scholarly journals Development of enzyme immunoassay for endogenous ouabain-like compound in human plasma

1997 ◽  
Vol 43 (5) ◽  
pp. 715-722 ◽  
Author(s):  
Steven Harwood ◽  
John A Little ◽  
Gerard Gallacher ◽  
David Perrett ◽  
Raymond Edwards ◽  
...  
Keyword(s):  
Detection Limit ◽  
Healthy Volunteers ◽  
Enzyme Immunoassay ◽  
Human Plasma ◽  
Hplc Analysis ◽  
Cross Reactivity ◽  
Standard Curve ◽  
Endogenous Ouabain ◽  
Endogenous Steroid ◽  
Hplc Study

Abstract Widespread evidence supports the existence of an endogenous digitalis-like compound in mammals. We report here the development of a novel enzyme immunoassay for ouabain that, in conjunction with a detailed HPLC study, identifies a ouabain-like compound (OLC) in extracted human plasma. The assay is sensitive—minimum detection limit for OLC 37 pmol/L (11 pmol/L in plasma)—and has a working range (between-assay CV <10%) of 180–10 000 pmol/L (54–3000 pmol/L in plasma). Mean recoveries of ouabain added to plasma ranged from 90% to 100%, and plasma extracts diluted in parallel to the standard curve. Plasma OLC concentrations in 10 healthy volunteers averaged 92 pmol/L (range 55–168), assuming 100% cross-reactivity of OLC in the ouabain assay. HPLC analysis with two distinct chromatographic conditions demonstrated that endogenous human plasma OLC co-eluted with authentic ouabain. The enzyme immunoassay is rapid and easy to perform and will support further investigation of the nature of this controversial endogenous steroid.

Evaluation of a solid-phase enzyme immunoassay for human choriogonadotropin beta subunit.

1983 ◽  
Vol 29 (11) ◽  
pp. 1964-1966 ◽  
Author(s):  
D D Davey ◽  
M M Sample ◽  
T O Oei
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Beta Subunit ◽  
Neoplastic Disease ◽  
Standard Curve ◽  
Human Choriogonadotropin ◽  
Beta Hcg ◽  
Substantial Problem ◽  
Sample Dilution

Abstract We evaluated a quantitative solid-phase enzyme immunoassay for human choriogonadotropin beta subunit (beta-HCG) with anti-beta-HCG:horseradish peroxidase conjugate, recently marketed by Abbott Laboratories. We compared results on 56 patients' serum specimens, obtained mostly for followup of neoplastic disease, with those by a competitive radioimmunoassay kit. The correlation was good, the differences being of little clinical significance. Linear regression in the low and intermediate ranges gave a slope of 0.93, a y-intercept of 0.34, and a correlation coefficient of 0.97. Precision studies yielded an interassay CV of 6.4% in the intermediate range and 13% in the low range. Sensitivity was 0.69 int. unit/L. Cross reactivity was 1 to 2% with specimens fortified with lutropin or follitropin. The only substantial problem was with linearity in the upper part of the standard curve, especially in the interval, 100-200 int. units/L. This problem is obviated by adequate sample dilution.

6 beta-hydroxycortisol in serum and urine as determined by enzyme immunoassay on microtitre plates.

1986 ◽  
Vol 32 (11) ◽  
pp. 2094-2097 ◽  
Author(s):  
A Zhiri ◽  
H A Mayer ◽  
V Michaux ◽  
M Wellman-Bednawska ◽  
G Siest
Keyword(s):  
Detection Limit ◽  
Healthy Volunteers ◽  
Enzyme Immunoassay ◽  
Oral Contraceptives ◽  
Excretion Rate ◽  
Routine Procedure ◽  
Mean Values ◽  
Analytical Recovery ◽  
Collection Period ◽  
Serum And Urine

Abstract We modified our previous enzyme immunoassay (Clin Chim Acta 1986;157:267-76) for estimating 6 beta-hydroxycortisol (6 beta-OHF) to provide a more routine procedure for measuring it in urine and to increase sensitivity for its measurement in serum. Precision, analytical recovery, specificity, and detection limit are reported. 6 beta-OHF and 17-hydroxycorticosteroids in 24-h and 4-h (08:00-12:00 h) urine collections from healthy volunteers showed no significant day-to-day differences in excretion rate for either collection period. Mean values for 6 beta-OHF in serum of men, women, and women taking oral contraceptives showed no significant differences among these groups.

