Heparin-binding protein immunoassay pair of antibodies and use thereof

Abstract

The application provides a heparin-binding protein immunodetection paired antibody and application thereof, and 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 the 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, the sequences of CDR1, CDR2 and CDR3 of the 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 CDR1, CDR2 and 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 CDR1, CDR2 and 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] This invention belongs to the field of bioimmunology technology, and in particular relates to a heparin-binding protein immunoassay paired antibody and its application. Background Technology

[0002] Heparin-binding protein (HBP) is a granular glycoprotein secreted by mature neutrophils. It has an extremely strong heparin-binding capacity, hence its name. Mature HBP consists of 222 amino acids, with a relative molecular weight of 37,000. It carries a positive charge and has bactericidal properties, therefore it is also known as CAP37 (cationic antimicrobial protein of 37,000). HBP is also known as azurocidin.

[0003] HBP can act on vascular endothelial cells, increasing their permeability, and can also activate monocytes / macrophages, amplifying the inflammatory response. In healthy individuals, the level of HBP in the blood is very low, generally less than 10 ng / mL. When an infection occurs, the level of HBP in the blood will rise, typically reaching 20-30 ng / mL during infection; in severe infections, it can even exceed 100 ng / mL.

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

[0005] HBP monoclonal antibodies are the core raw material of HBP immunoassay reagents, determining their performance. Currently, most HBP monoclonal antibodies suffer from narrow detection ranges and low sensitivity, necessitating the development of high-performance HBP monoclonal antibodies. Summary of the Invention

[0006] This invention provides a paired antibody for heparin-binding protein immunoassay and its application, which can effectively solve the above-mentioned problems.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a heparin-binding protein immunoassay pairing antibody, wherein the heparin-binding protein immunoassay pairing 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 in 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 in 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 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 of the second anti-heparin-binding protein monoclonal antibody are shown in SEQ.ID No.18, SEQ.ID No.19, and SEQ.ID No.20, respectively.

[0011] Secondly, the present invention provides a monoclonal antibody against heparin-binding protein, which can specifically bind to heparin-binding protein. The sequences of CDR1, CDR2, and CDR3 of the 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 of the light chain variable region are shown in SEQ.ID No.8, SEQ.ID No.9, and SEQ.ID No.10, respectively.

[0012] Thirdly, the present invention provides an anti-heparin-binding protein monoclonal antibody that specifically binds to heparin-binding protein. 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.ID No.19, and SEQ.ID No.20, respectively.

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

[0014] Fifthly, 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 this invention are:

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

[0017] This invention provides a heparin-binding protein immunoassay kit, comprising heparin-binding protein immunoassay paired antibodies, 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. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is an SDS-PAGE protein gel electrophoresis image of HBP antigen expressed in 293F cells according to an embodiment of the present invention.

[0020] Figure 2 This is a linear graph showing the activity of paired antibodies Ab1 and Ab2 detected by ELISA in an embodiment of the present invention.

[0021] Figure 3 This is a linear graph showing the detection of clinical samples using the heparin-binding protein immunoassay kit according to an embodiment of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Reagent sources in the embodiments of this invention:

[0024] 293F cells were obtained from Xiamen University;

[0025] The feeding medium was OPM293 ProFeed medium, purchased from Shanghai OPM 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 Wu's Animal Experiment Center in Fuzhou.

[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 obtained from Xiamen University;

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

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

[0035] Example 1

[0036] Preparation of HBP monoclonal antibodies

[0037] 1. Preparation of HBP recombinant antigen

[0038] The amino acid sequence encoding the mature HBP protein was obtained from NCBI: NP_001691. Adding a His tag to the C-terminus yielded the amino acid sequence shown in SEQ ID No. 21.

[0039] SEQ ID No. 21:

[0040] IVGGRKARPRQFPFLASIQNQGRHFCGGALIHARFVMTAASCFQSQNPGVSTVVLGAYDLRRRERQSRQTFSISSMSENGYDPQQNLNDLMLLQLDREANLTSSVTILPLPLQN ATVEAGTRCQVAGWGSQRSGGRLSRFPRFVNVTVTPEDQCRPNNVCTGVLTRRGGICNGDGGTPLVCEGLAHGVASFSLGPCGRGPDFFTRVALFRDWIDGVLNNPGPHHHHHHH

[0041] The nucleotide sequence was optimized according to the codon preferences of the human genome, and the resulting gene sequence is shown in SEQ ID No. 22. The sequence was then synthesized into the pTT5 vector by Genewiz in Suzhou.

