Production and characterization of monoclonal antibodies against human type K pyruvate kinase.
K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.
van Erp
H E
HE
Department of Haematology, University Hospital, Utrecht, The Netherlands.
Roholl
P J
PJ
Rijksen
G
G
Sprengers
E D
ED
van Veelen
C W
CW
Staal
G E
GE
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D000917:Antibody Formation; D005910:Glioma / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000276:immunology*; D011770:Pyruvate Kinase / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011770:Pyruvate Kinase