Production of antibodies to amanitins as the basis for their radioimmunoassay

Summary The production of antibodies against amanitins is described. By means of these antibodies, a radioimmunoassay was developed which allows detection of as little as 0.5 ng of amanitins in 1 ml of serum. By this method, the clearance of α-amanitin from the blood of poisoned mice was measured. Acknowledgments. We thank ProfessorT. Wieland and Dr.H. Faulstich (Heidelberg) for their generous gift of β-amanitin and [³H]O-methyl-demethyl-γ-amanitin. This work was supported by grants from C.N.R., Rome.     

Production of antibodies to amanitins as the basis for their radioimmunoassay.

The production of antibodies against amanitins is described. By means of these antibodies, a radioimmunoassay was developed which allows detection of as little as 0.5 ng of amanitins in 1 ml of serum. By this method, the clearance of alpha-amanitin from the blood of poisoned mice was measured.     

Vitamin A antibodies: Application to radioimmunoassay

Summary A radioimmunoassay for serum vitamin A is described which can detect as little as 1 ng of retinol. The statistical characteristics of this assay are presented and its use in a nutritional experiment is discussed.This work was supported by United States Public Health Service grant 2 RO1 AM19716.     

Production of antibodies against bradykinin

Summary High-titer antibodies against bradykinin were raised in rabbits. 2 different conjugates of bradykinin were used for immunization: bradykinin coupled to human serum albumin via 1,5-difluoro-2,4-dinitrobenzene and bradykinin coupled to edestin via 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide. The sensitivity of the radioimmunoassay method is in the range of 1–50 pg of bradykinin. Cross-reaction of anti-bradykinin antisera occurred with kallidin and met-lysbradykinin.     

Production of antibodies against bradykinin.

High-titer antibodies against bradykinin were raised in rabbits. 2 different conjugates of bradykinin were used for immunization: bradykinin coupled to human serum albumin via 1,5-difluoro-2,4-dinitrobenzene and bradykinin coupled to edestin via 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide. The sensitivity of the radioimmunoassay method is in the range of 1-50 pg of bradykinin. Cross-reaction of anti-bradykinin antisera occurred with kallidin and met-lys-bradykinin.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B₃ to bovine serum albumin. Aflatoxin B₃ was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B₁ (AFB₁) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B₁, B₂, G₁, and G₂ when tritiated AFB₁ was used as the marker ligand. The concentrations causing 50% inhibition of binding of³H-AFB₁ to the antibodies by unlabeled aflatoxins B₁, B₂, G₁, G₂ and B₃ were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B₁ and G₁, two of the most important toxic metabolites produced byAspergillus flavus andA. parasiticus.     

Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B3 to bovine serum albumin. Aflatoxin B3 was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B1 (AFB1) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B1, B2, G1, and G2 when tritiated AFB1 was used as the marker ligand. The concentrations causing 50% inhibition of binding of 3H-AFB1 to the antibodies by unlabeled aflatoxins B1, B2, G1, G2 and B3 were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B1 and G1, two of the most important toxic metabolites produced by Aspergillus flavus and A. parasiticus.     

Phenobarbital specific antisera and radioimmunoassay

Résumé On a développé un anticorps d'une grande affinité et spécifité envers le phénobarbital en inoculant des lapins avec une solution de barbiturateprotéide, synthétisée d'acide barbiturique de 5-phényl-5-(4-aminobutyl) et d'albumine de sérum bovin par du carbodiimide. Usant de cet anticorps comme radioimmunoeassai, on peut mesurer jusqu'à 1 picomole de phénobarbital par ml de fluides biologique sans que d'autres barbiturates y opposent une réaction significative.

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We express our appreciation to the Lilly Research Laboratories for the donation of amobarbital and secobarbital.     

Production of antibodies against synthetic angiotensin II in various animal species

Zusammenfassung Bei Ratten und Kaninchen, jedoch nicht bei Hunden und Katzen, konnten durch wiederholte Injektion von Angiotensin-II-Amid-Carbodiimid-Proteinkomplex in Freund'schem Adjuvans zirkulierende Antikörper gegen Angiotensin erzeugt werden. Durch diese Immunisierung wurde die pressorische Wirkung von exogenem Angiotensin aufgehoben bzw. abgeschwächt, während die von Noradrenalin oder Vasopressin unbeeinflusst blieb. Der Reningehalt der Nieren von Ratten und Kaninchen wurde durch die Immunisierung nicht verändert.     

Production of monoclonal anti-Schistosoma mansoni antibodies. Preliminary study of their biological activities]

Monoclonal antibodies against Schistosoma mansoni have been produced by fusion of splenic lymphocytes from S. mansoni infected Rats and P3-X63-Ag8 BALB/c cells. In vitro and in vivo studies of the biological activities of these antibodies have led to the identification of IgE antibodies with a high reaginic activity and antibodies which in a complement dependent or eosinophil dependent system were shown to have a marked cytotoxicity for schistosomula in vitro. This methodology seems to open new perspectives for the study of antibody function in immunity against parasites as well as for the isolation of the corresponding target antigens.     

