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Immunological assessment of low molecular weight marine toxins
Tenth World Congress
483
inactivation characteristics of sodium channels, and those that create transmembrane pores. Scorpion venoms and sea anemones are rich sources of toxins affecting the kinetics of sodium channels. Other toxins block neuronal potassium channels, and consequently facilitate transmitter release. These include snake toxins with phospholipase activity (e.g. //-bungarotoxin, crotoxin, notexin, and taipoxin), the dendrotoxins from mamba snakes, noxiustoxin from a Mexican scorpion, and MCDP from bee venom. These probably affect different subtypes of voltage-dependent potassium currents in nerve terminals. More recently, potassium channel blocking toxins have been found in sea anemones and in other scorpion venoms. Some of their effects on neuromuscular transmission will be described.
Immunological assessment of low molecular weight marine toxins. Y. HOKAMA (Department of Pathology, JABSOM, University of Hawaii, Honolulu, HI 96822, U.S.A.). A nglEF resum6 of the recent immunological analysis of low molecular weight marine toxins is presented, with emphasis on the detection of ciguatoxin (CTx) and its related polyethers. Palytoxin (PTx) and paralytic shellfish poison (PSP) immunological test systems are also discussed in brief. The competitive radioimmunoassay procedures have been established for PbTx, PSP, PTx and OA using goat-anti-PbTx and rabbit-anti-PTx. These competitive RIA systems detected pmole and nmole quantities of the toxins. The use of enzyme immunoassay systems with monoclonal antibodies is becoming most popular. An enzyme linked immunoabsorbent assay (ELISA) has been developed for OA, PbTx and PSP. The OA immunoassay is being used for screening shellfish suspected of causing diarrhetic shellfish poisoning (DSP). Several test systems have been developed and used in the screening of fish in Hawaii for ciguatera poisoning. A radioimmunoassay using sheep anti-CTx was devised in 1977 for CTx. This system proved successful in the screening of Seriola dumerili for commercial fisheries. Subsequently, a simplified stick enzyme immunoassay (SEIA) was developed and used extensively for catches by sports fishermen. Six-hundred and eighty-one fish were examined and no false negatives were reported. More recently, a highly simplified procedure using colored beads in a solid-phase immunobead assay (SPIA) was developed for field use. Preliminary data are encouraging, with no false negatives to date. This paper summarizes our immunological studies on ciguatera, including recent polyether cross-activity in the stick enzyme system. (Supported in part by the Hawaii State Department of Land and Natural Resources and the Hokama-Yagawa Memorial Fund, UHF.)
Modern approaches to generate antisera against toxic proteins. Use of free synthetic peptides, toxin-specific monoclonal antibodies and genetically engineered toxins. A. Mlh~z (Service de Biochimie des Prot6ines, CEA, Saclay, 91191 Gif-sur Yvette, Cedex, France). Toxic proteins from animal venoms have been widely studied. They are often small in size, folded in compact structures rich in disulphides. They exhibit a multiplicity of actions on various physiological systems. Serotherapy is currently used to protect humans against envenomations by animal bites or stings. This treatment, however, still needs to be improved. With the view to increase (i) paraspecificity of antitoxin antisera, (ii) protecting titers, and (iii) immunogenicity of toxic proteins, we have considered the possibility of using three types of different immunogeus, namely free synthetic peptides, anti-toxin monoclonal antibodies (MAbs) and genetically engineered toxins. The model toxins used during these investigations are curaremimetic toxins from snake venom. They are single chain proteins of 60-74 residues and 4/5 disulphides, which block specifically the nicotinic aeetylcholine receptor (AcChoR). (1) Use of free synthetic peptides: antibody response is known to be under the control of T-cell specific stimulation. We therefore identified, in BALB/c mice (H-2 ~ haplotype), the dominant T-cell epitope(s) of toxin ~t, a short-chain curaremimetic toxin (61 residues) from venom of the spitting cobra N. nigricollis. This was achieved using five synthetic peptides encompassing the whole toxin sequence. Only the fragment 24-41 was capable of stimulating T-cells harvested from mice immunized with the native toxin. Accordingly, only the fragment 24-41 elicited an antibody response. However, the immune response was directed against the cognate sequence of the unfolded toxin. In order to bring conformational restriction to the fragment 24-41, we made it cyclic. The antisera raised against the free cyclic peptide reacted with the native toxin as well as with a large set of toxin variants, and was capable of inhibiting the binding of neurotoxins to AcChoR. (2) Use of monoclonal antibodies (MAbs): anti-idiotypic response: MST2 is a murine MAb whose epitope overlaps the 'toxic' site of curaremimetic toxins (66-74 residues). It recognizes all variants of the long-chain toxins and (-) nicotine, an agonist of AcChoR. MST2 therefore has binding properties that mimic, at least partially, those of AcChoR. Injection in rabbits of MST2 mixed with adjuvant, elicited anti-idiotypic (anti-Id) antibodies that inhibited binding of the toxin to AcChoR. A small proportion of these anti-Id antibodies specifically bound to AcChoR and thereby mimicked the toxin. These data provide a molecular explanation for the previously reported signs of myasthenia gravis, as triggered by antibodies raised against cholinergic antagonists, including snake toxins. The anti-Id response is accompanied by elicitation of auto-anti-anti-Id antibodies. These Ab3 antibodies are capable of both binding and neutralizing the toxin. Clearly, stimulation of the idiotypic network is of interest to elicit toxin-specific neutralizing antibodies. (3) Use of recombinant toxin hybrids: we built a plasmid encoding a hybrid protein composed of the successive sequences of protein A and
483
inactivation characteristics of sodium channels, and those that create transmembrane pores. Scorpion venoms and sea anemones are rich sources of toxins affecting the kinetics of sodium channels. Other toxins block neuronal potassium channels, and consequently facilitate transmitter release. These include snake toxins with phospholipase activity (e.g. //-bungarotoxin, crotoxin, notexin, and taipoxin), the dendrotoxins from mamba snakes, noxiustoxin from a Mexican scorpion, and MCDP from bee venom. These probably affect different subtypes of voltage-dependent potassium currents in nerve terminals. More recently, potassium channel blocking toxins have been found in sea anemones and in other scorpion venoms. Some of their effects on neuromuscular transmission will be described.
