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Detection of ricin by colorimetric and chemiluminescence ELISA
Eleventh European Mating
40 5
has the potential to provide large amounts of recombinant XTx for further biochemical and toxicological analysis . Fxor~tert et al. (1988) PNAS ~, 8998-9002. Detection oJriciit by colorimetric and chemilumüeescence ELISA . M. A. Poly' V. R. RrveRw,' J. F. Iiswsrsort' and
G. A. M~rwu.2 ('Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, U.S .A .; and'Brooke Army Medical Center, San Antonio, TX, U.S .A .) . A Enaro.Y sensitive and specific calorimetric ELISA was developed to detect ricin in biological fluids. The assay utilizes a high amity (Kd = 10 - '°-10- " M) goat polyclonal antibody to adsorb ricin from solution . Biotinylated antibody is then used to form a sandwich, and avidin-linked alkaline phoaphatase allows color development and measurement of optical density at 405 nm . The standard assay utilizes a standard curve over the range of 1-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in our assay buffer as well as in 1 :10 human urine and 1 : 50 human serum. Measuring ricin in spiked samples (n = ~ at three different cancentration points within the standard curve demonstrated accuracy typically within 5% of the expected value (range = 0-12%) . Accuracy was not affectod by matrix . Coefficient of variation ringed from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml . Two variations on the standard calorimetric assay were also investigated . First, lengthening incubation times and allowing additional time for color development allowod accurate quantitation of ricin in serum dilutions as low as 1 :2 . Second, increasing the concentrations of biotinylated second antibody and avidin-linked alkaline phosphatase from 1 :250 to 1:70 increased the sensitivity of the assay by an order of magnitude, to at kast 100 pg/ml (10 pg/well) . Additional sensitivity could likely be achieved through additional manipulation . We modified this assay into a chemiluminescence format with Lumi-Phos 530 (Lumigen, Inc.) as substrate. This chemiluminescence format allowed accurate quantitation in the 0 .1-1 ng/ml range, but was subject to slightly greater day-today variability than the calorimetric assay. The assays described here are the most sensitive for ricin reported to date and are at kast 10-50-fold more sensitive than other ricin ELISAs . Further, the high sensitivity and accuracy of these assays in serum should support in vfvo distribution studies. The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted herein were performed according to the principles set forth in the Current edition of the Guide jor the Care and Use ojLaboratory Animals, Institute of Laboratory Animal Resources, National Research Council. Cloning and sequencing of cDNNs encoding tquinatoxins from the European sea mremoru Actinic equine. J. Puxo>:a~re,' B. $rxurv:u,' P. Mr,C~Z and F. Gtr' ('Department of Biochemistry and Molecular Biology, Jo~ef Stefan Institute, Jamova 39, P.O . Box 100, 61111 Ljubljana, Slovenia ; and =Department of Biochemistry, Biotechnical Faculty, University of Ljubljana, 61000 Ljubljana, Slovenia). Srr+e~tot~s possess a number of biologically active Compounds including some potent toxic polypeptides and proteins. One of the more characterized classes of these toxins are membrane-active cytolysins which are basic proteins devoid of cysteine residues with molecular mass in the range 15,000 to 20,000 . Cytolysins with similar cardiatic stimulatory and haemolytic activities were found in different species of sea anemones, such as Actinic equine, A . tenebrosa, A . cmi and Stichodacryle helianthus. On the basis of nearly perfect protein sequence identity between equinatoxin II from the European sea anemone A . equine and tenebrosin-C from the Australian red warath sea anemone A . torebrasc and almost identical biological activities, it has been suggested that these two anemones represent closely related species or subspecies . In order to get more information about the taxonomic relationship between these two species of sea anemones and to get more insight into the not yet Completely understood mode of action of these interesting membrane-active cytotoxins, we started with molecular cloning of complementary DNA (cDNA) of equinatoxins from A . equine. Total RNA was isolated from a single sea anemone specimen and poly(A)+ RNA separated on an oligo(dT}cellulose column . The first strand of cDNA was synthesized by reverse tranacriptase and amplified by a polymerise chain reaction (PCR) using two mixed oligonucleotide primers with degeneracy of 128 and 1024, respectively, deducod from a part of the primary structure of equinatoxin II . A cDNA library in bacteriophage lambda gtl l was prepared from the poly(A)+ RNA and screened for positive clones using the positive PCR product of about 300 bp, encoding a part of the equinatoxin cDNA sequence, as a probe. Positive cDNA clones were subcloned into a plasmid vector and Complete proton sequences of equinatoxins deducod from their cDNA sequences. Nucleotide sequencing of two different cDNA Clones revealed that they Code for equinatoxin II and for a new equinatoxin showing most similarities with the N-terminal sequence of tenebrosin-B from A . terttbrosa, respectively. Their cDNA sequences indicate that these proteins are probably synthesized as larger precursor molecules, in which their mature protein sequence is preceded by a relatively hydrophobic signal peptide and a more hydrophilic propeptide . The Afrkan vipers Echis ocellatus hase a selective predetion, possibly inJheencing their encounter with man. P .
Goyffon), Muséum national d'Histoire naturelle, 57, rue Cuvier, F-75005 Paris, France). IN rte Sudan and Sahelian Savanna, vipers from the genus Echis Contribute largely to human enveaomation (Hnara, 1992; ROUteN, 1968). Predation plays a major role in the dynamic and the organization of the tropical REVAULT (L .E.R .A .L : (M .
