PRODUCTION AND CHARACTERIZATION OF MONOCLONAL-ANTIBODIES AGAINST PANULIRUS-INTERRUPTUS HEMOCYANIN

Since the primary and higher-order structures of hemocyanin from the c rustacean arthropod Panulirus interruptus have been elucidated complet ely, it should be possible to determine which regions of this immunoge nic molecule are recognized most often by antibodies. Monoclonal antib odies were raised against subunits (a) under bar and (b) under bar of this hemocyanin, and fourteen of them were further characterized. The produced antibodies were of class IgG, subclasses 1 or 2a, Most of the m had dissociation constants on the order of magnitude 10(-8)-10(-10), a few had lower affinities. Most clones showed no or negligible cross -reactivity with other crustacean hemocyanins. The reactivity of most other clones diminished with increasing sequence difference between th e investigated hemocyanins. However, in a few instances a stronger rea ctivity with other hemocyanins was observed than with that from Panuli rus interruptus. After complete denaturation of the hemocyanin there w as no reaction with the monoclonal antibodies, indicating that the lat ter recognize conformational epitopes. Only one monoclonal antibody re acted with denatured hemocyanin, This antibody was also the only one w hich reacted with a CNBr digest, which means that it recognizes a sequ ential epitope, Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure, Limi ted proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment repre senting domains 2 and 3. It was determined on Western blots of the dig est on which fragment epitopes for eleven of the monoclonal antibodies were located.