Rosie B. Wong

CAPPING OF IMMUNE COMPLEXES BY SPOROZOITES OF EIMERIA TENELLA

Sporozoites of Eimeria tenella were incubated for 10, 20, or 30 min with parasite-specific monoclonal IgG antibody 3D3II from mice and then rinsed in a Tris-buffered glucose saline solution (TBGS). Some sporozoites were then incubated for 10, 20, or 30 min with ferritin- or colloidal gold-conjugated goat anti-mouse IgG antibody and then fixed in 2.5% glutaraldehyde and prepared for transmission (TEM) or scanning (SEM) electron microscopy. Other sporozoites that had been previously exposed to monoclonal antibody were prefixed with 0.25% glutaraldehyde, incubated with ferritin- or colloidal gold-conjugated anti-mouse IgG antibody and then fixed and prepared for TEM or SEM. Control preparations consisted of sporozoites exposed only to TBGS, monoclonal antibody 3D3II or to ferritin- or colloidal gold-conjugated anti-mouse IgG antibody. Capping of immune complexes occurred only on the surface of those sporozoites exposed to monoclonal antibody 3D3II followed by ferritin- or gold-conjugated antibody. Immune complexes moved laterally and posteriorly on the outer surface of the parasite plasma membrane to form a cap at the posterior end of the sporozoite. Capping did not occur in TBGS controls nor in sporozoites treated with monoclonal antibody 3D3II and prefixed in 0.25% glutaraldehyde before exposure to ferritin- or gold-conjugated antibody. Thus, capping of surface antigens did not occur in the presence of monoclonal 3D3II antibody only, whereas specimens exposed to both monoclonal and ferritin- or colloidal gold-conjugated antibodies were able to cap immune complexes.

Reusable fiber-optic-based immunosensor for rapid detection of imazethapyr herbicide

Abstract Imazethapyr is a herbicide belonging to the imidazolinone class of compounds. It is the active ingredient of a commercial herbicide PURSUIT®. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of imidazolinone compounds. Using the immune rabbit serum, an enzyme-linked immunosorbent assay (ELISA) has been developed for the entire class of commercial imidazolinone herbicides including imazaquin, imazapyr, imazethapyr and imazamethabenz methyl. The quantitation of imidazolinones is soil requires a certain amount of sample pretreatment, thus the through-put is not ideal. A simpler immunoassay method for screening large amount of soil samples economically would be useful. Taking the sheep polyclonal immune serum, we used a fluorescent immunoassay employing optical fiber and fluorescence (US Patent 4,582,809, 1986), to assay for imazethapyr. Purified sheep antibody was immobilized on quartz fibers. A mixture of fluorescein-labelled imazethapyr analog and free imazethapyr was presented to the fiber for direct competition of the antibody binding sites or displacement of a previously fluorescein labelled fiber. The response time for the detection of imazethapyr ranged from seconds to minutes. The sensitivity of the assay was 1 nM. This binding of the fluorochrome to the fiber was reversible by washing with a phosphate buffered saline. Multiple measurements were easily processed with a single fiber over the course of several hours. Analysis of imazethapyr residue in soil can be accomplished by subjecting a clarified soil extract solution directly for analysis without further treatment. The crossreactivity data indicates that the assay is apparently specific for the imidazolinone class of compounds.

Comparison of liposome amplification and fluorophor detection in flow-injection immunoanalyses

Abstract A flow-injection liposome immunoanalysis (FILIA) system was developed for the measurement of imazethapyr herbicide. The liposome is a spherical vesicle encapsulating many marker molecules in an aqueous interior. By incorporating analyte-phospholipid conjugates into the bilayer, the liposome can competitively bind to anti-analyte antibody, immobilized in the immunoreactor column of the FILIA system. In this study, the analyte-tagged liposome containing a fluorescent dye, carboxyfluorescein, was compared to a single fluorophor-tagged analyte used for the generation of the analytical signal. The liposome enhanced sensitivity by 1000-fold compared to the single fluorescent molecule-tagged analyte. This is the first report to demonstrate directly the amplification of the response by liposomes in a flow-injection immunoassay.

Development of flow-injection liposome immunoanalysis (FILIA) for imazethapyr

Imazethapyr is the herbicide developed for use in leguminous crops. In this study, flow-injection liposome immunoanalysis (FILIA) has been shown to be capable of measuring imazethapyr in a buffered solution with a detection limit of 0.1 ppb through the optimization process. Protein A coated glass beads covalently conjugated with antibody were contained in a glass column, and this column was used as an immunoreactor. Liposomes which encapsulated a fluorescent dye, sulforhodamine B (SRB) or carboxyfluorescein (CF), generated the analytical signal. By loading larger volumes of sample onto the column, it was shown that the detection limit could be lowered. Liposomes containing carboxyfluorescein gave more sensitive response and a lower detection limit than those with sulforhodamine B. Also, improved response was obtained by using a smaller flow cell in the fluorescence detector due to the reduced dilution effect.

Effects of monoclonal IgG antibodies on Eimeria tenella (coccidia) sporozoites.

