Novel routine assay of thyroperoxidase autoantibodies.

1988 ◽  
Vol 34 (11) ◽  
pp. 2231-2234 ◽  
Author(s):  
J Ruf ◽  
B Czarnocka ◽  
M Ferrand ◽  
F Doullais ◽  
P Carayon
Keyword(s):  
Monoclonal Antibody ◽  
Large Scale ◽  
Solid Phase ◽  
Hemagglutination Test ◽  
Routine Measurement ◽  
Coefficients Of Variation ◽  
Assay Design ◽  
Routine Assay ◽  
Thyroid Microsomal Antigen ◽  
Microsomal Antigen

Abstract This radioimmunoassay was developed for specific and large-scale routine measurement of autoantibodies to thyroperoxidase (TPO), an enzyme recently identified as the thyroid microsomal antigen. Because of the scarcity of purified thyroperoxidase, we did not base the assay on the antigen-coated method but rather on autoantibody inhibition of the binding of labeled TPO to a solid-phase-bound monoclonal antibody to TPO. This assay design ensured highly specific measurements without interference from irrelevant thyroid antigens and autoantibodies. When we used affinity-purified autoantibodies to TPO as standards, the range of the curve extended over 10(3)-fold differences in the autoantibodies' concentrations, which allowed us to assay most sera without dilution. Within- and between-assay coefficients of variation (CVs) ranged from 6.1% to 11.5% and from 6.6% to 12.0%, respectively. The correlation between anti-TPO and antimicrosomal autoantibodies, as assessed by hemagglutination test, was highly significant (r = 0.90, P less than 0.0001). This assay is sensitive, easy to perform, and requires only trace amounts of purified TPO.

scholarly journals A monoclonal antibody against Echinococcus multilocularis Em2 antigen

Parasitology ◽  
1991 ◽  
Vol 103 (1) ◽  
pp. 41-49 ◽  
Author(s):  
P. Deplazes ◽  
B. Gottstein
Keyword(s):  
Monoclonal Antibody ◽  
Echinococcus Multilocularis ◽  
Large Scale ◽  
Solid Phase ◽  
Affinity Purification ◽  
Sandwich Elisa ◽  
Binding Activity ◽  
Classical Means ◽  
Species Specific

A monoclonal antibody (MAb G11) species-specific to the Em2 antigen of Echinococcus multilocularis was generated for (i) further biological characterization of the Em2 antigen, (ii) easy affinity-purification of Em2 antigen for immunodiagnostic and immunological investigations and (iii) development of a sandwich-ELISA for the detection of Em2 antigen in diagnostic samples and thus species-specific identification of E. multilocularis metacestode material. The MAb G11 was used in an antibody sandwich-ELISA to detect soluble Em2 antigen with a methodical sensitivity of 80 ng E. multilocularis antigen/ml of solution. MAb G11 specifically detected Em2 antigen in all of 15 E. multilocularis-isolates originating from various geographical areas and in none of other helminth isolates (e.g. Echinococcus granulosus, E. vogeli, and others). Further biological analysis by FITC-labelled MAb G11 demonstrated unique binding activity to the laminated layer of the metacestode. Also, oncospheres were binding FITC-labelled MAb G11 on an outer layer synthesized during cultivation in vitro for 13 days after hatching. Application of the MAb G11 antibody sandwich-ELISA for investigation of solubilized oncospheres confirmed the in vitro synthesis of Em2 antigen by oncospheres on day 13 p.i. Adult stages (somatic antigens) and freshly hatched oncospheres were always MAb G11 negative. Solid-phase MAb G11 was used for purification of the corresponding Em2 antigen by affinity chromatography. A preliminary serological evaluation of the Em2(G11) antigen by ELISA revealed identical immunodiagnostic characteristics, compared to Em2 obtained by classical means, thus suggesting the presented method for future isolation of large-scale Em2 antigen.

Chemiluminescence enzyme immunoassay for thyroxin with use of glucose oxidase and a bis(2,4,6-trichlorophenyl)oxalate-fluorescent dye system.

