Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

Direct chemiluminescence immunoassay of estradiol in saliva

1990 ◽  
Vol 36 (12) ◽  
pp. 2036-2041 ◽  
Author(s):  
J De Boever ◽  
F Kohen ◽  
J Bouve ◽  
D Leyseele ◽  
D VandeKerckhove
Keyword(s):  
Detection Limit ◽  
Incubation Time ◽  
Solid Phase ◽  
Chemiluminescence Immunoassay ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Plate Reader ◽  
Steroid Binding ◽  
Method R ◽  
Direct Solid

Abstract A sensitive and simple direct solid-phase chemiluminescence immunoassay is described for estradiol in saliva. In this assay, a second antibody is bound to the wells of microtiter plates. Either buffer with standards or saliva (100 microL) is incubated in these wells with monoclonal anti-estradiol antibody and with estradiol-isoluminol conjugate. Incubation time is 2 h. Chemiluminescence of the bound fraction is measured in a manually operated luminometer (Biocounter). The assay has a detection limit of 3.8 pmol/L; analytical recovery of added estradiol is 96.8% (SD 7.0%); within- and between-assay CVs range between 2.5% and 12.7%. Forty unknown saliva samples can be assayed and results calculated within 4.5 h. Results of a slightly modified procedure-with black microtiter plates and a prototype of an automated plate reader (Luminoskan)--compare well with those of the described method (r = 0.97). Because steroid-binding globulins have been found in saliva, the effect of displacing agents on the results of the direct chemiluminescence assay is described.

Direct chemiluminescence immunoassay for estradiol in serum.

1986 ◽  
Vol 32 (10) ◽  
pp. 1895-1900 ◽  
Author(s):  
J De Boever ◽  
F Kohen ◽  
C Usanachitt ◽  
D Vandekerckhove ◽  
D Leyseele ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Chemiluminescence Immunoassay ◽  
Binding Reaction ◽  
High Affinity ◽  
Microtiter Plates ◽  
Analytical Recovery

Abstract In this simple, reliable, fast solid-phase chemiluminescence immunoassay for directly measuring (i.e., without prior extraction) estradiol-17 beta in serum, a monoclonal antibody is used that binds estradiol with high affinity (Ka = 10(10) L/mol), and does not bind other steroids tested, the highest cross reactivity observed being 0.1% for estradiol-17 alpha. In this system the monoclonal antibody is bound to the wells of microtiter plates via a second antibody directed against the monoclonal antibody. Fifty microliters of serum and estradiol-displacing agents are added, followed by 100 pg of estradiol-isoluminol conjugate, and the label is measured by luminometry after the binding reaction. The sensitivity of the assay is 180 pmol per liter of serum, and the effective working range at less than or equal to 10% CV is 270 to 6700 pmol/L. Analytical recovery of added estradiol averaged 99.7% (SD 6.5%). Within- and between-assay CVs ranged between 5 and 12.7%. Thirty-five unknown serum samples can be assayed within 4 h. Results correlated well with those obtained with a direct RIA: r = 0.94 (n = 149). This assay opens new perspectives for chemiluminescence immunoassays.

A receptor-antibody sandwich assay for teicoplanin.

1987 ◽  
Vol 33 (9) ◽  
pp. 1615-1618 ◽  
Author(s):  
A Corti ◽  
L Cavenaghi ◽  
E Giani ◽  
G Cassani
Keyword(s):  
Detection Limit ◽  
Dose Response ◽  
Response Curve ◽  
Synthetic Analog ◽  
Sandwich Complexes ◽  
Sandwich Assay ◽  
Receptor Antibody ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Biological Target

Abstract We have developed a new method for quantifying teicoplanin in complex matrixes, a receptor-antibody sandwich assay (RASA). The method is based on bioselective adsorption of teicoplanin onto microtiter plates coated with albumin-epsilon-aminocaproyl-D-alanyl-D-alanine, a synthetic analog of its biological target, and reaction with anti-teicoplanin antibodies. The sandwich complexes are detected by incubation with peroxidase-labeled goat antibodies to rabbit IgGs and chromogenic reaction with o-phenylenediamine. The dose-response curve was linear for teicoplanin concentrations in the range from 0 to 0.15 mg/L. We used the assay to measure teicoplanin concentrations in various biological matrixes. Analytical recovery from serum was 99.5%, the interassay CV was 5.1%, and the detection limit was 30 micrograms/L (P less than 0.01). Mean analytical recoveries from other biological specimens were 98% from ascitic fluid, 100% from pleuric liquid, 104.8% from prostate homogenate, and 98.5% from bronchial expectorate.