40 5
has the potential to provide large amounts of recombinant XTx for further biochemical and toxicological analysis . Fxor~tert et al. (1988) PNAS ~, 8998-9002. Detection oJriciit by colorimetric and chemilumüeescence ELISA . M. A. Poly' V. R. RrveRw,' J. F. Iiswsrsort' and
G. A. M~rwu.2 ('Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, U.S .A .; and'Brooke Army Medical Center, San Antonio, TX, U.S .A .) . A Enaro.Y sensitive and specific calorimetric ELISA was developed to detect ricin in biological fluids. The assay utilizes a high amity (Kd = 10 - '°-10- " M) goat polyclonal antibody to adsorb ricin from solution . Biotinylated antibody is then used to form a sandwich, and avidin-linked alkaline phoaphatase allows color development and measurement of optical density at 405 nm . The standard assay utilizes a standard curve over the range of 1-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in our assay buffer as well as in 1 :10 human urine and 1 : 50 human serum. Measuring ricin in spiked samples (n = ~ at three different cancentration points within the standard curve demonstrated accuracy typically within 5% of the expected value (range = 0-12%) . Accuracy was not affectod by matrix . Coefficient of variation ringed from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml . Two variations on the standard calorimetric assay were also investigated . First, lengthening incubation times and allowing additional time for color development allowod accurate quantitation of ricin in serum dilutions as low as 1 :2 . Second, increasing the concentrations of biotinylated second antibody and avidin-linked alkaline phosphatase from 1 :250 to 1:70 increased the sensitivity of the assay by an order of magnitude, to at kast 100 pg/ml (10 pg/well) . Additional sensitivity could likely be achieved through additional manipulation . We modified this assay into a chemiluminescence format with Lumi-Phos 530 (Lumigen, Inc.) as substrate. This chemiluminescence format allowed accurate quantitation in the 0 .1-1 ng/ml range, but was subject to slightly greater day-today variability than the calorimetric assay. The assays described here are the most sensitive for ricin reported to date and are at kast 10-50-fold more sensitive than other ricin ELISAs . Further, the high sensitivity and accuracy of these assays in serum should support in vfvo distribution studies. The views of the author do not purport to reflect the positions of the Department of the Army or the Department of Defense. The experiments conducted herein were performed according to the principles set forth in the Current edition of the Guide jor the Care and Use ojLaboratory Animals, Institute of Laboratory Animal Resources, National Research Council. Cloning and sequencing of cDNNs encoding tquinatoxins from the European sea mremoru Actinic equine. J. Puxo>:a~re,' B. $rxurv:u,' P. Mr,C~Z and F. Gtr' ('Department of Biochemistry and Molecular Biology, Jo~ef Stefan Institute, Jamova 39, P.O . Box 100, 61111 Ljubljana, Slovenia ; and =Department of Biochemistry, Biotechnical Faculty, University of Ljubljana, 61000 Ljubljana, Slovenia). Srr+e~tot~s possess a number of biologically active Compounds including some potent toxic polypeptides and proteins. One of the more characterized classes of these toxins are membrane-active cytolysins which are basic proteins devoid of cysteine residues with molecular mass in the range 15,000 to 20,000 . Cytolysins with similar cardiatic stimulatory and haemolytic activities were found in different species of sea anemones, such as Actinic equine, A . tenebrosa, A . cmi and Stichodacryle helianthus. On the basis of nearly perfect protein sequence identity between equinatoxin II from the European sea anemone A . equine and tenebrosin-C from the Australian red warath sea anemone A . torebrasc and almost identical biological activities, it has been suggested that these two anemones represent closely related species or subspecies . In order to get more information about the taxonomic relationship between these two species of sea anemones and to get more insight into the not yet Completely understood mode of action of these interesting membrane-active cytotoxins, we started with molecular cloning of complementary DNA (cDNA) of equinatoxins from A . equine. Total RNA was isolated from a single sea anemone specimen and poly(A)+ RNA separated on an oligo(dT}cellulose column . The first strand of cDNA was synthesized by reverse tranacriptase and amplified by a polymerise chain reaction (PCR) using two mixed oligonucleotide primers with degeneracy of 128 and 1024, respectively, deducod from a part of the primary structure of equinatoxin II . A cDNA library in bacteriophage lambda gtl l was prepared from the poly(A)+ RNA and screened for positive clones using the positive PCR product of about 300 bp, encoding a part of the equinatoxin cDNA sequence, as a probe. Positive cDNA clones were subcloned into a plasmid vector and Complete proton sequences of equinatoxins deducod from their cDNA sequences. Nucleotide sequencing of two different cDNA Clones revealed that they Code for equinatoxin II and for a new equinatoxin showing most similarities with the N-terminal sequence of tenebrosin-B from A . terttbrosa, respectively. Their cDNA sequences indicate that these proteins are probably synthesized as larger precursor molecules, in which their mature protein sequence is preceded by a relatively hydrophobic signal peptide and a more hydrophilic propeptide . The Afrkan vipers Echis ocellatus hase a selective predetion, possibly inJheencing their encounter with man. P .
Goyffon), Muséum national d'Histoire naturelle, 57, rue Cuvier, F-75005 Paris, France). IN rte Sudan and Sahelian Savanna, vipers from the genus Echis Contribute largely to human enveaomation (Hnara, 1992; ROUteN, 1968). Predation plays a major role in the dynamic and the organization of the tropical REVAULT (L .E.R .A .L : (M .