Development of Murine Monoclonal Antibodies to Pneumocystis carinii

Abstract

Relatively little is known about the antigenic structure of Pneumocystis carinii and the immunopathogenesis of pneumonitis caused by P. carinii. To begin to define the antigenic character of the surface of this organism, we have produced murine monoclonal antibodies that react with the surface of P. carinii (obtained from rats), as detected by immunofluorescence and immunoelectron microscopy. Immunoblot analysis revealed that the six antibodies described in this report bound an antigen with an apparent molecular mass of 90,000-95,000 daltons. Although all six monoclonal antibodies bound P. carinii obtained from rats, only one (5E12) was also able to bind P. carinii obtained from rabbits, ferrets, and a human; this result demonstrated that isolates of P. carinii obtained from different species are not antigenically identical.

The role of alveolar macrophages in Pneumocystis carinii degradation and clearance from the lung.

“…Monoclonal antibody 5E12 recognizing P. carinii gpA was provided by Dr. Frank Gigliotti (University of Rochester, Rochester, New York) (35). Concanavalin A conjugated to horseradish peroxidase was obtained from E.Y.…”

“…Fractions containing the complex migrating at 120 kD were pooled, dialyzed, and concentrated. Immunoblotting of the purified gpA complex was performed using monoclonal antibody 5E12 to verify the identity of the product (20,35). Monoclonal 5E12 is a mouse anti-P. carinii IgM-recognizing gpA from rodents, ferrets, and humans (35).…”

“…Next, radiolabeled P. carinii were washed with DME and BSA (1 mg/ml) to remove unincorporated label. 35 Slabeled P. carinii (4 ϫ 10 6 /well) were then allowed to attach to macrophages in DME and BSA (1 mg/ml) at 37 Њ C for 0-6 h. Unattached P. carinii were removed by washing, and the percentage of organisms associated with macrophages quantified.…”

“…The supernatant was spun again (2,000 g for 10 min) to bring down nonbound P. carinii . P. carinii degradation was determined by the amount of soluble 35 S-label released into the medium. To correct for spontaneous release of 35 S-label from P. carinii , radiolabeled organisms were treated in an identical fashion without macrophages.…”

“…P. carinii degradation was determined by the amount of soluble 35 S-label released into the medium. To correct for spontaneous release of 35 S-label from P. carinii , radiolabeled organisms were treated in an identical fashion without macrophages. Spontaneous release of 35 S was subtracted from the amount released in the presence of macrophages to yield a net measurement of P. carinii degradation.…”