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.

scholarly journals The nylon bag technique in the determination of ruminal feed protein degradation

1983 ◽  
Vol 55 (1) ◽  
pp. 1-78 ◽  
Author(s):  
Jouko Setälä
Keyword(s):  
Particulate Matter ◽  
Pore Size ◽  
Control Sample ◽  
Sample Treatment ◽  
Sample Weight ◽  
Factors Affecting ◽  
Experimental Animals ◽  
Incubation Periods ◽  
Feed Protein

The investigation included experiments in which factors affecting the reliability of the nylon bag method were studied. The possibility of applying the feed protein degradabilities to practical feeding conditions was also examined. In the experiments concerning reliability, such factors as bag porosity, sample weight, sample treatment, washing procedure, diets, and differences between animals and incubation days were studied. The feed protein degradabilities were also determined by using as incubation periods the ruminal retention times for particulate matter of different feeds, evaluated as a function of DM intake/100 kg liveweight in different diets. A nylon bag, with a pore size of 40 µm and internal dimensions of 6 X 12 cm was selected for the degradability determinations. The sample weight used in incubations was 57 —60  mg DM/cm2. In the determination of feed protein degradability, when sheep are used as experimental animals, it is recommended that for routine determinations only one animal be used, analyzing the contents of two bags for each incubation period during two successive days. A control sample of which degradability is determined in advance in many sheep, should be used in all incubations in order to control the digestive processes in the rumen of the experimental sheep. The actual degradabilities analyzed by the bag method are applicable in practise, if they are determined using animals at similar feeding levels and on diets similar to those prevailing under the conditions in which the degradabilities are going to be used.

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.

Spectrophotometric Determination of Factor Xa Generation in Factor IX Concentrates

1977 ◽  
Vol 37 (03) ◽  
pp. 535-540 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
Ann Howie
Keyword(s):  
Intrinsic Factor ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor V ◽  
Factor X ◽  
Generation Times ◽  
Incubation Periods ◽  
Multiple Sample ◽  
Recording Spectrophotometer

SummaryPrevious work from this department, concerned with testing the potential thrombogenicity of therapeutic factor IX concentrates, demonstrated that following recalcification of factor IX concentrates thrombin was generated within 3-30 minutes of incubation (Sas et al. 1975). The test developed (known as the TGt 50 test) is a two-stage assay and was thus found to be time consuming, tedious and tended to become inaccurate with long incubation periods and a large number of samples. A semiautomatic version of the test is reported in which the synthetic peptide Bz-ILE-GLU-GLY-ARG-pNA (S-2222) is added to recalcified, diluted factor IX concentrate in the micro-cuvette of a multiple sample recording spectrophotometer. Information can be obtained on (a) the amount of Xa (if any) present prior to recalcification (b) the initial amount of Xa formed and (c) the time taken to activate all factor X to Xa. Direct graphical interpretation shows a number of qualitative differences between commercial preparations, but by either of the criteria (b) or (c) above, it is possible to place the different products into “activated” and “non activated” groups such that both the Xa generation times and TGt 50 tests identify the same two groups of products. This agreement also indicates that the TGt 50 test is independent of the intrinsic factor V levels in the various concentrates.

scholarly journals Effect of packing on changes in erythrocyte osmotic fragility and malondialdehyde concentration in donkeys administered with ascorbic acid

2012 ◽  
Vol 79 (1) ◽  
Author(s):  
Folashade Olaifa ◽  
Joseph O. Ayo ◽  
Suleiman F. Ambali ◽  
Peter I. Rekwot
Keyword(s):  
Oxidative Stress ◽  
Ascorbic Acid ◽  
Osmotic Fragility ◽  
Blood Samples ◽  
Experimental Animals ◽  
Erythrocyte Osmotic Fragility ◽  
Nacl Concentration ◽  
Experimental Subjects ◽  
Apparently Healthy

Experiments were performed with the aim of investigating the effect of packing on erythrocyte osmotic fragility (EOF) and malondialdehyde (MDA) concentration in donkeys, and the effect of ascorbic acid (AA). Twelve apparently healthy donkeys raised under the traditional extensive system served as experimental subjects. Six donkeys administered orally with AA (200 mg/kg) and subjected to packing were used as experimental animals, whilst six others not administered with AA served as controls. Blood samples were collected pre- and post-packing from all the donkeys for the determination of MDA and EOF. At 0.3% Sodium Chloride (NaCl) concentration, the percentage haemolysis was 93.69% ± 2.21% in the control donkeys and the value was significantly (P < 0.05) higher than the value of 71.31% ± 8.33%, recorded in the experimental donkeys. The post-packing MDA concentration obtained in the control donkeys was 39.62 µmol ± 4.16 µmol, and was not significantly different (P > 0.05) from the value of 35.97 µmol ± 2.88 µmol recorded in the experimental donkeys. In conclusion, the increase in haemolysis obtained in the donkeys suggested that packing induced oxidative stress, which was ameliorated by AA administration.

