SOME FACTORS OF SIGNIFICANCE IN THE DOUBLE ANTIBODY IMMUNOASSAY OF INSULIN

1967 ◽  
Vol 54 (2) ◽  
pp. 347-361 ◽  
Author(s):  
K. Brunfeldt ◽  
K. R. Jorgensen
Keyword(s):  
Guinea Pig ◽  
Insulin Concentration ◽  
Precipitation Reaction ◽  
Antibody Technique ◽  
Complement Activity ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
Antibody Method ◽  
Heparin Preparation

ABSTRACT Differences were demonstrated between 4 insulin antibody (guinea pig) precipitating antisera (rabbit), int. al. in their ability to cross react with human γ-globulin components. This cross reaction is presumably the reason why »negative« insulin values can be found at higher concentrations of certain precipitating antisera. Independently of the precipitating antiserum it was possible, in some cases, to demonstrate a difference between the insulin concentration in serum and in heparinized plasma. This difference could be eliminated by the addition of heparin, by long-lasting freezing, and by dilution of the serum. Hence, the higher values found in certain cases in serum, as compared with plasma are presumably due to the presence of complement. It is essential in the use of the double antibody technique for the determination of insulin in plasma and serum that a heparin preparation with anti-complement activity should be used and that the concentration range of the precipitating antiserum should be fixed. The double antibody method can be used with or without pre-precipitation if the above mentioned criteria are fulfilled. Pre-precipitation, however, is to be preferred, as the precipitation reaction here occurs under constant conditions.

EVALUATION OF THE DOUBLE ANTIBODY RADIOIMMUNOASSAY OF INSULIN AND THE DETERMINATION OF INSULIN IN PLASMA AND URINE IN NORMAL SUBJECTS

1969 ◽  
Vol 60 (2) ◽  
pp. 327-351 ◽  
Author(s):  
Kai R. Jørgensen
Keyword(s):  
Extreme Values ◽  
Degradation Products ◽  
Insulin Response ◽  
Normal Subjects ◽  
Precipitation Reaction ◽  
Fasting Insulin ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
The Subject

ABSTRACT The use of the double antibody method for radio-immunological determination of insulin in plasma was evaluated on the basis of dilution and recovery experiments, as well as by the investigation of the reproducibility and accuracy of the method. Given the optimum conditions for the precipitation reaction, the method appears from the present investigations to be well suited for plasma insulin determinations. With the technique used for the separation of free and antibody bound insulin, the results of the insulin determination were found to be independent of the radioactive degradation products present in the tracer insulin. It was not possible to demonstrate any increased degradation of the tracer insulin by incubation in plasma or urine as compared with incubation in 0.5 % human albumin buffer. The absolute insulin values were found to be independent of the length of the incubation period. No difference was found between the fasting insulin concentrations in a group of new-born infants of non-diabetic mothers and a group of adult normal subjects. Similarly, the fasting insulin values were found to be independent of the sex of the subject investigated. After oral administration of glucose a considerable variation was found in the insulin response in a group of normal subjects. This variation, within the weight limits used, was found to be independent of the sex, age and weight of the subject investigated. A corresponding condition was found to be valid for the insulin excretion in the urine of normal subjects. It was concluded that the large variation in the insulin response in a group of normal subjects did not allow conclusions about the clinical significance of the extreme values, but that the variation alone must be taken as an expression for a pronounced biological variation.

DETERMINATION OF HUMAN GROWTH HORMONE (HGH) IN PLASMA BY A DOUBLE ANTIBODY RADIOIMMUNOASSAY

1966 ◽  
Vol 53 (1) ◽  
pp. 101-120 ◽  
Author(s):  
E. Cerasi ◽  
L. Della Casa ◽  
R. Luft ◽  
A. Roovete
Keyword(s):  
Present Method ◽  
Human Growth ◽  
Antibody Technique ◽  
Double Antibody ◽  
Insulin In Plasma ◽  
Assay Procedure ◽  
Absolute Measure ◽  
Assay Methods ◽  
Per Se

