INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

Giardiasis, caused by the protozoan parasite Giardia lamblia, is the most commonly diagnosed parasitic infection in the United States (5). This parasite exists in two main life forms: trophozoites and cysts. Infection typically occurs following the ingestion of water or foods contaminated with fecal material containing cysts, and the infective dose may be as low as 10 cysts (15). The most common symptoms are diarrhea, abdominal cramps, bloating, flatulence, fatigue, and weight loss resulting from malabsorption.

Several Giardia antigens have been identified and partially characterized (2, 11, 14, 17, 19). GSA65 is a glycoprotein of M_r 65,000 that reportedly is present in trophozoites and cysts (16). It is resistant to proteolysis and heat but is sensitive to sodium periodate oxidation. According to the manufacturer, this is the antigen detected by the Alexon ProSpecT Giardia microplate assay (Alexon, Inc., Sunnyvale, Calif.). Giardia cyst wall proteins 1 and 2 (CWP1 and CWP2, respectively) which have M_rs of 26,000 and 39,000, respectively, are other antigens that have been studied (9, 12). The genes for these proteins have been sequenced, and both proteins contain stretches of leucine-rich repeats. These proteins can form a stable CWP1-CWP2 65-kDa heterodimer (9).

The diagnosis of giardiasis is based on clinical history, symptoms, and the presence of the organism or its antigens in stool specimens from suspected individuals. Several types of tests, including microscopic examination, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA), are utilized as diagnostic aids for testing stool specimens. In the following study, we identified and characterized the antigen detected by two commercial ELISA kits: the TechLab Giardia Test (TechLab, Inc., Blacksburg, Va.) and the Alexon test. Both of these tests are monoclonal antibody (MAb)-based ELISAs used as in vitro diagnostics for giardiasis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Preparation of Giardia cysts. G. lamblia WB (ATCC 30957) trophozoites were axenically cultured in Keister's modified TYI-33 medium at pH 7.1 and incubated as previously described (7). Growing trophozoites were synchronized by washing a confluent monolayer five times with phosphate-buffered saline (PBS) at pH 7.4. Trophozoites were chilled on ice for 20 min, pelleted by centrifugation, and suspended in TYI-33 medium containing 10 mg of bovine bile per ml (Sigma Chemical Co., St. Louis, Mo.) at pH 7.8 to trigger encystment without a preencystment incubation (4, 6, 7). Cysts were harvested by centrifugation and suspended in deionized water overnight at room temperature to lyse residual trophozoites (7). Cysts were collected by centrifugation and washed with sterile deionized water, and counts were done by trypan blue exclusion (11). Encystment culture supernatants were passed through a 0.2-µm-pore-size filter (Nalgen Co., Rochester, N.Y.) as instructed by the manufacturer, and the culture filtrates were stored at 4°C.

Immunoassays. The Giardia Test, the ProSpecT Giardia microplate assay, and the Crypto/Giardia IF (immunofluorescence) test (TechLab, Inc.) were performed as instructed by the manufacturer.

Immunoaffinity chromatography. Giardia antigen was purified by immunoaffinity chromatography with a MAb immobilized on Affi-Gel 15 (Bio-Rad Laboratories, Melville, N.Y.) as instructed by the manufacturer. The MAb used for purification was the same MAb used in the TechLab test. Culture filtrate from Giardia cultures was loaded onto a 2-ml MAb-Affi-Gel 15 column, and the gel was washed with sterile PBS (pH 7.4). Bound antigen was eluted with 100 mM glycine buffer (pH 2.5) containing 10% ethylene glycol and 0.5 M sodium chloride. Purified Giardia antigen was concentrated and washed with PBS by centrifugation with a Centri-plus concentrator (10-kDa size exclusion; Amicon, Beverly, Mass.).

Western blot analysis. Protein concentration was determined by the method of Bradford (1). Molecular weight was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (8). Protein staining was done with Coomassie brilliant blue R-250 and SYPRO orange protein stain (Bio-Rad Laboratories). Immunoblot analysis was done by the method of Towbin et al. (18). The primary antibody consisted of either (i) the TechLab test MAb; (ii) ascites fluid containing the 7D2 MAb, which binds specifically to CWP2 (9); or (iii) the Alexon test MAb conjugate.

N-terminal sequencing. Samples of purified antigen (1 µg) were transferred to polyvinylidene difluoride membrane for sequencing on an Applied Biosystems Procise sequencer (Perkin-Elmer Corp., Norwalk, Conn.). Sequence homologies were determined with the FASTA program (12).

Assessment of antigen stability. For stability testing, purified antigen (0.2 µg/ml) was heated at 100°C for 5 min. Serial dilutions were tested by ELISA and immunoblotting. The effect of proteolysis was determined by incubating purified antigen (0.2 µg/ml) with a mixture of trypsin and chymotrypsin (0.2 µg/ml) in 0.1 M Tris-HCl buffer (pH 8.0) and with pronase (2 mg/ml) in 0.1 M Tris-HCl buffer (pH 7.5) containing 0.01 M EDTA and 0.5% SDS. Azocasein was used as a substrate to compare the activities of the protease solutions (3).

Expression and purification of rCWP1. Recombinant CWP1 (rCWP1) was expressed in a glutathione S-transferase gene fusion system in Escherichia coli BL21 (12) (kindly supplied by the National Institutes of Health). Expression was induced in 6-h (37°C) shaking cultures with 200 µM isopropyl--D-thiogalactopyranoside. Cells were lysed by sonication, clarified by centrifugation, and applied to glutathione-Sepharose 4B (Pharmacia Biotech, Piscataway, N.J.). CWP1 was released from the column by cleavage with bovine thrombin (25 U in PBS [pH 7.5]).

