The sandwich ELISA measures the amount of antigen between two layers of antibodies. The antigens to be measured must contain at least two antigenic sites, capable of binding to antibody, since at least two antibodies act in the sandwich. So sandwich assays are restricted to the quantitation of multivalent antigens such as proteins or polysaccharides. Sandwich ELISAs for quantitation of antigens are especially valuable when the concentration of antigens is low and/or they are contained in high concentrations of contaminating protein.
1. A capture antibody is first diluted in 0.1M Bicarbonate buffer, pH 9.2 and then 50 µl is added to each well of the microtiter plate.
2.The antibody coated plate is covered with Parafin and incubated in the cold room overnight in a moist box containing a wet paper towel or at room temperature and humidity for two hours.
3.The plate is emptied and the unoccupied sites are blocked with 100 µl of blocking buffer containing 100 mM phosphate buffer, pH 7.2, 1% BSA, 0.5% Tween-20 and 0.02% Thimerosol for 30 min at room temperature.
4. The plate is emptied and washed three times with wash buffer(100 mM phosphate buffer, 150 mM NaCl, 0.2% BSA and 0.05% Tween 20).
5. The antigen solution is first diluted in antigen buffer (100 mM phosphate buffer, 150mM NaCl) and then added to the plate in a volume of 50 µl per well. The plate is incubated at room temperature for 45 min to one hour.
6. The plate is emptied again and washed three times with wash buffer.
7. The enzyme-labeled antibody against antigen is diluted appropriately in 0.1M Bicarbonate buffer,pH 9.2 and then 50 µl is added to each well and incubated at room temperature for 30 min.
8. The plate is emptied again and washed three times with wash buffer.
9. The color development system is added and the color intensities are measured.
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