Uncategorized Wednesday, 2015/11/25
The development of ELISA is based on the immobilization and enzyme labeling of antigen or antibody. The immunological activity of antigen or antibody remains after the immobilization. And enzyme-labeled antigen or antibody remains both their immunological activity and enzymatic activity. Materials: Antigen Serum Reagent and kit: Carbonate coating buffer PBST BSA Equipment and Supplies: 96-Well ELISA plates 4℃refrigerator Incubator Spectrophotometer Procedure: (1) Coating antigen 1. Dilute the antigen into 10-20μg/ml with 50 mM carbonate coating buffer (pH9.6) ; add 100μl per well to the 96-Well ELISA plate; incubate at 4 ℃ overnight; 2. Discard the supernatant; wash with PBST for three times; add 150 μl 1％ BSA to each well and block for 1 hour at 37℃; 3. Wash with PBST for three times; add 100 μl serum of gradient concentration and also add the control; incubate at 37℃ for 2 hours; 4. Wash with PBST for five times; add 100μl diluted HRP-labeled secondary antibody; incubate at 37℃ for 1 hour; 5. Wash with PBST for five times; add chromogenic reagents to react protecting from light; measure OD at 450 nm with a microplate reader 20 min later. (2) Coating cell 1. Seed the cell into 96-well plate at an initial concentration of 1 x 104 cells per well and culture at 37℃ overnight; 2. Wash the plates with PBS 2-3 times the next day; 3. Add 125 μl/well 10% Formalin (1:10 dilution); fix at room temperature for 15 min; 4. Wash the plates with ddH2O for three times and dry it; store at 2-8 ℃ for spare; 5. Wash with PBST for three times; add 150μl 1% BSA to each well and incubate for 1 hour; 6. Wash with PBST for three times; add 100 μl serum of gradient concentration and also add the control; incubate at 37℃ for 2 hours; 7. Wash with PBST for five times; add 100μl diluted HRP-labeled secondary antibody; incubate at 37℃ for 1 hour; 8. Wash with PBST for five times; add chromogenic reagents to react protecting from light.
Note One: 50mM carbonate coating buffer: 0.05mol/L carbonate buffer (pH9.6), store at 4℃; Na2CO3 15g, NaHCO3 0.293g; dilute to 100ml with distilled water. Note Two: ABTS substrate in color reaction (10ml): 0.2M Na2HPO4 2.4ml 0.1M citrate 2.6ml ddH2O 5ml ABTS 5mg H2O2(30%) 4 ul (added before use)
ELISA used in Creative BioMart: Enzyme/Antibody conjugation Host Cell Protein Mitigation Epitope Mapping Pharmaceutical Stability Analysis Biopharmaceutical Process and Product Related Impurities Analysis