ELISA

If the sample gave a negative result for the disease-causing agent, dies this mean that you do not have the disease? What reasons could there be for a negative result when you actually do have the disease?

A negative result does not mean that you do not have the disease. It could be a false negative. The ELISA may not be sensitive enough to detect very low levels of the disease agent, as might occur if one is tested soon after infection before a proper immune response occurs. Another cause of false negatives is experimental error, such as putting a negative control in a well where you thought you were putting an experimental sample.

What are the reagents?

Antigen: Chicken gamma globulin Primary antibody (PA): Polyclonal anti-chicken antibody made by rabbits Secondary antibody (enzyme-linked) SA: Polyclonal anti-rabbit antibody made by goats linked (conjugated) to horseradish peroxidase (HRP) Enzyme substrate (SUB): 3,3',5,5' - tetramethylbenzidine (TMB) - a colorless solution that when oxidized by HRP turns blue

Why did you assay your samples in triplicate?

Assaying the samples in triplicate is another form of control. If you do not get the same result in all three wells you have a problem with your experimental technique or you have made a pipetting error. In a clinical laboratory, the experiment would have to be repeated.

Why do you need to assay positive and negative control samples as well as your own experimental samples?

Controls are needed to make sure the experiment worked. if there are no positive controls and the sample is negative, we can't know if the sample was truly negative or if the assay process didn't work. Conversely, without a negative control, there is no way of knowing if all samples (positive or not) would have given a positive result.

What does ELISA stand for?

Enzyme-linked immunosorbent assay

Why are enzymes used in this immunoassay?

Enzymes provide a way to see whether the primary antibody has attached to its target (antigen) in the microplate well. Primary and secondary antibodies are invisible, so a detection method is necessary. The enzyme HRP is linked to the second antibody. HRP reacts with a colorless substrate in a chemical reaction that turns blue. If the secondary antibody is present in the well, the blue color change indicates a positive result.

If you tested positive for disease exposure during this lab experiment, did you have direct contact with one of the original infected students? If not what conclusions can you reach about transmissibility of disease in a population?

Having intimate contact with another person means that you are exposed to any germs that a person may have contracted from any previous intimate contacts.

When you added primary antibody to the wells, what happened if your sample contained the antigen?

If the sample contained the antigen, the primary antibody bound the antigen

When you added secondary antibody to the wells, what happened if your sample contained the antigen?

If the sample contained the antigen, the secondary antibody bound to the primary antibodies already bound to the antigen in the wells.

When you added primary antibody to the wells, what happened if your sample did not contain the antigen?

If the sample did not contain the antigen, the primary antibody did not bind and wash flushed out in the wash step.

When you added secondary antibody to the wells, what happened if your sample did not contain the antigen?

If the test sample did not contain antigen, primary antibody did not bind to the wells, so the secondary antibody did not have anything to bind to and was flushed out in the wash step.

The samples that you added to the microplate strip contain many proteins and may or may not contain the disease antigen. What happened to the proteins in the plastic well if the sample contained the antigen? What happened if it did not contain the antigen?

In either case, all of the proteins present in the sample bind to the plastic wells.

What antibody-based tests can you buy at your local pharmacy?

Test kits that are based on the same principles as the ELISA include home pregnancy tests, ovulation tests, and tests for illegal drugs like marijuana and cocaine.

Buffer wash

Wash buffer contains phosphate buffer saline (PBS) to keep antibodies in a stable environment that helps keep their structure

Why did you need to wash the wells after each step?

Washing removes any proteins that have not bound to the plastic wells and any antibodies that have not bound to their targets, thus preventing unbound proteins (either antigen or antibodies) from giving false positive results.