Comparison of an established antibody sandwich method with an inhibition method of Histoplasma capsulatum antigen detection

Abstract

The Histoplasma antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis. Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements. We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay. Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods. The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%, P = 0.500) and serum (82.5% versus 80.0%, P = 1. 000). With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%, P = 1.000), and with urine, the 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 with Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Penicillium 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.

FIG. 1

Antibody sandwich EIA and inhibition EIA reactivity. Graphs: A, antibody sandwich EIA; B, inhibition EIA. The dotted line represents the cutoff point (antibody sandwich EIA, 1.00 EU; inhibition EIA, 0.080 [serum] and 0.010 [urine] μg/ml). An open circle indicates a value with excessive duplicate variation (CV, >20.0%). For graph A, n is the number of results contained within the darkened box. For graph B, n is the number of results that are ≤0.005 μg/ml.

FIG. 2

Antibody sandwich EIA reproducibility with serum and urine specimens. Graphs: A, serum specimens; B, urine specimens. Closed circles represent specimens from culture-proven histoplasmosis cases. Open circles represent control specimens. The dotted line represents the cutoff point (1.00 EU). The ICC of serum specimens is 0.9949. The ICC of urine specimens is 0.9975.

FIG. 3

Inhibition EIA reproducibility with serum and urine specimens. Graphs: A, serum specimens; B, urine specimens. Closed circles represent specimens from culture-proven histoplasmosis cases. Open circles represent control specimens. The dotted line represents the cutoff point (serum, 0.080 μg/ml; urine, 0.010 μg/ml). The ICC of serum specimens is 0.9850. The ICC of urine specimens is 0.9736. n is the number of reproducible results that are ≤0.005 μg/ml.

FIG. 4

Cross-reactivity of the antibody sandwich EIA and inhibition EIA. Graphs: A, antibody sandwich EIA; B, inhibition EIA. All of the specimens tested were urine. The dotted line represents the cutoff point (antibody sandwich EIA, 1.00 EU; inhibition EIA, 0.010 μg/ml). The other mycoses are coccidioidomycosis (n = 5), candidiasis (n = 5), cryptococcosis (n = 6), aspergillosis (n = 5), and sporotrichosis (n = 2). An open circle indicates a value with excessive duplicate variation (CV, >20.0%).