ABSTRACT The double antibody technique of Hales & Randle (1963) for determination of insulin in plasma was developed for the assay of HGH in plasma. The precision and reproducibility of the method as well as the recovery of HGH added to plasma were highly satisfactory. Increased HGH values were obtained in acromegaly and during insulin hypoglycaemia, while decreased values were observed during glucose infusion. Successful treatment of acromegaly was accompanied by the return of plasma HGH levels toward normal. The plasma HGH values obtained with the present method cannot be considered as reflecting the absolute amounts of HGH, since several factors in the assay procedure, mainly the incubation time and the concentration of HGH antibodies used, markedly influenced the values obtained. It is questioned whether the radioimmunological assay methods for HGH per se give an absolute measure of this hormone in plasma.

An improved double antibody radioimmunoassay for the determination of insulin in serum, plasma, and tissue incubation media

1982 ◽  
Vol 60 (10) ◽  
pp. 962-966 ◽  
Author(s):  
Marthe Dalpé-Scott ◽  
H. M. C. Heick ◽  
Nicole Bégin-Heick
Keyword(s):  
Experimental Animals ◽  
Limited Size ◽  
Double Antibody ◽  
Incubation Periods ◽  
Free Insulin ◽  
Antibody Method ◽  
Nonspecific Interactions ◽  
Insulin Radioimmunoassay ◽  
Pig Serum

We have modified the double antibody method of insulin radioimmunoassay to allow relatively short incubation periods and the use of centrifugation to separate bound from free insulin. This was achieved by altering the order of addition of reagents and by adding normal guinea-pig serum to reduce nonspecific interactions. The method allows for precise measurements in the range of 0–3.2 ng insulin. Both serum and plasma give consistent values. The technique is useful for the measurement of insulin levels in samples of limited size such as those from small experimental animals.

OBSERVATIONS ON THE PRECIPITATION REACTION IN A DOUBLE-ANTIBODY IMMUNOASSAY FOR INSULIN

1968 ◽  
Vol 59 (1) ◽  
pp. 139-149 ◽  
Author(s):  
D. B. Grant
Keyword(s):  
Human Serum ◽  
Rabbit Antiserum ◽  
Precipitation Reaction ◽  
Double Antibody ◽  
Antibody Method

ABSTRACT The effects of human serum and ethylenediaminetetraacetate (EDTA) on the precipitation stage of a double-antibody immunoassay for insulin have been studied. Human serum consistently reduced the rate of precipitation of bound insulin by rabbit antiserum and the results obtained with modified human serum suggested that this effect was due to complement. Under certain conditions, EDTA was found to accelerate the precipitation of antibody-bound insulin and some implications of this finding to the double-antibody method of assay are considered.

RADIOIMMUNOASSAY OF HUMAN GROWTH HORMONE USING THE DOUBLE ANTIBODY TECHNIQUE

1968 ◽  
Vol 57 (4) ◽  
pp. 549-556 ◽  
Author(s):  
F. Melani ◽  
U. Gröschel-Stewart ◽  
J. Lawecki
Keyword(s):  
Growth Hormone ◽  
Human Growth ◽  
Biologically Active ◽  
List Type ◽  
Purification Step ◽  
Antibody Technique ◽  
Double Antibody ◽  
Starch Gel ◽  
Radio Immunoassay

ABSTRACT The method of Roos et al. (1963) for the preparation of HGH yields a highly purified biologically active hormone preparation suitable for clinical purposes and, after one additional purification step (chromatography on Sephadex G-200 according to Hunter (1965), for radioiodination. In the double antibody technique described, the labelled hormone can be used for radio-immunoassay without starch-gel or Sephadex purification. The method is simple and sensitive enough to permit the determination of HGH in serum.

