Antibodies glucose oxidase

Adsorbed antigen Gelatin Antibody-glucose oxidase (sacrificial antibody) conjugate... 

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). 

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. 

Enzyme labels are usually coupled to secondary antibodies or to (strept)avidin. The latter is used for detection of biotinylated primary or secondary antibodies in ABC methods (see Sect. 6.2.1). Enzyme labels routinely used in immunohisto-chemistry are horseradish peroxidase (HRP) and calf intestinal alkaline phosphatase (AP). Glucose oxidase from Aspergillus niger and E. coli (3-galactosidase are only rarely applied. 

Antigen-Antibody Immobilization of Glucose Oxidase. Kinetic Analysis... 

FIGURE 5.26. Antigen-antibody construction of a monolayer glucose oxidase electrode with an attached ferrocenium cosuhstrate and cyclic voltammetric response in a phosphate buffer (pH 8) at 25°C and a scan rate of 0.04 V/s. a Attached ferrocene alone, h Addition of the substrate, c Primary plots, d Secondary plot. The numbers on the curves in parts h and c are the values of the substrate concentration in mM. Adapted from Figure 2 in reference 24, with permission from the American Chemical Society. 

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... 

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). 

Enzymes that are glycosylated (i.e., HRP and glucose oxidase) may be oxidized according to the following method to produce aldehyde groups for reductive amination coupling to an antibody molecule. 

---