The method used for production of the NaOH extract was that of Reiss et al. specificity of the antibody sandwich method was superior (97.5% versus 80.0%,P= 0.039). While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = 0.0152, serum = 0.5595. Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470. Cross-reactivity was observed in specimens from patients infected withBlastomyces dermatitidis,Paracoccidioides brasiliensis, andPenicillium marneffei. In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range. TheHistoplasmaantigen immunoassay is usually a well-established method for the rapid diagnosis of histoplasmosis (11,13,14). In addition, the assay is usually semiquantitative and can be used to monitor treatment and identify relapse (6,9). The method used is an antibody sandwich enzyme immunoassay (EIA) employing anti-Histoplasma capsulatumpolyclonal antibody for both the capture and detection actions. The sensitivities in serum and urine for patients with disseminated disease are 82 and 92%, respectively (14), specificity is usually >98% (12), and reproducibility is excellent (2,12). However, production of polyclonal antibody of sufficient sensitivity and specificity toward the carbohydrate antigen has had limited Rabbit polyclonal to FN1 success and has proven to be time consuming, laborious, and expensive. Beaucage reagent Also, a cross-reacting antigen may be detected in samples from patients infected withBlastomyces dermatitidis,Paracoccidioides brasiliensis, andPenicillium marneffei, but a previous study decided that theHistoplasmaantigen immunoassay could be used to assist in the diagnosis of these cross-reacting mycoses because the treatment regimens for all are similar and the epidemiological and clinical differences among these mycoses can be used for differential diagnosis (8). An inhibition assay would enable more precise quantitation of theHistoplasmaantigen (HcAg) and reduce the amount of antibody required for testing. An inhibition EIA has been described that uses a murine monoclonal antibody to detect the presence of anH. capsulatumprotein antigen in the serum and urine of patients with histoplasmosis (4; L. J. Wheat, Letter, J. Clin. Microbiol.37:2387, 1999). The overall sensitivity in serum was 71.4% for all those clinical forms of histoplasmosis. However, antigen was not detected well in urine (44.0% overall sensitivity) and cross-reactivity occurred in sera from patients with aspergillosis, cryptococcosis, and tuberculosis, features not observed in theHistoplasmaantigen immunoassay. In this paper, we report the development of an inhibition EIA capable of detecting HcAg in both serum and urine specimens. The sensitivity, specificity, reproducibility, and cross-reactivity of the inhibition EIA are compared to those of the standard antibody sandwich EIA. == MATERIALS AND METHODS == == Specimens. == All specimens were stored at or below 4C. Forty serum specimens and 40 urine specimens from patients with culture-proven histoplasmosis, collected from 1992 through 1997, and 40 serum controls and 40 urine controls, originally requisitioned for pregnancy or toxicology screening, were tested concurrently in a blind fashion by the antibody sandwich EIA and the inhibition EIA. Previous studies have shown that antigen levels from histoplasmosis cases are unevenly distributed over the complete result range of unfavorable (<1.0 EIA units [EU]) (2) to highly positive (>10.0 EU) (7,10). For this study, specimens from culture-proven histoplasmosis cases were selected so that the distribution of the original results mirrored that of untreated patients with disseminated histoplasmosis. Of the 40 serum specimens from culture-proven cases, 16 were highly positive (>10 EU), 14 were moderately positive (2.1 to 10.0 EU), 4 were weakly positive (1.0 to 2.0 EU), and 6 were unfavorable (<1.0 EU) when originally tested by theHistoplasmaantigen immunoassay. Of the 40 urine specimens from culture-proven cases, 20 were highly positive, 14 were moderately positive, 4 were weakly positive, and 2 were nega- tive when originally tested. Serum and urine specimens were tested by individual assays. Fifty Beaucage reagent urine specimens from patients with other fungal infections or tuberculosis, collected from 1983 through 1997, were tested separately using both methods. The mycoses evaluated were blastomycosis, paracoccidioidomycosis, coccidioidomycosis, aspergillosis, candidiasis, cryptococcosis, penicilliosis, and sporotrichosis. == Antibody sandwich EIA. == The antibody sandwich EIA method used has been Beaucage reagent previously described (2). == Inhibition EIA. == The procedures for preparing the inhibition plate and making standards and dilution buffer are layed out below and are based upon procedures described by Gomez.