Hybridoma cells capable of secreting anti-h-fabp monoclonal antibody, monoclonal antibody, preparation method and application thereof
A monoclonal antibody and hybridoma cell technology, applied in the biological field, can solve the problems of unstable performance, long time-consuming ELISA detection, low concentration of H-FAPB, etc., and achieve high specificity, valuable treatment time, and high affinity.
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Embodiment 1
[0054] Establishment of hybridoma cell line and preparation of anti-H-FABP monoclonal antibody.
[0055] 1. Antigen immunity.
[0056] Mix the recombinant heart-type fatty acid binding protein (H-FABP) antigen (1.2mg / mL, produced by Shenzhen Feipeng Biological Co., Ltd., product name: K1-FABP) with complete Freund's adjuvant (SIGMA, F5881) in equal volumes , to obtain an oily emulsion. The emulsion was subcutaneously administered to the back site of BALB / c mice (Guangdong Provincial Medical Experimental Animal Center: No. 119, Poyang Road, Huangqi, Nanhai, Foshan City, Guangdong Province, 6-week-old female, 5) with a dose of 0.2 ml each. Fourteen days after the first immunization, intraperitoneal booster immunization (antigen mixed with incomplete Freund's adjuvant (SIGMA, F5506) in equal volumes), after booster immunization to four injections, tail blood was collected for titer detection, and the titer reached the fusion requirement.
[0057] Three days before the fusion, t...
Embodiment 2
[0078] Preparation of H-FABP detection test paper.
[0079] 1. Preparation of nitrocellulose membrane.
[0080] Coating buffer preparation: 0.01M PBS buffer solution containing 6% methanol with a pH of 7.2 is the coating buffer, filtered through a 0.22μ membrane, placed at 4°C for later use, and valid for one week. 1000mL 0.01M pH 7.2 PBS buffer solution of 6% methanol Recipe: NaCL 8g, KCL 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60mL, distilled deionized water to 1000mL.
[0081] Preparation of nitrocellulose membrane: Dilute the anti-heart fatty acid binding protein (H-FABP) monoclonal antibody 1F38 to 1 mg / mL-5 mg / mL with coating buffer, adjust the machine, and draw the line as T1 line, and the T1 line is For the detection line, the T1 line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad. Dilute the goat anti-mouse IgG antibody (manufactured by Shenzhen Feipeng Biological Co., Ltd., product name: go...
Embodiment 3
[0106] H-FABP detection kit
[0107] 1. H-FABP detection kit includes:
[0108] ①A pack of test strips (10 strips / pack)
[0109] ②One bottle of sample diluent (10mL / bottle)
[0110] Preparation of relevant solutions.
[0111] Sample diluent: The sample diluent is 8% NaCl solution. Preparation method: 80gNaCl, add distilled water to make up to 1000mL.
[0112] 2. Detection of H-FABP content by colloidal gold method
[0113] (1) Directly put 20 μL of collected venous whole blood into a plastic test tube containing 180 μL of diluent, mix well, take 120 μL of dissolved sample and add it to the sample hole of the test paper card, wait for 15 minutes and then observe the result.
[0114] (2) Judgment of results: When the test strip has a purple-red quality control line visible to the naked eye, there is no visible purple-red detection line (see Figure 4 ), the result was negative. When the test strip shows a purple-red quality control line visible to the naked eye, the first t...
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