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

FG PERTON, W BARON, AJ SCHEFFER, JJ BEINTEMA

Research output: Contribution to journalComment/Letter to the editorAcademicpeer-review

5 Citations (Scopus)

Abstract

Since the primary and higher-order structures of hemocyanin from the crustacean arthropod Panulirus interruptus have been elucidated completely, it should be possible to determine which regions of this immunogenic molecule are recognized most often by antibodies. Monoclonal antibodies 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 them 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 the investigated hemocyanins. However, in a few instances a stronger reactivity with other hemocyanins was observed than with that from Panulirus interruptus.

After complete denaturation of the hemocyanin there was no reaction with the monoclonal antibodies, indicating that the latter recognize conformational epitopes. Only one monoclonal antibody reacted with denatured hemocyanin, This antibody was also the only one which reacted with a CNBr digest, which means that it recognizes a sequential epitope, Several antibodies showed a faint reaction on Western blots, indicating the presence of some refolded native structure, Limited proteolysis of the hemocyanin molecule results in the formation of a 18 kDa fragment, representing domain 1, and a 55 kDa fragment representing domains 2 and 3. It was determined on Western blots of the digest on which fragment epitopes for eleven of the monoclonal antibodies were located.

Original languageEnglish
Pages (from-to)243-247
Number of pages5
JournalBiological chemistry hoppe-Seyler
Volume376
Issue number4
Publication statusPublished - Apr-1995

Keywords

  • CRUSTACEANS
  • EPITOPES
  • HEMOCYANIN
  • MONOCLONAL ANTIBODIES
  • PANULIRUS INTERRUPTUS
  • AMINO-ACID-SEQUENCE
  • LINKED IMMUNOSORBENT-ASSAY
  • LIMITED PROTEOLYSIS
  • SUBUNIT
  • PROTEINS
  • SITE

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