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A sensitive enzyme immunoassay for measurements of rat parvalbumin was established using antibodies raised in rabbits with parvalbumin purified from skeletal muscles. Antibodies in the antiserum were purified with a parvalbumin-coupled Sepharose column. The sandwich-type immunoassay system for parvalbumin was composed of polystyrene balls with immobilized purified antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The assay was highly sensitive and the minimum detection limit was 1 pg parvalbumin/tube. The assay did not cross-react with other calcium binding proteins, including human S-100a0 and S-100b proteins, rat 28-kDa calbindin-D, and bovine calmodulin. High concentrations of parvalbumin were observed in the skeletal muscles, especially in those composed of fast-twitch fibers, and in the diaphragm and tongue, but not in heart muscle. A relatively high concentration was estimated in the central nervous tissue. Parvalbumin was detected in the cerebral cortex and cerebellum of gestational 15-day fetuses. However, the levels of parvalbumin in the muscle tissues and central nervous tissue were very low in rats before 1 week of age. Thereafter, they increased sharply, reaching the adult levels by 5 weeks in most of the tissues. Parvalbumin concentrations in adult rat soleus muscle increased less than 20-fold within 10 days after transection of the ipsilateral sciatic nerve, while the concentrations in the extensor digitorum longus muscle did not change in the same period.

In order to determine the quantitative distribution of alpha B crystallin (alpha B) in non-lenticular tissues, we have established a sensitive immunoassay system for specific measurement of alpha B. Antisera were raised in rabbits by injecting alpha B purified from bovine lenses, or C-terminal decapeptide (KPAVTAAPKK) of alpha B (alpha Bpep). The antibodies to alpha B and alpha Bpep were purified by the use of alpha B-coupled Sepharose column. The F(ab')2 fragments of antibody IgG to alpha B were immobilized on polystyrene balls and the Fab' fragments of antibody IgG to alpha Bpep were labeled with beta-D-galactosidase from Escherichia coli. The sandwich-type enzyme immunoassay consisted of the above two antibodies was sensitive, and the minimum detection limit of the assay was 10 pg alpha B without any crossreactivity with alpha A. By using the assay method, it is revealed that the alpha B was distributed in most of the tissues examined. Among the non-lenticular tissues, alpha B was present at high levels in the heart and striated muscles, especially in the soleus muscle, and kidney. High levels of alpha B in the muscle tissues were also seen in various animals. Developmental increases of alpha B in rat muscle tissues and kidney were observed from 16 days of gestational age to 1 or 5 weeks of postnatal age. In contrast, the alpha B in the brain kept a low level during the same period. After 5 weeks of age, alpha B concentrations in the brain increased sharply, reaching the adult levels at 9 weeks of age. Immunohistochemical staining with anti-alpha Bpep revealed that alpha B was positive not only in glial cells, in the central nervous tissues, but also in some neurons of spinal cord, brainstem, hippocampus, and olfactory bulb. Spermatocytes in the testis were also immunopositive for alpha B.

A sensitive sandwich-type enzyme immunoassay for the aldolase isozyme, A4, was developed using purified antibodies specific to the A subunit of aldolase. The antibodies were raised in sheep being immunized with purified aldolase A4 and then purified by immunoaffinity chromatography on a column of aldolase A4-coupled Sepharose. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody F(ab')2 fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was sensitive enough to detect 10 pg/tube of aldolase A4. The assay was specific to the A subunit of aldolase (aldolase A). It cross-reacted about 40% to aldolase A3C, 7% to A2C2 and 0.3% to AC3, but not cross-reacted with C4 nor B4. Coefficients of variation in intra- and inter-assay were less than 16%. Serum aldolase A levels were determined in healthy adults, which were about 200 ng/ml. The distribution and concentrations of immunoreactive aldolase A in various human tissues were also determined. High concentrations of aldolase A were found in skeletal muscle, heart muscle, cerebrum and lymphatic tissue.

Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity-purified with a column of brain phosphorylase-coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase-coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich-type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross-reactivity of the assay with muscle phosphorylase was less than 1%. Tissue concentrations of immunoreactive brain-type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.