Conjugated enzyme preparations enzymes

Tests for extents of coupling are conveniently carried out by molecular-sieve chromatography of conjugated enzyme preparations on appropriate gel columns, and comparison of the elution... 

One limitation to this method should be noted. If the antibody-enzyme conjugate is prepared using antibody fragments such as Fab or F(ab )2, then nickel-chelate affinity chromatography will not work, since the requisite Fc portion of the antibody necessary for complexing with the metal is not present. 

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. 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

We have examined in detail the properties of the conjugated enzymes we have synthesized. The results of carbohydrate attachment may be illustrated by considering typical properties that are changed by the modification process, with appropriate illustrations from the range of conjugates we have prepared and characterized. 

Effect of Protein Denaturants. Most of the conjugated enzymes we have prepared show greater resistance to inactivation than do the corresponding native enzymes when treated with protein... 

The formation of the GSH conjugate (or the J4 isomer of androstene-3,17-dione) is monitored at 30°C in a jacketed spectrophotometer micro-cell. The GSH-containing sodium phosphate buffer (0.1 M, 0.9 ml) of appropriate GSH concentration, is added first to the cuvette followed by the substrate in 0.05 ml ethanol (or methanol for d5-an-drostene-3,17-dione). The reaction is initiated by the addition of the enzyme preparation in the appropriate sodium phosphate buffer and... 

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