Centrifugal analyzer used for enzyme immunoassay of progesterone and choriomammotropin.

1983 ◽  
Vol 29 (2) ◽  
pp. 302-304 ◽  
Author(s):  
B Terouanne ◽  
J Marchand ◽  
C Calzolari ◽  
J Monnier ◽  
J C Nicolas ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Enzyme Immunoassay ◽  
Gel Filtration ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Rapid Separation ◽  
Enzyme Conjugate ◽  
Enzyme Label ◽  
Free Antigen

Abstract Recently we developed an enzyme immunoassay involving the use of steroid delta-isomerase (EC 5.3.3.1) as enzyme label and exclusion-affinity chromatography for rapid separation of free antigen-enzyme conjugate that bound to antibodies (J. Immunol. Methods 35: 267-284, 1980). Here we describe an automated version of this procedure, for immunoassay of progesterone and choriomammotropin (human placental lactogen) in serum with the use of a centrifugal analyzer. After incubation, suitable dilutions of sera or extract plus antiserum, conjugate, and double antibody were filtered on an estradiol affinity gel-filtration column; the enzyme activity of the filtrates was determined with the centrifugal analyzer. Results correlate well with those obtained by radioimmunoassay: r (progesterone) = 0.980, r (choriomammotropin) = 0.940. The within-run and between-run precision, specificity, sensitivity, accuracy, and speed of this system make it a useful tool for immunoassay.

Effect of Spacer and the Enzyme-Linked Immunosorbent Assay

2016 ◽  
Vol 66 (5) ◽  
pp. 471
Author(s):  
Manisha Sathe ◽  
Shruti Srivastava ◽  
Sumit Agrawal ◽  
Ramrao Ghorpade
Keyword(s):  
Immunosorbent Assay ◽  
Inhibitory Concentration ◽  
Methylphosphonic Acid ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunoassay ◽  
Ic50 Value ◽  
Lower Detection ◽  
Enzyme Conjugate ◽  
Enzyme Label ◽  
Free Antigen

The effect of spacers and the enzyme-linked immunosorbent assay (ELISA) formats on the functional parameters of assays such as lower detection limit, inhibitory concentration at 50 per cent (IC50), and specificity were studied. Enzyme conjugates having hydrophobic and hydrophilic spacers were prepared using O-isopropyl methylphosphonic acid (IMPA) and horseradish peroxidase (HRP) as an enzyme label. Comparison was made with reference to enzyme conjugate without any spacer. The present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate significantly improves the sensitivity of assays. An enhanced IC50 value achieved was 0.01 μg mL−1 for free antigen detection by direct immunoassay using hydrophilic spacers and precoating of ELISA plates by secondary antibody. The use of a hydrophilic spacer might have helped in projecting the hapten in the aqueous phase, leading to enhanced antibody binding signal and improved sensitivity of the assay.

scholarly journals Enhanced plasma persistence of therapeutic enzymes by coupling to soluble dextran

1977 ◽  
Vol 164 (2) ◽  
pp. 461-464 ◽  
Author(s):  
R F Sherwood ◽  
J K Baird ◽  
T Atkinson ◽  
C N Wiblin ◽  
D A Rutter ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Single Peak ◽  
Tumour Bearing ◽  
Enzyme Conjugate ◽  
Covalently Linked ◽  
Therapeutic Enzymes

Conjugation of carboxypeptidase G and arginase, two enzymes of therapeutic interest, to a soluble dextran significantly enhanced plasma persistence in normal and tumour-bearing mice. A prolonged decrease in arginine concentrations in plasma of tumour-bearing mice was demonstrated by using the dextran-linked arginase. Gel filtration of dextran-enzyme conjugate showed that enzyme activity co-chromatographed as a single peak with carbohydrate, and enzyme was shown to be covalently linked to the dextran.

A new enzyme immunoassay for prolactin in serum or plasma

1990 ◽  
Vol 36 (1) ◽  
pp. 76-80 ◽  
Author(s):  
R Babiel ◽  
P Willnow ◽  
M Baer ◽  
M van Gent ◽  
V Ehrhardt
Keyword(s):  
Polyethylene Glycol ◽  
Horseradish Peroxidase ◽  
Enzyme Immunoassay ◽  
World Health ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Analytical Recovery ◽  
Human Prolactin ◽  
Health Organization ◽  
Measurable Range

Abstract This enzyme immunoassay (EIA) of human prolactin (hPRL) involves incubation of sample and anti-hPRL antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) in tubes coated with a second antibody to hPRL. The test can be performed within 60 min. No reaction of the antibodies with human placental lactogen and human somatotropin is detectable. The presence of detergent allows assay of both serum and plasma. Precision was improved by including polyethylene glycol in the reaction mixture. To optimize analytical recovery, we added protease inhibitor. Assay of the EIA standards shows good correlation with results for World Health Organization reference preparations. The measurable range is 1 to 400 micrograms/L. Intra- and interassay CVs are about 5%. Comparisons with two RIAs and two other EIAs show reasonably good correlations. The components of our EIA are stable for 18 months.

scholarly journals Purification of phosphatidylinositol kinase from bovine brain myelin

1987 ◽  
Vol 241 (3) ◽  
pp. 759-763 ◽  
Author(s):  
A R Saltiel ◽  
J A Fox ◽  
P Sherline ◽  
N Sahyoun ◽  
P Cuatrecasas
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Bovine Brain ◽  
Anion Exchange Column ◽  
Ionic Detergent ◽  
Membrane Bound ◽  
Phosphatidylinositol 4 ◽  
Pi Kinase

A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme.

Enzyme-immunoassay of human placental lactogen

1979 ◽  
Vol 91 (3) ◽  
pp. 309-316 ◽  
Author(s):  
H. Van Hell ◽  
J.A.M. Brands ◽  
A.H.W.M. Schuurs
Keyword(s):  
Enzyme Immunoassay ◽  
Placental Lactogen ◽  
Human Placental Lactogen

scholarly journals Observations on the use of solid-phase-coupled antibodies in the radioimmunoassay of human placental lactogen

1974 ◽  
Vol 137 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Jacqueline Gardner ◽  
Gordon Bailey ◽  
Timothy Chard
Keyword(s):  
Solid Phase ◽  
Coupling Reaction ◽  
High Sensitivity ◽  
Comparative Assessment ◽  
Placental Lactogen ◽  
Affinity Constant ◽  
Considerable Proportion ◽  
Lower Sensitivity ◽  
Human Placental Lactogen ◽  
Free Antigen

A detailed comparative assessment was made of the use of solid-phase-coupled antibodies in radioimmunoassay, by using an assay for human placental lactogen as a model system. The major advantages of the solid-phase technique are: (1) in common with the use of a second antibody, it is universally applicable; (2) separation can be carried out rapidly; (3) in contrast with some other techniques, the separation of antibody-bound and free antigen is virtually complete. The disadvantages when compared with other procedures are: (1) a considerable proportion of the antibody may be lost during the initial coupling reaction; (2) the tubes must be continuously mixed during incubation, and much effort is expended in removing and replacing the caps; (3) there is a decrease in the apparent affinity constant of the antibody after coupling, which is reflected in a lower sensitivity of the assay system. It is concluded that solid-phase antibodies are of greatest value in those systems in which the supply of antiserum is abundant, and in which the achievement of high sensitivity is not a requirement.

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