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A mixture of two fluorescein isothiocyanate-conjugated monoclonal antibodies (MAbs) was used to optimize a direct specimen test (Chlamydia Direct Specimen Test IF; Clonatec, Paris, France) for detection of chlamydial elementary bodies in clinical specimens. One MAb reacted with a subspecies-specific epitope of the major outer membrane protein (molecular weight 43,000) of Chlamydia trachomatis, whereas the other reacted with the periodate-sensitive genus-specific antigen (molecular weight 11,000) of Chlamydia spp. Nonfat dry milk was the most efficient additive at suppressing the fluorescent background and was included in the antibody preparation. Fc-dependent binding of fluorescein-conjugated MAbs to protein A-containing Staphylococcus aureus was inhibited by addition of purified rabbit immunoglobulin. The Chlamydia Direct Specimen Test IF was compared with tissue culture isolation by using 309 genital specimens. The sensitivity and specificity were 77.4 and 98%, respectively.