![]() ![]() ![]() The plates were washed in wash buffer (PBS with 0.05% Tween-20) and blocked with blocking buffer (wash buffer with 5% nonfat milk) (200 μL/well) for 2 h at room temperature. Duplicate wells of a 96-well plate were coated with antigen (1 μg/mL, 100 μL/well in PBS pH 7.4 overnight. A standard serum sample, obtained from a healthy donor with known reactivity to MsgA, MsgB, and MsgC in Western blot, is run on each plate as a control. Serum specimens were tested against the following antigens: recombinant Msg fragments Escherichia coli extract expressing the pET vector without insert as a vector control tetanus toxoid (TT) as a positive control phosphate-buffered saline (PBS) without antigen (negative control). ![]() We present three different analyses of the ELISA data and compare the results to those obtained by Western blot. We developed an ELISA using recombinant Msg fragments as antigens and analyzed antibody responses of healthy blood donors and HIV-positive patients (including PCP-positive and PCP-negative patients). ELISA overcomes this problem and is better suited for population surveys. The data showed significant differences in the frequency of reactivity to the Msg constructs, not only between the two groups but also between PCP-positive and PCP-negative patients with HIV.Īlthough Western blot is a valuable serologic technique, its major limitation is that it is not quantitative. jiroveci Msg and used these antigens to measure serum antibodies by Western blot in healthy blood donors and HIV patients ( 27). We recently developed three overlapping recombinant fragments of Msg that span the length of P. An enzyme-linked immunosorbent assay (ELISA) using the carboxyl fragment showed significantly higher antibody levels in HIV-negative patients with Pneumocystis pneumonia (PCP) compared with healthy controls, but it could not distinguish among HIVpositive, PCP-positive and HIV-positive, PCP-negative patients and controls. In Western blot analysis, the carboxyl fragment was recognized more frequently by serum specimens than the amino fragment but did not distinguish between HIV+ patients and healthy donors. One group of investigators has developed two recombinant fragments that correspond to the amino and carboxyl halves of Msg ( 25, 26). Recombinant Pneumocystis antigens offer a viable approach to developing novel reagents for use in immunologic assays ( 25– 27). jiroveci Msg is in short supply and contains multiple isoforms of this glycoprotein, which complicates immunologic studies of this antigen ( 23, 24). The Pneumocystis antigen that has received the most attention is the 95- to 140-kDa major surface glycoprotein (Msg or gpA), which contains shared and species-specific epitopes, elicits humoral and cellular protective immune responses, and plays a central role in the interaction of Pneumocystis with its host ( 2– 8, 22). Thus, no standardized antigen preparations are useful for antigen-specific immunologic studies of Pneumocystis infection in humans. Conflicting results have been obtained in attempts to distinguish past from present infection or colonization from active disease ( 16– 21). The frequency or level of serum antibodies to Pneumocystis among HIV-positive patients and other immunocompromised hosts has usually been similar to the corresponding value in healthy controls ( 6, 8– 15). Serologic surveys using crude Pneumocystis antigen preparations are generally not effective as clinical or epidemiologic tools. The use of human Pneumocystis has been further hindered by the small amounts of material that can be obtained from clinical specimens. Since a reliable in vitro culture system for Pneumocystis has not been developed, antigens used in these studies consisted mainly of whole or fractionated organism preparations derived from infected human or rodent lungs. Seroepidemiologic surveys have shown that exposure to Pneumocystis occurs early in life by 2 to 4 years of age, most children have antibodies to the organism ( 4– 8). jiroveci for human-derived organisms ( 3). Pneumocystis nomenclature, which is evolving and somewhat controversial, has designated two species so far: P. However, data over the past decade have shown that Pneumocystis is genetically diverse and host specific, suggesting that studies of immune responses to Pneumocystis are best performed by using organisms or organism products that are specific for that host ( 1). Since Pneumocystis organisms in humans and animals are very similar, and the pneumonia that develops has many common features, these microbes were originally thought to belong to a single genus and species. Pneumocystis also infects a variety of animals and causes pneumonia in those that are immunodeficient or immunosuppressed ( 2). Pneumocystis is an opportunistic fungus of worldwide distribution that can cause lethal pneumonia in persons infected with HIV and in other persons with depressed immune function ( 1). ![]()
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