Introduction of Direct Elisa Protocol
ELISA is a highly sensitive test technique based on immunological reactions that combine the specific reaction of antigens and antibodies with the efficient catalytic action of enzymes on substrates. Since the reaction of the antigen and the antibody is carried out in the well of a solid phase carrier-polystyrene microtiter plate, after each reagent is added, the excess free reactant can be removed by washing to ensure the specificity and stability of the test result. In ELISA, direct ELISA is useful for qualitative or quantitative antigen detection in a sample, antibody screening, and epitope mapping since only one antibody is involved.
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Bicarbonate/carbonate coating buffer (100 mM)
Antigen or antibody should be diluted in coating buffer to immobilize them to the wells:
3.03 g Na2CO3,
6.0 g NaHCO3
1000 ml distilled water
1.16 g Na2HPO4,
0.1 g KCl,
0.1 g K3PO4,
4.0 g NaCl (500 ml distilled water) pH 7.4.
Commonly used blocking agents are 1% BSA, serum, non-fat dry milk, casein, gelatin in PBS.
Usually PBS or Tris-buffered saline (pH 7.4) with detergent such as 0.05% (v/v) Tween20 (TBST).
Antibody dilution buffer
Primary and secondary antibody should be diluted in 1x blocking solution to reduce non-specific binding.
Coating antigen to microplate
Test samples containing pure antigen are usually pipeted onto the plate at less than 2ug/ml. Pure solutions are not essential, but as a guideline, over 3% of the protein in the test sample should be the target protein. (antigen). Antigen protein concentration should not be over 20ug/ml as this will saturate most of the available sites on the microtitre plate.
Add 150 µl of blocking solution to each well. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or overnight at 4°C. Wash 4 times in wash buffer. The process blocks the remaining protein-binding sites in the coated wells.
Incubation with the antibody
Analysis of data
Prepare a standard curve from the data produced from the serial dilutions with concentration on the x axis (log scale) vs absorbance on the Y axis (linear). Read the concentration of the sample from this standard curve by the OD of sample.