Principle and Protocol of Enzyme-linked Immunosorbent Assay (ELISA)

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

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