The development of monoclonal antibodies to the isozymes of malate dehydrogenase, with laboratory applications

Malate dehydrogenase (MDH) is a well characterized pyridine nucleotide linked enzyme which catalyzes the ordered single displacement reaction: L-malate + NAD$\sp+\rightleftharpoons$ oxaloacetate + NADH + H$\sp+$. In higher eukaryotic cells, the enzyme is present in two forms, cytoplasmic and mitochondrial. The mitochondrial isozyme (m-MDH) is a key constituent of the tricarboxylic acid cycle. The cytoplasmic isozyme (c-MDH) is a member of the malate-aspartate shuttle, which bidirectionally permits the flow of NADH reducing equivalents into and out of the mitochondria. This paper will describe the development of two monoclonal antibodies, each with high affinity to one isozyme and low or no cross reactivity to the other. The monoclonals were then characterized and purified. Their effect on the enzymatic activity of the isozymes was investigated. Finally, two immunofluorescent assays were developed using the antibodies.