Avidin-Biotin Enzyme Immunoassay of Osteocalcin in Serum or Plasma

1992 ◽  
Vol 38 (10) ◽  
pp. 1968-1974 ◽  
Author(s):  
J Jaouhari ◽  
F Schiele ◽  
S Dragacci ◽  
P Tarallo ◽  
J P Siest ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Healthy Subjects ◽  
Limit Of Detection ◽  
Serum Concentrations ◽  
Calcium Dependent ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
High Concentrations

Abstract We describe a competitive enzyme immunoassay, the ExtrAvidin-biotin system, for determining osteocalcin in human serum or plasma. Antibodies were raised against bovine osteocalcin. Binding of the antibodies to osteocalcin was calcium-dependent. Limit of detection is 0.07 nmol/L (0.4 microgram/L). The standard curve for method is linear between 0.3 and 17.6 nmol/L (1.9 and 100 micrograms/L). Interassay CV over the range 0.9 to 14.8 nmol/L (5.3 to 84 micrograms/L) is 7.5% to 11.7%. Analytical recovery is 105% +/- 5% (mean +/- SD). The measurement, which is adapted to microtiter plates, requires only 20 microL of serum and 5 h. The coefficient of correlation between the concentrations measured by this method and by a commercially available radioimmunoassay kit (CIS Biointernational) is 0.91. Osteocalcin can be measured in serum or heparinized plasma. Hemolysis (174 mumol/L hemoglobin) reduces osteocalcin concentration by 54%. High concentrations of triglycerides (7 mmol/L) give an overestimation of 63%. Serum concentrations of osteocalcin measured in 130 healthy subjects (ages 15-64 years) and 86 children (ages 4-14 years) were 1.4 +/- 0.8 and 4.0 +/- 1.5 nmol/L (8.1 +/- 4.6 and 22.5 +/- 8.6 micrograms/L), respectively (mean +/- SD).

Highly sensitive enzyme immunoassay of proinsulin immunoreactivity with use of two monoclonal antibodies

1993 ◽  
Vol 39 (10) ◽  
pp. 2146-2150 ◽  
Author(s):  
L L Kjems ◽  
M E Røder ◽  
B Dinesen ◽  
S G Hartling ◽  
P N Jørgensen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Enzyme Immunoassay ◽  
Healthy Subjects ◽  
Sandwich Elisa ◽  
C Peptide ◽  
Peptide Antibody ◽  
Analytical Recovery ◽  
Highly Sensitive

Abstract A highly sensitive two-site sandwich ELISA measuring total proinsulin immunoreactive material in serum or plasma was developed. The assay was based on two monoclonal antibodies, an anti-C-peptide antibody bound to a microtest plate and a biotin-labeled anti-insulin antibody. The detection limit (3 SD above zero value) in buffer was 0.05 pmol/L, corresponding to 0.25 pmol/L in human serum (diluted 1:5). The linear calibrator range was 0.05-20 pmol/L. Interassay CVs were 4.7% at a median (range) of 2.3 pmol/L (1.4-2.8 pmol/L, n = 8), 6.7% at 5.1 pmol/L (3.3-8.0 pmol/L, n = 8), and 8.7% at 10.0 pmol/L (8-12 pmol/L, n = 10). Mean analytical recovery of added human proinsulin (hPI) (2, 5, and 10 pmol/L) to serum was 84% (range 68-128%, n = 9). Human insulin and human C-peptide did not cross-react at 5000 and 10,000 pmol/L, respectively. The four major proinsulin conversion intermediates reacted 65-99%: split(32-33)hPI 74%, des-(31,32)hPI 65%, split(65-66)hPI 78%, and des(64,65)hPI 99%. All serum values from 38 fasting healthy subjects were above the detection limit: median (range) 4.0 (2.1-12.6) pmol/L.

Results with six "kit" radioimmunoassays for primary bile acids in human serum intercompared.

1980 ◽  
Vol 26 (12) ◽  
pp. 1677-1682 ◽  
Author(s):  
A Roda ◽  
E Roda ◽  
R Aldini ◽  
M Capelli ◽  
D Festi ◽  
...  
Keyword(s):  
Human Serum ◽  
Bile Acids ◽  
Cholic Acid ◽  
Chenodeoxycholic Acid ◽  
Healthy Subjects ◽  
Liquid Scintillation ◽  
Normal Values ◽  
Affinity Constant ◽  
Analytical Recovery ◽  
High Concentrations

Abstract We examined six radioimmunoassay procedures for measuring primary bile acids in human serum (two 3H-labeled and four 125I-labeled). A significant (p < 0.01) correlation was observed between measurements in the assay both for cholic acid and chenodeoxycholic acid, at low and high concentrations of serum bile acids. All kits were acceptable with respect to accuracy, precision, stability, and analytical recovery. All six procedures gave similar results for chenodeoxycholic and cholic acid in sera of 80 healthy subjects; the agreement was also close when the two primary bile acids were compared with their sum in serum. Normal values ranged from 0.4 to 2.5 mumol/L for conjugated chenodeoxycholic acid and from 0.3 to 1.5 mumol/L for conjugated cholic acid. The 125I assays do not require liquid-scintillation equipment but 125I induces a decrease in the affinity constant of antibody. The sensitivity of the assays was still adequate for measuring bile acids in the serum of healthy fasting persons and liver-disease patients.