Direct solid-phase enzymoimmunoassay of testosterone in saliva.

1989 ◽  
Vol 35 (10) ◽  
pp. 2044-2047 ◽  
Author(s):  
K Howard ◽  
M Kane ◽  
A Madden ◽  
J P Gosling ◽  
P F Fottrell
Keyword(s):  
Detection Limit ◽  
Analytical Procedure ◽  
Solid Phase ◽  
Medical Applications ◽  
Microtiter Plates ◽  
Large Numbers ◽  
Coefficients Of Variation ◽  
Serial Sampling ◽  
Direct Solid

Abstract This competitive, solid-phase enzymoimmunoassay for testosterone in saliva is carried out on microtiter plates and involves no chromatographic or extraction steps. With an overnight incubation the detection limit of the assay is 230 fg per well (16.1 pmol/L). There was a good correlation (correlation coefficient 0.95) between testosterone concentrations measured with and without prior extraction of the saliva samples. Repeated assay of three control saliva samples containing a range of testosterone concentrations (200-1000 pmol/L) gave within- and between-assay coefficients of variation of 5.5-13.2%. The analytical procedure is simple and closely resembles already published procedures for the determination of progesterone and estrone (with extraction) in saliva. One person can assay 200 samples in 24 h and the assay is suitable for reproductive and sports medical applications, particularly for projects involving serial sampling and yielding large numbers of samples.

Enzyme immunoassay for intact human insulin in serum or plasma

1993 ◽  
Vol 39 (4) ◽  
pp. 578-582 ◽  
Author(s):  
L Andersen ◽  
B Dinesen ◽  
P N Jørgensen ◽  
F Poulsen ◽  
M E Røder
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Clinical Importance ◽  
Porcine Insulin ◽  
Microtiter Plates ◽  
Relative Response ◽  
Normal Individuals ◽  
Insulin Dependent ◽  
Murine Monoclonal Antibodies

Abstract We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.

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.

scholarly journals Direct Assay for Cobalamin Bound to Transcobalamin (Holo-Transcobalamin) in Serum

2002 ◽  
Vol 48 (3) ◽  
pp. 526-532 ◽  
Author(s):  
Marius Ulleland ◽  
Ingar Eilertsen ◽  
Edward V Quadros ◽  
Sheldon P Rothenberg ◽  
Sergey N Fedosov ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Quantitative Determination ◽  
Reliable Method ◽  
Solid Phase ◽  
Reference Interval ◽  
Magnetic Microspheres ◽  
Affinity Constant ◽  
Capture Assay

Abstract Background: Only cobalamin carried by transcobalamin (holo-transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-transcobalamin are available. We report a novel holo-transcobalamin assay based on solid-phase capture of transcobalamin. Methods: A monoclonal antibody specific for human transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate transcobalamin. The cobalamin bound to transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-transcobalamin was accomplished using calibrators composed of recombinant, human holo-transcobalamin. Results: The assay was specific for holo-transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well (r2 = 0.87) with total cobalamin, holo-transcobalamin correlated poorly (r2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-transcobalamin in serum.

scholarly journals Simple and Fast Methods Based on Solid-Phase Extraction Coupled to Liquid Chromatography with UV Detection for the Monitoring of Caffeine in Natural, and Wastewater as Marker of Anthropogenic Impact

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Sònia Moret ◽  
Manuela Hidalgo ◽  
Juan M. Sanchez
Keyword(s):  
Detection Limit ◽  
Solid Phase Extraction ◽  
Natural Waters ◽  
Wastewater Treatment Plants ◽  
Solid Phase ◽  
Sample Volume ◽  
Uv Detection ◽  
Phase Extraction ◽  
Method Detection Limit ◽  
Spe Method