Optimization of Radioimmunoassay for Human Growth Hormone by the Charcoal-Dextran Technique

1970 ◽  
Vol 16 (10) ◽  
pp. 845-848 ◽  
Author(s):  
Joseph C Meek ◽  
Mary M Stoskopf ◽  
Robert E Bolinger
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Guinea Pig ◽  
Human Growth Hormone ◽  
Serum Proteins ◽  
Human Growth ◽  
Molecular Fragments ◽  
Antibody Technique ◽  
Double Antibody

Abstract The effect of serum on the radioimmunoassay for human growth hormone was tested by use of the double-antibody technique and the charcoal-coated dextran technique. With the double-antibody technique, serum effects a falsely high reading for unknowns, while with the charcoal-dextran technique it causes falsely low values to be obtained. Part of the anomaly with the charcoal-dextran technique is the result of adsorption of small molecular fragments of radiolabeled hormone onto serum proteins of higher molecular weight. These fragments can be eliminated by frequent repurification of the labeled hormone. Another part is not related to purity of the label but is corrected by subtracting values for serum blanks that contain no antibody. The use of antiserum to human growth hormone prepared in the goat is more sensitive than that prepared in the guinea pig.

INHIBITION OF LIPOLYTIC ACTIVITY OF SYNTHETIC β1–24 AND β1–39 CORTICOTROPHIN BY ANTI-ACTH ANTISERUM

1966 ◽  
Vol 51 (1) ◽  
pp. 88-94 ◽  
Author(s):  
A. Villanueva ◽  
S. J. H. Ashcroft ◽  
J. P. Felber
Keyword(s):  
Guinea Pig ◽  
Lipolytic Activity ◽  
Peptide Hormones ◽  
Fat Pad ◽  
Double Antibody ◽  
Epididymal Fat ◽  
Antibody Complex ◽  
Radio Immunoassay ◽  
Antigen Antibody

ABSTRACT The synthetic ACTH peptides β1–39 and β1–24 stimulated lipolysis as determined by the rat epididymal fat pad in vitro. The stimulating effect of these peptides was diminished by prior incubation of the peptides with antibodies produced by the guinea-pig against ACTH. The stimulating effect of these hormones was also diminished by the double antibody system used in the radio-immunoassay of ACTH and other peptide hormones, in which incubation with antiserum is followed by precipitation of the antigen-antibody complex by rabbit anti-guinea-pig-γ-globulin.

Rapid quantification and in situ detection of nitrifying bacteria in biofilms by monoclonal antibody method

2000 ◽  
Vol 41 (4-5) ◽  
pp. 301-308 ◽  
Author(s):  
N. Noda ◽  
H. Ikuta ◽  
Y. Ebie ◽  
A. Hirata ◽  
S. Tsuneda ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fluorescent Antibody ◽  
Nitrifying Bacteria ◽  
Fluorescent Antibody Technique ◽  
Antibody Technique ◽  
Antibody Method ◽  
In Situ Detection ◽  
Rapid Quantification

Fluorescent antibody technique by the monoclonal antibody method is very useful and helpful for the rapid quantification and in situ detection of the specific bacteria like nitrifiers in a mixed baxterial habitat such as a biofilm. In this study, twelve monoclonal antibodies against Nitrosomonas europaea (IFO14298) and sixteen against Nitrobacter winogradskyi (IFO14297) were raised from splenocytes of mice (BALB/c). It was found that these antibodies exhibited little cross reactivity against various kinds of heterotrophic bacteria. The direct cell count method using monoclonal antibodies could exactly detect and rapidly quantify N. europaea and N. winogradskyi. Moreover, the distribution of N. europaea and N. winogradskyi in a biofilm could be examined by in situ fluorescent antibody technique. It was shown that most of N. winogradskyi existed near the surface part and most of N. europaea existed at the inner part of the polyethylene glycol (PEG) gel pellet, which had entrapped activated sludge and used in a landfill leachate treatment reactor. It was suggested that this monoclonal antibody method was utilized for estimating and controlling the population of nitrifying bacteria as a quick and favorable tool.

Determination of amino- and carboxyl-terminal sequences of guinea pig liver transglutaminase: evidence for amino-terminal processing

Biochemistry ◽  
1989 ◽  
Vol 28 (5) ◽  
pp. 2344-2348 ◽  
Author(s):  
Koji Ikura ◽  
Hiroyuki Yokota ◽  
Ryuzo Sasaki ◽  
Hideo Chiba
Keyword(s):  
Guinea Pig ◽  
Pig Liver ◽  
Amino Terminal ◽  
Carboxyl Terminal ◽  
Guinea Pig Liver
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