A RADIOIMMUNOASSAY FOR THE DETECTION OF DELTA CRYSTALLIN IN CHICK LENS DIFFERENTIATION.

Abstract

Delta crystallin was isolated from 10-13 day chick embryo lens fiber cells. The lens fiber cell extract was isoelectrically precipitated at pH 5.1 to remove alpha and beta crystallins. Further purification by filtration through Sephadex G-150 and then acrylamide gel electrophoresis yielded a single, homogeneous preparation of delta crystallin, as characterized by gel electrophoresis. This purified delta crystallin was injected into rabbits to produce a potent antiserum to chick lens delta crystallin. The purified delta crystallin was iodinated with 125 Iodine, using the chloramine-T procedure. A radioimmunoassay for delta crystallin was then developed, using the principles of competitive protein binding analysis. The radioimmunoassay developed here had a minimum sensitivity of 50 nanograms, and effectively ranged to 1000 nanograms. Developing lens rudiments from early chick embryos, beginning from 24 hr incubation up to 72 hr were examined at 6 hr intervals. All determinations from 24 hr through the 48 hr sample showed less than 10 nanograms per 100 lens rudiments. This was below the effective minimum detection limits of the assay. The first accumulation of delta crystallin was detected in the 54 hr sample, and increased thereafter.