCTGCAGAAT GCTACTGTGGAAGCCGGTACGCGATGTCAGGTGGCTGGTTGGGGATCACAGCGCAGTGGAGGACGACTTAGCCGGTTCCCGAGGTTTGTCAATGTGACAGTGACGCCAGAGGATCAGTGCCGGCCAAACAACGTGTGCACCGGCGTCTTGACCCGGAGGGGGGGGATATGC AACGGGGACGGGGGGACACCTCTGGTGTGTGAAGGCTTGGCCCACGGGGTCGCTAGCTTCTCCCTTGGCCCCTGCGGCCGAGGACCCGATTTCTTCACTCGCGTGGCCCTCTTTCGGGACTGGATAGATGGCGTGCTCAACAATCCCGGCCCTCACCACCACCACCACCAC

[0044] The constructed pTT5-HBP plasmid was transfected into 293F cells by PEI transfection method. After successful transfection, the cells were placed in a shaker at 37°C, 8% CO2, 120 rpm and cultured for 24 h, and then 3.5% volume ratio of feed medium was added, and the cells were returned to the shaker and cultured for 96 h to obtain 293F cell culture fluid expressing HBP. The cell culture fluid was centrifuged at 4°C, 12000 rpm for 30 min, the supernatant was collected, and filtered with a 0.22 μm filter membrane to obtain the cell supernatant.

[0045] After the NiFF column was equilibrated with Buffer A (20mM PB, pH7.4), the cell supernatant was loaded onto the NiFF column for purification. After loading, the column was rinsed and equilibrated with Buffer A, impurities were eluted with Buffer B (20mM PB, 10mM imidazole, pH7.4), and the target protein was eluted with Buffer C (20mM PB, 200mM imidazole, pH7.4). The purified HBP recombinant protein was ultrafiltered, concentrated, stored in pH7.4 PBS, and diluted to 1mg / mL. The purification results are shown in Figure 2. Figure 1 shown.

[0046] 2. Mouse immunization

[0047] The above HBP recombinant antigen was emulsified with an equal volume of Freund's complete adjuvant and Week-old SPF Balb / c mice were injected subcutaneously at multiple points with 200 μg / mouse. Two weeks later, an equal volume of Freund's incomplete adjuvant was used to emulsify the antigen and 100 μg / mouse was injected subcutaneously at multiple points. The immunization was boosted twice and 100 μg / mouse was injected intraperitoneally 3 days before fusion for shock.

[0048] 3. Preparation of feeder cells

[0049] Balb / c mouse peritoneal macrophages were used as feeder cells. One day before cell fusion, the mice were killed by pulling the neck, and the whole body was soaked in 75% alcohol. Under sterile conditions in a clean bench, the abdominal skin was cut open with scissors to expose the peritoneum. 5 mL of RPMI1640 basal culture medium was injected into the peritoneal cavity with a syringe, and the cells were repeatedly rinsed. The rinsing fluid was recovered and centrifuged at 1000 rpm for 5 minutes. The precipitate was retained and resuspended in RPMI 1640 complete culture medium containing 1% HAT to adjust the cell concentration to 1×10 5 / mL, added to 96-well cell culture plates, 150 μL / well, and cultured overnight at 37°C, 5% CO2.

[0050] 4. Preparation of immune spleen cells

[0051] Three days after the last immunization of the above mice, the spleen was removed under sterile conditions, placed in a plate, rinsed once with RPMI1640 basal culture medium, and then ground and filtered on a nylon mesh (the nylon mesh was placed in a small beaker) to prepare a cell suspension. Centrifugation was performed, the supernatant was discarded, and the cell suspension was resuspended in RPMI 1640 basal culture medium. This was repeated three times to obtain a suspension for cell counting.

[0052] 5. Cell fusion

[0053] (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12000) solution and preheat them in a 37°C water bath.

[0054] (2) Mouse myeloma cells Sp2 / 0 (2×10 7 ) and the above suspension (immune spleen cells 1×10 8 ), add to a 50mL centrifuge tube and mix well, add DMEM serum-free culture medium to 40mL, centrifuge for 10min, pour off the supernatant and mix well.

[0055] (3) Place the above centrifuge tube in 37°C preheated water, add 0.7 mL of 37°C preheated 50% PEG solution, let stand for 90 seconds, and immediately drop 15 mL of 37°C preheated serum-free culture medium.

[0056] (4) Add DMEM serum-free culture medium to the above centrifuge tube to make up to 40 mL. Centrifuge for 10 min, pour off the supernatant, add 40 mL of HAT culture medium containing 15%-20% fetal bovine serum, mix with a pipette, and drop into the wells of the above 96-well cell culture plate containing feeder cells, 2 drops per well, and culture in an incubator at 37°C and 7% CO2.