Determination of dehydroepiandrosterone sulfate in plasma by a one-step enzyme immunoassay with a microtitre plate.

1985 ◽  
Vol 31 (11) ◽  
pp. 1876-1879 ◽  
Author(s):  
T K Dhar ◽  
C Müller ◽  
M Schöneshöfer
Keyword(s):  
Hydrogen Peroxide ◽  
Detection Limit ◽  
Enzyme Immunoassay ◽  
Cost Effective ◽  
Dehydroepiandrosterone Sulfate ◽  
Turnaround Time ◽  
Microtitre Plate ◽  
Standard Curve ◽  
One Step

Abstract We have developed a rapid and cost-effective enzyme immunoassay for dehydroepiandrosterone sulfate (DHEA-S) in plasma, performed with samples on a microtitre plate within 2.5 h. No extraction or centrifugation steps are involved. The 3-hemisuccinate of dehydroepiandrosterone is labeled with horseradish peroxidase, then mixed with hydrogen peroxide substrate in the presence of the chromogen, tetramethylbenzidine. The detection limit of the assay is 12.5 pg of DHEA-S per well. Intra- and interassay CVs at three steroid concentrations (12.8, 1.28, and 0.16 mumol/L) ranged from 2.3 to 5.4% and 6.1 to 8.4%, respectively. Results correlated well (r = 0.95) with those of a radioimmunoassay with iodinated DHEA-S. The turnaround time for 41 samples (in duplicate) is 2.5 h, which includes 2 h of incubation time. The sensitivity of this one-step version and the linearity of its standard curve are equivalent to those of a less practicable two-step version. This technique may replace coated-tube enzyme immunoassays for routine use.

Automated in-line ratio-correcting filter fluorometer.

1981 ◽  
Vol 27 (9) ◽  
pp. 1586-1591 ◽  
Author(s):  
O S Khalil ◽  
W S Routh ◽  
K Lingenfelter ◽  
D B Carr ◽  
P Ladouceur
Keyword(s):  
Detection Limit ◽  
Enzyme Immunoassay ◽  
Light Source ◽  
Fluorescence Intensity ◽  
Correlation Equation ◽  
Line Ratio ◽  
Fluorescence Immunoassay ◽  
Transmitted Beam ◽  
Standard Curve ◽  
Excitation Geometry

Abstract We have developed a novel optical arrangement for the Abbott VP bichromatic analyzer, converting it into a fluorometer upon insertion of a specially designed filter carriage. Fluorescence intensity is measured by using straight-through excitation geometry and is corrected for fluctuations in light-source intensity by using the attenuated transmitted beam through the solution as a reference. The modification allows switching from the absorbance to fluorescence mode without an auxiliary light source or a major hardware change. The detection limit for an experimental prototype was estimated as 192 pmol of fluorescein per liter. In an application to a fluorescence immunoassay for theophylline in patients' sera, the data (y) correlated well with results by an enzyme immunoassay procedure (EMIT), giving a correlation equation of y = 1.849 + 0.988x (r = 0.969). A fluorometric standard curve for glucose and some performance data are also presented.