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.

scholarly journals A simple method for the analysis of urinary sucralose for use in tests of intestinal permeability

2005 ◽  
Vol 42 (3) ◽  
pp. 224-226 ◽  
Author(s):  
A D G Anderson ◽  
P Poon ◽  
G M Greenway ◽  
J MacFie
Keyword(s):  
Refractive Index ◽  
Limit Of Detection ◽  
Internal Standard ◽  
Simple Method ◽  
Standard Curve ◽  
Additional Information ◽  
Accuracy And Precision ◽  
Analytical Recovery ◽  
Peak Areas ◽  
Syringe Filter

Background: Sucralose is a unique disaccharide probe which is stable in the colon and can be used to assess permeability over the whole gut. Additional information can be gained when sucralose is administered in combination with lactulose and a monosaccharide such as L-rhamnose in the form of a 'triple sugar test.' We describe a simple assay for urinary sucralose by HPLC with refractive index detection (HPLC-RI). Methods: Phenyl-β-D-glucopyranoside (internal standard) was added to 10 mL of urine, which was then passed through a 0.45 μm syringe filter. Elution was with 30% methanol (1 mL/min) on a reverse-phase C18 column. Detection was by refractive index, and integration based upon peak areas. Sixty standards of sucralose in human urine were analysed in order to quantify analytical variation. Results: The standard curve for urinary sucralose was linear from 25 to 500 mg/L ( r>0.99). The limit of detection was 11 mg/L. Analytical recovery of sucralose at concentrations of 25, 50 and 100 mg/L was 101.5% (CV 7.59%), 102.9% (CV 5.82%) and 105.0% (CV 4.26%), respectively Conclusions: The technique described represents a simple assay for urinary sucralose which performed with acceptable accuracy and precision and should facilitate the use of the triple sugar test in clinical research.

Evaluation of a commercial enzyme immunoassay for insulin in human serum, and its clinical application.

1979 ◽  
Vol 25 (1) ◽  
pp. 35-38 ◽  
Author(s):  
M Yoshioka ◽  
H Taniguchi ◽  
A Kawaguchi ◽  
T Kobayashi ◽  
K Murakami ◽  
...  
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Clinical Laboratory ◽  
Reduced Glutathione ◽  
Serum Insulin ◽  
Aminosalicylic Acid ◽  
Hydrogen Donors ◽  
Sandwich Method ◽  
High Concentrations ◽  
Commercial Enzyme Immunoassay

Abstract We applied a "sandwich" method, with use of beads coated with anti-insulin serum and of peroxidase-labeled anti-insulin serum, to an enzyme immunoassay of insulin in human serum. 5-Aminosalicylic acid was used as the substrate for the enzymic reaction. As little as 5 milli-int. units of insulin per liter of serum insulin was detectable. Reproducibility was satisfactory, but extraordinarily high concentrations of proinsulin and of hydrogen donors such as reduced glutathione affect results of the assay. Values determined by our enzyme immunoassay and by double-antibody radioimmunoassay correlated highly (r = 0.938, p less than 0.001, n = 216). We recommend this method for use in the clinical laboratory.

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.

Homogeneous enzyme immunoassay for cortisol with a centrifugal analyzer.

1984 ◽  
Vol 30 (11) ◽  
pp. 1824-1826 ◽  
Author(s):  
J M Izquierdo ◽  
A Quirós ◽  
J Alvarez-Uría ◽  
P Sotorrío
Keyword(s):  
Enzyme Immunoassay ◽  
Protein Complex ◽  
Acidic Solution ◽  
Serum Cortisol ◽  
Standard Curve ◽  
Analytical Recovery ◽  
Target Values ◽  
Assay Precision ◽  
Homogeneous Enzyme Immunoassay ◽  
Control Samples

