scholarly journals Development of Monoclonal Antibody-Based EIA for Tetranor-PGDM which Reflects PGD2 Production in the Body

2021 ◽  
Vol 2021 ◽  
pp. 1-6
Author(s):  
Nanae Nagata ◽  
Sakura Masuko ◽  
Rikako Inoue ◽  
Tatsuro Nakamura ◽  
Kosuke Aritake ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Ionic Strength ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Dilution Effect ◽  
Cross Reactivity ◽  
The Body ◽  
Inhibition Concentration ◽  
Artificial Urine ◽  
Inter Assay Variation

Tetranor-PGDM is a metabolite of PGD2. Urinary tetranor-PGDM levels were reported to be increased in some diseases, including food allergy, Duchenne muscular dystrophy, and aspirin-intolerant asthma. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength, pH, precision, and reliability. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited a half-maximal inhibition concentration (IC50) of 1.79 ng/mL, limit of detection (LOD) of 0.0498 ng/mL, and range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The intra- and inter-assay variation for tetranor-PGDM was 3.9–6.0% and 5.7–10.4%, respectively. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA. The established EIA would be useful for routine detection and monitoring of tetranor-PGDM in research or diagnostic body fluids.

scholarly journals Molecularly Imprinted Nanoparticles Based Sensor for Cocaine Detection

Biosensors ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 22 ◽  
Author(s):  
Roberta D’Aurelio ◽  
Iva Chianella ◽  
Jack A. Goode ◽  
Ibtisam E. Tothill
Keyword(s):  
Electrochemical Impedance ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Cost Effective ◽  
Cross Reactivity ◽  
P Value ◽  
Sensing Element ◽  
Molecularly Imprinted ◽  
Cost Effective Method ◽  
Cocaine Detection

The development of a sensor based on molecularly imprinted polymer nanoparticles (nanoMIPs) and electrochemical impedance spectroscopy (EIS) for the detection of trace levels of cocaine is described in this paper. NanoMIPs for cocaine detection, synthesized using a solid phase, were applied as the sensing element. The nanoMIPs were first characterized by Transmission Electron Microscopy (TEM) and Dynamic Light Scattering and found to be ~148.35 ± 24.69 nm in size, using TEM. The nanoMIPs were then covalently attached to gold screen-printed electrodes and a cocaine direct binding assay was developed and optimized, using EIS as the sensing principle. EIS was recorded at a potential of 0.12 V over the frequency range from 0.1 Hz to 50 kHz, with a modulation voltage of 10 mV. The nanoMIPs sensor was able to detect cocaine in a linear range between 100 pg mL−1 and 50 ng mL−1 (R2 = 0.984; p-value = 0.00001) and with a limit of detection of 0.24 ng mL−1 (0.70 nM). The sensor showed no cross-reactivity toward morphine and a negligible response toward levamisole after optimizing the sensor surface blocking and assay conditions. The developed sensor has the potential to offer a highly sensitive, portable and cost-effective method for cocaine detection.

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.

Detection of bisphenol A in water samples using ELISA determination method

2011 ◽  
Vol 11 (1) ◽  
pp. 55-60 ◽  
Author(s):  
J. Zheng ◽  
S. Q. Zhao ◽  
X. T. Xu ◽  
K. Zhang
Keyword(s):  
Drinking Water ◽  
Bisphenol A ◽  
Water Samples ◽  
Limit Of Detection ◽  
Industrial Effluent ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Spectrophotometry ◽  
Inhibition Concentration

In order to study whether bisphenol A (BPA) can pass into drinking water from polycarbonate barrel and exist in the river and industrial effluent the indirect competitive enzyme-linked immunosorbent assay (ELISA) for the determination of BPA was established. The results presented an inhibition concentration at 50% absorbance (IC50) of 0.123 mg L−1, and the limit of detection (LOD) is 9.934 μg L−1. The specificity of antiserum was proved well because the cross-reactivity with benzene, tert-butylbenzene, hydroquinone and o-hydroxybenzoic acid were found lower than 0.01%, except phenol was 0.26%. The method was found to be reliable and repeatable. It was used for monitoring the concentration of BPA in the barreled drinking water. The results confirmed BPA can pass into barreled drinking water from the polycarbonate barrel and concentration increased as days went on. A certain content of BPA was found in industrial effluent. The results of ELISA were consistent with the results of UV spectrophotometry. BPA could not be found in the water samples obtained from Zhujiang River. The established method shows specific recognition of BPA and could be applied in detection of environmental BPA.

scholarly journals Design of Novel Haptens and Development of Monoclonal Antibody-Based Immunoassays for the Simultaneous Detection of Tylosin and Tilmicosin in Milk and Water Samples

