Conjugate, enzyme immunoassay

Enzyme Immunoassay. In EIA, antibody (or antigen) is labeled with (or conjugated to) an enzyme, and this reagent is used to complex and quantify the target antigen (or antibody) in a sample. Conjugation may utilize a variety of chemical methods. 

Figure 1 Schematic of an enzyme immunoassay. (1, 2) The test solution and enzyme conjugate are added to a tube or well pre-coated with anti-anal) e antibodies. (3) After the inhibition step, the solid phase is washed, and only antibody-bound material is retained. (4A-C) Colorless substrate is added and is converted to a visible color in inverse proportion to the amount of analyte in the sample...

Hashida, S., and Ishikawa, E. (1985) Use of normal IgG and its fragments to lower the nonspecific binding of Fab -enzyme conjugates in sandwich enzyme immunoassay. Anal. Lett. 18(B9), 1143-1155. 

Jeanson, A., Cloes, J.M., Bouchet, M., and Rentier, B. (1988) Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent. Anal. Biochem. 172, 392. 

Kitagawa, T., Kawasaki, T., and Munechika, H. (1982) Enzyme immunoassay of blasticidin S with high sensitivity a new and convenient method for preparation of immunogenic (hapten-protein) conjugates. J. Biochem. (Tokyo) 92, 585-590. 

O Sullivan, M., Gnemmi, E., Morris, D., Chieregatti, G., Simmonds, A., Simmons, M., Bridges, J., and Marks, V. (1979) Comparison of two methods of preparing enzyme—antibody conjugates Application of these conjugates for enzyme immunoassay. Anal. Biochem. 100, 100-108. 

Tussen, R, and Kurstak, E. (1984) Highly efficient and simple methods for the preparation of peroxidase and active peroxidase—Antibody conjugates for enzyme immunoassays. Anal. Biochem. 136, 451-457. 

Yoshitake, S., Imagawa, M., and Ishikawa, E. (1982b) Efficient preparation of rabbit Fab -horseradish peroxidase conjugates using maleimide compounds and its use for enzyme Immunoassay. Anal. Lett. 15(B2), 147-160. 

The Abbott IMx , a dedicated commercial immunoassay analyzer that employs FPIAs for small molecules, can also determine larger analytes by a fluorescence-based microparticle capture enzyme immunoassay (MEIA).(44) In this system, antibody-coated0.47- mlatexparticles are used for both sandwich and competitive assays, and alkaline phosphatase conjugates that bind to the particles cleave 4-methylumbelliferyl phosphate to generate the fluorophore. 

Avrameas, S. and Guilbert, B. (1972) Enzyme-immunoassay for the measurement of antigens using peroxidase conjugates. Biochemie 54, 837. 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

Immunoassays employ monoclonal or polyclonal antibody preparations to detect and quantify the product. The specificity of antibody-antigen interaction ensures good assay precision. The use of conjugated radiolabels (radioimmunoassay RIA) or enzymes (enzyme immunoassay EIA) to allow detection of antigen-antibody binding renders such assays very sensitive. Furthermore, when compared to bioassays, immunoassays are rapid (undertaken in minutes to hours), inexpensive and straightforward to undertake. 

A competitive enzyme immunoassay for the quantification of ivermectin residues in bovine liver has also been reported recently (85). This method uses a polyclonal antiserum raised in rabbits against 5-O-succinoylivermectin-trans-ferrin conjugate. Cross-reactivity was demonstrated with doramectin, a member... 

A test strip enzyme immunoassay that could not discriminate between the 17 - and 17 -stereoisomers but allowed on-site screening of urine samples within 45-60 min was also reported (118). In this assay, 17, 19-nortestosterone was coupled to bovine serum albumin through a hemisuccinate bridge at the 17-position (119). This conjugate was used to raise polyclonal antibodies that would... 

Instead of using human serum albumin as a carrier protein, other workers (135) utilized ovalbumin for preparing the diazotized clenbuterol antigen in an enzyme immunoassay developed for screening of clenbuterol residues in bovine urine, liver, and eye. Alkaline phosphatase rather than -galactosidase was also used as an enzyme label in the preparation of the enzyme-clenbuterol conjugate. 

A generic enzyme immunoassay for the determination of several synthetic corticosteroids including dexamethasone, betamethasone, flumethasone, triamcinolone, prednisolone, and methylprednisolone in milk, liver, kidney, and muscle samples was recently developed (156). Antibodies raised against dexamethasone-21-hemisuccinate-bovine serum albumin were used in this assay, whereas dexa-methasone-horseradish peroxidase was the label conjugate. Skimmed milk could be directly screened for the presence of corticosteroids at limits of detection of 0.1 ppb for dexamethasone, betamethasone, and flumethasone, 0.3 ppb for triamcinolone and 0.5 ppb for prednisolone. Tissue samples were submitted, prior to the immunoassay, to an extraction/cleanup procedure involving liquid-liquid partitions with acetonitrile-water followed by hexane-chloroform. Background values for bovine liver, swine kidney, and calf muscle were determined to be 0.26, 0.26, and 0.07 ppb, respectively, of dexamethasone equivalents. 

Figure 5.8 depicts a typical immunoassay-based LAPS. The reaction of an antibody to its antigen will immobilise the species and after washing, only those species that have been successfully bound will be left onto the LAPS surface. Here, a conjugated enzyme, e.g., urease, will change the pH value of the test sample by enzymatic catalysis after injection of urea. 

Figure 6. Diagrammatic scheme for performance of a solid-phase enzyme immunoassay ((0) juice sample (limonin) (%-E) tracer (limonin-enzyme conjugate))...

Sensitivity of immunoassays is largely conditioned by markers applied for conjugation with the antibody. Traditional immunodetection methods ELISA with radioactive markers (radioimmunoassay—RIA), enzymatic markers (enzyme immunoassays—El A), or fluorescent markers (fluoroenzyme immunoassays FEIA) are currently the most widely used techniques in laboratory analysis of allergens as well as in clinical studies for determination of general and specific IgE and other subclasses of immunoglobulines, e.g., IgG4 in the Immuno-CAP system (Samson, 2001 Duran-Tauleria et al., 2004 Lidholm et al., 2006). 

Since the first report on enzyme immunoassay appeared in 1971 (E5, V2) relevant techniques have been developed and applications are increasing. There are many reports on new coupling agents for preparing conjugates of... 

Aizawa et al. reported the development of a competitive enzyme immunoassay using electrodes (A2). In this method, antibody immobilized on the membrane is attached to the electrodes for oxygen. When catalase-antigen conjugate binds to the immobilized antibody, production of oxygen can be... 

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