1985 ◽  
Vol 31 (3) ◽  
pp. 430-434 ◽  
Author(s):  
H Arakawa ◽  
M Maeda ◽  
A Tsuji
Keyword(s):  
Glucose Oxidase ◽  
Enzyme Immunoassay ◽  
Sulfonic Acid ◽  
Large Scale ◽  
Solid Phase ◽  
Preliminary Screening ◽  
Naphthalene Sulfonic Acid ◽  
Coefficients Of Variation ◽  
Enzyme Label ◽  
Chemiluminescence Enzyme Immunoassay

Abstract In this chemiluminescence enzyme immunoassay for thyroxin, glucose oxidase (EC 1.1.3.4) is the enzyme label and the bound and free fractions are separated by the double-antibody solid-phase technique. In the assay of enzyme activity, hydrogen peroxide generated from glucose substrate is measured by its chemiluminescence reaction with bis(2,4,6-trichlorophenyl)oxalate and 8-anilino-1-naphthalene-sulfonic acid. The measurable range of thyroxin is 2.5 to 200 micrograms/L. The coefficients of variation for thyroxin concentrations of 43.2 and 12.8 micrograms/L were 7.0% and 8.6% (intra-assay), and 6.4% and 12.3% (interassay), respectively. The proposed method is applicable to large-scale preliminary screening of neonates for congenital hypothyroidism, with samples consisting of dried blood spotted on filter paper.

scholarly journals Development and evaluation of a monoclonal antibody-based competitive ELISA for the detection of antibodies against H7 avian influenza virus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yuan Li ◽  
Hongliu Ye ◽  
Meng Liu ◽  
Suquan Song ◽  
Jin Chen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Large Scale ◽  
Operation Time ◽  
Reference Method ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration

Abstract Background H7 subtype avian influenza has caused great concern in the global poultry industry and public health. The conventional serological subtype-specific diagnostics is implemented by hemagglutination inhibition (HI) assay despite lengthy operation time. In this study, an efficient, rapid and high-throughput competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of antibodies against H7 avian influenza virus (AIV) based on a novel monoclonal antibody specific to the hemagglutinin (HA) protein of H7 AIV. Results The reaction parameters including antigen coating concentration, monoclonal antibody concentration and serum dilution ratio were optimized for H7 antibody detection. The specificity of the cELISA was tested using antisera against H1 ~ H9, H11 ~ H14 AIVs and other avian viruses. The selected cut-off values of inhibition rates for chicken, duck and peacock sera were 30.11, 26.85 and 45.66% by receiver-operating characteristic (ROC) curve analysis, respectively. With HI test as the reference method, the minimum detection limits for chicken, duck and peacock positive serum reached 20, 21 and 2− 1 HI titer, respectively. Compared to HI test, the diagnostic accuracy reached 100, 98.6, and 99.3% for chicken, duck and peacock by testing a total of 400 clinical serum samples, respectively. Conclusions In summary, the cELISA assay developed in this study provided a reliable, specific, sensitive and species-independent serological technique for rapid detection of H7 antibody, which was applicable for large-scale serological surveillance and vaccination efficacy evaluation programs.

scholarly journals A simple and efficient automated microvolume radiosynthesis of [18F]Florbetaben

2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Ksenia Lisova ◽  
Jia Wang ◽  
Philip H. Chao ◽  
R. Michael van Dam
Keyword(s):  
Large Scale ◽  
Solid Phase ◽  
Acetonitrile Solution ◽  
Synthesis Methods ◽  
Pet Tracers ◽  
Molar Activity ◽  
Alternative Approaches ◽  
High Yields ◽  
Sufficient Concentration