[0042] SEQ ID No. 22:

[0043] ATTGTGGGAGGAAGAAAAGCTAGACCCAGACAGTTCCCGTTCCTGGCATCTATTCAGAACCAGGGGCGACACTTCTGCGGGGGCGCCTTGATCCATGCAAGGTTCGTGATGACGGCGGCCTCATGTTTCCAATCCCAGAACCCCGGTGTGAGCACAGTCGTTCTTGGAGCA TACGATCTGCGCCGCCGGGAACGGCAAAGCAGGCAGACATTCAGCATCAGCTCCATGTCCGAGAACGGTTACGACCCCCAACAGAATCTCAACGATCTGATGCTGCTGCAACTGGACCGGGAAGCTAACCTGACCTCCTCAGTGACTATCCTCCCGCTTCCCCTGCAGAAT GCTACTGTGGAAGCCGGTACGCGATGTCAGGTGGCTGGTTGGGGATCACAGCGCAGTGGAGGACGACTTAGCCGGTTCCCGAGGTTTGTCAATGTGACAGTGACGCCAGAGGATCAGTGCCGGCCAAACAACGTGTGCACCGGCGTCTTGACCCGGAGGGGGGGGATATGC AACGGGGACGGGGGGACACCTCTGGTGTGTGAAGGCTTGGCCCACGGGGTCGCTAGCTTCTCCCTTGGCCCCTGCGGCCGAGGACCCGATTTCTTCACTCGCGTGGCCCTCTTTCGGGACTGGATAGATGGCGTGCTCAACAATCCCGGCCCTCACCACCACCACCACCAC

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

[0045] After equilibrating the NiFF column with Buffer A (20 mM PB, pH 7.4), the cell supernatant was loaded onto the NiFF column for purification. After loading, the column was washed with Buffer A for equilibration, eluted with Buffer B (20 mM PB, 10 mM imidazole, pH 7.4) to remove contaminating proteins, and eluted with Buffer C (20 mM PB, 200 mM imidazole, pH 7.4) to remove the target protein. The purified HBP recombinant protein was then ultrafiltered, concentrated, and stored in pH 7.4 PBS, diluted to 1 mg / mL. The purification results are shown below. Figure 1 As shown.

[0046] 2. Mouse immunization

[0047] The HBP recombinant antigen was emulsified with an equal volume of Freund's complete adjuvant and homogenized. 200 μg / mouse was injected subcutaneously at multiple sites in 6-8 week old SPF-grade Balb / c mice. Two weeks later, 100 μg / mouse was injected subcutaneously at multiple sites in an equal volume of Freund's incomplete adjuvant to emulsify the antigen. Two booster immunizations were given. Three days before fusion, 100 μg / mouse was injected intraperitoneally for shock treatment.

[0048] 3. Preparation of feeder cells

[0049] Balb / c mouse peritoneal macrophages were used as feeder cells. One day before cell fusion, mice were euthanized by cervical dislocation, immersed in 75% alcohol, and under sterile conditions in a laminar flow hood. The abdominal skin was cut open to expose the peritoneum, and 5 mL of RPMI 1640 basal culture medium was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, and the rinsing fluid was collected. The cells were centrifuged at 1000 rpm for 5 min, and the pellet was resuspended in RPMI 1640 complete culture medium containing 1% HAT. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Add 150 μL / well to a 96-well cell culture plate and incubate overnight at 37°C with 5% CO2.

[0050] 4. Preparation of immune spleen cells

[0051] Three days after the last immunization of the mice, the spleens were removed under aseptic conditions, placed in a petri dish, and rinsed once with RPMI 1640 basal culture medium. The rinsed spleens were then ground and filtered through a nylon mesh placed in a small beaker to prepare a cell suspension. The suspension was centrifuged, the supernatant discarded, and the cells resuspended in RPMI 1640 basal culture medium. This process was repeated three times to obtain a cell 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) Take mouse myeloma cells Sp2 / 0 (2×10⁻⁶) respectively 7 (10) and the above suspension (1 × 10 immune spleen cells) 8 Add the sample (number of samples) to a 50 mL centrifuge tube and mix well. Add DMEM serum-free culture medium to a final volume of 40 mL. Centrifuge for 10 min, discard the supernatant, and mix well.

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

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

[0057] 6. Selection, culture, and clone screening of hybridoma cells

[0058] Cells were cultured in HAT medium containing 15%-20% fetal bovine serum on days 1, 3, 5, and 7 after fusion. Positive wells that produced the target antibody were selected, and three rounds of subcloning were performed to screen for monoclonal cell lines that specifically recognized the HBP antigen. Finally, 15 hybridoma monoclonal cell lines were selected.

