Practical Applications of Immunology

16. Describe the direct and indirect fluorescent-antibody techniques and give an example of each.
17. Describe the enzyme-linked immunosorbent assay (ELISA) procedure.

Fluorescent-Antibody Techniques

Fluorescent-antibody techniques use antibodies labeled with fluorescent dyes.

Direct Fluorescent-Antibody Tests

Direct fluorescent-antibody tests are used to identify specific microorganisms (antigens).

Antibodies directed against antigens on the surface of a specific microorganism are labeled with fluorescent dye.

Fluorescent antibodies are incubated with the sample and antigen-specific binding allowed to occur

Excess and non-specifically attached antibodies are washed from the sample

The sample is viewed with a fluorescence viewer, whether a fluorescence microscope or plate reader or even fluorescence-activated flow cytometer

Indirect Fluorescent-Antibody Tests

Indirect fluorescent-antibody tests are used to demonstrate the presence of antibodies against a specific antigen in serum.

Antigen or the microorganism itself is incubated with the patient's serum

Excess serum is washed away, leaving only antibodies specific for the antigen (or antigenic portion of the microorganism) present in the patient's serum bound

The sample (lab stock of antigen or organism) is then incubated with antibodies labeled with fluorescent dye that are specific for human antibodies (fluorescent aniti-human antibody antibodies)

(inject human immunoglobulins into another species and it will produce anti-human immunoglobulin antibodies)

The sample is viewed with a fluorescence viewer, exactly like the direct antibody tests

Flow Cytometry

A fluorescence-activated cell sorter can be used to detect, count, and seperate cells labeled with fluorescent antibodies.

Enzyme-Linked Immunosorbent Assay (ELISA)

ELISA techniques use antibodies linked to an enzyme, such as horseradish peroxidase or alkaline phosphatase.

Antigen – antibody reactions are detected by enzyme activity.
An antibody linked to the indicator enzyme is added to the test well and is bound in the well if the antigen it is specific for is present.
To determine whether or not the enzyme-linked antibody is bound in the well substrate for the enzyme is added.
If the enzyme linked antibody is present the substrate is converted to a product that causes a color change.

Direct

The direct ELISA is used to detect specific antigens bound in a test well.

Say you want to know if a patient's serum contains a specific antigen.

First you coat the bottom of the test well with an antibody against that antigen. Then you add patient serum. If the antigen you're looking for is present in the patient's serum it will stick to the antibody that you coated the bottom of the well with.

So let the serum sit in the well for a few minutes to give the antigen a chance to stick and then wash the excess serum away.

Next you add your enzyme-linked antibody, which is also specific for the antigen - its the same antibody that you coated the bottom of the well with except you linked and enzyme to it. If the antigen is present you'll end up with a sort of antibody-antigen-enzyme-linked antibody sandwich.

You have to wash again to get rid of any enzyme-linked antibody that didn't bind to antigen.

Now add substrate. If there is any enzyme-linked antibody present (and it should only be there if it is bound to antigen) a product will be formed that causes the color change.

Indirect

The indirect ELISA is used to detect antibodies against an antigen bound in a test well.

In this case you want to know if a patient's serum contains an antibody against a specific antigen (the opposite situation from the one in the direct ELISA test).

First coat the bottom of the well with the antigen that the antibody would be specific for.

Next add patient serum and allow it to sit in the well for a few minutes to allow any antibody a chance to bind to the antigen.

Now you have to wash away the serum and any unbound antibodies.

Add your enzyme-linked antibody. This time the enzyme-linked antibody is specific for human immunoglobulins (its an anti-human antibody antibody). Let it sit for a few minutes and wash the excess away. If the patient's serum had antibodies against the antigen you coated the well with you'll end up with an antigen-antibody-enzyme-linked anti-antibody antibody sandwich.

Now add substrate and look for the color change just like in the direct ELISA.

ELISA kits are available for both clinical diagnostics and home use. These tests are used for everything from screening blood for anti-HIV antibodies to home pregnancy tests.

Fluorescence is much easier and more sensitive for lab work but not practical for use outside of a well equipped lab.

Western Blotting (Immunoblotting)

Much like Southern blotting but uses enzyme linked antibodies to detect specific proteins rather than labeled probes to detect specific nucleic acid sequences.

The most common application is in a confirmatory test for HIV.

The Future of Diagnostic and Therapeutic Immunology

Although the use of monoclonal antibodies has made serologic diagnostic tests more sensitive, rapid, and simpler to use, most new diagnostic tests will probably be nonimmunological. PCR and microarrays (DNA chips) can be used for much more rapid screening and can be automated.