Indirect Fluorescent Antibody (IFA) Test

The indirect fluorescent antibody test (IFA) is a semi-quantitative, sensitive, and rapid test to detect specific antibodies or antigens present in the samples.

Fluorescent antibody methods
Direct and indirect immunofluorescence for the detection of antigen
(Image source: Brock Biology of Microorganisms)

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:

  1. Rabies
  2. Syphilis
  3. Toxoplasmosis
  4. Leishmaniasis
  5. Legionellosis


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.

IFA for antibodies detection
IFA for antibodies detection

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.

  1. An unlabeled primary antibody which binds to a specific antigen, and
  2. 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.  

DFA and IFA for the detection of viral antigen
DFA and IFA for the detection of viral antigen

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

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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