Lab 3 - Enzyme-Linked ImmunoSorbant Assay

Question 1

antigen

Answer 1

protein or carbohydrate found on an invading pathogen such as a bacterium or virus

Question 2

antibody

Answer 2

y-shaped protein produced by white blood cells that binds specifically to a foreign substance known as an antigen. Antibody targets antigen for destruction by the immune system.

Question 3

aflatoxin

Answer 3

powerful carcinogenic produced by mold Aspergillus

Question 4

primary antibody

Answer 4

a rabbit is injected with aflatoxin repeatedly over several weeks; it then produces anti-aflatoxin primary antibodies that bind to the aflatoxin; these are harvested from the rabbit’s blood

Question 5

secondary antibody

Answer 5

the rabbit antibodies are injected into a goat repeatedly over several weeks; the goat produces antibodies that bind to the rabbit antibodies known as “goat anti-rabbit secondary antibody”; this antibody is harvested from the goat’s blood

Question 6

HRP

Answer 6

the antibody harvested from the goat is attached to an enzyme like horseradish peroxidase; this is “HRP-conjugated goat anti-rabit secondary antibody”

HRP catalyzes a reaction that produces a naturally colored product - IT DOES NOT REQUIRE FLUORESCENT LIGHT

Question 7

What happens during the five minute incubation?

Answer 7

in wells containing antigen, the antigen is adsorbing to the wells

Question 8

adsorption

Answer 8

proteins stick by hydrophobic interactions to the surface of the well

Question 9

Blocking

Answer 9

Detergent in the blocking agent is binding to the hydrophobic plastic surface of the wells. This prevents antibodies in future steps from binding to the wells directly. After rinsing out the antibody, it should only remain in the wells if it stuck to its antigen, not the wells themselves.

Question 10

addition of primary antibody to Positive control well

Answer 10

The positive control well has antigen adsorbed to its surface. The primary antibody is binding to that antigen.

Question 11

addition of primary antibody to Negative control well

Answer 11

In the negative control well, the primary antibody has no antigen to bind to and it can’t bind to the well directly because of the blocking step. It will all be rinsed out in the next step.

Question 12

addition of secondary antibody to the Positive control

Answer 12

the secondary antibody is binding to the primary antibody which, in turn, is bound to the antigen stuck to the well

Question 13

Suppose you skipped the blocking step and used a regular saline solution, not blocking/wash buffer, for all the rinses. Could that have created false negatives or false positives? Would you have detected that error with your positive control or your negative control?

Answer 13

false positive detected by negative control

Question 14

Suppose the antigen did not stick to the wells and all of it got rinsed out. Would that have created false negatives or false positives? Would you have detected the error with your positive control or your negative control?

Answer 14

false negative detected by positive control

Question 15

steps for procedure

Answer 15

• add antigen
• blocking
• rinsing
• primary antibody
• rinsing x 2
• secondary antibody
• rinsing x 2
• substrate - enzyme to cause color change