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Development of hybridomas secreting monoclonal antibodies against hemorrhagic factors in Crotalus atrox venom
30 2. 3. 4. 5. 6.
First American Symposium on Animal, Plant and Microbial Toxins NAHAs, L., DENSON, K. W. E. and McFARLANE R. G. (1964) Thromb. Diath. haemorrh . NIEW!AROWSm, C., KIRBY, E. P. and STOCKER, K. (1977) Thromb . Res. 10, 863. HOFMANN, H., DUMAREY, C. and BON C. (1983) Biochimie 65, 201. KORNALIK . F. an d BLOMBACK, B. (1975) Thromb . Res. 6, 53 . MORITA, T., IWANAGA, S. and SUZUKI, T. (1976) J. Biochem. 79, 1089 .
12,
355.
Evaluation of the enzyme immunoassay for the detection of ciguatoxin from fish tissue.
YOSHITsuoi HOKAMA, MARY A. ABAD, LANCE K. SHIRAI, WENDELL HINO and LUCILLE KIMURA (Department of Pathology, University of Hawaii, Honolulu, Hawaii, U .S .A .) . AN ENZYME immunoassay (EIA) procedure for the detection of ciguatoxin (CTX) in fish tissues has been utilized to examine fish from clinically documented ciguatera cases and a variety of fish species from the nearshore waers of the northwestern Hawaiian Islands (NWHI) . Results of 15 toxic and 86 non-toxic fishes demonstrated that the EIA procedure distinguished documented toxic from non-toxic tissues . Evaluation of 511 samples of fishes of various species from the NWHI survey by the EIA method indicated that tissues from a number of species contained CTX-like toxin (11 .9%) . Species having higher percentages of EIA positive and borderline levels included Cheilinus uWasciatus, a frequently implicated species in ciguatera poisoning, Caranx ignoblis and Acanthurus triostegus . Of the larger fishes (Caranx sp .) examined, liver samples gave significantly higher EIA values than their corresponding muscle tissues . Studies with Acanthurus striostegus showed that the male species gave higher EIA values than the females. A slight negative correlation between fish weight and EIA levels was demonstrated . Analysis of the findings of this study showed that the EIA procedure was sensitive (minimum detectable level of CTX at approximately 2-5 pg), practical and specific for CTX-like toxins . The procedure has value in routine examination of fish tissues for the assessment of developing fishing grounds and for the analysis of suspected fishes in ciguatera poisoning outbreaks.
Acknowledgements - Supported in
part by the Division of Aquatic Resources, Hawaii State Department of Land and Natural Resources and The Hokama-Yagawa Fund, University of Hawaii Foundation .
Purification and characterization of a cholera-toxin crossreactive factor produced by
Aeromonas hydrophila . CLIFFORD W. HOUSTON, JAMES D. CAMPBELL, CHARLES GENAUX, FELIX C. W. KOO and ALEXANDER KUROSKY (Departments of Microbiology and Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, U.S .A .) .
Aeromonas hydrophila is
responsible for a wide variety of human diseases, including acute bacterial diarrhea and wound infections . The symptoms of these diseases are in part related to the elaboration of toxic extracellular products . We have discovered that A. hydrophila isolates, grown in Casamino acid-yeast (CYE) broth, can produce high levels of a toxic extracellular product that antigenically crossreacts with cholera enterotoxin, as determined by the enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to produce, isolate and purify this cholera-toxin crossreactive (GTC) factor and to further characterize this toxic virulence factor antigenically, biologically and biochemically. The CTC factor was purified by affinity immunosorbent column chromatography utilizing cholera antitoxin as the immobilized ligand . The results suggest that both cytotoxic and enterotoxic activities are associated with this factor . Sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions revealed a major band with a molecular weight between 45,000 and 50,000 . Isoelectric focusing electrophoresis indicated that the factor has an approximate isoelectric point of 5.1 .
Development of hybridomas secreting monoclonal antibodies against hemorrhagic factors in Crotalus atrox venom . SHYI HUANG and JOHN C. PEREZ (Biology Department, Texas A&I University, Kingsville, TX 78363, U.S .A .) .
CROTALID venoms have cytotoxic properties which could be useful in medical research once they are better characterized. C. atrox venom-hyperimmunized spleen cells were fused with SP2/0 myeloma cells. Forty-one wells containing the hybridoma cells were positive for C. atrox venom, as determined by the ELISA. Cell line 1-e12 was cloned and used to produce ascites tumors in BALB/c mice. The monoclonal antibody produced by cloned and subcultured 1-e12 cells reacted with both C. atrox venom and seven other snake venoms, as well as DEAF A-50 isolated hemorrhagic fractions of C. atrox in the ELISA. Supernatants of the ascites fluid and 1-ell cell culture medium neutralized the hemorrhagic activity of crude C. atrox venom. Antivenon monoclonal antibodies are useful in affinity chromatography for isolation of venom toxins and enzymes.
The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper). AURA S. KAMIGuTI, MARIA CRISIINA C. SOUSA E SILVA, PASQUALE MORENA and LINDA NAHAS (Laboratory of Hematology, Instituto Butantan, Sâo Paulo, Brazil) .
THE ACTION of Bothrops castelnaudi venom on blood coagulation differs from the clot-promoting activity of other Bothrops venoms in that it has an inhibitory effect upon factor X-activation and/or prothrombin activationl'1. In order to understand this rare property among Bothrops venoms, the interference of B. castelnaudi venom on some blood coagulation tests was investigated . An inhibitory effect was observed with 100 mg venom on the following systems: prothrombin time, activated partial thromboplastin time (APTT),
First American Symposium on Animal, Plant and Microbial Toxins NAHAs, L., DENSON, K. W. E. and McFARLANE R. G. (1964) Thromb. Diath. haemorrh . NIEW!AROWSm, C., KIRBY, E. P. and STOCKER, K. (1977) Thromb . Res. 10, 863. HOFMANN, H., DUMAREY, C. and BON C. (1983) Biochimie 65, 201. KORNALIK . F. an d BLOMBACK, B. (1975) Thromb . Res. 6, 53 . MORITA, T., IWANAGA, S. and SUZUKI, T. (1976) J. Biochem. 79, 1089 .
12,
355.
Evaluation of the enzyme immunoassay for the detection of ciguatoxin from fish tissue.
YOSHITsuoi HOKAMA, MARY A. ABAD, LANCE K. SHIRAI, WENDELL HINO and LUCILLE KIMURA (Department of Pathology, University of Hawaii, Honolulu, Hawaii, U .S .A .) . AN ENZYME immunoassay (EIA) procedure for the detection of ciguatoxin (CTX) in fish tissues has been utilized to examine fish from clinically documented ciguatera cases and a variety of fish species from the nearshore waers of the northwestern Hawaiian Islands (NWHI) . Results of 15 toxic and 86 non-toxic fishes demonstrated that the EIA procedure distinguished documented toxic from non-toxic tissues . Evaluation of 511 samples of fishes of various species from the NWHI survey by the EIA method indicated that tissues from a number of species contained CTX-like toxin (11 .9%) . Species having higher percentages of EIA positive and borderline levels included Cheilinus uWasciatus, a frequently implicated species in ciguatera poisoning, Caranx ignoblis and Acanthurus triostegus . Of the larger fishes (Caranx sp .) examined, liver samples gave significantly higher EIA values than their corresponding muscle tissues . Studies with Acanthurus striostegus showed that the male species gave higher EIA values than the females. A slight negative correlation between fish weight and EIA levels was demonstrated . Analysis of the findings of this study showed that the EIA procedure was sensitive (minimum detectable level of CTX at approximately 2-5 pg), practical and specific for CTX-like toxins . The procedure has value in routine examination of fish tissues for the assessment of developing fishing grounds and for the analysis of suspected fishes in ciguatera poisoning outbreaks.
Acknowledgements - Supported in
part by the Division of Aquatic Resources, Hawaii State Department of Land and Natural Resources and The Hokama-Yagawa Fund, University of Hawaii Foundation .
Purification and characterization of a cholera-toxin crossreactive factor produced by
Aeromonas hydrophila . CLIFFORD W. HOUSTON, JAMES D. CAMPBELL, CHARLES GENAUX, FELIX C. W. KOO and ALEXANDER KUROSKY (Departments of Microbiology and Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, U.S .A .) .
Aeromonas hydrophila is
responsible for a wide variety of human diseases, including acute bacterial diarrhea and wound infections . The symptoms of these diseases are in part related to the elaboration of toxic extracellular products . We have discovered that A. hydrophila isolates, grown in Casamino acid-yeast (CYE) broth, can produce high levels of a toxic extracellular product that antigenically crossreacts with cholera enterotoxin, as determined by the enzyme-linked immunosorbent assay (ELISA). The purpose of this study was to produce, isolate and purify this cholera-toxin crossreactive (GTC) factor and to further characterize this toxic virulence factor antigenically, biologically and biochemically. The CTC factor was purified by affinity immunosorbent column chromatography utilizing cholera antitoxin as the immobilized ligand . The results suggest that both cytotoxic and enterotoxic activities are associated with this factor . Sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions revealed a major band with a molecular weight between 45,000 and 50,000 . Isoelectric focusing electrophoresis indicated that the factor has an approximate isoelectric point of 5.1 .
Development of hybridomas secreting monoclonal antibodies against hemorrhagic factors in Crotalus atrox venom . SHYI HUANG and JOHN C. PEREZ (Biology Department, Texas A&I University, Kingsville, TX 78363, U.S .A .) .
CROTALID venoms have cytotoxic properties which could be useful in medical research once they are better characterized. C. atrox venom-hyperimmunized spleen cells were fused with SP2/0 myeloma cells. Forty-one wells containing the hybridoma cells were positive for C. atrox venom, as determined by the ELISA. Cell line 1-e12 was cloned and used to produce ascites tumors in BALB/c mice. The monoclonal antibody produced by cloned and subcultured 1-e12 cells reacted with both C. atrox venom and seven other snake venoms, as well as DEAF A-50 isolated hemorrhagic fractions of C. atrox in the ELISA. Supernatants of the ascites fluid and 1-ell cell culture medium neutralized the hemorrhagic activity of crude C. atrox venom. Antivenon monoclonal antibodies are useful in affinity chromatography for isolation of venom toxins and enzymes.
The anticoagulant effect of Bothrops castelnaudi snake venom (Castelnaud's pit viper). AURA S. KAMIGuTI, MARIA CRISIINA C. SOUSA E SILVA, PASQUALE MORENA and LINDA NAHAS (Laboratory of Hematology, Instituto Butantan, Sâo Paulo, Brazil) .
THE ACTION of Bothrops castelnaudi venom on blood coagulation differs from the clot-promoting activity of other Bothrops venoms in that it has an inhibitory effect upon factor X-activation and/or prothrombin activationl'1. In order to understand this rare property among Bothrops venoms, the interference of B. castelnaudi venom on some blood coagulation tests was investigated . An inhibitory effect was observed with 100 mg venom on the following systems: prothrombin time, activated partial thromboplastin time (APTT),