Production and Evaluation of Reagents for Detection of Histoplasma capsulatum Antigenuria by Enzyme Immunoassay

Abstract

The detection of urinary Histoplasma capsulatum polysaccharide antigen (HPA) by enzyme immunoassay (EIA) has proven useful for the presumptive diagnosis of histoplasmosis in AIDS patients. Assay limitations include (i) detection of a largely uncharacterized antigen and (ii) difficulty in reproducibly generating antibodies for use in the EIA. To improve antibody production for use in this test and to better understand the antigen being detected, we compared rabbit antibodies elicited using various immunization schedules, routes, and H. capsulatum-derived antigens. Antibodies were evaluated by EIA for their ability to detect purified H. capsulatum C antigen (C-Ag) and antigenuria. Reported as enzyme immunoassay (EI)

Development and Evaluation of an Enzyme-Linked Immunosorbent Assay To DetectHistoplasma capsulatumAntigenuria in Immunocompromised Patients

“…In the first trial, rabbits (n ϭ 6) were exsanguinated on day 49. Five of these rabbits (83%) showed hyperimmunity greater than or equal to that of the positive control rabbit immunized previously (16). When sera from blood samples drawn on day 42 were compared by ELISA to the exsanguination sera (day 49), rabbits showed greater reactivity to C-Ag at day 42.…”

“…(i) Immunogen. H. capsulatum yeast-phase cells (Thon strain) were cultured at 37°C for 72 h in Pine's liquid medium (20) and then killed with thimerosal as described by Lindsley et al (16). Yeast cells were harvested by centrifugation at 1,500 ϫ g and rinsed three times in 0.01 M phosphate-buffered saline, pH 7.2 (PBS).…”

“…Female New Zealand White rabbits (n ϭ 33) weighing 2 to 4 kg were immunized intravenously with 0.5 ml of the 0.4% H. capsulatum immunogen on days 0, 1,4,8,10,15,18,23,29, and 32 (10 injections in three separate trials), as previously described by Lindsley et al (16), but no subsequent intramuscular injections were given. During the first trial performed at the CDC (n ϭ 6 rabbits), postimmunization test bleeds (5 ml) were taken on days 14, 28, and 42 and rabbits were exsanguinated on day 49.…”

“…Absorbance was read at 450 nm, using a SpectraMax 250 microplate spectrophotometer and SoftMax Pro data acquisition software (Molecular Devices, Toronto, Canada). The reactivities of individual rabbit sera from all three trials were compared on each plate to the reactivity of an unimmunized rabbit as well as to that of a positive control rabbit that had been immunized using a similar protocol in a previous trial (16). Sera from rabbits with the highest reactivities to C-Ag were precipitated individually, using three rounds of 35% saturated ammonium sulfate as described by Hebert (10), and dialyzed twice against a 1,000ϫ volume of PBS at 4°C for 24 h. The resulting PAb were quantitated using the Bradford protein assay (1) with a gamma globulin standard, diluted in PBS-40% glycerol, and stored at Ϫ20°C for later evaluation in an antigen-capture ELISA format.…”

“…Prior work in our laboratory resulted in the selection of a polyclonal antibody (PAb) reagent generation protocol for use in an H. capsulatum enzyme immunoassay (EIA) to detect antigenuria (16). The EIA has since been modified, resulting in increased assay sensitivity and reduction of the nonspecific background.…”