Production of antibodies of high binding affinities to glucagon in rabbits

Worobec, R.B.; Locke, R. F.; Hall, A. Rupert; Ertl, Reinhard; Ernst, K; Deininger, Eva

doi:10.1016/0006-291x(67)90471-8

Cited by 10 publications

(3 citation statements)

References 8 publications

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“…L'immunisation ultgrieure a ~tg poursuivie en administrant tousles 2 mois des doses de rappel par vole intramusculaire. Chacune de ces doses de rappel gtai~ eomposge de 5 injections, effectuges s 2 jours d'intervalle, de 1 mg de glueagon gmulsionng dans un mglange d'adjuvant de Freund et de cire d'abeille liqu~fige selon la technique de Worobee et al [24].…”

Section: Antis@rum Anti-glucagonunclassified

Une m�thode de dosage radioimmunologique du glucagon comportant une s�paration par le charbon-dextran

1970

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Prof. P.A. 13astenie) l~e~u le 80ctobre 1969Dextran.coated charcoal radioimmunoassay of glucagon Summary. A dextran-coated charcoal radioimmunoassay of glucagon is described. Antigen (1~1 or 12~I-glucagon, 0.060 ng) --antibody (rabbit antiserum 1 : 550, final dilution 1:6600) reactions reached equilibrium within 3 days at 4 ~ C. The use of a proteinase inhibitor (Trasylol) prevented incubation damage of the immunoreaetive glueagon. The sensitivity of the assay (< to 0.020 ng) corresponds to about 0.100 ng/ml of plasma. The precision, the reproducibility, the specificity of the assay and some problems related to the dextran-chareoal separation have been studied. Recovery of ghcagon added go plasma was approximatively 90%. The mean glucagon concentration measured in peripheral venous plasma was 0.208 ng/ml in normal human subjects after an overnight fast, 0.396 ng/ml in anaesthetized dogs after an overnight fast, 0.354 ng/ml in fed rats and 0.909 ng/ml in the overnightfasted duck. Up go now, the use of the assay seems go be restricted go studies of glueagon secretion in vitro since our antisermn cross-reacts with a material extracted from the duodenum. The relative contribution of this material in the plasma glucagon determinations remains, however, go be established.

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Section: Antis@rum Anti-glucagonunclassified

Une m�thode de dosage radioimmunologique du glucagon comportant une s�paration par le charbon-dextran

1970

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“…When the concentration of glucose was 30 mg/100 ml the average rate of glucagon secretion was 72 ng/lO islets/hr (p<O.OOS). Although many investigators studied the distribution of antibody producing cells in the lymphoid tissues of rodents during the primary antibody response ( 1-5) , few studied the distribution of antigen reactive cells (6)(7)(8). Recent reports showed that the distribution of the latter cells in unimmunized animals varies within the same species depending upon the antigen-antibody system studied.…”

Section: Materials and Methods Glucagon Standardsmentioning

confidence: 98%

A Simplified Glucagon Immunoassay and Its Use in a Study of Incubated Pancreatic Islets

Nonaka

Foà

1969

Experimental Biology and Medicine

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Several methods for the radioimmunoassay of glucagon have been described (1-9). Although, in general, these methods are sufficiently sensitive and specific for most physiologic studies, sometimes they are lengthy (3-6 days) and too cumbersome for the simultaneous analysis of many samples. The purposes of this study were to develop a simple and rapid assay method and to study the secretion of glucagon by isolated rat pancreatic islets, incubated under various experimental conditions. Materials and Methods. Glucagon standards.A stock solution of crystalline glucag o d (lot 258-234B-167-1; 1 mg/ml), prepared with distilled water adjusted to pH 9.0 with NaOH, was divided into 0.2-ml portions and stored at -20'.When needed, working standards were prepared by diluting the stock solution with 0.05 M Verona1 buffer at pH 8.0, containing 0.25% bovine serum albumin2 (hereafter, this buffer will be called BSAVB). The BSAVB was used also for the dilution of antiglucagon serum (AGS) and for the preparation of the 1251-labeled glucagon solutions.A ntiglucagon sera. An tiglucagon sera were prepared according to the method of Assan et al. ( l o ) , with slight modifications (1 1) and, more recently, according to the method of Worobec et QZ. (S), using polyvinyl pyrrolidone instead of bees wax. With this method, after 13-16 injections, 8 rabbits out of 15 produced sera with a binding capacity sufficient to warrant further exploration. The suitability of a serum was determined in the following manner: 0.1 ml of serum was incubated overnight at 4' with 50 pg of labeled Bovine serum albumin from other sources caused a flattening of the standard curve. and 5 ng of nonlabeled glucagon, in 1.1 ml of BSAVB containing 1000 KIU of Trasyl01.~ After incubation, the free (F) and antibodybound ( B ) glucagon were separated by the addition of 0.5 ml of a 10% cellulose suspen-sion4, a technique previously used for the separation of insulin (12-14). Sera giving a ( B / B + F) ratio greater than 60%, were used to prepare standard curves. The selection of the serum and of the optimum amount to be used in the a s a y (in general 1-8 pl) was based on the slope of the standard curve, as determined for each batch of serum. Glucagon was labeled with 125iodine using the method of Greenwood and Hunter (15) and purified by means of a column of Whatman CF 1 1 cellulose powder (12). The specific activity of labeled glucagon varied from 198 to 43 7 mCi/mg. Immunologically inactive or damaged glucagon ranged between 3 and SF, at the time of iodination. IMMUNOASSAY O F GLUCAGON (G) 1 ml diluted AGS with Trasylol (2000 KIU) + 0.1 ml glucagon standard or sample + 0.1 ml glucagon-=I (50 pg) 5-First reaction (binding of G by AGS) 4" overnight (16-18 hr) . 1 0.5 ml cold cellulose powder suspension (10% w/v) 5-Sccoiid reaction (adsorption of free glucagon) 4" within 5 min 4 Centrifuge 4", 2500 rpm, 3 mixi

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“…However, when the dose of glucagon was increased and the interval between injections shortened to two weeks, detectable titers were observed; but it was not until after the tenth immunization that a reasonably high titer was found in one of the guinea pigs. Table 1 shows Studies by Assan et al (1965) and Worobec et al (1967) As suspected, the antibody preparation contained antibody to insulin (Table 1). In fact the insulin antibody titer was higher than that of glucagon.…”

Section: Resultsmentioning

confidence: 99%

Nutritional regulation of insulin and glucagon secretion in sheep

Kamalu

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Production of antibodies of high binding affinities to glucagon in rabbits

Cited by 10 publications

References 8 publications

Une m�thode de dosage radioimmunologique du glucagon comportant une s�paration par le charbon-dextran

Une m�thode de dosage radioimmunologique du glucagon comportant une s�paration par le charbon-dextran

A Simplified Glucagon Immunoassay and Its Use in a Study of Incubated Pancreatic Islets

Nutritional regulation of insulin and glucagon secretion in sheep

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