Study sites and stool specimens. Three separate studies evaluating CWP1 as a diagnostic marker of giardiasis were performed. Study 1, performed at Sacred Heart Medical Center (Spokane, Wash.), compared both ELISAs to ova and parasite examination (O&P). Study 2, performed at DeKalb Medical Center (Decatur, Ga.), compared the TechLab test to the Alexon test and included stool samples from SmithKline Laboratory (Atlanta, Ga.). Study 3, performed at the Virus Reference Laboratory (VRL; San Antonio, Tex.) resembled study 1. All specimens were preserved in 10% buffered formalin and submitted for routine O&P. Discrepant results were resolved by repeat testing by ELISA and immunofluorescent antibody (IFA) analysis.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Characterization of purified Giardia antigen. The antigen purified by immunoaffinity chromatography with the MAb used in the TechLab ELISA consisted of two proteins with sizes of 22 and 26 kDa (Fig. 1A) that reacted intensely with both of the MAbs from the TechLab and Alexon tests (Fig. 1B). Preincubation of immunoblots with the TechLab MAb reduced the binding of the Alexon MAb conjugate, demonstrating competitive inhibition by the TechLab MAb. Under nonreducing conditions, a major 50-kDa band and a minor 65-kDa band were detected with both MAbs. Further analysis with a more sensitive fluorescent staining procedure revealed two minor proteins with sizes of 32 and 39 kDa that did not react with these MAbs.

View larger version (56K): [in this window] [in a new window]   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.

View larger version (35K): [in this window] [in a new window]   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.

Clinical evaluation of CWP1 as a diagnostic marker. Nine Giardia-positive stool specimens, all of which were positive by both commercial ELISAs, were analyzed by immunoblotting with the MAb from the TechLab test. We detected the 26-kDa CWP1 in eight specimens (data not shown). The stool specimen that did not react by immunoblotting gave an absorbance value of <0.5 in the TechLab test, indicating that it contained only low levels of antigen. Nine stool specimens that were negative by the ELISAs also were negative by immunoblot analysis (data not shown).

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

The encystment process of G. lamblia remains one of the least understood phases of its life cycle, but during the past decade, researchers have begun to understand some of the intriguing effects of this process. The cysts contain a network of filaments of different sizes that begin at multiple points on the outer membrane of encysting trophozoites known as cap structures (4). These cap structures generate cyst filaments of multiple diameters that elongate and proceed to fully encase the trophozoite. Gas chromatographic and mass spectrometric analyses of the cyst wall indicate the presence of protein and carbohydrate, which is primarily N-acetylgalactosamine (10). Although some progress has been made, actually very little is known about the composition of the cyst wall, the mechanism of construction, and the function of known cyst wall proteins.

The purpose of our study was to identify and characterize the cyst antigen detected by the TechLab test. By utilizing immunoaffinity chromatography, we were able to obtain highly purified antigen and identify it as CWP1. When we examined highly purified CWP1 for cross-reactivity in the Alexon test, we found that CWP1 reacted at roughly the same level in both ELISAs. Supporting these observations was our finding that rCWP1, which has an M_r of 26,000, also reacted strongly in both tests. In addition, the TechLab MAb competitively inhibited the binding of the Alexon MAb conjugate, indicating a similar or identical epitope. Thus, our results demonstrate that both commercial tests detect CWP1, contrary to reports that the Alexon test detects GSA65. Some of the confusion may result from the ability of these proteins to form heterodimers. Heterodimer formation of Giardia cyst wall proteins has been described previously (9), and our findings showing low levels of CWP2 in our CWP1 preparations can lead to heterodimer formation. Whether this explains the confusion with GSA65, however, is not clear, since CWP1 is easily distinguished from GSA65 by SDS-PAGE in the presence of reducing agents (16). In addition, our findings suggest that CWP1 is an unglycosylated protein. This result also is contradictory, based on earlier observations that GSA65 is sensitive to periodate oxidation (16).

The identical homologies of the N-terminal sequences of the 22- and 26-kDa forms of CWP1 suggest that cleavage occurs at the C terminus of the molecule. Both forms appear to be highly stable and retain their immunoreactivity. Interestingly, CWP1 was detected by both ELISAs in cultures undergoing encystment but not in synchronized trophozoite cultures. Synchronization of growing trophozoites is done in vitro by eliminating unattached trophozoites that are beginning the encystment process. During an infection, trophozoites and encysting trophozoites are both present in the small intestine. Thus, the absence of CWP1 in synchronized trophozoite cultures does not diminish the diagnostic value of this antigen. In fact, because of the secretion of CWP1 early in encystment and the high sensitivity of the ELISAs, there may be cases in which the antigen is detected in the absence of mature cysts.

In the clinical evaluation, the presence of CWP1 correlated well with Giardia-positive specimens as determined by ELISA, O&P, and IFA. The correlation between ELISAs was 98.7%, which is not surprising since they both detect the same antigen. Our findings provide additional evidence demonstrating the stability of CWP1 in human feces. The detection of CWP1 in clinical specimens from various study sites suggests that this antigen is conserved among Giardia isolates throughout the United States. These results further support its use as a diagnostic marker.

In conclusion, our results show that CWP1 is a highly stable cyst wall protein produced and released by encysting trophozoites. The combination of encysting Giardia in the gastrointestinal tracts of infected persons, high-level secretion of the antigen early in the encystment process, and a high correlation with O&P in patients suffering from giardiasis makes CWP1 a useful marker of giardiasis.