Genotype and environment in the determination of minor skeletal variants and body weight in mice

Development ◽  
1967 ◽  
Vol 17 (2) ◽  
pp. 283-292
Author(s):  
W. L. Howe ◽  
P. A. Parsons
Keyword(s):  
Maternal Age ◽  
Maternal Diet ◽  
Heterogeneous Material ◽  
Inbred Strains ◽  
Relative Importance ◽  
Maternal Weight ◽  
Detailed Understanding ◽  
Experimental Animals ◽  
Percentage Incidence

Numerous minor skeletal variants have been described in the mouse (Grüneberg, 1963), other small mammals (Berry & Searle, 1963) and in man (Comas, 1960; Brothwell, 1963; Grüneberg, 1963). In genetically heterogeneous material such as man it is very difficult to sort out the factors causing these variants. However, in experimental animals such as the mouse the use of inbred strains and crosses derived from them permits a more detailed understanding of these factors, in particular the relative importance of heredity and environment. Grüneberg (1963) cites numerous references showing very great differences in the percentage incidence of many minor variants in mice between inbred strains and in some cases between hybrids. The conclusion is that much of this variation is genetic in origin. Even so, certain environmental factors have been shown to be of importance, such as maternal age, parity, maternal weight and maternal diet.

Enzyme immunoassay of free thyroxin in dried blood samples on filter paper.

1985 ◽  
Vol 31 (5) ◽  
pp. 750-753 ◽  
Author(s):  
N Hata ◽  
K Miyai ◽  
M Ito ◽  
Y Endo ◽  
Y Iijimi ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Filter Paper ◽  
Room Temperature ◽  
Normal Subjects ◽  
Blood Samples ◽  
Double Antibody ◽  
Coefficients Of Variation ◽  
The Mean ◽  
Measurable Range

Abstract We describe a double-antibody enzyme immunoassay for determination of free thyroxin (FT4) in dried blood samples on filter paper, with use of a T4-beta-D-galactosidase complex. The measurable range of FT4 concentration in two 3-mm blood discs, each of which contained about 2.7 microL of blood, was 1.9 to 93 ng/L, as determined by comparison with concentrations of FT4 in known serum standards. FT4 in blood samples dried on filter paper was stable for at least four weeks when kept dry at -20 degrees C, room temperature, or 37 degrees C. The mean coefficients of variation were 7.6% (within assay) and 6.4% (between assays). Results for FT4 by this method correlated well with those for serum determined by radioimmunoassay (r = 0.98). The proposed method can be used to differentiate persons with hyper- and hypothyroidism from normal subjects and those with abnormal concentrations of thyroxin-binding globulin. The procedure seems suited for screening studies.

Double-antibody method and the protein-A-bearing Staphylococcus aureus cells method compared for separating bound and free antigen in radioimmunoassay.

1979 ◽  
Vol 25 (5) ◽  
pp. 752-756 ◽  
Author(s):  
R K Gupta ◽  
D L Morton
Keyword(s):  
Staphylococcus Aureus ◽  
Protein A ◽  
Human Albumin ◽  
Precipitation Method ◽  
High Adsorption ◽  
Time Saving ◽  
Double Antibody ◽  
Antibody Method ◽  
Free Antigen ◽  
Formalin Fixed

Abstract We compared the protein-A-bearing Staphylococcus aureus immunoadsorbent to the double-antibody precipitation method for separating bound and free radiolabeled antigen in a radioimmunoassay. With human albumin (antigen) and rabbit anti-human albumin (antibody) as a model, our results indicate that formalin-fixed, heat-killed S. aureus cells could be substituted for the double-antibody precipitation method. Ease of preparation and high adsorption capacity of protein-A-bearing S. aureus for most mammalian IgG make this method economical and time saving.

Modification of the choriogonadotropin beta-subunit radioimmunoassay for determination of urinary choriogonadotropin.

1978 ◽  
Vol 24 (11) ◽  
pp. 1958-1961 ◽  
Author(s):  
J McCready ◽  
G D Braunstein ◽  
D Helm ◽  
M E Wade
Keyword(s):  
Pregnant Women ◽  
Radioreceptor Assay ◽  
Beta Subunit ◽  
Urine Samples ◽  
Human Choriogonadotropin ◽  
Analytical Recovery ◽  
Antibody Method ◽  
Serum And Urine ◽  
Serum And Urine Samples

Abstract The choriogonadotropin beta-subunit radioimminoassay has been used extensively to measure human choriogonadotropin in the sera of pregnant women and individuals with trophoblastic and nontrophoblastic tumors. Unmodified, this method cannot be used to measure choriogonadotropin in urine because of interfering substances. We circumvented the non-parallelism between the standards and serial dilutions of urine containing choriogonadotropin by adding pooled urine from men to the standard tubes and limiting the volume of urine to 100 microliter. This modified assay has a sensitivity of 3 int. units/liter of urine and is specific for choriogonadotropin concentrations of 40 int. units/liter of urine. Analytical recovery of choriogonadotropin added to urine ranged from 96 to 105%. The within-assay CV was 7.6%; the between-assay CV was 11.8%. Concentrations of choriogonadotropin in concurrently collected serum and urine samples from pregnant women correlated well. The test can be performed within 24 h by using the double-antibody method for separating bound from free hormone, or in 3 h with a dioxane method. The assay is about 20-fold more sensitive than the 2-min or 2-h slide and tube pregnancy tests, and seven-to 12-fold more sensitive than the radioreceptor assay.

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