[0057] 6. Hybridoma cell selection, culture and cloning screening

[0058] On the 1st, 3rd, 5th and 7th days after cell fusion, the culture medium was replaced with HAT culture medium containing 15%-20% fetal bovine serum, and the positive wells producing the target antibody were selected, and three rounds of subcloning were performed to screen out the monoclonal cell line that specifically recognized the HBP antigen. Finally, 15 hybridoma monoclonal cell lines were selected.

[0059] 7. Ascites expression and purification

[0060] Balb / c mice were intraperitoneally injected with 0.5 mL of liquid paraffin. Ten days later, the selected hybridoma cell lines were inoculated into the abdominal cavity of the mice. After about 10 days, the abdomen of the mice began to swell. The mice were killed by dislocating the cervical vertebrae, and then immersed in 75% alcohol for 5 minutes for disinfection. The ascites was extracted once. The ascites was purified with a Protein A affinity column to obtain monoclonal antibodies.

[0061] Example 2

[0062] ELISA method to screen paired antibodies

[0063] (1) Dilute the coating antibody to 1 μg / mL using 20 mM PB, pH 7.4 coating solution, add 100 μL per well to coat a 96-well ELISA plate (i.e., 100 ng / well), and incubate at 4°C overnight.

[0064] (2) Aspirate the liquid in the wells, wash each well three times with 300 μL PBST washing buffer, add 300 μL blocking solution (5% skim milk powder, prepared in PBST), and incubate at 37°C for 1 h. Add 100 μL of 2-fold serially diluted HBP recombinant antigen sample to each well and incubate at 37°C for 1 h.

[0065] (3) Aspirate the liquid in the wells and wash each well three times with 300 μL of washing buffer. Dilute the detection antibody to 1 μg / mL with 20 mM PB, pH 7.4, and add 100 μL of detection antibody to each well.

[0066] (4) Aspirate the liquid in the wells, wash each well three times with 300 μL of washing buffer, add 100 μL of HRP-labeled goat anti-mouse antibody (1:5000, diluted in PBST), and incubate at 37°C for 30 min.

[0067] (5) Aspirate the liquid in the wells, wash each well with 300 μL of washing buffer 5 times, add 100 μL of color development solution, develop at 37°C for 10 min, add 50 μL of 2 mol / L H2SO4 stop solution, and read the OD value on a microplate reader within 20 min after adding the stop solution. 450 Numeric value.

[0068] The obtained monoclonal antibodies were paired in pairs and used as coating antibodies and detection antibodies respectively. The diluted HBP recombinant antigen samples were detected by double sandwich ELISA method. Finally, a pair of suitable paired antibodies were screened and named Ab1 and Ab2.

[0069] The detection results of paired antibodies Ab1 and Ab2 are shown in Table 1 and Figure 2 shown.

[0070] Table 1

[0071]

[0072] From Table 1 and Figure 2 It can be seen that when the HBP concentration is in the range of 0.39-400.00 ng / mL, the paired antibodies Ab1 and Ab2 have a good linear relationship, R 2 =0.9889.

[0073] Antibody Ab1:

[0074] The heavy chain sequence is shown as SEQ.ID No.1.

[0075] SEQ.ID No.1:

[0076] QVQLQDSGAQLVKPGISVKLSCRASGYTFTEYTNHTWVKQAMGRSLEWLGTITESGGYTDYEQKTKKSTLTADRSSSVTYIQLGSLTSEDTAFYYCARDYEGVSLTYWGQGTLLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK The heavy chain variable region sequence is shown in SEQ.ID No.2.

[0077] SEQ.ID No.2:

[0078] QVQLQDSGAQLVKPGISVKLSCRASGYTFTEYTNHTWVKQAMGRSLEWLGTITESGGYTDYEQKTKKSTLTADRSSSVTYIQLGSLTSEDTAFYYCARDYEGVSLTYWGQGTLLTVSS

[0079] The heavy chain CDR1 sequence is shown in SEQ.ID No.3.

[0080] SEQ.ID No.3:

[0081] EYTNHT

[0082] The heavy chain CDR2 sequence is shown in SEQ.ID No.4.

[0083] SEQ.ID No.4:

[0084] TITESGGYTDYEQKTK

[0085] The heavy chain CDR3 sequence is shown in SEQ.ID No.5.

[0086] SEQ.ID No.5:

[0087] DYEGVSLTY

[0088] The light chain sequence is shown in SEQ.ID No.6.