scholarly journals Determination of salivary cortisol in donkey stallions

2018 ◽  
Author(s):  
MICAELA SGORBINI ◽  
Francesca Bonelli ◽  
Alessandra Rota ◽  
Christine Aurich ◽  
Natascha Ille ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Salivary Cortisol ◽  
Circadian Variation ◽  
Cross Reactivity ◽  
Commercial Enzyme ◽  
Standard Curve ◽  
Non Invasive ◽  
Detectable Concentration ◽  
Commercial Enzyme Immunoassay

Salivary cortisol provides information about free plasma cortisol concentration and salivary sampling is a non-invasive well-tolerate procedure. The aim of this study was to validate a commercial enzyme immunoassay for the determination of salivary cortisol in donkeys. Saliva samples were collected in 4 donkey stallions on thirteen non-consecutive days at 8:30 AM to avoid circadian variation. Animals were already accustomed to be handled. Saliva was collected by using a swab inserted at the angle of the lips, placed onto the tongue for 1 min and returned into a polypropylene tube. Tubes were centrifuged and at least 1 ml of saliva was aspirated from each sample and frozen at −20° C until analysis. A commercial enzyme immunoassay kit without extraction was used for determination of cortisol in saliva. Median cortisol concentrations with minimum and maximum value were calculated. Recovery of cortisol standard in donkey saliva was 107.9% and serial dilution of donkey saliva samples with assay buffer resulted in changes in optical density parallel to the standard curve. Cross-reactivity of the antiserum was 10.4% with 11-deoxycortisol, 5.2% with corticosterone, 0.4% with 11-deoxycorticosterone, 0.2% with cortisone and <0.1% with testosterone, progesterone and estradiol. The intra-assay coefficient of variation was 10.7%, the inter-assay variation was 8.0% and the minimal detectable concentration was 0.01 ng/ml. The results of the present study demonstrate the validity of a commercial kit to determine the concentration of cortisol in donkey saliva, as already reported in other species.

scholarly journals ACTH Adrenal Cell Bioassay: Improved Sensitivity (12 ng/L) Achieved by Immunoextraction of Acth from Human Plasma by a Monoclonal Antibody

1990 ◽  
Vol 27 (1) ◽  
pp. 59-64
Author(s):  
R Mitchell ◽  
A Lambert ◽  
S R Crosby ◽  
A White ◽  
W R Robertson
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Human Plasma ◽  
Calf Serum ◽  
Plasma Acth ◽  
Human Pituitary ◽  
Adrenal Cells ◽  
Standard Curve ◽  
Cell Dispersion ◽  
Increased Sensitivity

We have previously reported a bioassay for human plasma ACTH based upon trypsin dispersed guinea-pig adrenal cells which was sensitive to 100 ng/L ACTH in unextracted human plasma when measured against human pituitary ACTH (1–39) standard 74/555. We now present a bioassay of increased sensitivity (12 ng/L) which incorporates three major changes. The trypsin/trypsin inhibitor step in the cell dispersion protocol has been replaced with collagenase, donor calf serum (3%) has been incorporated into the standard curve and ACTH has been extracted from human plasma and dilutions of standard hormone by a sephacryl bound monoclonal antibody (2A3) directed towards the 25–39 sequence. The extracted standard curve has a detection limit of 6 ng/L and the cells can tolerate up to 50% plasma equivalent concentration. Thus, the improved assay has a detection limit of 12 ng/L ACTH in plasma. The assay can now measure bioactive plasma ACTH levels reliably in the normal range.

scholarly journals Protein C in human plasma determined by homogeneous enzyme immunoassay with use of a centrifugal analyzer.

