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.

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.

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.

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, Simplified, and Sensitive Assay of Angiotensin II in Plasma Extracts Performed with a High-Affinity Monoclonal Antibody

1992 ◽  
Vol 38 (10) ◽  
pp. 1963-1967 ◽  
Author(s):  
D Simon ◽  
B Romestand ◽  
H Huang ◽  
G Badouaille ◽  
J A Fehrentz ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
High Performance ◽  
Solid Phase ◽  
High Performance Liquid Chromatographic ◽  
Reversed Phase ◽  
Cross Reactivity ◽  
Ang Ii ◽  
High Affinity ◽  
Before And After

Abstract A very simple, fast, and sensitive RIA of angiotensin (Ang) II has been developed, based on a monoclonal antibody with high affinity and specificity, making possible the direct measurement of circulating Ang II in human plasma after solid-phase extraction. The purified monoclonal antibody 4D8 has an association constant of 1.3 x 10(11) L/mol with Ang II and a cross-reactivity of < 1% for Ang I. The assay can detect as little as 0.8 fmol of Ang II in 2 mL of plasma and is not influenced by the presence of Ang I. Analytical recoveries between 112% and 116% were obtained for Ang II added to human plasma at physiological concentrations. Comparison of the RIA with a reversed-phase, high-performance liquid chromatographic method followed by RIA to measure Ang II in human plasma samples from normal and hypertensive subjects--and from normotensive subjects before and after an acute inhibition of angiotensin-converting enzyme with captopril (50 mg)--showed a high degree of correlation (r2 = 0.93) between the two methods.

Highly sensitive assays of autoantibodies to thyroglobulin and to thyroid peroxidase.

1989 ◽  
Vol 35 (9) ◽  
pp. 1949-1954 ◽  
Author(s):  
K Beever ◽  
J Bradbury ◽  
D Phillips ◽  
S M McLachlan ◽  
C Pegg ◽  
...  
Keyword(s):  
Solid Phase ◽  
Protein A ◽  
Cross Reactivity ◽  
Thyroid Peroxidase ◽  
Scatchard Analysis ◽  
Thyroid Autoantibodies ◽  
Serum Samples ◽  
Highly Sensitive ◽  
Phase Protein ◽  
High Concentrations

Abstract These highly sensitive assays are based on the interaction between thyroid autoantibodies and 125I-labeled autoantigens. Serum samples are incubated with labeled thyroid peroxidase (TPO) or thyroglobulin (Tg) to allow the formation of antibody-labeled antigen complexes. The complexes are then precipitated by addition of solid-phase Protein A. In the presence of high concentrations of TPO antibody or Tg antibody, more than 50% of the respective labeled antigen was precipitated, whereas only 1-2% was precipitated in the absence of autoantibody. Interassay CVs were 3.2% and 5.7%, respectively, for the anti-TPO and anti-Tg assays. There was no cross-reactivity between Tg antibody and TPO antibody. Results correlated highly significantly with results from other assay systems based on antigen-coated cells or plastic supports, but the assays described here were considerably more sensitive. Scatchard analysis of the assay data provided information on the affinity and serum concentration of TPO autoantibodies (ka approximately 10(9) L/mol and concentrations up to 1 g/L) and Tg autoantibodies (ka approximately 4 x 10(10) L/mol and concentrations up to 1 g/L). Overall, these assays provide a sensitive, precise, and convenient system for measuring and investigating the properties of thyroid autoantibodies.

scholarly journals Hemoglobin-Specific Antibody in a Multiply Transfused Patient With Sickle Cell Disease

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2155-2158 ◽  
Author(s):  
Peter A. Noronha ◽  
Loyda N. Vida ◽  
C. Lucy Park ◽  
George R. Honig
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Cell Disease ◽  
Serum Samples ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Level Of Activity

Abstract Human hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to κ chains, the anti-Hb antibody demonstrated specificity for the region of the Hb β chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.

Amplified Enzyme-Linked Immunoassay of Human Proinsulin in Serum (Detection Limit: 0.1 pmol/L)

1992 ◽  
Vol 38 (2) ◽  
pp. 227-232 ◽  
Author(s):  
F J Dhahir ◽  
D B Cook ◽  
C H Self
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Cross Reactivity ◽  
C Peptide ◽  
Human Proinsulin ◽  
Microtiter Plates ◽  
Enzyme Amplification ◽  
Fold Excess ◽  
Amplification Procedure ◽  
Serum Detection

Abstract We describe an amplified enzyme-linked immunoassay of human proinsulin in serum that detects intact proinsulin and both the 32/33 and 65/66 split forms. The method uses the IgG fraction of a polyclonal antibody raised in a guinea pig against intact proinsulin, which we used to coat plastic microtiter plates. A sandwich was formed with proinsulin by using a monoclonal antibody against C-peptide labeled with alkaline phosphatase. We quantified the reaction by using the enzyme amplification procedure, which detected as little intact proinsulin as 0.1 pmol/L. We found no cross-reactivity with C-peptide in the assay, and decreased recovery attributable to the presence of insulin could be demonstrated only with a 30-fold excess of this hormone over proinsulin.

