Highly sensitive enzyme immunoassay systems for three forms (alpha alpha, alpha gamma, and gamma gamma) of rat brain enolase were prepared by use of specific antisera to two distinct subunits (alpha and gamma) of the isozymes and beta-D-galactosidase from Escherichia coli as label. Less than fmol-levels of the homologous dimer forms (alpha alpha and gamma gamma) could be determined with the corresponding antibody F(ab')2-bound solid-phase and the antibody Fab'-beta-D-galactosidase complex. The hybrid form (alpha gamma) could also be assayed specifically by use of the antibody to one subunit as solid-phase, and the antibody to another subunit as labelled complex with a minimum detectable sensitivity of 1 fmol.
Three forms of rat brain enolase (Fletcher, L., Rider, C.C., & Taylor, C.B. (1976) Biochim. Biophys. Acta 452, 245-252), which were separable by DEAE-cellulose chromatography (referred as enolase I, enolase II, and enolase III in order of the elution), were purified with a high yield by use of column chromatographies of Sephadex G-150, Blue Sepharose CL-6B, and hydroxylapatite. About ten mg of each isozyme was obtained from 220 g of the brain with a yield of 30-50%. Sodium dodecyl sulfate gel electrophoresis of purified enolase I and enolase III showed single bands with relative mobilities corresponding to molecular weights of 49,000 (enolase I) or 46,000 (enolase III), and that of purified enolase II showed two bands corresponding to the above molecular sizes. Amino acid analysis of three forms of enolase revealed that the amount of each amino acid enolase II was midway between those of enolase I and enolase III. Antiserum to enolase I or enolase III was raised in New Zealand white rabbits. Results of immunochemical neutralization studies and immunolectrophoresis indicated that anti-enolase III was specific to the subunit of enolase III and reacted with enolase II and enolase III. However, anti-enolase I reacted not only with enolase I and enolase II, but also with enolase in various other tissues. These results support previous reports on crude preparations that enolase II was a hybrid molecule consisting of one enolase I subunit and one enolase III subunit.
We describe a "sandwich-type" enzyme immunoassay for insulin in serum, in which antibody Fab'-beta-D-galactosidase conjugate and an antibody-immobilized silicone rubber solid-phase are used. The interference by serum factors with the solid-phase enzyme immunoassay can now be removed by using a buffer containing gelatin. Serum samples of 50 microL can be analyzed by the enzyme immunoassay, which is as sensitive as radioimmunoassay for human insulin. Our results correlate well with those for radioimmunoassay (r = 0.97, slope = 0.92, y-intercept = 4.6 milli-int. units /L for 181 samples). Between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (5--160 milli-int. units/L).
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