IMMUNOREACTIVE ALPHA A CRYSTALLIN IN RAT NONLENTICULAR TISSUES DETECTED WITH A SENSITIVE IMMUNOASSAY METHOD

For the quantitative analysis of the A subunit of alpha-crystallin (alpha-A) in the lens and for the survey of possible existence of alpha-A in the non-lenticular tissues, we have established a highly sensitive and specific immunoassay method for alpha-A. Antisera to alpha-A were raised in rabbits with alpha-A purified from bovine lens, or the C-terminal decapeptide (EEKPSSAPSS) of alpha-A (alpha-A(pep)). The antibodies to alpha-A and alpha-A(pep) were purified by the use of an alpha-A-coupled Sepharose 4B column. The F(ab')2 fragments of purified anti-alpha-A IgG were immobilized on polystyrene balls and the Fab' fragments of purified anti-alpha-A(pep) IgG were labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the sandwich-type immunoassay using the two antibody preparations was < 10 pg alpha-A without any cross-reactivity with alpha-B. By employing the present methods, it was found that a significant amount of immunoreactive alpha-A was present in rat spleen and thymus. Very low levels of immunoreactive alpha-A were detected in the rectum, caecum, liver, kidney, adrenal, cerebellum and brainstem. The immunoreactive alpha-A in the spleen extract was purified partially (about 50% purity) by the use of anti-alpha-A(pep)-coupled Sepharose. The concentration of alpha-A in the spleen was < 1 ng / mg protein before 3 weeks of age. After 5 weeks of age, however, it increased lineally reaching about 20 ng / mg protein by 18 weeks of age. Immunohistochemically, the alpha-A was localized in the reticular cells in the spleen and thymus.