TechLab and alexon Giardia enzyme-linked immunosorbent assay kits detect cyst wall protein 1

Abstract

A Giardia lamblia antigen detected by the TechLab Giardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.) was purified by immunoaffinity chromatography from supernatant fluids of encystment cultures. Two major proteins (Mr 22,000 and 26,000) were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie staining that did not resemble the GSA65 antigen reportedly detected by the Alexon test. These proteins reacted intensely with the monoclonal antibodies used in both commercial enzyme-linked immunosorbent assays (ELISAs). Both proteins had identical N-terminal amino acid sequences and were identified as cyst wall protein 1 (CWP1). The 26-kDa form appeared early during encystment followed by the appearance of the 22-kDa form. Recombinant CWP1 (Mr 26,000) was strongly positive in both commercial tests. CWP1 was stable in human stool specimens, resistant to degradation by proteases and N- and O-glycanases, and unaffected by oxidation with sodium periodate. Two minor proteins with Mrs of 32,000 and 39,000 were detected in CWP1 preparations by using a sensitive fluorescent protein stain. Both were identified as CWP2, and neither reacted with the monoclonal antibodies from the commercial tests. We analyzed 535 stool specimens for CWP1 by using both commercial ELISAs and resolved discrepant results by using routine ova and parasite examination (O&P) and on immunofluorescence antibody assay. The presence of CWP1 correlated well between both ELISAs (98.7% correlation). Our results demonstrate that both commercial ELISAs detect CWP1, which is a useful diagnostic marker because it is highly stable, is secreted in large amounts by encysting trophozoites, and correlates well with O&P.

FIG. 1

(A) Analysis by SDS-PAGE of Giardia CWP1 purified by immunoaffinity chromatography with the immobilized TechLab test MAb. Lanes: 1, starting culture filtrate (5 μg); 2, purified Giardia CWP1 (1 μg). Note the presence of 22- and 26-kDa protein bands in the purified preparation. (B) Analysis of CWP1 by immunoblotting with the TechLab test MAb (lane 1) and Alexon test MAb conjugate (lane 2). Both lanes show the presence of the 22- and 26-kDa protein bands. Molecular mass markers are shown with each gel.

FIG. 2

Appearance of CWP1 during encystment. Culture supernatant fluids were sampled at the time points listed and assayed by immunoblotting and ELISA. (A) Immunoblot analysis of culture supernatant fluids. The MAb from the TechLab test was used as the detecting antibody. The 26-kDa band initially was detected at 18 h, and the 22-kDa band initially was detected at 42 h. (B) Analysis of culture supernatant fluids by the TechLab test and by the Alexon test. CWP1 initially was detected by both ELISAs at 10 h. CWP1 was not detected by ELISA or immunoblotting at any time in cultures of synchronized trophozoites.