Abstract We determine serum cortisol by a homogeneous enzyme immunoassay in the Cobas Bio centrifugal analyzer. To unbind cortisol from its protein complex, serum is treated for 15 min with an acidic solution. The reaction then proceeds automatically in the analyzer at 37 degrees C. To 50 microL of sample mixture is added 125 microL of reagent (cortisol antibodies, glucose 6-phosphate, and NAD+). This mixture is incubated for 60 s, after which 25 microL of a cortisol derivative labeled with glucose 6-phosphate is added; the increase in absorbance is monitored at 340 nm. The standard curve was linear from 10 to 500 micrograms of cortisol per liter. Within-assay precision (CV) varied from 0.2 to 0.6%, between-assay precision from 6.2 to 10.6%. Analytical recovery ranged from 100 to 103%. Results for control samples deviated from target values by 1.4 to 7.8%. Results compared well with those by radioimmunoassay. The method is reliable and practicable and will usefully replace previous routine methods for serum cortisol.

RADIOIMMUNOASSAY OF 3,3′-DI-IODOTHYRONINE IN UNEXTRACTED SERUM: THE EFFECT OF ENDOGENOUS TRI-IODOTHYRONINE

1978 ◽  
Vol 79 (3) ◽  
pp. 357-362 ◽  
Author(s):  
T. J. VISSER ◽  
L. M. KRIEGER-QUIST ◽  
R. DOCTER ◽  
G. HENNEMANN
Keyword(s):  
Human Serum ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Transport Proteins ◽  
Normal Serum ◽  
Serum Concentrations ◽  
Specific Radioimmunoassay ◽  
Highly Sensitive ◽  
The Mean ◽  
Mean Concentration

The development of a highly sensitive and specific radioimmunoassay for 3,3′-di-iodothyronine (3,3′-T2) is described. The assay was applied to the measurement of 3,3′-T2 in unextracted human serum and used 8-anilino-l-naphthalene-sulphonic acid to inhibit the binding of 3,3′-T2 to serum transport proteins. The lower limit of detection of the assay was 2 fmol 3,3′-T2 per tube, which corresponded to 10 pmol 3,3′-T2/l serum. The mean concentration of 3,3′-T2 in normal serum was found to be 23 pmol/l, which is considerably lower than most values reported previously. Evidence is presented which suggests that the cross-reactivity of tri-iodothyronine with the antiserum to 3,3′-T2 is an important factor in the measurement of serum concentrations of 3,3′-T2 by radioimmunoassay.

Evaluation of four assay methods for determination of tobramycin in human serum.

1982 ◽  
Vol 28 (1) ◽  
pp. 177-180 ◽  
Author(s):  
W J Acton ◽  
O M Van Duyn ◽  
L V Allen ◽  
D J Flournoy
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Correlation Coefficient ◽  
Clinical Laboratory ◽  
The Other ◽  
Serum Enzyme ◽  
Analytical Recovery ◽  
Assay Methods

Abstract Four assay procedures for tobramycin in serum--enzyme immunoassay (I), substrate-labeled fluorescent immunoassay (II), radioimmunoassay (III), and bioassay (IV)--were compared and evaluated by replicate and analytical recovery studies. I and II were about 50% more precise than III and IV. II was substantially more nearly accurate than the other methods and also gave the best reproducibility (correlation coefficient 0.992 between-day). The least expensive method was IV. Ease of handling favored I and II. Overall, we find II to be the most acceptable procedure for use in the clinical laboratory.

Enzyme-amplified immunoassays: a new ultrasensitive assay of thyrotropin evaluated.

1986 ◽  
Vol 32 (1) ◽  
pp. 88-92 ◽  
Author(s):  
P M Clark ◽  
C P Price
Keyword(s):  
Healthy Subjects ◽  
Normal Values ◽  
External Quality Assessment ◽  
Limit Of Detection ◽  
Microtiter Plate ◽  
Front Line ◽  
External Quality ◽  
Analytical Recovery ◽  
Ultrasensitive Assay ◽  
Line Test

Abstract An enzyme-amplified immunoassay for thyrotropin has been evaluated. The lower limit of detection of the assay (mean + 3 SD of the zero standard) was 0.037 milli-int.unit/L and the precision was good, giving a working range of 0.13-24.0 milli-int.units/L. The accuracy of the assay was good, as judged from analytical-recovery experiments, analysis of external quality-assessment samples and comparison with in-house assays. No significant interferences or cross reactivities were identified. Assay of a 25-microL serum sample on the microtiter plate takes 3 h. Values for healthy subjects ranged from 0.4 to 4.0 milli-int.units/L. For 32 thyrotoxic patients thyrotropin concentrations were clearly suppressed and completely distinct from normal values. Patients taking triiodothyronine also showed lower concentrations. The enzyme-amplified immunoassay may thus be a suitable "front line" test for assessing thyroid function.

Sign in / Sign up

Export Citation Format

Share Document