[0089] SEQ.ID No.6:

[0090] DIVLTQSPSLMSASPGERVTISCRASEDYNYVMAWYQQKPGQSPRLWIYDASNIYSGVPERFSGSGSGSSYTLTISRVEAEDVASYYCEQKTHSPKTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0091] The light chain variable region sequence is shown in SEQ.ID No.7.

[0092] SEQ.ID No.7:

[0093] DIVLTQSPSLMSASPGERVTISCRASEDYNYVMAWYQQKPGQSPRLWIYDASNIYSGVPERFSGSGSGSSYTLTISRVEAEDVASYYCEQKTHSPKTFGGGTKLEIK

[0094] The light chain CDR1 sequence is shown in SEQ.ID No.8.

[0095] SEQ.ID No.8:

[0096] RASEDYNYVMA

[0097] The light chain CDR2 sequence is shown in SEQ.ID No.9.

[0098] SEQ.ID No.9:

[0099] DASNIYS

[0100] The light chain CDR3 sequence is shown in SEQ.ID No.10.

[0101] SEQ.ID No.10:

[0102] EQKTHSPKT

[0103] Antibody Ab2:

[0104] The heavy chain sequence is shown as SEQ.ID No.11.

[0105] SEQ.ID No.11:

[0106] EVQLQESGAQLVKPGISLKLSCAASGYTFTEYSENHWIKQAPGRSLEWVGTLSETGGYTDTEQRTKRSTTLTRDRSSSVTYIQLGSLRSEDTAVYYCARETESVSLTWGQGTLLTVSAAKTTP PSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLT ITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK The heavy chain variable region sequence is shown in SEQ.ID No.12.

[0107] SEQ.ID No.12:

[0108] EVQLQESGAQLVKPGISLKLSCAASGYTFTEYSENHWIKQAPGRSLEWVGTLSETGGYTDTEQRTKRSTTLDRSSSVTYIQLGSLRSEDTAVYYCARETESVSLTWWGQGTLLTVSA

[0109] The heavy chain CDR1 sequence is shown as SEQ.ID No.13.

[0110] SEQ.ID No.13:

[0111] EYSENH

[0112] The heavy chain CDR2 sequence is shown in SEQ.ID No.14.

[0113] SEQ.ID No.14:

[0114] TLSETGGYTDTEQRTK

[0115] The heavy chain CDR3 sequence is shown as SEQ.ID No.15.

[0116] SEQ.ID No.15:

[0117] ETESVSLTW

[0118] The light chain sequence is shown in SEQ.ID No.16.

[0119] SEQ.ID No.16:

[0120] DVLLTQSPSLMSASPGERVTITCKASEDTDYLVAWFDQKPGTSPRLIIYEASNLTSGVPDRFTGSGSGSYTTLKISRMEAEDVAVYYCQQRTHSPRTFGAGTKLEVK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0121] The sequence of the light chain variable region is shown in SEQ.ID No.17.

[0122] SEQ.ID No.17:

[0123] DVLLTQSPSLMSASPGERVTITCKASEDTDYLVAWFDQKPGTSPRLIIYEASNLTSGVPDRFTGSGSGSYTTLKISRMEAEDVAVYYCQQRTHSPRTFGAGTKLEVK

[0124] The light chain CDR1 sequence is shown in SEQ.ID No.18.

[0125] SEQ.ID No.18:

[0126] KASEDTDYLVA

[0127] The light chain CDR2 sequence is shown in SEQ.ID No.19.

[0128] SEQ.ID No.19:

[0129] EASNLTS

[0130] The light chain CDR3 sequence is shown in SEQ.ID No.20.

[0131] SEQ.ID No.20:

[0132] QQRTHSPRT

[0133] Example 3

[0134] Paired antibodies Ab1 and Ab2 for use in immunofluorescence kits

[0135] Ab1 antibody and HBP recombinant antigen were streaked on nitrocellulose membrane at a dosage of 0.04 μg / cm membrane and 0.09 μg / cm membrane respectively, and dried at 55°C for 3 days. At the same time, Ab2 antibody was coupled and labeled with fluorescent microspheres, sprayed on the sample pad at a dosage of 0.02 μg antibody / cm binding pad, and vacuum-dried for 3 hours to assemble into a reagent strip.

[0136] Then, in a normal temperature and humidity environment (25°C, humidity 50%-60%), add sample diluent to the sample pad of the reagent strip, the amount is 130 μL, and then drip different concentrations of clinical samples to the sample pad of the reagent strip, the amount is 15 μL, read the detection fluorescence value, the results are shown in Table 2 and Figure 3 shown.

[0137] The sample buffer includes the following components by mass fraction: HEPES 1.5%, perfluorooctanoic acid 2.0%, sodium chloride 2.0%, S9 1.0%, and the pH value is 7.0.

