Fluorescent Antibody Technique (Direct, Indirect)

What is Fluorescent Antibody Technique (Direct, Indirect)?

Test for tissue antigen using either a direct method, by conjugation of antibody with fluorescent dye (fluorescent antibody technique, direct) or an indirect method, by formation of antigen-antibody complex which is then labeled with (fluorescent antibody technique, indirect).

Direct Fluorescent Antibody Test

Principles
Fluorescence microscopy requires a special type of light source, usually a mercury lamp. The light from the lamp passes through special colored filters which only allow light with distinct wavelengths to pass. When this narrow band of light hits the specimen, certain compounds in the specimen (either natural compounds or added fluorescent chemicals) capture the light and reflect it back up as light with a lower energy. This reflected light is detected either with the viewer’s eye, or with sensitive detectors. Because this type of microscopy uses reflected light on a dark background, very small amounts of light (and of your sample) can be seen. Fluorescent compounds include natural compounds such as chlorophyll, as well as certain DNA-binding dyes such as ethidium bromide and DAPI.

The Direct Fluorescent Antibody Test detects the presence of a particular antigen (typically a specific protein on the surface of a virus, bacterium or other microbe).

Fluorescent chemicals are attached to the constant region of an antibody. If the antigen is present, the antibody binds to generate a very specific, very sensitive protein tag.

Direct Fluorescent Antibody Procedure

This antibody must be specific for the organism or protein you are trying to detect.

1. Prepare your sample by fixing it to the slide.

2. Give them time to bind.

3. Rinse off unbound antibody and observe the slide under a fluorescent microscope. If the sample contains the antigen of interest, it will emit light.

Advantages

• Both sensitive and specific (need mono-clonal antibodies)

• Could use on microbes that can’t be easily cultured

• Could label single cells

• Could view cells in natural environment

• Could use different types of fluorescent-labeled antibodies, each with different dye, to see multiple cell types in one sample

Disadvantages

• Cross reactivity may be a problem — often difficult to develop the monoclonal antibody that works well

• Must run careful controls to assure no false positives or negatives

Indirect Fluorescent Antibody (IFA) Assay

IFA is an assay which uses fluorescent microscopy to detect antibodies to specific antigenic material.This test is often used to confirm positive results obtained by ELISA(Enzyme Linked Immunosorbent Assay) or MFIA(Multiplexed Flurometric ImmunoAssay).It is typically used as a confirmation test as the location of antibody-antigen reactions can be visualized within an infectef cell.

Advantages

• Inexpensive to perform

• The morphology and location of fluorescence can be evaluated to differentiate from non-specific reactions

Disadvantages

• Fluorescent microscope is required.

• Due to efficiency limitations, IFA is not primary serological screening tool.

• Interpretation is subjective.

• Results are not quantiative.

• Limited to one antigen per slide.

• Nonspecific fluorescence is common and intensity of fluorescence is common and intensity of fluorescence is variable.

• As with all serological tests, IFA does not detect the infectious organism.  IFA only provides a historical indication of infection (antibodies).

• As they do not produce antibodies, IFA is not suitable for use with immunodeficient animals.

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