Immunoreactive alpha A crystallin in rat non-lenticular 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 Apep). The antibodies to alpha A and alpha Apep 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 Apep 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 less than 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 Apep-coupled Sepharose. The concentration of alpha A in the spleen was less than 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.
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
K
K
Kato
H
H
Shinohara
N
N
Kurobe
S
S
Goto
Y
Y
Inaguma
K
K
Ohshima
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D003429:Cross Reactions; D003459:Crystallins / Q000032:analysis* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G; D007150:Immunohistochemistry; D007908:Lens, Crystalline / Q000737:chemistry*; D046911:Macromolecular Substances; D008297:Male; D009928:Organ Specificity; D051381:Rats; D011919:Rats, Inbred Strains; D013154:Spleen / Q000166:cytology*; D013950:Thymus Gland / Q000166:cytology*
D003459:Crystallins; D007074:Immunoglobulin G; D046911:Macromolecular Substances