Question

Please answer the following questions about the lab:

1) Explain each step of a Direct ELISA

2) What is the importance of using blocking buffers?

3) Explain each step of a Competitive ELISA?

4) Explain what separates this from a Direct ELISA?

Answer 1

(1)Direct Elisa is one wherein there is only one set of antigens and one set of antibodies to react. In this ELISA method, antigens from the patient sample fixed to the Elisa plates are made to react with an antibodies sample which is tagged to a marker enzyme. I.e. directly to the antigen in the test an enzyme-linked antibody is added to produce a color reaction with externally added substrate i.e. Elisa reagent.

Ag or Ab + Ab or Ag-(e) −−−−−→ Reaction color.

(2)A blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate that is not occupied by the coated protein. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether, without altering or obscuring the epitope for antibody binding.

(3)competative ELISA

Procedure

1. Antibody is incubated with sample containing antigen.
2. Antigen-antibody complex are added to the microtitre well which are pre-coated with the antigen.
3. Wash the plate to remove unbound antibody.
4. Enzyme linked secondary antibody which is specific to the primary antibody is added.
5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
6. Add substrate which is converted by the enzyme into a fluorescent signal.

(4)

The difference between indirect and direct elisa

Indirect and direct elisa are both typically performed in 96-well polystyrene plates or 8-well strip tubes of the same material. The polystyrene is used because proteins bind well to the surface. The 96 well plates are used to ensure that plenty of space is available for determining optimum dilution for the reaction, creating standard curves, and running samples in quadruplicate or more. The 8-well strips are available for finely-tuned elisa processes where a 96-well plate seems excessive.

Indirect and direct elisa have antigen bound directly to the polystyrene well. These assays differ only in the detection antibody used. In a direct elisa only one antibody is used—this single antibody is conjugated directly to the detection enzyme. The indirect elisa requires two antibodies—a primary antibody and an enzyme-linked secondary antibody that is complementary to the primary antibody. Aside from this difference, the two protocols are similar in that a substrate is added to the well to be acted on by the detection enzyme to produce a color change, a fluorescence, or a light emission (chemiluminescence).