Glucose oxidase conjugation

Beyond polyclonal antibodies, monoclonal antibodies to isoxazolyl penicillins were recently produced by immunization of mice with a cloxacillin-human serum albumin conjugate prepared by a mixed anhydride procedure (35). Sensitivity and specificity of these antibodies were tested in an indirect ELISA in which a cloxacillin-glucose oxidase conjugate prepared by an activated ester procedure served as a coating agent. It was found diat die prepared antibodies could be... 

An example of a monomolecular layer electrode coating obtained by this technique is sketched in Sch. 3. Adsorption of rabbit IgG is followed by adsorption of gelatin and specific binding of a glucose oxidase conjugated antibody [57]. 

Schemes Antigen-antibody construction of a monolayer enzyme electrode the adsorbed rabbit IgG (antigen) is coupled with a glucose oxidase conjugated antibody, the antirabbit IgG (whole molecule) produced in goats.

Fig. 9 Binding of polyclonal antibody glucose oxidase conjugates (in the presence of0.1 M glucose and 0.1 mM ferrocene methanol) to a saturated monolayer of whole antigen deposited on the surface of a GC rotating disk electrode. Variation of the coverage with time at three rotation rates (G 1600,...

The major enzymes used in ELISA technology include horseradish peroxidase (HRP), alkaline phosphatase (AP), (3-galactosidase (P-gal), and glucose oxidase (GO). See Chapter 26 for a detailed description of enzyme properties and activities. HRP is by far the most popular enzyme used in antibody-enzyme conjugates. One survey of enzyme use stated that HRP is incorporated in about 80 percent of all antibody conjugates, most of them utilized in diagnostic assay systems. 

Perhaps the most common conjugates of (strept)avidin involve attaching enzyme molecules for use in ELISA systems. As in the case of antibody-enzyme conjugation schemes (Chapter 20), by far the most commonly used enzymes for this purpose are HRP and alkaline phosphatase. Other enzymes such as (3-galactosidase and glucose oxidase are used less often, especially with regard to assay tests for clinically important analytes (Chapter 26). 

If P-galactosidase is used to conjugate with an SMCC-activated (strept)avidin, then there is no need to thiolate the enzyme, since it contains sulfhydryls in its native state (Fujiwara et al., 1988 Sivakoff and Janes, 1988). For conjugations using HRP, alkaline phosphatase, or glucose oxidase, however, thiolation is necessary to add the requisite sulfhydryls. 

Yoshitake, S., Yamada, Y., Ishikawa, E., and Masseyeff, R. (1979) Conjugation of glucose oxidase from Aspergillus niger and rabbit antibodies using N-hydroxysuccinimide ester of N-(4-carboxycyclohexyl methyljmaleimide. Eur.J. Biochem. 101, 395-399. 

W. Sung and Y. Bae, A glucose oxidase electrode based on electropolymerized conducting polymer with polyanion-enzyme conjugated dopant. Anal. Chem. 72, 2177-2181 (2000). 

Selected entries from Methods in Enzymology [vol, page(s)j Detection and quantification, 74, 608-616 effect of antigen/anti-body ratio, 74, 613, 614 applications, 74, 614-615 controls, 74, 612 data analysis, 74, 612-613 immunoglobulin G [conjugation to glucose oxidase, 74, 610 heat aggregation, 74, 609-610 heat denaturation, 74, 610 immobilization, 74, 610] precision, 74,... 

Thus, glycoproteins such as horseradish peroxidase, glucose oxidase, or most antibody molecules can be activated for conjugation by brief treatment with periodate. Cross-linking with an amine-containing protein takes place under alkaline pH conditions through the formation of Schiff base intermediates. These relatively labile intermediates can be stabilized by reduction to a secondary amine linkage with sodium cyanoborohydride (Fig. 303). 

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