Monoclonal antibody for resisting to cyprinus carpio haematopterus immune globulin (IgM) and application of monoclonal antibody
A monoclonal antibody and immunoglobulin technology, applied in the biological field, can solve problems such as single method, false negative, difficult quantitative detection and diagnosis of the disease
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Embodiment 1
[0034] Embodiment 1 Preparation and purification of anti-koi immunoglobulin IGM monoclonal antibody
[0035] Materials and Methods
[0036] Experimental Materials
[0037] The test koi came from the Pearl River Fisheries Research Institute of the Chinese Academy of Fishery Sciences, with a body length of 20 cm; other chemical reagents were of analytical grade, provided by Sinopharm Chemical Reagent Co., Ltd.
[0038] 1 Koi IgM purification
[0039] 1.1 Take the blood of koi carp by docking the tail, put it in a glass tube, solidify it at room temperature, place it at 37°C for 2 hours, then place it at 4°C overnight, after the serum is fully analyzed, centrifuge at 3000g / min for 10min, and take the supernatant .
[0040] 1.2 Purification: Centrifuge the fish serum at 12000rpm for 20min, take the supernatant; measure the volume of the supernatant; slowly add saturated ammonium sulfate solution to the supernatant while stirring, until the final saturation is 33%; put the solut...
Embodiment 2
[0070] Example 2 Establishment of Indirect ELISA Detection Method for Koi Herpes Virus
[0071] 1. Determination of antigen working concentration
[0072] Dilute the purified koi herpes virus with coating solution (pH8.6) to a concentration gradient of 50 μg / mL, 30 μg / mL, and 10 μg / mL, coat a 96-well microtiter plate, add 100 uL to each well, and pack in a wet box at 4 °C be overnight. Add 300 μL of 10% skim milk to each well to block, incubate in a 37°C incubator for 1 h, and wash with PBST 3 times, each time for 3-5 min. Dilute anti-KHV positive serum and koi negative serum 1:300 times, add 100uL to each well for ELISA square test, incubate in 37°C incubator for 1h, wash with PBST 3 times, each 3-5min. The above prepared mouse anti-koi IgM antibody 1:2000, 100uL per well, incubated in a 37°C incubator for 1h, washed 3 times with PBST, 3-5min each time. Add 50 μL chromogenic substrate (TMB), and develop color at 37°C in the dark for 10 minutes. With 50μL 2mol / LH 2 SO 4 ...
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