Anti-DNA antibodies: aspects of structure and pathogenicity

Anti-DNA antibodies contribute to the pathology of systemic lupus erythematosus. Their depositon in tissue lesions could result from localization of preformed immune complexes of antibodies with DNA or nucleosomes, or from cross-reaction of anti-DNA antibodies directly with tissue proteins. Structural analyses contribute to understanding their pathogenic potential. Primary structures of lupus immunoglobulin G double-stranded DNA-binding autoantibodies are determined by immunoglobulin genes with mutated variable region segments, indicative of selection by immunizing antigen. Arginine, lysine and asparagine residues in complementarity-determining region favor DNA binding. Heavy-chain variable regions make major contributions to DNA binding; affinity and specificity of binding are modulated or can be abrogated by the light-chain variable domain. Crytallographic structure is known for a few antibody-DNA complexes and several ligand-free Fab fragments. Computer modeling supplements this limited information. Structural information of lupus antibody interactions with both DNA and cross-reacting molecules will support use of ligands to inhibit tissue deposition of the antibodies and prevent lesion formation in lupus. Received 4 July 2002; accepted 23 July 2002 RID="*" ID="*"Corresponding author.     

The lipids of hydrogen-oxidizing bacteria: Occurrence ofcis-9,10-methylene hexadecanoic acid inHydrogenomonas H 16

Zusammenfassung Knallgasbakterien (Hydrogenomonas H 16) wurden unter Speicherbedingungen in Submerskultur herangezogen. Die dabei gespeicherte Poly--hydroxybuttersäure wurde zusammen mit anderen «lokker gebundenen » Lipiden mit Chloroform extrahiert. Die daraus abgetrennten polaren Lipide bestanden ausschliesslich aus Phosphatidyläthanolamin. Die Analyse der Fettsäurezusammensetzung dieses Phosphatids ergab einen Gehalt von 42% (bezogen auf das Gewicht der Gesamtfettsäuren) einer C₁₇-Cyclopropansäure. Durch spektroskopische Untersuchungen und oxydativen Abbau wurde letztere alscis-9,10-Methylenhexadecansäure identifiziert.

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The author is grateful to Prof.H. G. Schlegel for the bacterial culture and to Prof.G. Spiteller for measuring the mass spectra. The technical aid of MissG. Thiele is gratefully acknowledged. The analytical GLC apparatus used has been borrowed from the Deutsche Forschungsgemeinschaft.     

Presence of digoxin detectable by radioimmunoassay inTetrahymena

Summary Digoxin was demonstrated inTetrahymena pyriformis by radioimmunoassay, at a concentration of 4.3 ng/100,000 cells. Pretreatment of the cells with digoxin or ouabain did not significantly alter the digoxin concentration of the progeny generations.     

Stereoselectivity of a radioimmunoassay for the insecticide S-bioallethrin

Summary The stereoselectivity of a radioimmunoassay (RIA) system using an S-bioallethrin specific antiserum was studied by observing the abilities of the 8 allethrin isomers and other selected compounds to compete with a radiolabelled S-bioallethrin tyramine derivative for antibody binding sites. The results demonstrate the feasibility of RIA as a rapid, sensitive and stereoselective residue technique for compounds difficult to analyze by classical methods.This work was supported in part by grant No. 5-R01-ES01260-02 from the National Institutes of Health.     

Production of porcine antibodies with high specificity to [8-D-arginine] deamino-vasopressin (dDAVP)

Summary The development of a specific radioimmunoassay for [8-D-arginine] deamino-vasopressin (dDAVP) is described.Acknowledgment. We wish to express our thanks to Drs I. Bláha, M. Zaoral, M. Flegel and K. Jot for generous gift of hormones and their analogues used in this study.     

Stereoselectivity of a radioimmunoassay for the insecticide S-bioallethrin

The stereoselectivity of a radioimmunoassay (RIA) system using an S-bioallethrin specific antiserum was studied by observing the abilities of the 8 allethrin isomers and other selected compounds to compete with a radiolabelled S-bioallethrin tyramine derivative for antibody binding sites. The results demonstrate the feasibility of RIA as a rapid, sensitive and steroselective residue technique for compounds difficult to analyze by classical methods.     

A technical improvement in the radioimmunoassay of cyclic-AMP.

An improvement in the technique for the radioimmunoassay of cyclic-AMP, wherein ammonium sulfate precipitation is replaced with zirconyl phosphate gel, is presented. This substitution produces a more stable pellet than that obtained with ammonium sulfate. This greatly reduces a potential source of error due to pellet instability.     

Comparison of intradermal pigeon crop-sac bioassay and double antibody radioimmunoassay for rat prolactin

Summary On an absolute basis, the intradermal pigeon crop-sac bioassay (PCA) gave results that were 20.5% higher than the radioimmunoassay (RIA) in rat anterior pituitary (AP) preparations. A highly significant correlation (r=0.87) was obtained between RIA (in g) and PCA (in Reece-Turner units) when 58 medium samples obtained by culturing rat APs in vitro were assayed for prolactin (PRL) content.Supported by the Charles and Johanna Busch Memorial Fund and by Hatch Amended Funds, Rutgers University-The State University of New Jersey, New Brunswick, NJ 08903, USA.The authors acknowledge the generous supply of rat PRL reference preparation and iodination material for the RIA by NIAMDD, NIH.