Specific Cross-Reactivity of Antibodies Raised Against Two Major Stress Proteins, Stress 70 and Chaperonin 60, in Diverse Species

Abstract

Immunoblot analysis using several antibodies raised against two major families of stress proteins, stress 70 and chaperomn 60 (cpn60), which are highly conserved in mammals, was carried out in diverse species often used in environmental research, including molluscs, annelids, crustaceans, echinoderms, and fish The study revealed surprisingly different patterns of antibody cross reactivity among species The monoclonal anti‐stress 70 antibody (mAb) C92 was the least cross reactive for all species tested The mAbs anti‐stress 70 N27, BRM‐22, and 3a3 were more broadly cross reactive, but their binding specificities to stress 70 isoforms in the diverse species tested did not correlate with one another or follow taxonomic lines The polyclonal anti‐stress 70 antibody reacted to proteins in the 70 to 74 kDa range in all fish examined and in most invertebrates When a polyclonal antibody (pAb) raised against cpn60 from a moth was used as a probe, specific binding was observed with proteins in the 60 to 64 kDa range in all fish examined and in most invertebrates However, the size and number of isoforms that reacted with the pAb were species specific These data suggest that these two major stress protein families are less highly conserved in invertebrates and fish than in mammals Therefore, to minimize misinterpretation when using antibodies in heterologous assays with species in which the stress response has not been well characterized, it is important to determine which isoforms of stress 70 react with a particular antibody and to take into account the differential regulation of each member of this multigene family

Does Stress Protein Induction by Copper Modify Natural Toxicity in Sponges?

“…The monoclonal antibody used, which was raised against bovine brain HSP70 (mAb), immunologically cross-reacted with both the HSP72 and the HSP54 kDa proteins. This antibody is known to cross-react with proteins of different mo-lecular weights: 70-and 72-kDa proteins in mammals and crustaceans, a 78-kDa in bivalves and echinoderms [34], and both 78-and 72-kDa proteins in the clam Ruditapes decussata [35]. Yet the cross-reaction of a specific antibody with proteins of two different HSP families is remarkable and had not been reported to date in sponges.…”

Biomarkers in Aquatic Plants: Selection and Utility

“…Furthermore, because stress-proteins are integrators of protein damage, they may provide added value to biomonitoring, by increasing the sensitivity of biological testing and complementing tests that measure more specific modes of toxic action (Bierkens 2000 ;Bierkens et al 1998 ;Sanders et al 1994) . Hsp70 is induced by a wide variety of chemicals, and evidence exists that measuring Hsp70 induction adds to detection sensitivity by a factor of 5-1,000, depending on the class of chemicals involved (Bierkens et al 1998) .…”

Two‐dimensional gel analysis of stress proteins identified in Chironomus flaviplumus (Diptera: Chironomidae) exposed to 4‐nonylphenol

“…Heat shock proteins increase resistance to potential toxicants most likely by stabilizing cellular proteins (Bendorf et al 2001). The separation of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970) followed by western blotting has been the most commonly used technique for monitoring expression of Hsp70 in aquatic organisms (Sanders et al 1994;Karouna-Renier & Zehr 1999).…”