Elisa Question Answers
Byakhya Lamichhaney
Immunology lab
03/13/13
Answer the following questions:
1) Describe the mechanism of indirect ELISA. Why is ELISA so sensitive?
The indirect ELISA maintains the following mechanism wherein the antigen which needs to test for first is added on to every well of the microtiter plate. Then a solution which contains the non reactive protein like casein or the bovine serum albumin is introduced to stop any further changes that had not drawn the interest protein, which is called the blocking step. Next the serum is introduced on which is known to contain the antibodies that is special only for the antigen that has been added on originally. This is called secondary antibody and it recognizes and binds to the heavy chain of primary antibody. Then the substrate for the enzymes is added on. Quite often, the substrate changes color on reaction to the enzyme. Finally, if the serum has greater concentration of the basic antibodies, the change in color would also be great. ELISA is so sensitive because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Even a tiny amount of antigen can trigger the color change and see the presence of the antigen.
2) Why is it necessary to block unoccupied binding sites on the microtiter wells?
It is necessary to block unoccupied binding sites on the microtiter wells to reduce nonspecific antibody binding. When the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces the chances of false reading or false results.
3) What can cause a false positive in ELISA? How can you control for this?
Contamination during any of the steps in the experiment will cause a false positive in ELISA and also similar antigens in blood serum also cause these false positive. The control for it would be is to compare it with a known sample both positive and negative.
Immunology lab
03/13/13
Answer the following questions:
1) Describe the mechanism of indirect ELISA. Why is ELISA so sensitive?
The indirect ELISA maintains the following mechanism wherein the antigen which needs to test for first is added on to every well of the microtiter plate. Then a solution which contains the non reactive protein like casein or the bovine serum albumin is introduced to stop any further changes that had not drawn the interest protein, which is called the blocking step. Next the serum is introduced on which is known to contain the antibodies that is special only for the antigen that has been added on originally. This is called secondary antibody and it recognizes and binds to the heavy chain of primary antibody. Then the substrate for the enzymes is added on. Quite often, the substrate changes color on reaction to the enzyme. Finally, if the serum has greater concentration of the basic antibodies, the change in color would also be great. ELISA is so sensitive because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. Even a tiny amount of antigen can trigger the color change and see the presence of the antigen.
2) Why is it necessary to block unoccupied binding sites on the microtiter wells?
It is necessary to block unoccupied binding sites on the microtiter wells to reduce nonspecific antibody binding. When the antibody is added, there is no room on the membrane for it to attach other than on the binding sites of the specific target protein. This reduces the chances of false reading or false results.
3) What can cause a false positive in ELISA? How can you control for this?
Contamination during any of the steps in the experiment will cause a false positive in ELISA and also similar antigens in blood serum also cause these false positive. The control for it would be is to compare it with a known sample both positive and negative.