Immunofluorescence (IFA) is a traditional laboratory technique that utilizes fluorescent dyes to identify the presence of antibodies bound to specific antigens. The IFA is very useful to detect the serologic response of a patient who has been exposed to certain infectious agents. The two classes of IFA are direct and indirect immunofluorescence. The Imugen laboratory performs an IFA procedure for the detection of IgG antibody to Babesia microti and Ehrlichia chaffeensis.
Direct IFA uses a single fluorophore linked antibody for the direct detection of antigen. Indirect IFA uses a primary antibody which recognizes the antigen and then a secondary fluorophore linked antibody which recognizes the bound primary antibody. Antigen preparation used in IFA can be infected cells, a native cell line, a tissue section, or recombinant antigen proteins.
In the case of IFA testing for the detection of antibody to Babesia microti, each well of a multiple well microscope slide is coated with a preparation of RBC containing B. microti organisms and permanently affixed. Patient serum specimen is prepared and added to a well of a microscope slide. If the patient has been exposed to B. microti and has produced an immune response to this organism, the resulting B. microti specific antibodies will bind to the antigens on the slide. A washing step will remove any unbound patient antibody. A fluorescent dye labeled IgG anti-human antibody is then added and binds to the patient specific anti-Babesia antibody. Bound antibody-antigen complexes will fluoresce when examined microscopically.
The Imugen laboratory utilizes an IFA as part of its B. microti serology testing. Specimens are screened at a single dilution and if positive at this dilution will be serially diluted to an endpoint titer. The final test result is reported as the highest dilution resulting in a positive fluorescent reaction. Certain titers are considered to be diagnostic of exposure and sero-conversion or a four-fold rise in titer is indicative of recent infection.