Immunological assessment of low molecular weight marine toxins. Y. HOKAMA (Department of Pathology, JABSOM, University of Hawaii, Honolulu, HI 96822, U.S.A.). A nglEF resum6 of the recent immunological analysis of low molecular weight marine toxins is presented, with emphasis on the detection of ciguatoxin (CTx) and its related polyethers. Palytoxin (PTx) and paralytic shellfish poison (PSP) immunological test systems are also discussed in brief. The competitive radioimmunoassay procedures have been established for PbTx, PSP, PTx and OA using goat-anti-PbTx and rabbit-anti-PTx. These competitive RIA systems detected pmole and nmole quantities of the toxins. The use of enzyme immunoassay systems with monoclonal antibodies is becoming most popular. An enzyme linked immunoabsorbent assay (ELISA) has been developed for OA, PbTx and PSP. The OA immunoassay is being used for screening shellfish suspected of causing diarrhetic shellfish poisoning (DSP). Several test systems have been developed and used in the screening of fish in Hawaii for ciguatera poisoning. A radioimmunoassay using sheep anti-CTx was devised in 1977 for CTx. This system proved successful in the screening of Seriola dumerili for commercial fisheries. Subsequently, a simplified stick enzyme immunoassay (SEIA) was developed and used extensively for catches by sports fishermen. Six-hundred and eighty-one fish were examined and no false negatives were reported. More recently, a highly simplified procedure using colored beads in a solid-phase immunobead assay (SPIA) was developed for field use. Preliminary data are encouraging, with no false negatives to date. This paper summarizes our immunological studies on ciguatera, including recent polyether cross-activity in the stick enzyme system. (Supported in part by the Hawaii State Department of Land and Natural Resources and the Hokama-Yagawa Memorial Fund, UHF.)
Modern approaches to generate antisera against toxic proteins. Use of free synthetic peptides, toxin-specific monoclonal antibodies and genetically engineered toxins. A. Mlh~z (Service de Biochimie des Prot6ines, CEA, Saclay, 91191 Gif-sur Yvette, Cedex, France). Toxic proteins from animal venoms have been widely studied. They are often small in size, folded in compact structures rich in disulphides. They exhibit a multiplicity of actions on various physiological systems. Serotherapy is currently used to protect humans against envenomations by animal bites or stings. This treatment, however, still needs to be improved. With the view to increase (i) paraspecificity of antitoxin antisera, (ii) protecting titers, and (iii) immunogenicity of toxic proteins, we have considered the possibility of using three types of different immunogeus, namely free synthetic peptides, anti-toxin monoclonal antibodies (MAbs) and genetically engineered toxins. The model toxins used during these investigations are curaremimetic toxins from snake venom. They are single chain proteins of 60-74 residues and 4/5 disulphides, which block specifically the nicotinic aeetylcholine receptor (AcChoR). (1) Use of free synthetic peptides: antibody response is known to be under the control of T-cell specific stimulation. We therefore identified, in BALB/c mice (H-2 ~ haplotype), the dominant T-cell epitope(s) of toxin ~t, a short-chain curaremimetic toxin (61 residues) from venom of the spitting cobra N. nigricollis. This was achieved using five synthetic peptides encompassing the whole toxin sequence. Only the fragment 24-41 was capable of stimulating T-cells harvested from mice immunized with the native toxin. Accordingly, only the fragment 24-41 elicited an antibody response. However, the immune response was directed against the cognate sequence of the unfolded toxin. In order to bring conformational restriction to the fragment 24-41, we made it cyclic. The antisera raised against the free cyclic peptide reacted with the native toxin as well as with a large set of toxin variants, and was capable of inhibiting the binding of neurotoxins to AcChoR. (2) Use of monoclonal antibodies (MAbs): anti-idiotypic response: MST2 is a murine MAb whose epitope overlaps the 'toxic' site of curaremimetic toxins (66-74 residues). It recognizes all variants of the long-chain toxins and (-) nicotine, an agonist of AcChoR. MST2 therefore has binding properties that mimic, at least partially, those of AcChoR. Injection in rabbits of MST2 mixed with adjuvant, elicited anti-idiotypic (anti-Id) antibodies that inhibited binding of the toxin to AcChoR. A small proportion of these anti-Id antibodies specifically bound to AcChoR and thereby mimicked the toxin. These data provide a molecular explanation for the previously reported signs of myasthenia gravis, as triggered by antibodies raised against cholinergic antagonists, including snake toxins. The anti-Id response is accompanied by elicitation of auto-anti-anti-Id antibodies. These Ab3 antibodies are capable of both binding and neutralizing the toxin. Clearly, stimulation of the idiotypic network is of interest to elicit toxin-specific neutralizing antibodies. (3) Use of recombinant toxin hybrids: we built a plasmid encoding a hybrid protein composed of the successive sequences of protein A and