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.

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).

A highly sensitive enzyme immunoassay of anti-insulin antibodies in human serum

1987 ◽  
Vol 1 (2) ◽  
pp. 170-174 ◽  
Author(s):  
Takeyuki Kohno ◽  
Eiji Ishikawa ◽  
Satoru Sugiyama ◽  
Syuji Nakamura ◽  
Yoshimasa Kanemaru
Keyword(s):  
Human Serum ◽  
Enzyme Immunoassay ◽  
Insulin Antibodies ◽  
Highly Sensitive

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.

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.

Two-site enzyme immunoassay of CD4 and CD8 molecules on the surface of T lymphocytes from healthy subjects and HIV-1-infected patients

1994 ◽  
Vol 40 (1) ◽  
pp. 30-37 ◽  
Author(s):  
D Carriere ◽  
C Fontaine ◽  
A M Berthier ◽  
A M Rouquette ◽  
P Carayon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Lymphocytes ◽  
Enzyme Immunoassay ◽  
Cd8 T Cells ◽  
Envelope Glycoprotein ◽  
Healthy Subjects ◽  
Gp 120 ◽  
Highly Sensitive ◽  
One Step ◽  
Hiv 1

Abstract A highly sensitive two-site enzyme immunoassay (Capcellia) was developed to determine the concentration of CD4 and CD8 molecules expressed on the surface of human T lymphocytes. This assay, performed in one step (20 min), involves the specific immunocapture of T lymphocytes and reaction of the CD4 or CD8 molecules with an enzyme-labeled monoclonal antibody (mAb). The results were expressed as molar concentrations of the T-cell markers on the basis of results obtained with calibrated CD4 and CD8 standards. The assay was sensitive enough to detect 0.4 pmol/L CD4 or 0.8 pmol/L CD8, which corresponded to approximately 20 x 10(6) CD4+ or CD8+ T cells per liter of blood. Mean concentrations in healthy adults were 17.2 pmol/L for CD4 and 22.1 pmol/L for CD8. The CD4 concentration was < 8 pmol/L in 50% of HIV-1-infected patients and in 95% of AIDS patients. Given the epitopic specificity of the mAb to CD4 we used, these values correspond to the concentration of CD4 molecules free of envelope glycoprotein (gp)120.

scholarly journals Highly specific radioimmunoassay for human insulin based on immune exclusion of all insulin precursors

1998 ◽  
Vol 44 (7) ◽  
pp. 1504-1513 ◽  
Author(s):  
Michelle Deberg ◽  
Paule Houssa ◽  
Bruce H Frank ◽  
Françoise Sodoyez-Goffaux ◽  
Jean-Claude Sodoyez
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Affinity Constant ◽  
Serum Samples ◽  
C Peptide ◽  
Specific Radioimmunoassay ◽  
Coefficients Of Variation ◽  
Patients With Diabetes ◽  
A Chain ◽  
Immune Exclusion

Abstract We describe a rapid and simple insulin RIA in which proinsulin and conversion intermediates do not interfere. Three monoclonal antibodies (S1, S2, and S53) were selected for their specificity (directed, respectively, against the B10 region, the junction between A chain and C-peptide, and the junction between B chain and C-peptide), their affinity constant (∼1010 L/mol), and their interactive properties in mixture. S2 and S53 were able to bind simultaneously to the same proinsulin molecule, whereas neither could bind simultaneously with S1. Preincubation of serum samples with an excess of S2 resulted in capture of proinsulin and conversion intermediates modified at the junction between B chain and C-peptide into immune complexes that no longer reacted with S1. Similarly, preincubation with S53 prevented proinsulin and conversion intermediates modified at the junction between A chain and C-peptide from reacting with S1. Preincubation with an excess of both S2 and S53 left insulin as the sole reactant with S1. Thus, separation of insulin precursors from insulin by mutually exclusive antibodies is feasible, and on the basis of this new principle, a highly specific RIA for insulin was designed. The detection limit was 11 pmol/L, and the inter- and intraassay coefficients of variation were 11% and 5%, respectively. The potential of the assay for use in clinical studies was verified by application to serum samples from control subjects and patients with diabetes or insulinoma.

Development of a Highly Sensitive Chemiluminescence Enzyme Immunoassay for the Determination of Bisphenol A

2013 ◽  
Vol 726-731 ◽  
pp. 1283-1286
Author(s):  
Li Rui Liu ◽  
Li Qin Liu ◽  
Xue Qing Chen ◽  
Guo Qing Shi
Keyword(s):  
Detection Limit ◽  
Bisphenol A ◽  
Enzyme Immunoassay ◽  
Rapid Determination ◽  
Secondary Antibody ◽  
Average Recovery ◽  
Highly Sensitive ◽  
Optimized Conditions ◽  
Chemiluminescence Enzyme Immunoassay

A highly sensitive chemiluminescence enzyme immunoassay (CLEIA) method for the determination of bisphenol A (BPA) was developed, which used a secondary antibody labeled with horseradish peroxidase detected with a luminol-based substrate. Under the optimized conditions, the linear range was 0.01μg/mL~0.74μg/mL, and the detection limit was 0.008μg/mL. The average recovery for BPA in barreled water was 104%. This developed method could be applied for the selective, high-throughput, and rapid determination of BPA in barreled water.

scholarly journals Development of Enzyme-Immunoassay for Bacillus anthracis Detection

2018 ◽  
pp. 78-82
Author(s):  
D. V. Pechenkin ◽  
O. O. Fomenkov ◽  
A. V. Eremkin ◽  
G. D. Elagin ◽  
G. V. Kuklina ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Enzyme Immunoassay ◽  
Specific Activity ◽  
Hybrid Cell ◽  
Sandwich Elisa ◽  
Central Research Institute ◽  
Exchange Chromatography ◽  
Central Research ◽  
Test Kit

Objective of the study was to develop enzyme-immunoassay test-kit for the detection of Bacillus anthracis spores. Materials and methods. Microbial cultures from the State Collection of Microorganisms at the premises of Affiliated Branch of the «48th Central Research Institute» of the Ministry of Defense of the Russian Federation and BALB/c mice were used in the research. Hybridization of B-lymphocytes with SP2/0-Ag14 myeloma cells was performed according to G. Kohler and C. Milstein procedure in De St. Fazekas and P. Scheidegger modification. Hybridomas were cultured in the peritoneal cavity of BALB/c mice. Ascitic fluids were isolated from mice, precipitated with ammonium sulfate and purified by means of ion-exchange chromatography for preparation of monoclonal antibodies. Specific activity of hybridoma’s supernatants, ascitic fluids, purified monoclonal antibodies was studied by «sandwich» ELISA. Specific components of test-kit were lyophilized in suitable cryoprotective medium. Results and conclusions.We have obtained new hybrid cell lines producing specific monoclonal antibodies against Bacillus anthracisspore antigens and ascitic fluids from which immunoglobulins were isolated. Optimum combinations of monoclonal antibodies as a sensitizer and a component of immunoperoxidase conjugates have been selected. Monoclonal antibodies 272E10G1-272F7A10 provide the highest sensitivity of ELISA for the detection of anthrax microbe spore antigens. Our enzyme-immunoassay test allows for identification of Bacillus anthracis spores in concentrations up to 5,0·105 spores per milliliter. No cross reaction with closely related saprophytes and other heterologous microorganisms in concentrations of 1,0·108 CFU per milliliter is observed.

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.

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.

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