Foot-and-mouth disease type a Wuhan strain monoclonal antibody and its composition and application
A technology of monoclonal antibody and foot-and-mouth disease, which is applied in the direction of anti-viral immunoglobulin, instruments, measuring devices, etc., can solve the problems of inability to distinguish and quantitatively detect, quantitatively detect, and indistinguishable from the foot-and-mouth disease virus
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Embodiment 1
[0045]Example 1. Screening of hybridoma cell lines for monoclonal antibodies of foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain)
[0046]It includes the following steps:
[0047]1) Using sucrose density gradient method to obtain foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain) (provided by China Animal Husbandry Co., Ltd.) 146S antigen, adjust the concentration of the antigen to 10 μg / ml, and use it as an immunogen;
[0048]2) The immunized animals are BALB / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), immunized 4 times in a row with an interval of 14 days, the first 3 times are multi-point subcutaneous immunization, and the fourth time The immunization method of intraperitoneal injection, each mouse is injected with 10 μg of 146S antigen;
[0049]3) Seven days after the last immunization, the mouse tail blood was taken to separate the serum, and then tested by indirect ELISA. After the titer was greater than 1:50000, the spleen ce...
Embodiment 2
[0052]Example 2. Pairing test of monoclonal antibodies against foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain)
[0053]1) Preparation of coated plates with specific monoclonal antibodies of foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain)
[0054]Dilute the purified specific monoclonal antibody with pH 9.6 carbonate solution into 0.5μg / ml, 1μg / ml, 2μg / ml, 4μg / ml coating working solution, and then add to 96-well polystyrase Link reaction plate, 100μl / well, place at 2~8℃ for 8~12 hours to make the specific monoclonal antibody fully bind to the enzyme-linked reaction plate, and then add 300μl / well containing 10mg / ml bovine serum albumin pH7.4 The PBS buffer is sealed at 37°C for 2 to 3 hours. After spinning, the enzyme-linked reaction plate is dried and sealed at 2 to 8°C for storage.
[0055]2) Preparation of horseradish peroxidase-labeled foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain) specific monoclonal antibody
[0056]The specific monoclonal antibody of foo...
Embodiment 3
[0073]Example 3. Gene sequencing of specific hybridoma cell lines of foot-and-mouth disease type A Wuhan strain (A / WH / 09 strain) monoclonal antibody and establishment of monoclonal antibody recombinant expression system
[0074]It includes the following steps:
[0075]1) Total RNA extraction, reverse transcription, PCR and sequence determination of specific positive clone hybridoma cell lines:
[0076]①Total RNA extraction: Take 250μl of hybridoma cell suspension, add 750μl of Trizol, mix upside down, add 200μl of chloroform, mix well, and centrifuge at 12000rpm at 4°C for 15min. Pipette the supernatant into a new 1.5ml EP tube, add 600μl of isopropanol, mix and centrifuge for 10min. The isopropanol was discarded, washed with 75% DEPC ethanol, and centrifuged. The ethanol was discarded, dried, and the RNA was dissolved in 20 μl RNase-free water.
[0077]②Reverse transcription: Use Invitrogen reverse transcription kit to perform reverse transcription according to the instructions to obtain cDNA ...
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