Although monoclonal antibodies have been generated against certain coccidia (Carosi et al., 1980, Boll. Inst. Sieroter Milan. 59: 25-30; Danforth, 1982, J. Parasitol. 68: 392-397; Sethi et al., 1980, J. Parasitol. 66:192-196), little is known about their effects on various stages of these parasites. In a previous study that used transmission electron microscopy (TEM), we found that 1A, 9D, and 3D3II monoclonal IgG antibodies caused complement-mediated lysis of Eimeria tenella sporozoites (Speer et al., 1983, J. Protozool. 30: 548-554). When viewed by scanning electron microscopy (SEM), we found that sporozoites of E. tenella exposed to these monoclonal antibodies without complement differed considerably in surface texture and size from normal sporozoites. Monoclonal antibodies were obtained as described previously (Kohler and Milstein, 1975, Nature 256: 495-497; Speer et al., 1983, loc. cit.). Sporozoites of E. tenella were incubated for 30 min at room temperature (22 C) with normal mouse ascites fluid or with 1A, 9D, or 3D3II monoclonal IgG antibodies (heat-inactivated) that had been diluted 1: 1 in a Tris-buffered glucose saline solution (pH 7.4) consisting of 10 mM Tris, 10 mM glucose, 170 mM NaCl, and 10% fetal calf serum. Sporozoites were then fixed in 2% glutaraldehyde in Millonigs phosphate buffer (pH 7.2). Sporozoites suspended in Millonigs buffer were captured on Nuclepore filters (0.6 Mm pore size) that had been coated previously with normal mouse serum in order to ensure adherence of the sporozoites. Glutaraldehyde fixative was then pulled through the filter to fix the sporozoites to the filter. Sporozoites on filters were rinsed in buffer, placed in 1% osmium tetroxide for 20 min, rinsed in buffer, dehydrated in ethanol, critical-point dried in a Samdri critical-point dryer, mounted on metal studs, coated with 25 nm of gold: palladium (60:40), and examined with a Zeiss Novascan 30 SEM. All measurements were made at a magnification of X7,000. Significant differences between dimensions of sporozoites exposed to normal ascites fluid and of those exposed to monoclonal antibody were determined by Students t-test. By using indirect fluorescent antibody assay, lA, 9D, and 3D3II monoclonal IgG antibodies were found to have titers of 1:1.3 X 106, 1:1.2 X 104, and 1:1.6 X 106, respectively. Sporozoites exposed to normal ascites fluid measured 10.01 X 2.02 ,m (9.5-10.5 X 1.8-2.2 Mm; n = 20), whereas those exposed to 9D, 1A, or 3D3II were 9.36 X 2.02 ,um (8-10 X 1.8-2.3 Am; n = 20), 7.97 X 2.18 Am (7-9 X 2-2.5 inm; n = 20), and 6.8 X 2.39 ,m (6-8 X 2.2-2.6 ,im; n = 20), respectively. Sporozoites incubated with 1A and 3D3II were significantly shorter than those incubated with normal ascites fluid (P < 0.0005); 9D caused no significant difference in length (Figs. 1-4). Sporozoites exposed to 1 A and 9D did not differ significantly in width when compared to those exposed to normal ascites fluid, whereas those exposed to 3D3II were significantly wider (P < 0.05). Sporozoites incubated with normal ascites fluid or 9D had relatively smooth surfaces with small protrusions (Figs. 1, 2), whereas those exposed to 1A or 3D3II had rough or wrinkled surfaces with blebs (Figs. 3, 4). Although immune sera have been found to have an immobilizing or lytic effect on sporozoites and merozoites of E. tenella (Bums and Challey, 1965, J. Parasitol. 51: 660-668; Herlich, 1965, J. Parasitol. 51: 847-851), the role that immune sera plays in immunity to this parasite is still unknown. By using SEM, Witlock and Danforth (1982, J. Protozool. 29: 441-445) found that immune chicken serum caused surface bulges, a fibrinous coat and complement-me-

Reusable fiber optic immunofluorosensor for rapid detection of pesticides

Quartz fibers coated with acetylcholinesterase (AChE) or antibody (Ab) are used as biosensors utilizing total reflectance fluorescence for the rapid detection of pesticides. The enzyme biosensor was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ AChE on quartz fibers. The fluorescent signal was generated by hydrolysis of acetylcholine (ACh) that is present in the perfusate. Organophosphate (OP) and carbamate anticholinesterase (AntiChE) insecticides inhibited AChE and reduced the fluorescent quenching resulting from AChE hydrolysis. A parathion biosensor was constructed by immobilizing casein-parathion on the quartz fibers, that bound rabbit antiparathion antibody. The optical signal was generated by perfusing the fibers with fluorescein-labeled goat antirabbit IgG. Free parathion inhibited the binding of antiparathion Abs and reduced the optical signal and provided the basis for detection of parathion. Another immunosensor developed detected the herbicide PursuitR by utilizing the reversible binding of a fluorescein-Pursuit derivative to antiPursuit Abs immobilized on the fiber. Unlabeled Pursuit competed effectively and displaced the bound fluorescent compound in a dose-dependent manner. The sensor discriminated effectively between Pursuit-like and structurally unrelated herbicides. The immunosensor offers the advantage of continuous monitoring, ease of operation, speed of detection, low cost, stability, specificity, matrix transparency, and reusability.