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Highly Sensitive Immunoassay for Rat Brain‐Type Creatine Kinase: Determination in Isolated Purkinje Cells
Author(s) -
Kato Kanefusa,
Suzuki Fujiko,
Shimizu Atsuko,
Shinohara Haruo,
Semba Reiji
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb08496.x
Subject(s) - microbiology and biotechnology , creatine kinase , antibody , enolase , polyclonal antibodies , antiserum , immunoassay , antigen , protein subunit , biology , chemistry , biochemistry , immunohistochemistry , immunology , gene
Ultrasensitive enzyme immunoassay method for the measurement of rat brain‐type creatine kinase BB (CK‐BB) was developed by use of purified antibodies specific to the B subunit of creatine kinase. The antibody immunoglobulin G was purified with immunoaffinity chromatography of the antiserum raised in rabbits by injecting the purified rat CK‐BB. The assay system consisted of polystyrene balls with immobilized antibody F(ab′) 2 fragments and the same antibody Fab' fragments labeled with β‐D‐galactosidase from Escherichia coli. The assay was specific to the B subunit of CK (CK‐B), showing about 10% cross‐reactivity with CK‐MB, but it did not cross‐react with CK‐MM and neuron‐specific γγ enolase. The minimum detection limit of the assay was 0.1 pg or 1 amol CK‐BB, being sufficiently sensitive for the measurement of CK‐B contents in the isolated Purkinje cell bodies at the level of single cells. The average content of CK‐B in a single Purkinje cell was 1.64 pg. The CK‐B concentration in rat cerebellum (about 22 μg/mg protein) was about twofold higher than that (about 13 μg/mg protein) in the cerebrum. High levels (> 5 μ/mg protein) of CK‐B were also found in the peripheral tissues such as gastrointestinal tract and urinary bladder, all of which are composed of smooth muscle. Immunohistochemical localization of CK‐B antigens in the CNS revealed that the antigens is distributed not only in the neurons but also in the glial cells.