1988 ◽  
Vol 34 (9) ◽  
pp. 1834-1838 ◽  
Author(s):  
M Shimamoto ◽  
T Yoshimura ◽  
N Kimura ◽  
M Kita ◽  
N Hoshino ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Human Plasma ◽  
Protein C ◽  
Standard Curve ◽  
Coagulation Protein ◽  
Adult Plasma ◽  
Antibody Conjugate ◽  
Antigen Protein ◽  
Antigen Antibody ◽  
Homogeneous Enzyme Immunoassay

Abstract We describe the simple and rapid enzyme immunoassay of protein C in human plasma with use of a Cobas Fara centrifugal analyzer. The antibody, labeled with horseradish peroxidase, is reacted with antigen (protein C) for 15 min. The peroxidase activity of the resulting antigen-antibody conjugate is measured at 500 nm for 5 min in the presence of excess H2O2, phenol, and 4-aminoantipyrine, as compared with that of free conjugates. Results are calculated from a stored standard curve and expressed as a percentage of the value determined for a pooled specimen of normal adult plasma. The standard curve is linear from 0% to 200%. The CV is generally less than 4% for different concentrations of protein C. In liver cirrhosis, hepatocellular carcinoma, therapy with warfarin, thrombosis, and disseminated intravascular coagulation, protein C concentrations are about 40-70% of normal. Results obtained with the present homogeneous enzyme immunoassay correlated well with those by enzyme-labeled immunosorbent assay (r = 0.97).

Novel canine anti-Coccidioides immunoglobulin G enzyme immunoassay aids in diagnosis of coccidioidomycosis in dogs

2019 ◽  
Vol 57 (7) ◽  
pp. 800-806 ◽  
Author(s):  
Eric D Holbrook ◽  
Russell T Greene ◽  
Stanley I Rubin ◽  
Janelle S Renschler ◽  
Bradley P Book ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Immunoglobulin G ◽  
Fungal Infections ◽  
Turnaround Time ◽  
Cross Reactivity ◽  
Serological Method ◽  
Standard Curve ◽  
Increased Sensitivity ◽  
And Control ◽  
Antigen Testing

AbstractThe diagnosis of coccidioidomycosis (CM) in dogs is typically based on clinical presentation, serology, and (less frequently) spherule identification. Agar gel immunodiffusion (AGID) is the most commonly employed serological method, but AGID is slow (requiring up to a week for titer). A Coccidioides antigen enzyme immunoassay (EIA) is also available; however, sensitivity is low in CM dogs. An antibody EIA was developed to detect canine immunoglobulin G (IgG) reacting to Coccidioides antigens. Serum was evaluated from dogs with pathology proven CM and/or AGID positive CM, as well as dogs with histoplasmosis, blastomycosis, non-fungal infections, or healthy dogs. A standard curve was used to convert optical density (OD) values into EIA units (EU). Serum and urine samples from CM dogs were also tested in the antigen EIA. Sensitivity and specificity for IgG were 89.2% and 97.2%, respectively, upon evaluation of dogs with proven or probable CM and control dogs. Cross-reactivity was observed in 7.7% and in 6.4% of dogs with histoplasmosis or blastomycosis, respectively. The antigen EIA alone was insensitive (33.8%). Combined IgG and antigen testing increased sensitivity to 93.2%, as three dogs were IgG-negative but had detectable serum or urine antigen. In 22 dogs with proven CM, sensitivity was statistically similar for antibody EIA and AGID (86% and 73%; P = .487). The MiraVista® canine Coccidioides antibody IgG EIA may aid in the diagnosis of CM by improving turnaround time with comparable sensitivity to AGID. Serial or concurrent testing by antibody and antigen EIAs may be beneficial when screening dogs for CM.

scholarly journals Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples

2016 ◽  
Vol 54 (1) ◽  
pp. 101-106 ◽  
Author(s):  
Shiomi Haraya ◽  
Koji Karasawa ◽  
Yoshihiro Sano ◽  
Kimiko Ozawa ◽  
Nobumasa Kato ◽  
...  
Keyword(s):  
Horseradish Peroxidase ◽  
Enzyme Immunoassay ◽  
Colorimetric Method ◽  
Peptide Hormone ◽  
Cross Reactivity ◽  
Microtitre Plate ◽  
Standard Curve ◽  
Highly Sensitive ◽  
Lysine Residues ◽  
The Central Nervous System

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

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