[0059] 7. Expression and purification of ascites fluid

[0060] Balb / c mice were injected intraperitoneally with 0.5 mL of liquid paraffin. Ten days later, the selected hybridoma cell lines were inoculated into the peritoneal cavity of the mice. After about 10 days, the mice's abdomens began to swell. The mice were then euthanized by cervical dislocation, disinfected by immersion in 75% alcohol for 5 minutes, and ascites fluid was extracted once. The ascites fluid was purified using a Protein A affinity column to obtain monoclonal antibodies.

[0061] Example 2

[0062] ELISA method for screening paired antibodies

[0063] (1) Dilute the coating antibody with 20 mM PB, pH 7.4 coating buffer to 1 μg / mL, add 100 μL to each well of a 96-well microplate (i.e., 100 ng / well), and incubate overnight at 4°C.

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

[0065] (3) Remove the liquid from 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) Remove the liquid from 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 with PBST), and incubate at 37°C for 30 min.

[0067] (5) Remove the liquid from the wells, wash each well 5 times with 300 μL of washing buffer, add 100 μL of colorimetric solution, and develop the color at 37℃ for 10 min. Then add 50 μL of 2 mol / L H2SO4 stop solution. Within 20 min after adding the stop solution, read the OD value using a microplate reader. 450 Numerical value.

[0068] The obtained monoclonal antibodies were paired up and used as coating antibodies and detection antibodies, respectively. The diluted HBP recombinant antigen samples were detected by double sandwich ELISA. Finally, a suitable pair of 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 As shown.

[0070] Table 1

[0071]

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

[0073] Antibody Ab1:

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

[0075] SEQ ID No. 1:

[0076] QVQLQDSGAQLVKPGISVKLSCRASGYTFTEYTNHTWVKQAMGRSLEWLGTITESGGYTDYEQKTKKSTLTADRSSSVTYIQLGSLTSEDTAFYYCARDYEGVSLTYWGQGTLLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0077] The heavy chain variable region sequence is shown in SEQ.ID No.2.

[0078] SEQ.ID No.2:

[0079] QVQLQDSGAQLVKPGISVKLSCRASGYTFTEYTNHTWVKQAMGRSLEWLGTITESGGYTDYEQKTKKSTLTADRSSSVTYIQLGSLTSEDTAFYYCARDYEGVSLTYWGQGTLLTVSS

[0080] The heavy chain CDR1 sequence is shown in SEQ.ID No.3.

[0081] SEQ.ID No.3:

[0082] EYTNHT

[0083] The heavy chain CDR2 sequence is shown in SEQ.ID No.4.

[0084] SEQ.ID No.4:

[0085] TITESGGYTDYEQKTK

[0086] The heavy chain CDR3 sequence is shown in SEQ.ID No.5.

[0087] SEQ ID No. 5:

[0088] DYEGVSLTY

[0089] The light chain sequence is shown in SEQ ID No. 6.

[0090] SEQ ID No. 6:

[0091] DIVLTQSPSLMSASPGERVTISCRASEDYNYVMAWYQQKPGQSPRLWIYDASNIYSGVPERFSGSGSGSSYTLTISRVEAEDVASYYCEQKTHSPKTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0092] The light chain variable region sequence is shown in SEQ ID No. 7.

[0093] SEQ ID No. 7:

[0094] DIVLTQSPSLMSASPGERVTISCRASEDYNYVMAWYQQKPGQSPRLWIYDASNIYSGVPERFSGSGSGSSYTLTISRVEAEDVASYYCEQKTHSPKTFGGGTKLEIK

[0095] The light chain CDR1 sequence is shown in SEQ ID No. 8.

[0096] SEQ ID No. 8:

[0097] RASEDYNYVMA

[0098] The light chain CDR2 sequence is shown in SEQ ID No. 9.

[0099] SEQ ID No. 9:

[0100] DASNIYS

[0101] The light chain CDR3 sequence is shown in SEQ ID No. 10.

[0102] SEQ ID No. 10:

[0103] EQKTHSPKT

[0104] Antibody Ab2:

[0105] The heavy chain sequence is shown in SEQ ID No. 11.

[0106] SEQ ID No. 11:

[0107] EVQLQESGAQLVKPGISLKLSCAASGYTFTEYSENHWIKQAPGRSLEWVGTLSETGGYTDTEQRTKRSTLTRDRSSSVTYIQLGSLRSEDTAVYYCARETESVSLTWWGQ GTLLTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0108] The heavy chain variable region sequence is shown in SEQ ID No. 12.