[0138] The concentration values ​​of clinical samples were calibrated using the Zhonghan Shengtai heparin binding protein detection kit.

[0139] Table 2

[0140] Sample No. Concentration value (ng / mL T line value C line value T / C 1 0.1 1184 62316 0.019 2 04 1463 54185 0.027 3 1.2 2234 53190 0.042 4 1.9 4358 50092 0.087 5 53 7269 49449 0.147 6 8.1 11317 47550 0.238 7 9.4 13054 40415 0.323 8 12.7 17876 37555 0.476 9 28.3 28465 37112 0.767 10 60.1 32201 24904 1.293 11 108.6 35563 18957 1.876 12 230.5 43879 12416 3.534 13 314.2 55451 11654 4.758 14 402.6 57379 9366 6.126 15 723.1 65354 6370 10.259

[0141] From Table 2 and Figure 3 It can be seen that the correlation between the fluorescence value detected by the reagent strip and the concentration value of the clinical sample is R 2 =0.9972, the detection sensitivity can reach 0.1ng / mL, and the linear detection range can reach 0.1-723.1ng / mL.

[0142] The Ab1 antibody and the Ab2 antibody provided in the embodiment of the present invention can also be used in pairs with other anti-heparin binding protein monoclonal antibodies, so as to be applied to other heparin binding protein immunoassay kits.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A heparin binding protein immunoassay paired antibody, characterized in that: The heparin binding protein immunoassay paired antibodies include a first anti-heparin binding protein monoclonal antibody and a second anti-heparin binding protein monoclonal antibody; The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the first anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.3, SEQ.ID No.4, and SEQ.ID No.5, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the first anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.8, SEQ.ID No.9, and SEQ.ID No.10, respectively; The sequences of CDR1, CDR2, and CDR3 of the heavy chain variable region of the second anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.13, SEQ.ID No.14, and SEQ.ID No.15, respectively, and the sequences of CDR1, CDR2, and CDR3 of the light chain variable region of the second anti-heparin binding protein monoclonal antibody are shown as SEQ.ID No.18, SEQ.ID No.19, and SEQ.ID No.20, respectively.

2. The heparin binding protein immunoassay paired antibody according to claim 1, characterized in that: The sequence of the heavy chain variable region of the first anti-heparin binding protein monoclonal antibody is shown as SEQ.ID No.2, and the sequence of the light chain variable region is shown as SEQ.ID No.

7.

3. The heparin binding protein immunoassay paired antibody according to claim 1, characterized in that: The sequence of the heavy chain variable region of the second anti-heparin binding protein monoclonal antibody is shown as SEQ.ID No.12, and the sequence of the light chain variable region is shown as SEQ.ID No.

17.

4. The heparin binding protein immunoassay paired antibody according to claim 1, characterized in that: The sequence of the heavy chain of the first anti-heparin binding protein monoclonal antibody is shown as SEQ.ID No.1, and the sequence of the light chain is shown as SEQ.ID No.

6.

5. The heparin binding protein immunoassay paired antibody according to claim 1, characterized in that: The sequence of the heavy chain of the second anti-heparin binding protein monoclonal antibody is shown as SEQ.ID No.11, and the sequence of the light chain is shown as SEQ.ID No.

16.

6. An anti-heparin binding protein monoclonal antibody, characterized in that: It can specifically bind to heparin-binding protein, and the sequences of CDR1, CDR2, and CDR3 in its heavy chain variable region are shown in SEQ.ID No.3, SEQ.ID No.4, and SEQ.ID No.5, respectively, and the sequences of CDR1, CDR2, and CDR3 in its light chain variable region are shown in SEQ.ID No.8, SEQ.ID No.9, and SEQ.ID No.10, respectively.

7. An anti-heparin binding protein monoclonal antibody, characterized in that: It can specifically bind to heparin-binding protein, and the sequences of CDR1, CDR2, and CDR3 in its heavy chain variable region are shown in SEQ.ID No.13, SEQ.ID No.14, and SEQ.ID No.15, respectively, and the sequences of CDR1, CDR2, and CDR3 in its light chain variable region are shown in SEQ.ID No.18, SEQ.ID No.19, and SEQ.ID No.20, respectively.

8. A heparin binding protein immunoassay kit, characterized in that: The invention comprises the heparin binding protein immunoassay paired antibody according to any one of claims 1 to 5.

9. The heparin binding protein immunoassay kit according to claim 8, characterized in that: The first anti-heparin binding protein monoclonal antibody is a coating antibody, and the second anti-heparin binding protein monoclonal antibody is a detection antibody.

10. A heparin binding protein immunoassay kit, characterized in that: The invention comprises the anti-heparin binding protein monoclonal antibody according to claim 6 or 7.

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