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Fc glycosylation of immunoglobulins is necessary for antibody effector functions. These glycans of immunoglobulins are often referred as glycoforms and they can be heterogeneous due to the variations in glycosylation machinery present in the endoplastic reticulum (ER) and in the Golgi. In the absence of a generic culturing protocol that can render a consistent glycosylation pattern, monitoring glycoforms of monoclonal antibodies from cultured cells is becoming essential. Accordingly, quantification of glycosylated and deglycosylated heavy chains of an IgG4 monoclonal antibody was accomplished using an Agilent Bioanalyzer Lab-On-the-Chip electrophoresis system. In addition to the native antibody, completely deglycosylated antibody prepared by treating with PNGase F and a F(ab’)2 fraction were evaluated for their antigen binding kinetics using Biacore surface Plasmon resonance (SPR). The equilibrium binding constants KD are found to be comparable at 1.81E-09M, 1.96E-09M, for the native and deglycosylated antibody, respectively, and 5.79E-10M for the F(ab’)2. An in vitro biological activity employing a competition binding assay was also developed to demonstrate the role of the Fc glycan. The results confirm that for a neutralizing antibody therapeutic the biological activity of the native MAb-1 and deglycosylated antibody are comparable, thus indicating that the Fc glycan does not contribute to the antigen binding or the biological function. The kinetics and competitive assays performed on an SPR instrument are quick and reliable. Combined with the on-chip electrophoresis method they can be used as monitoring methods for process development and quality control.

A set of haptens structurally resembling the herbicide imazethapyr (PURSUIT) was synthesized and used to derive monoclonal antibodies (MAbs) and direct and indirect competition enzyme immunoassays (EIAs) which could detect imazethapyr, imazaquin (SCEPTER), imazapic (CADRE), and imazamox (RAPTOR) in the 3−30 ng/mL (parts per billion) range, and imazapyr (ARSENAL) and imazamethabenz-methyl (ASSERT) in the 300−500 ppb range. Two MAbs, 3A2 and 3A5, had affinities of 10−75 nM for imazethapyr. MAbs 1A5, 1D2, and 3A5 were specific for the S isomers of the herbicides. Some MAbs were stable in solutions containing up to 15% methanol and 5% acetonitrile in indirect EIAs. Plates coated with hapten conjugates for indirect EIA could be stored frozen. Selectivity for the imidazolinones by some MAbs varied with different coating conjugates. These MAbs and haptens should prove useful in immunochemical analysis and residue recovery methods for imazethapyr and other imidazolinone herbicides. Keywords: Immunoassay; immunoaf...

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.

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.

Imazethapyr is the active ingredient of PURSUIT herbicide. This herbicide belongs to the imidazolinone class of compounds. Polyclonal antibodies have been prepared in rabbits and sheep which specifically recognize this class of compounds. Using immune sera, ELISAs have been developed for imazaquin, imazapyr, imazethapyr, and imazmethabenz methyl (the active ingredient for SCEPTER, ARSENAL, PURSUIT, and ASSERT herbicides, respectively) with sensitivity at low nanogram per milliliter levels. The quantitation of imidazolinones in soil requires a certain amount of sample pretreatment, thus, the throughput is not ideal. A simpler immunoassay method for screening large amounts of soil samples economically would be useful

A sensitive immunoassay system was developed for the quantitation of imazaquin, the active ingredient for SCEPTER herbicide. The assay required a polyclonal antibody generated by immunizing rabbits with a bovine serum albumin conjugate of 5-(2-formylvinyl)-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)benzoic acid. A competitive ELISA system employing horseradish peroxidase conjugated 6-isothiocyanatoimazaquin as a competing molecule was shown to be best for the quantitation of imazaquin

Isomerization of a monoclonal antibody is one of the common routes of protein degradation. An isomerization in the complementarity-determining region (CDR) was found previously and is investigated in depth in this work. Affinity analysis proves that the antibody with one isomerized heavy chain has lower binding. Binding constants were determined, and exhibited a slower on-rate in conjunction with a faster off-rate for this isomerization. To determine the role of the buffer on the rate of isomerization, this antibody was incubated in various matrices and the amount of isomerized antibody was determined by hydrophobic interaction chromatography (HIC). The rate was found to be dependent on the pH as well as the net negative charge of the buffer components that can act as proton acceptors. An Arrhenius plot was performed to predict the levels of isomerization and a comparison of real samples proved the model was correct. This work affirms that isomerization in the CDR of a therapeutic antibody is important to monitor and the formulation buffer plays a significant role in the rate of the isomerization.

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.

Impact of Emerging Technologies on Immunochemical Methods for Environmental Analysis Recombinant Antibodies Against Haptenic Mycotoxins Sequence Analysis of Individual Chains of Antibodies to Triazine Herbicides Recombinant Antibodies to Diuron: A Model for the Phenylurea Combining Site Antidioxin Monoclonal Antibodies: Molecular Modeling of Cross-Reactive Congeners and the Antibody Combining Site Antibody Mimics Obtained by Noncovalent Molecular Imprinting Type Reactivity for Analyte Profiling Analytical Representation and Prediction of Macroscopic Properties: A General Interaction Properties Function Strategies for Immunoassay Hapten Design Hapten Versus Competitor Design Strategies for Immunoassay Development Miniaturized Microspot Multianalyte Immunoassay Systems Very Sensitive Antigen Detection by Immuno-Polymerase Chain Reaction Self-Regenerating Fiber-Optic Sensors Fiber-Optic Immunosensors for Detection of Pesticides Liposome-Amplified Immunoanalysis for Pesticides Polarization Fluoroimmunoassay for Rapid, Specific Detection of Pesticides Immunoaffinity Chromatography Applications in Pesticide Metabolism and Residue Analysis Development of Assay for Analysis of Hg^2]+ Based on Sulfur-Containing Ligands Interpretation of Immunoassay Data Guidelines to the Validation and Use of Immunochemical Methods for Generating Data in Support of Pesticide Registration Quality Standards for Immunoassay Kits Immunochemical Approach for Pesticide Waste Treatment Monitoring of s-Triazines Evaluation and Application of Immunochemical Methods for Mycotoxins in Food Immunodetection of Ecosystem Contaminants: Research, Application, and Acceptance in Canada

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.© (1993) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Monoclonal IgG antibodies against sporozoites of Eimeria tenella were obtained from the ascites fluid of BALB/c mice. Oocysts, sporocysts, and sporozoites were exposed to medium 199, normal ascites fluid, or monoclonal antibodies 1A, 9D, 3D3II, or 2G8f. Specimens were then incubated with ferritin-conjugated goat anti-mouse IgG antibody. Ferritin was uniformly distributed over the surface of sporozoites exposed to 1A, 9D, or 3D3II; ferritin was localized in patches on sporozoites exposed to 2G8f. A uniform layer of ferritin was present on the inner layer of oocyst walls and on the Stieda body and outer surface of sporocysts exposed to 1A, 9D or 3D3II. In specimens treated with 2G8f, ferritin was present on the inner layer of the oocyst wall and the Stieda body, but not on the sporocyst wall. No ferritin was found on specimens exposed to medium 199 or normal ascites fluid. Monoclonal antibodies 1A, 9D, and 3D3II, but not 2G8f, caused complement-mediated lysis of sporozoites. These findings indicate that oocysts, sporocysts, and sporozoites of E. tenella contain common antigens specific for each monoclonal antibody tested.

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.

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 Millonig's phosphate buffer (pH 7.2). Sporozoites suspended in Millonig's 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 Student's 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