This paper describes methods to improve the use of immunoassays for quantification of soilborne fungal antigens. Calibration curves, prepared by diluting known quantities of an antigen into soil extracts and into soil, were described by a four-parameter logistic curve from which two principal criteria, the lower detection limit and the horizontal locational parameter, were used to summarize the sensitivity and bias of an immuno-assay. We identify two sources of bias, retention of the antigen in soil due to bonding and interference of soluble soil components in plate-trapped—antigen, enzyme-linked immunosorbent assays. Using a monoclonal antibody that recognizes a putative catechol oxidase secreted by hyphae of Rhizoctonia solani, we show that bias due to retention of the antigen in soil is substantially greater than bias due to interference. Three soils were compared: a sand, a clay, and a loam. The degree of retention varied with soil type, with more than a 1,000-fold reduction in sensitivity in the clay soil. Addition of CuSO4 to the extraction solution and optimizing the volume of extractant reduced the bias and increased the sensitivity of the assay for all three soils. Possible mechanisms for the effect due to Cu2+ and the implications for the design and use of calibration curves for assays involving quantification of fungal antigens in soil are discussed.