[0109] SEQ ID No. 12:

[0110] EVQLQESGAQLVKPGISLKLSCAASGYTFTEYSENHWIKQAPGRSLEWVGTLSETGGYTDTEQRTKRSTTLDRSSSVTYIQLGSLRSEDTAVYYCARETESVSLTWWGQGTLLTVSA

[0111] The heavy chain CDR1 sequence is shown in SEQ ID No. 13.

[0112] SEQ ID No. 13:

[0113] EYSENH

[0114] The heavy chain CDR2 sequence is shown in SEQ ID No. 14.

[0115] SEQ ID No. 14:

[0116] TLSETGGYTDTEQRTK

[0117] The heavy chain CDR3 sequence is shown in SEQ ID No. 15.

[0118] SEQ ID No. 15:

[0119] ETESVSLTW

[0120] The light chain sequence is shown in SEQ ID No. 16.

[0121] SEQ ID No. 16:

[0122] DVLLTQSPSLMSASPGERVTITCKASEDTDYLVAWFDQKPGTSPRLIIYEASNLTSGVPDRFTGSGSGSYTTLKISRMEAEDVAVYYCQQRTHSPRTFGAGTKLEVK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0123] The light chain variable region sequence is shown in SEQ ID No. 17.

[0124] SEQ ID No. 17:

[0125] DVLLTQSPSLMSASPGERVTITCKASEDTDYLVAWFDQKPGTSPRLIIYEASNLTSGVPDRFTGSGSGSYTTLKISRMEAEDVAVYYCQQRTHSPRTFGAGTKLEVK

[0126] The light chain CDR1 sequence is shown in SEQ ID No. 18.

[0127] SEQ ID No. 18:

[0128] KASEDTDYLVA

[0129] The light chain CDR2 sequence is shown in SEQ ID No. 19.

[0130] SEQ ID No. 19:

[0131] EASNLTS

[0132] The light chain CDR3 sequence is shown in SEQ ID No. 20.

[0133] SEQ ID No. 20:

[0134] QQRTHSPRT

[0135] Example 3

[0136] Paired antibodies Ab1 and Ab2 are used in an immunofluorescence kit.

[0137] Ab1 antibody and HBP recombinant antigen were streaked onto nitrocellulose membranes at doses of 0.04 μg / cm membrane and 0.09 μg / cm membrane, respectively, and dried at 55°C for 3 days. Simultaneously, Ab2 antibody was conjugated with fluorescent microspheres and sprayed onto the sample pad at a dose of 0.02 μg antibody / cm binding pad. The sample pads were then vacuum dried for 3 hours to assemble the reagent strips.

[0138] Subsequently, in a normal temperature and humidity environment (25℃, 50%-60% humidity), 130 μL of sample diluent was added to the sample pad of the test strip. Then, clinical samples of different concentrations were added to the sample pad of the test strip, with a volume of 15 μL. The fluorescence values ​​were then read, and the results are shown in Table 2. Figure 3 As shown.

[0139] The sample buffer comprises the following components by mass fraction: HEPES 1.5%, perfluorooctanoic acid 2.0%, sodium chloride 2.0%, S9 1.0%, with a pH of 7.0.

[0140] The concentration values ​​of clinical samples were calibrated using the Zhonghanshengtai Heparin Binding Protein Detection Kit.

[0141] Table 2

[0142]

[0143] From Table 2 and Figure 3 It can be seen that the correlation R between the fluorescence value detected by the reagent strip and the concentration value of the clinical sample is [missing information]. 2 =0.9972, the detection sensitivity can reach 0.1 ng / mL, and the linear detection range can reach 0.1-723.1 ng / mL.

[0144] The Ab1 and Ab2 antibodies provided in this invention can also be paired with other anti-heparin-binding protein monoclonal antibodies for use in other heparin-binding protein immunoassay kits.

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A paired antibody for the immunoassay of heparin-binding protein, 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 in 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 in 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 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 of the second anti-heparin-binding protein monoclonal antibody are shown in SEQ.ID No.18, SEQ.ID No.19, and SEQ.ID No.20, respectively.

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

7.

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

17.

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

6.

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

16.

6. A heparin-binding protein immunoassay kit, characterized in that, Includes the heparin-binding protein immunoassay paired antibody as described in any one of claims 1-5.

7. The heparin-binding protein immunoassay kit as described in claim 6, 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.

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