Difference between Direct ELISA and Indirect ELISA
Direct ELISA
1. Well coated with Ab
2. Washed with buffer to remove free or excess Ab
3. Test solution with Ag added to well and incubated at 37 degree Celsius for 2 hour's. If Ag corresponds to Ab, Ag- Ab complex is formed.
4. Well washed to remove any free or unbound Ag
5. Enzyme linked second antibody is added and incubated at 37 degree Celsius for 1 hours.
6. Wells washed to remove excess enzyme linked second Ab
7. Substrate added
8. Substrate hydrolyzed to colored product
9. Color intensity measured by spectrophotometer.
Indirect ELISA
1. Well coated with Ag
2. Washed with buffer to remove free or excess Ag
3. Test serum added to well and incubated at 37 degree Celsius for 2 hour's. If Ag and Ab correspond to each -other.
Ag-Ab complex is formed.
4. Well washed to remove any free or unbound Ab
5. Enzyme linked second Ab is added and incubated at 37 degree Celsius for 1 hours.
6. Wells washed to remove excess enzyme linked second Ab
7. Substrate added
8. Substrate hydrolyzed to colored product.
9. Colour intensity measured by spectrophotometer.
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