Detection of Antibodies to Pneumocystis carinii by Enzyme-Linked Immunosorbent Assay in Experimentally Infected Mice

Abstract

Pulmonary pneumocystosis, caused by Pneumocystis carinii is one of the most important opportunistic infections in man and animals under immunosuppressed state (Arean, 1972. Pathology of protozoal and helminthic disease. Williams & Wilkins Co., Baltimore, 291 p.; Frenkel et al., 1966, Laboratory Investigation 15: 1559-1577). In such immunosuppressive hosts, serum antibody to P. carinii is generally produced at a low level (Kagan and Norman, 1976, National Cancer Institute Monograph 43: 121125). Therefore the development of a highly specific and sensitive method to measure antibody would be required to elucidate the relationship between progression of infection and antibody response and to evaluate the plausibility of serodiagnosis (Pifer et al., 1978, Pediatrics 61: 3541) as well as for epidemiological survey. In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies to P. carinii, and the sensitivity was compared with an indirect immunofluorescence (IF) test routinely used in our laboratory (Furuta et al., 1984, Japanese Journal of Experimental Medicine 54: 57-64). The P. carinii, isolated from a naturally infected nu/nu mouse, has been maintained by the intranasal (i.n.) passage in nu/nu mice in our laboratory (Furuta et al., 1984, loc. cit.) and organisms from the sixth passage were used in this experiment. The standard ELISA technique for various viruses was modified for P. carinii as follows (Voller et al., 1976. Microplate enzyme immunoassays for immunodiagnosis of virus infection. American Society of Microbiology, Washington, D.C., 506 p.). Pneumocystis carinii cyst suspension was prepared from infected mouse lungs as described previously (Furuta et al., 1984, loc. cit.). The sample was sonicated at 200 W (Kubota Insonator, Model 200) for 50 min, and diluted to 5 ,tg/ml with phosphate buffered saline (PBS) pH 7.2. Fifty 1l of the cyst suspension was added to each well in a polystyrene microplate (Nunc-Immuno Plate 96-F, Nunc Inter Med, Roskilde) and the plate was incubated at room temperature for 1 hr. The plate was washed 3 times with 0.05% Tween 20 in PBS (Tween 20-PBS), and 50 ,ul of serum dilution was added to each well. After further incubation at room temperature for 1 hr, the plate was washed 3 more times with Tween 20-PBS. Subsequently, 50 ,l of 1:1,000 rabbit anti-mouse IgG or IgM serum which were both conjugated with horseradish peroxidase (specific to gamma or mu chain, respectively; Cappel Labs., Cochranville, Pennsylvania) was added to the well. After incubation at room temperature for 1 hr, the residual conjugate was removed by washing 3 times with Tween 20-PBS, and 200 ,dl of substrate solution consisting of 40 mg o-phenylenediamine dihydrochloride and 10 ,l H202 in 100 ml of 0. 1 M citrate buffer, pH 4.7 was added to each well. The plate was incubated in the dark for 1 hr at 37 C, and 50 ,1 of 6 N sulfuric acid was added to each well to stop the enzyme reaction. The absorbance of the reaction mixture was measured at 492 nm by a spectrophotometer (Titertek Multiskan; Flow Labs., Inc., Rockville, Maryland) and the reading of a given diluted serum higher than 1.5 times of equivalent dilution of normal serum was taken as a positive reaction. Outbred ICR mice were obtained from a commercial breeder (Charles River Japan Co., Atsugi). The colony had no detectable antibodies to P. carinii by our routine IF test. The mice were maintained in cages with a filter cap (Sankikagaku Co., Tokyo) and placed in laminar airflow racks. Twenty mice were inoculated i.n. with 104 P. carinii cysts in 0.05 ml PBS (Furuta et al., 1984, loc. cit.) and 5 mice were killed at weekly intervals. The number of cysts in the lung preparations stained with toluidine blue-O (Chalvardjian and Grawe, 1963, Journal of Clinical Pathology 16: 383-384) was counted under the microscope (Furuta et al., 1984, loc. cit.). As