Biomolecules ◽  
2019 ◽  
Vol 9 (12) ◽  
pp. 770 ◽  
Author(s):  
Jian-Xin Huang ◽  
Chan-Yuan Yao ◽  
Jin-Yi Yang ◽  
Zhen-Feng Li ◽  
Fan He ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Water Samples ◽  
Simultaneous Detection ◽  
Aldehyde Group ◽  
Cross Reactivity ◽  
Side Chain ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibition Concentration ◽  
Ic50 Values ◽  
Optimized Conditions

In this work, a monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was established to detect tylosin and tilmicosin in milk and water samples. A sensitive and specific monoclonal antibody was prepared by rational designed hapten, which was achieved by directly oxidizing the aldehyde group on the side chain of tylosin to the carboxyl group. Under the optimized conditions, the linear range of icELISA for tylosin and tilmicosin were 1.3 to 17.7 ng/mL and 2.0 to 47.4 ng/mL, with half-maximal inhibition concentration (IC50) values of 4.7 and 9.6 ng/mL, respectively. The cross-reactivity with other analogues of icELISA was less than 0.1%. The average recoveries of icELISA for tylosin and tilmicosin ranged from 76.4% to 109.5% in milk and water samples. Besides, the detection results of icELISA showed good correlations with HPLC-MS/MS. The proposed icELISA was satisfied for rapid and specific screening of tylosin and tilmicosin residues in milk and water samples.

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.

Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

1997 ◽  
Vol 78 (04) ◽  
pp. 1262-1267 ◽  
Author(s):  
Claudia C Folman ◽  
Albert E G K von dem Borne ◽  
Irma H J A M Rensink ◽  
Winald Gerritsen ◽  
C Ellen van der Schoot ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Platelet Transfusion ◽  
Large Scale ◽  
Limit Of Detection ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Reliable Tool

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

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.

Enzyme-linked immunosorbent assay for triclocarban in aquatic environments

2015 ◽  
Vol 72 (10) ◽  
pp. 1682-1691 ◽  
Author(s):  
Kun Zeng ◽  
Yanmin Zou ◽  
Jianxia Liu ◽  
Wei Wei ◽  
Meng Zhang ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Limit Of Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Aquatic Environments ◽  
Satisfactory Accuracy ◽  
Good Linear ◽  
High Affinity ◽  
Inhibition Concentration ◽  
Optimized Conditions

A sensitive, competitive indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of triclocarban (TCC) in waters and sediments. Haptens were synthesized by derivatizing the paraposition of a phenyl moiety of TCC. The synthesized hapten was then coupled to bovine thyroglobulin to be used as an immunogen, based on which, a high affinity monoclonal antibody 4D5 was produced with the hybridoma technique. Under the optimized conditions, using the monoclonal antibody, excellent performances of the assay were obtained: satisfactory sensitivity (IC50 (50% inhibition concentration) value, 0.43 ng/mL; limit of detection, 0.05 ng/mL); good linear range (0.05–10 ng/mL); and satisfactory accuracy (recoveries 70.7–107% in waters; 74.8–98.3% in sediments). Furthermore, TCC was found with the concentration ranging from not detected to 422.12 ng/L in waters and from 6.68 ng/g to 78.67 ng/g in sediments in Yunliang River, Ancient Canal and Hongqiao Port in Zhenjiang City. In conclusion ELISA could be applied for monitoring TCC in aquatic environments.

Enzyme-linked immunoabsorbant assay of apolipoprotein AII in plasma, with use of a monoclonal antibody.

1986 ◽  
Vol 32 (6) ◽  
pp. 967-971 ◽  
Author(s):  
E A Stein ◽  
L DiPersio ◽  
A J Pesce ◽  
M Kashyap ◽  
J T Kao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cross Reactivity ◽  
Lipoprotein Subfractions ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Human Apolipoprotein ◽  
Plasma High Density

Abstract We produced a monoclonal antibody (C2-22) to human apolipoprotein (Apo) AII and describe its use in an enzyme-linked immunoabsorbant assay (ELISA) for Apo AII in human plasma and lipoprotein subfractions. No cross reactivity of the antibody with Apo CI, CII, CIII, E, or ablumin was detected. Apo AI and low- and very-low-density lipoprotein cross reacted by 0.25%, less than 0.2%, and less than 0.3%, respectively. Whole plasma high-density lipoprotein (HDL) and HDL subfractions (HDL2 and HDL3) produced parallel displacement curves. This quantitative ELISA is based on competition between solid-phase-bound Apo AII and free Apo AII. Bound C2-22 is detected by alkaline-phosphatase-labeled second antibody. The standard curve for the assay is linear for plasma diluted 500-fold originally containing 140 to 1140 mg of Apo AII per liter. Delipidation of plasma samples exposed no additional antigenic sites. Within- and between-run CVs were respectively 8.4% and 8.7% at 327 mg/L of Apo AII, and 6.8% and 7.4% at 587 mg/L. Results correlated well with those by a polyvalent-antisera-based RIA procedure: r = 0.916, p less than 0.01, RIA = 0.896 ELISA -19.1 mg/L.

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