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.

Highly specific enzyme immunoassay for the beta-subunit of human thyrotropin with use of monoclonal antibodies.

1988 ◽  
Vol 34 (1) ◽  
pp. 98-102 ◽  
Author(s):  
Y Iijima ◽  
Y Endo ◽  
N Hata ◽  
H Fujita ◽  
M Unoki ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Pituitary Tumors ◽  
Cross Reactivity ◽  
Beta Subunit ◽  
Specific Enzyme ◽  
Human Choriogonadotropin ◽  
Pituitary Thyroid Axis ◽  
Thyroid Axis ◽  
Hypothyroid Patients

Abstract A highly specific enzyme-linked "sandwich" immunoassay is described for determining free human thyrotropin (hTSH) beta-subunit in serum by using a anti-hTSH beta-subunit monoclonal antibody conjugated with beta-D-galactosidase (EC 3.2.1.23) and a solid phase consisting of silicone rods coated with another monoclonal antibody. We could detect as little as 0.04 ng of beta-subunit per assay. The measurable range of hTSH beta-subunit concentrations in serum was 0.4 to 50 micrograms/L. The assay demonstrated little or no cross reactivity with intact hTSH, hTSH alpha-subunit, or human choriogonadotropin. The mean CVs were 12.2% within assay, 13.9% between assay. The hTSH beta-subunit was not detectable in sera from healthy subjects, patients with hyperthyroidism, or two patients with pituitary tumors producing TSH. It was measurable (at concentrations of 0.65 to 2.70 micrograms/L) in sera from eight of 23 hypothyroid patients. In five of the hypothyroid patients examined, the concentration of hTSH beta-subunit in serum increased after administration of thyroliberin. This method may be useful in elucidating the physiological and pathological significance of the hTSH beta-subunit and in examining the function of the hypothalamus-pituitary-thyroid axis.

An evaluation of four serum tests for pregnancy.

1983 ◽  
Vol 29 (3) ◽  
pp. 561-563 ◽  
Author(s):  
K W Ryder ◽  
R A Munsick ◽  
T O Oei ◽  
P C Young ◽  
H F Blackford
Keyword(s):  
Positive Result ◽  
Early Pregnancy ◽  
Beta Subunit ◽  
The Other ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
Human Choriogonadotropin ◽  
Beta Hcg

Abstract We evaluated four pregnancy tests (Biocept-G, Beta-CG, Preg/Stat, and HCG-Beta Screen), using sera from 59 nonpregnant subjects and 77 patients with serum human choriogonadotropin beta-subunit (beta-hCG) concentrations ranging from 4 to 100 000 int. units/L. The results obtained for each test were compared with the results predicted on the basis of the sample's beta-hCG concentration and the beta-hCG concentration the manufacturer claimed necessary for a positive result (the test's analytical sensitivity). Biocept-G had the best sensitivity (100%), specificity (98.9%), and accuracy (99.2%). Beta-CG had the poorest sensitivity (86.4%), Preg/Stat the poorest specificity (87.5%), and accuracy (92.6%). We confirmed the manufacturer's claimed analytical sensitivity (200 int. units/L) for the Biocept-G procedure, but our calculated analytical sensitivity for the other tests was significantly different from that claimed by their manufacturers. Best results were obtained with Biocept-G, but with its analytical sensitivity of 200 int. units/L, samples from early pregnancy will give negative results. None of the pregnancy tests evaluated here will establish the presence or absence of early pregnancy with certainty.

An accelerated enzyme immunoassay for human choriogonadotropin in urine, involving reflow of specimen through capillary tubes.

1987 ◽  
Vol 33 (4) ◽  
pp. 498-501 ◽  
Author(s):  
H M Chandler ◽  
S A Fuller ◽  
C H Nakagawa ◽  
P A Nagainis ◽  
J G Hurrell
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Enzyme Immunoassay ◽  
Test Specimen ◽  
Capillary Tube ◽  
Test Procedure ◽  
Beta Subunit ◽  
Sensitive Assay ◽  
Substrate Solution ◽  
Human Choriogonadotropin

Abstract A very rapid and sensitive assay for human choriogonadotropin (hCG) has been developed involving two beta-subunit-specific monoclonal antibodies. In the assay the test specimen is passed backward and forward (reflow) through a monoclonal-antibody-coated capillary tube for 1 min, then incubated for 1 min with a second monoclonal antibody conjugated to urease (EC 3.5.1.5). After addition of a urease substrate solution, 10 int. units of hCG per liter can be detected visually within 5 min, which compares very favorably with other currently available hCG assay procedures. Advantages of the reflow/capillary tube assay system and optimization of the test procedure are discussed.