Two concentration methods for fast and routine determination of caffeine (using HPLC-UV detection) in surface, and wastewater are evaluated. Both methods are based on solid-phase extraction (SPE) concentration with octadecyl silica sorbents. A common “offline” SPE procedure shows that quantitative recovery of caffeine is obtained with 2 mL of an elution mixture solvent methanol-water containing at least 60% methanol. The method detection limit is 0.1 μg L−1 when percolating 1 L samples through the cartridge. The development of an “online” SPE method based on a mini-SPE column, containing 100 mg of the same sorbent, directly connected to the HPLC system allows the method detection limit to be decreased to 10 ng L−1 with a sample volume of 100 mL. The “offline” SPE method is applied to the analysis of caffeine in wastewater samples, whereas the “on-line” method is used for analysis in natural waters from streams receiving significant water intakes from local wastewater treatment plants.

Rapid, competitive enzymoimmunoassay for albumin in urine.

1986 ◽  
Vol 32 (4) ◽  
pp. 669-671 ◽  
Author(s):  
J Chesham ◽  
S W Anderton ◽  
C F Kingdon
Keyword(s):  
Diabetic Nephropathy ◽  
Human Serum Albumin ◽  
Horseradish Peroxidase ◽  
Serum Albumin ◽  
Human Serum ◽  
Human Urine ◽  
Solid Phase ◽  
Low Concentrations ◽  
Initiation Of Therapy ◽  
Assay Protocol

Abstract In this solid-phase competitive enzymoimmunoassay for albumin in human urine, antiserum to human serum albumin labeled with horseradish peroxidase (EC 1.11.1.7) is incubated with solid-phase-bound human serum albumin in the presence of sample or standard. Results obtained correlate well (r = 0.96) with those of an established fluoroimmunoassay. The present assay covers the range 0.9 to 200 mg/L and can be performed within 1 h. These characteristics, together with the simplicity of the assay protocol, make it very useful for monitoring low concentrations of albumin in urine. Detection of such minimal albuminuria allows initiation of therapy that may prevent development of clinical proteinuria and associated diabetic nephropathy.

Solid-phase enzyme-receptor assay (SPERA): a competitive-binding assay for glycopeptide antibiotics of the vancomycin class.

1985 ◽  
Vol 31 (10) ◽  
pp. 1606-1610 ◽  
Author(s):  
A Corti ◽  
C Rurali ◽  
A Borghi ◽  
G Cassani
Keyword(s):  
Human Serum ◽  
Binding Assay ◽  
Solid Phase ◽  
Quantitative Detection ◽  
Synthetic Analog ◽  
Bacterial Cells ◽  
Glycopeptide Antibiotics ◽  
Competitive Binding Assay ◽  
Analytical Recovery ◽  
Receptor Assay

Abstract A solid-phase enzyme-receptor assay (SPERA) has been developed for glycopeptide antibiotics of the vancomycin class such as teicoplanin, vancomycin, ristocetin, avoparcin, actaplanin, A-47934, A-41030, and A-35512-B. The assay exploits the mechanism of most action of these antibiotics, which is based on their interaction with acyl-D-alanyl-D-alanine, a constituent of the walls of most growing bacterial cells. The antibiotics and enzyme-labeled teicoplanin compete for a synthetic analog of the biological receptor, albumin-epsilon-aminocaproyl-D-alanyl-D-alanine. The various antibiotics produced different competition curves, 50% displacement being obtained with antibiotic concentrations ranging from 0.04 to 4 mg/L, vancomycin and actaplanin being the weakest and strongest competitors, respectively. For teicoplanin in human serum the intra-assay CV was 7.2%, the interassay CV was 11.2%, and the analytical recovery 94%. Teicoplanin concentrations obtained by SPERA (chi) correlated well with those obtained by microbiological assay (y): y = 1.03 chi + 0.053 (r = 0.943; n = 60). We conclude that SPERA is a powerful tool for identification and quantitative detection of glycopeptide antibiotics, even in complex media.

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