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.

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.

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.

Homogeneous nonisotopic assay for phenytoin evaluated.

1982 ◽  
Vol 28 (5) ◽  
pp. 1190-1191 ◽  
Author(s):  
S Dugan ◽  
J Bailey ◽  
J W Wu ◽  
S Hoskin ◽  
S Riebe ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Rabbit Antiserum ◽  
Test Sample ◽  
Subsequent Reaction ◽  
Immunoprecipitation Assay ◽  
Coefficients Of Variation ◽  
Serum Test ◽  
Sample Dilution

Abstract We evaluated a new homogeneous immunoprecipitation assay for phenytoin in human serum. No sample dilution or pretreatment is required. The new method is based on spectrophotometry of the inhibition by free phenytoin of the precipitating reaction between anti-phenytoin antibody and a phenytoin-human serum albumin conjugate. A serum test sample is simultaneously mixed with the phenytoin-albumin conjugate and rabbit antiserum to phenytoin in a centrifugal analyzer, and the subsequent reaction is monitored at 3 min. Within-run and between-run coefficients of variation were well below 7%. The relation between results for patients' test sample as determined by immunoprecipitation assay (y) and an enzyme immunoassay (x) can be expressed as y = 1.10x + 1.1 (r = 0.966, n = 66).

Human Saliva and Semen: Evidence for Proteins Common to Both Which Do Not Occur in Serum

1981 ◽  
Vol 60 (2) ◽  
pp. 179-184 ◽  
Author(s):  
P. D. Eckersall ◽  
J. A. Beeley
Keyword(s):  
Antibacterial Activity ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Serum Protein ◽  
Serum Proteins ◽  
Rabbit Antiserum ◽  
Human Saliva ◽  
Cross Reactions ◽  
Whole Saliva ◽  
The Cross

1. Rabbit antiserum to human whole saliva cross-reacts with both human serum and semen. After absorption of the antiserum by affinity chromatography on a column of immobilized serum protein, the cross-reactions with serum were eliminated. 2. The absorbed antiserum, however, still cross-reacted with semen thus indicating the presence of proteins with immunological similarity in both saliva and semen, but which did not occur in serum. 3. Some of these proteins clearly showed a reaction of complete immunological identity between saliva and semen. 4. The presence of the non-serum proteins in both saliva and semen might be related to common functions in both such as lubrication or antibacterial activity.

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.

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.

Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum.

1982 ◽  
Vol 28 (6) ◽  
pp. 1314-1318 ◽  
Author(s):  
W A Ratcliffe ◽  
J E Corrie ◽  
A H Dalziel ◽  
J S Macpherson
Keyword(s):  
Human Serum ◽  
Binding Proteins ◽  
Serum Progesterone ◽  
Double Antibody ◽  
Analytical Recovery ◽  
Conventional Methods

Abstract We compared two direct radioimmunoassays for progesterone in 50 microL of unextracted serum or plasma with assays involving extraction of serum. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11 alpha hemisuccinyl conjugate and the radioligand 125I-labeled progesterone 11 alpha-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r greater than 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum.

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