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.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland

1993 ◽  
Vol 39 (11) ◽  
pp. 2322-2325 ◽  
Author(s):  
S M Yie ◽  
E Johansson ◽  
G M Brown
Keyword(s):  
Pineal Gland ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Linear Regression Analysis ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Rat Serum ◽  
Practical Applications ◽  
Pineal Function ◽  
Basic Studies

Abstract We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.

Choriogonadotropin and its beta subunit separated by hydrophobic-interaction chromatography and quantified in serum during pregnancy by time-resolved immunofluorometric assays.

1988 ◽  
Vol 34 (9) ◽  
pp. 1758-1762 ◽  
Author(s):  
H Alfthan ◽  
J Schröder ◽  
R Fraser ◽  
A Koskimies ◽  
H Halila ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Menstrual Period ◽  
Beta Subunit ◽  
Last Menstrual Period ◽  
Time Resolved ◽  
Vitro Fertilization ◽  
Human Choriogonadotropin ◽  
Beta Hcg

Abstract Concentrations of human choriogonadotropin (hCG) and its free beta subunit (beta hCG) were measured in serum by highly sensitive and specific time-resolved immunofluorometric assays (IFMAS). The results were confirmed by completely separating beta hCG and hCG by a novel method based on hydrophobic-interaction chromatography. We used three monoclonal antibodies in two different combinations. In both assays an antibody reacting with both free beta hCG and with intact hCG was immobilized onto the wall of a microtiter strip well. For assay of intact hCG we used as the indicator antibody an antibody against the alpha subunit, labeled with a europium chelate. For assay of beta hCG we used an indicator antibody that reacted only with the free beta subunit. hCG cross-reacted in the assay of beta hCG by 0.6%. Quantifying hCG in serum after in vitro fertilization showed that, seven to eight days after embryo transfer, the hCG concentration started to increase, thereafter increasing with a doubling time of 1.9 days during the following three weeks. hCG concentrations in serum peaked six to 10 weeks later, corresponding to eight to 12 weeks after the last menstrual period. Throughout pregnancy, measurable amounts of beta hCG were present in serum. The highest beta hCG/hCG ratio (maximum 7.3%, median 3.0%) was observed during early gestation. During the fourth to 13th weeks after the last menstrual period the ratio of beta hCG/hCG decreased gradually, being 1.0% during the second and third trimesters.

Bioelectrochemical enzyme immunoassay of human choriogonadotropin with magnetic electrodes.

1985 ◽  
Vol 31 (9) ◽  
pp. 1449-1452 ◽  
Author(s):  
G A Robinson ◽  
H A Hill ◽  
R D Philo ◽  
J M Gear ◽  
S J Rattle ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzyme Immunoassay ◽  
Magnetic Particles ◽  
Solid Phase ◽  
Model System ◽  
Antigen Binding ◽  
Immunoradiometric Assay ◽  
Human Choriogonadotropin ◽  
Assay Time ◽  
Rapid Quantification

Abstract We describe an amperometric technique for quantification of an enzyme immunoassay in which we use a magnetic working electrode, both to separate bound and free analyte and to monitor the electrochemical response. We used a "two-site" immunometric assay with monoclonal antibodies for human choriogonadotropin (hCG) as a model system in which magnetic particles were used as the solid phase. Separation of bound and free label is readily achieved by localizing the particles at the electrode. Activity of the bound enzyme in the environment of the electrode is determined electrochemically, permitting rapid quantification of the analyte without the need for a separate incubation step to measure enzyme activity. The sensitivity of the system is 150 int. units of hCG per litre (1st Int. Ref. Preparation). Correlation between the amperometric measurement of urinary hCG and data for an immunoradiometric assay was r = 0.9. The assay is rapid, requiring a total assay time for each sample of 20 min, which includes 15 min for antibody/antigen binding.

Automated and manual quantitative assays of choriogonadotropin in serum compared.

1987 ◽  
Vol 33 (1) ◽  
pp. 167-169 ◽  
Author(s):  
C J Beinlich ◽  
R H Carpenter
Keyword(s):  
Enzyme Immunoassay ◽  
Quantitative Agreement ◽  
Cross Reactivity ◽  
Analytical Performance ◽  
Good Quantitative Agreement ◽  
Correlation Studies ◽  
Automated Assay ◽  
Human Choriogonadotropin ◽  
Interassay Precision

Abstract We found the analytical performance of a rapid, automated assay of human choriogonadotropin (hCG) in serum, the Stratus hCG Fluorometric Enzyme Immunoassay, superior to a widely used manual assay for hCG (Hybritech Tandem-E hCG). The two assays were comparable in sensitivity; recovery; cross reactivity with lutropin, follitropin, and thyrotropin; and freedom from interference from hemoglobin and bilirubin. Patient-correlation studies indicated good quantitative agreement [Stratus hCG = (1.08 X Tandem hCG) - 4.3 int. units/L]. However, intra- and interassay precision was substantially better with the Stratus hCG assay, and this may allow earlier confirmation of pregnancy.

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.

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