The indirect fluorescent antibody test (IFA) is a semi-quantitative, sensitive, and rapid test to detect specific antibodies or antigens present in the samples.
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IFA for the detection of Antibodies
Indirect fluorescent antibody (IFA) test can be used to detect specific antibodies against various etiological agents present in patient serum and cerebral spinal fluid (CSF) samples.
IFA is being used for the diagnosis of:
Known antigen is immobilized on a glass slide (slides smeared with cells carrying known antigens are commercially available). Test sample (patient serum) is added over the smear. If specific antibodies are present in the serum, the antigen-antibody complex is formed. The serum is washed off and a secondary antihuman immunoglobulin conjugated to a fluorochrome (fluorescein isothiocyanate or rhodamine B) is added. On examination, bacteria will only be visible if they have been bound by the antibodies from the patient’s serum.
Fluorescent dyes; rhodamine B fluoresces red and fluorescein isothiocyanate fluoresces yellow-green.
For example, an IFA test for the diagnosis of syphilis uses Treponema pallidum isolated from a lab animal and a smear is prepared on a glass slide. Patient serum is spread over the smear and anti-treponemal antibodies, if present, are allowed to bind. The serum is washed off and a secondary antibody labeled with fluorescein isothiocyanate (FITC) is added. On examination, the T. pallidum bacteria will only be visible if they have been bound by the antibodies from the patient’s serum.
IFA for the detection of Antigens
Unlike direct fluorescence assay, the immunofluorescence assay for the detection of the antigens is a two-step or sandwich procedure that uses two specific antibodies.
- An unlabeled primary antibody which binds to a specific antigen, and
- FITC labeled anti-species secondary antibody which binds to the primary antibody-antigen complex.
Virus-infected cells or samples are fixed using acetone, methanol, or paraformaldehyde; to preserve cell morphology or tissue architecture. After incubation of the sample with the appropriate antibody (primary antibody), the excess antibody is removed by washing. A secondary antibody labeled with fluorochrome is added and incubated. Again the excess antibody is removed by washing and the smear is visualized using a fluorescence microscope.
Comparison between IFA and DFA
IFA is more sensitive than Direct Fluorescent Assay (DFA) because the second antibody is coupled to the indicator. The second antibody recognizes a common epitope on the first antibody bound with antigen. Multiple second antibodies can bind to the first antibody, leading to an increased signal from the indicator compared to direct immunofluorescence.
The turnaround time of IFA is longer compared to DFA because it has two incubation steps.
References and further readings
- Detecting viral proteins in infected cells or tissues by immunostaining. virology blog. Retrieved June 7, 2020.
- Tille, P. (2017). Bailey & Scott’s Diagnostic Microbiology (14 edition). Mosby.
- Madigan Michael T, Bender, Kelly S, Buckley, Daniel H, Sattley, W. Matthew, & Stahl, David A. (2018). Brock Biology of Microorganisms (15th Edition). Pearson.