Rat liver alcohol dehydrogenase. II. Quantitative enzyme-linked immunoadsorbent assay.
Monospecific rabbit antibodies against purified Fischer-344 rat liver alcohol dehydrogenase were produced and used to develop an enzyme-linked immunoadsorbent assay for alcohol dehydrogenase. The assay is based upon the competitive inhibition of specific antibody binding to antigen (alcohol dehydrogenase adsorbed onto plastic microtiter plates) by soluble alcohol dehydrogenase (contained in unknown sample extracts or in known standard solutions). The amount of bound antibody is determined following incubation with peroxidase-linked second antibody (goat anti-rabbit IgG antibody-peroxidase conjugate) by colorimetric measurements of peroxidase activity at 490 nm in the presence of O-phenylenediamine. The assay is highly sensitive (it detects 10-1000 ng alcohol dehydrogenase/50 microliter) and it offers a precise (interexperimental variations in samples were less than 10%), rapid (6-8 h), and specific method for measurements of alcohol dehydrogenase in tissue homogenates or cultured hepatocytes. The assay was used to study changes in alcohol dehydrogenase levels during the growth cycle of cultured hepatocytes over a 2-week period and in rat liver homogenates after starving the animals for 72 h. In cultured hepatocytes, alcohol dehydrogenase activity and immunoassayable enzyme levels decreased coordinately during lag and early log phase, from 13.2 +/- 1.2 to 5.0 +/- 1.0 micrograms enzyme/mg protein, respectively. In mid-log phase, the enzyme levels were very low (1.3 +/- 0.4 micrograms enzyme/mg protein). During stationary phase, the levels (5.7 +/- 0.6 micrograms enzyme/mg protein) increased to 35% of the levels of freshly isolated hepatocytes (15.6 +/- 1.4 micrograms enzyme/mg protein). In starved animals, the enzyme levels decreased from 7.56 +/- 0.55 to 2.97 +/- 0.27 mg enzyme/liver. These changes also coincided with decreases in activity from 8.84 +/- 0.35 to 6.56 +/- 0.68 microM/min/liver.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000918:Antibody Specificity; D002453:Cell Cycle; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D007124:Immunoenzyme Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344; D013217:Starvation
D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase