- Email: [email protected]
The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to retronecine
32
9th World Congress
The development o f an enzyme-linked immunosorbent assay ( ELISA ) for the detection o f antibodies to retronecine. MARY A. BOBER,1 MARK J. KURTH,1 BRIAN MILLER1 and MICHAEL MOUNT2 (~Departments of Chemistry and 2Clinical Pathology, University of California, Davis, CA 95616, U.S.A.). A CONVENIENTsynthetic source of retronecine (155 mol.wt) can be obtained by hydrolyzing the commercially available moncrotaline. Suecinylated retronecine was conjugated to bovine serum albumin (BSA) using the direct coupling carbodiimide method. The molar concentration of hapten was 2.5 times that of the number of moles of primary amino groups for BSA. The coupling efficiency of 12 haptens per BSA molecule was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS). Rabbits were immunized subcutaneously with 1 mg/kg of the retronecine - BSA conjugate. Blood samples were collected 6 days after a second booster injection (1 mg/ml) and assayed for the presence of antibodies directed against retronecine. Retronecine and a representative macrocyclic naturally occurring pyrrolizidine alkaloid (PA), riddelliine, were conjugated to ovalbumin and conalbumin, respectively, for coating antigens in the ELISA indirect antibody detection assay. Three concentrations of retronecine - ovalbumin and riddelliine - conalbumin (i, 10, 100/~g/ml) were used to coat the microtiter plates. A blocking step to decrease non-specific binding was included in the assay. The blocking step consisted of the addition to each well (200#1) of a 1% solution of a non-dairy creamer. Antibodies to retronecine can be detected using the indirect ELISA method. Microtiter plates coated with retronecine - ovalbumin at concentrations of 10 and 100 #g/ml had absorbance values of 0.78 and 1.49 at a l : 100 titer of antiretronecine, and 0.60 and 1.01 with an antibody titer of I : I000, respectively. Microtiter plates coated with riddelliine - conalbumin (1 #g/ml) were able to detect antibodies to retronecine indicating absorbance values of 0.447 and 0.446 at antibody titers of I : 100 and 1 : 1000, respectively.
lntraspecific variability in acclimated cell toxicities among Atlantic and Pacific clones of the ciguatera-causing dinoflagellate Gambierdiscus toxicus. JEFFREYW. BOMnER,~ DONALD R. TINDALL1 and DONALD M. MILLER2 (Departments of ~Botany and 2Physiology, Southern Illinois University, Carbondale, IL 62901, U.S.A.). T.E CIRCUMTROPICALDISEASEciguatera in humans is caused by ingesting fish containing toxins produced by Gambierdiscus toxicus and associated dinoflagellates. Until now, the variability in the number of cases of ciguatera has been an enigma, e.g. despite the presence of G. toxicus in the Florida Keys and Bermuda the number of ciguatera cases in these areas is small relative to the Caribbean. This study indicates that some Caribbean isolates of G. toxicus are inherently more toxic than isolates from Florida and Bermuda. One Caribbean clone yielded 102.25 MU(mouse units)/mg of dried cells, whereas clones from Bermuda, the Bahamas and Florida ranged from only 2.07 to a maximum of 9.85 MU/mg of dried cells. The toxicity values are derived only from cultures which are physiologically adapted to the same environment over several months. This work indicates that once the variation in toxicity due to the non-acclimated condition is eliminated, the comparisons can be used chemosystematically and may help identify separate races of G. toxicus. The magnitude of the clonal heterogeneity helps explain the wide geographic differences in the incidence of the disease. There is no clear relationship between toxicity and acclimated reproduction rates, nitrate nor ammonia uptake rates. Supported by U.S. Army Medical Research Institute of Infectious Diseases (DAMD- 17-85-R-012 l), Illinois Indiana Sea Grant Program and Myron Hokin.
Approach of the mode of action of crotoxin, a neurotoxic phospholipase A2. CASSlANBON, FRANqOISRADVANYI, GRAZYNA FAURE and BERNARD SALIOU (Laboratoire des Venins, Unit6 associ6e Pasteur/INSERM N. 285, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France).
CROTOXIN, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a potent neurotoxin which possesses a phospholipase A2 activity and blocks neuromuscular transmission. Crotoxin consists of two nonidentical subunits: a basic component B, which carries the phospholipase A2 activity of the toxin and possesses a low toxicity, and an acidic component A, which has no enzymatic activity although it resembles a phospholipase A2 in its primary structure. Component A, is not toxic by itself but considerably enhances the lethal potency of the phospholipase component B. Upon interaction with biological or artificial membranes, the two subunits dissociate: component A is released free in solution and component B is bound. The isolated component B possesses a low affinity for unilamellar vesicles constituted of zwitterionic phospholipids, but binds with a high affinity to negatively charged phospholipids. The non enzymatic component A enhances the selectivity of compound B for negatively charged phospholipids since it completely inhibits the low affinity binding of component B to vesicles of zwitterionic phospholipids. These observations strongly suggest that negatively charged phospholipids are the physiological target of crotoxin or at least constitute an important component of this target. This hypothesis implies that, at variance
9th World Congress
The development o f an enzyme-linked immunosorbent assay ( ELISA ) for the detection o f antibodies to retronecine. MARY A. BOBER,1 MARK J. KURTH,1 BRIAN MILLER1 and MICHAEL MOUNT2 (~Departments of Chemistry and 2Clinical Pathology, University of California, Davis, CA 95616, U.S.A.). A CONVENIENTsynthetic source of retronecine (155 mol.wt) can be obtained by hydrolyzing the commercially available moncrotaline. Suecinylated retronecine was conjugated to bovine serum albumin (BSA) using the direct coupling carbodiimide method. The molar concentration of hapten was 2.5 times that of the number of moles of primary amino groups for BSA. The coupling efficiency of 12 haptens per BSA molecule was determined using 2,4,6-trinitrobenzenesulfonic acid (TNBS). Rabbits were immunized subcutaneously with 1 mg/kg of the retronecine - BSA conjugate. Blood samples were collected 6 days after a second booster injection (1 mg/ml) and assayed for the presence of antibodies directed against retronecine. Retronecine and a representative macrocyclic naturally occurring pyrrolizidine alkaloid (PA), riddelliine, were conjugated to ovalbumin and conalbumin, respectively, for coating antigens in the ELISA indirect antibody detection assay. Three concentrations of retronecine - ovalbumin and riddelliine - conalbumin (i, 10, 100/~g/ml) were used to coat the microtiter plates. A blocking step to decrease non-specific binding was included in the assay. The blocking step consisted of the addition to each well (200#1) of a 1% solution of a non-dairy creamer. Antibodies to retronecine can be detected using the indirect ELISA method. Microtiter plates coated with retronecine - ovalbumin at concentrations of 10 and 100 #g/ml had absorbance values of 0.78 and 1.49 at a l : 100 titer of antiretronecine, and 0.60 and 1.01 with an antibody titer of I : I000, respectively. Microtiter plates coated with riddelliine - conalbumin (1 #g/ml) were able to detect antibodies to retronecine indicating absorbance values of 0.447 and 0.446 at antibody titers of I : 100 and 1 : 1000, respectively.
lntraspecific variability in acclimated cell toxicities among Atlantic and Pacific clones of the ciguatera-causing dinoflagellate Gambierdiscus toxicus. JEFFREYW. BOMnER,~ DONALD R. TINDALL1 and DONALD M. MILLER2 (Departments of ~Botany and 2Physiology, Southern Illinois University, Carbondale, IL 62901, U.S.A.). T.E CIRCUMTROPICALDISEASEciguatera in humans is caused by ingesting fish containing toxins produced by Gambierdiscus toxicus and associated dinoflagellates. Until now, the variability in the number of cases of ciguatera has been an enigma, e.g. despite the presence of G. toxicus in the Florida Keys and Bermuda the number of ciguatera cases in these areas is small relative to the Caribbean. This study indicates that some Caribbean isolates of G. toxicus are inherently more toxic than isolates from Florida and Bermuda. One Caribbean clone yielded 102.25 MU(mouse units)/mg of dried cells, whereas clones from Bermuda, the Bahamas and Florida ranged from only 2.07 to a maximum of 9.85 MU/mg of dried cells. The toxicity values are derived only from cultures which are physiologically adapted to the same environment over several months. This work indicates that once the variation in toxicity due to the non-acclimated condition is eliminated, the comparisons can be used chemosystematically and may help identify separate races of G. toxicus. The magnitude of the clonal heterogeneity helps explain the wide geographic differences in the incidence of the disease. There is no clear relationship between toxicity and acclimated reproduction rates, nitrate nor ammonia uptake rates. Supported by U.S. Army Medical Research Institute of Infectious Diseases (DAMD- 17-85-R-012 l), Illinois Indiana Sea Grant Program and Myron Hokin.
Approach of the mode of action of crotoxin, a neurotoxic phospholipase A2. CASSlANBON, FRANqOISRADVANYI, GRAZYNA FAURE and BERNARD SALIOU (Laboratoire des Venins, Unit6 associ6e Pasteur/INSERM N. 285, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France).
CROTOXIN, the major toxic component of the South American rattlesnake, Crotalus durissus terrificus, is a potent neurotoxin which possesses a phospholipase A2 activity and blocks neuromuscular transmission. Crotoxin consists of two nonidentical subunits: a basic component B, which carries the phospholipase A2 activity of the toxin and possesses a low toxicity, and an acidic component A, which has no enzymatic activity although it resembles a phospholipase A2 in its primary structure. Component A, is not toxic by itself but considerably enhances the lethal potency of the phospholipase component B. Upon interaction with biological or artificial membranes, the two subunits dissociate: component A is released free in solution and component B is bound. The isolated component B possesses a low affinity for unilamellar vesicles constituted of zwitterionic phospholipids, but binds with a high affinity to negatively charged phospholipids. The non enzymatic component A enhances the selectivity of compound B for negatively charged phospholipids since it completely inhibits the low affinity binding of component B to vesicles of zwitterionic phospholipids. These observations strongly suggest that negatively charged phospholipids are the physiological target of crotoxin or at least constitute an important component of this target. This hypothesis implies that, at variance