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.

scholarly journals No Specific Reactivity to E. Coli Glutamic Acid Decarboxylase from Sera of Newly-Diagnosed Insulin Dependent Diabetic Patients

2003 ◽  
Vol 16 (2) ◽  
pp. 129-138
Author(s):  
C. Konidaris ◽  
P.G. Mitlianga ◽  
G.K. Papadopoulos
Keyword(s):  
Monoclonal Antibody ◽  
Glutamic Acid ◽  
Glutamic Acid Decarboxylase ◽  
Solid Phase ◽  
Normal Subjects ◽  
Cross Reactivity ◽  
Acid Decarboxylase ◽  
Diabetic Patients ◽  
Dependent Manner ◽  
E Coli

The 65 kD isoform of Glutamic Acid Decarboxylase (GAD), is one of the major autoantigens in human type 1 diabetes mellitus. This enzyme shares aminoacid identity, in select regions already determined as antigenic with its counterpart from E. coli. We tested the reactivity of diabetic and normal sera and an E. coli GAD-specific monoclonal antibody (2D9) to E. coli GAD by solid phase and competition ELISA, as well as immunoblotting to check for cross-reactivity of autoantibodies to the two antigens. Specific antibodies for E. coli GAD are present in diabetics and normal subjects without any differences in frequency and titer. The reactivity of such antibodies in ELISA could be blocked in a dose-dependent manner by the addition of excess antigen in the liquid phase. Furthermore, the monoclonal antibody against E. coli GAD does not recognise human recombinant GAD65 in an ELISA. We conclude that there is no basis for cross-reactivity between the two antigens, and antibody reactivity to GAD65 in man cannot arise from cross-reactivity to the E. coli enzyme.

scholarly journals Development of a Sensitive Bead-Based Assay for Enhanced Monoclonal Antibody Detection

2011 ◽  
Vol 1346 ◽  
Author(s):  
Manuel E. Ruidíaz ◽  
Natalie Mendez ◽  
Ana B. Sanchez ◽  
Bradley T. Messmer ◽  
Andrew C. Kummel
Keyword(s):  
Monoclonal Antibody ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Fluorescent Labeling ◽  
Antibody Detection ◽  
Serum Samples ◽  
Mimetic Peptide ◽  
Mimetic Peptides ◽  
Zero Response

ABSTRACTMonoclonal antibodies are increasingly used in the treatment of cancer due to their enhanced targeting and immune system stimulation properties. Dosage guidelines typically do not account for personal cancer load or metabolism, thereby possibly affecting treatment outcome or causing unwanted side effects. The requirement for an assay that can quickly and precisely measure the concentration of the monoclonal antibody in a serum sample of a patient during therapy is unmet. A bead-based assay with peptide antigen mimetics has been developed to rapidly determine the concentration of antibody drug present in serum specimens with high sensitivity. Alemtuzumab (anti-CD52) and rituximab (anti-CD20) antigen mimetic peptides, as discovered by phage display, were synthesized on 10 um TentaGel resin beads using conventional solid phase peptide synthesis techniques. The beads were modified to allow for multiplexing and microfluidic handling via fluorescent labeling and magnetic functionalization. The antigen-displaying fluoromagnetic particles were incubated with spiked serum samples which allowed free antibody to be captured. Primary antibody detection was performed on alemtuzumab while rituximab detection was used to compensate for non-specific serum binding to the beads. After washing, the beads were incubated with a fluorescently tagged secondary label for detection by flow cytometry. (Results) A fast, low cost, specific assay has been developed with several key techniques which allows detection at low concentration (0.1ug/ml) of spiked samples. Primary to achieving this detection limit was the implementation of a compensation scheme where two antigen mimetic peptides behave linearly (R2=0.996) which enables the calculation of the zero response of the antigen mimetic peptide of interest (alemtuzumab antigen mimetic) while measuring the zero response of the compensatory antigen mimetic peptide (rituximab antigen mimetic) during primary assay measurement. This reduces fluorescence response variation due to variations present due to sample preparation, storage and different patients because of the equivalent interactions these effects have on the compensatory beads. The developed assay is therefore robust against serum variation and enables a lower limit of detection.

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