Fluorescein-labeled anti-human globulins were examined to determine the need for standardization of conjugates used in the fluorescent treponemal antibody-absorption (FTA-ABS) test. Twenty-one of 33 conjugates submitted by commercial manufacturers to the Reagents Control Activity, Venereal Disease Research Laboratory, for evaluation in the FTA-ABS test were available for study. Conjugates, after evaluation in FTA-ABS performance tests, were examined by immunoelectrophoresis, by titration against immunoglobulins G and M (IgG, IgM) with FTA-ABS techniques, and by the biuret protein and fluorescein diacetate methods for determining fluorescein to protein (F/P) ratios. The conjugates were predominately anti-IgG globulin with anti-light-chain activity. Differences were noted in the ability of some conjugates to detect IgM antibody. The F/P ratios of those conjugates that could be determined varied from 2.6 to 17.8 μg of fluorescein per mg of protein. The need to identify and standardize both the immunologic capabilities and the optimum F/P ratio for FTA-ABS test conjugates is presented.

The fluorescent treponemal antibody-absorption double-staining step-by-step procedure and proposed reference reagents for the test are described. The test and reagents were evaluated in two separate laboratories on 265 fresh sera, and test results were compared with the reference fluorescent treponemal antibody-absorption test results performed in a third laboratory. The data indicate that the tests are comparable in the areas where the test is recommended for use. Problems with inadequate light filtration occurred, but these could be resolved. This test is recommended for use with microscopes equipped with incident illumination.

A double-staining procedure for the fluorescent treponemal antibody-absorption test, using fluorescein isothiocyanate as a label for the class-specific anti-human globulin and tetramethylrhodamine isothiocyanate as a label for a counterstain reagent, has been described. This method requires the addition of a KP560 barrier filter, with a microscope equipped with vertical illumination, to exclude the rhodamine emission in reading the fluorescein fluorescence. The present study evaluated reversing the dye label for each conjugate in the double-staining procedure, thus eliminating the need for the KP560 filter. It also considered the possibility of shortening the counterstaining time and compared various methods for preparing antigen slides in an attempt to establish a method that increases the number of treponemes per microscopic field. The results indicate that a rhodamine-labeled class-specific anti-human globulin as a primary stain, and a fluorescein-labeled anti-treponemal globulin as a counterstain, provide an acceptable method for performing the fluorescent treponemal antibody-absorption double-staining procedure. Nonfixed antigen slides were held for 16 days in a desiccator or stored in plastic bags with silica gel for 3 weeks; then, with methanol fixation, they were used satisfactorily in the double-staining procedure. A shortened incubation time for the counterstain allowed more rapid slide processing.

Immunofluorescent staining of Treponema pallidum was studied to clarify the effect of three factors on the results of the fluorescent treponemal antibody-absorption test: (i) heat inactivation of sera at 56 degrees C for 30 min before testing, (ii) use of multicircle slides, and (iii) tungsten illumination to visualize and assess unstained treponemes on reactive as well as nonreactive smears. It was found that serum inactivation before testing was not necessary for detection of immunoglobin G antibody, but an immunoglobulin M prozone was detected in unheated serum. On multicircle slides, it was demonstrated that a false-positive reaction could be obtained in 30 s at 37 and 25 degrees C if a smear where a nonreactive serum had been placed was crossed by a strongly reactive serum from another circle. Tungsten illumination proved necessary for correct assessment of unstained treponemes on all fluorescent treponemal antibody-aborption test smears, reactive or nonreactive. The possible role of these factors in incorrect fluorescent treponemal antibody-absorption test results is discussed.