Abstract Background Current automated radiosynthesizers are generally optimized for producing large batches of PET tracers. Preclinical imaging studies, however, often require only a small portion of a regular batch, which cannot be economically produced on a conventional synthesizer. Alternative approaches are desired to produce small to moderate batches to reduce cost and the amount of reagents and radioisotope needed to produce PET tracers with high molar activity. In this work we describe the first reported microvolume method for production of [18F]Florbetaben for use in imaging of Alzheimer’s disease. Procedures The microscale synthesis of [18F]Florbetaben was adapted from conventional-scale synthesis methods. Aqueous [18F]fluoride was azeotropically dried with K2CO3/K222 (275/383 nmol) complex prior to radiofluorination of the Boc-protected precursor (80 nmol) in 10 μL DMSO at 130 °C for 5 min. The resulting intermediate was deprotected with HCl at 90 °C for 3 min and recovered from the chip in aqueous acetonitrile solution. The crude product was purified via analytical scale HPLC and the collected fraction reformulated via solid-phase extraction using a miniature C18 cartridge. Results Starting with 270 ± 100 MBq (n = 3) of [18F]Fluoride, the method affords formulated product with 49 ± 3% (decay-corrected) yield,> 98% radiochemical purity and a molar activity of 338 ± 55 GBq/μmol. The miniature C18 cartridge enables efficient elution with only 150 μL of ethanol which is diluted to a final volume of 1.0 mL, thus providing a sufficient concentration for in vivo imaging. The whole procedure can be completed in 55 min. Conclusions This work describes an efficient and reliable procedure to produce [18F]Florbetaben in quantities sufficient for large-scale preclinical applications. This method provides very high yields and molar activities compared to reported literature methods. This method can be applied to higher starting activities with special consideration given to automation and radiolysis prevention.

scholarly journals Characterization of Pharmaceutical Tablets Using UV Hyperspectral Imaging as a Rapid Line to Line Analysis Tool

Sensors ◽  
2021 ◽  
Vol 21 (13) ◽  
pp. 4436
Author(s):  
Mohammad Al Ktash ◽  
Mona Stefanakis ◽  
Barbara Boldrini ◽  
Edwin Ostertag ◽  
Marc Brecht
Keyword(s):  
Hyperspectral Imaging ◽  
Large Scale ◽  
Solid Phase ◽  
Ultra Violet ◽  
Principal Component ◽  
Ccd Camera ◽  
Conveyor Belt ◽  
Hyperspectral Data ◽  
Analysis Tool ◽  
Sample Set

A laboratory prototype for hyperspectral imaging in ultra-violet (UV) region from 225 to 400 nm was developed and used to rapidly characterize active pharmaceutical ingredients (API) in tablets. The APIs are ibuprofen (IBU), acetylsalicylic acid (ASA) and paracetamol (PAR). Two sample sets were used for a comparison purpose. Sample set one comprises tablets of 100% API and sample set two consists of commercially available painkiller tablets. Reference measurements were performed on the pure APIs in liquid solutions (transmission) and in solid phase (reflection) using a commercial UV spectrometer. The spectroscopic part of the prototype is based on a pushbroom imager that contains a spectrograph and charge-coupled device (CCD) camera. The tablets were scanned on a conveyor belt that is positioned inside a tunnel made of polytetrafluoroethylene (PTFE) in order to increase the homogeneity of illumination at the sample position. Principal component analysis (PCA) was used to differentiate the hyperspectral data of the drug samples. The first two PCs are sufficient to completely separate all samples. The rugged design of the prototype opens new possibilities for further development of this technique towards real large-scale application.

scholarly journals Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C

2003 ◽  
Vol 47 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Sylvie Larrat ◽  
Françoise Stanke-Labesque ◽  
Agnès Plages ◽  
Jean-Pierre Zarski ◽  
Germain Bessard ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Combination Treatment ◽  
High Performance ◽  
Solid Phase ◽  
Ammonium Phosphate ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Immunodeficiency Virus

ABSTRACT Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C18-bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra- and interassay accuracies, precision, and stability at 4°C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 ± 1.06 μg/ml (n = 24) at week 12 of treatment (W12) and 3.24 ± 1.35 μg/ml (n = 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n = 11) than in patients with treatment failure (n = 13), with significant intergroup differences at W12 (P = 0.030) and W24 (P = 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

scholarly journals Epitope mapping by cDNA expression of a monoclonal antibody which inhibits the binding of von Willebrand factor to platelet glycoprotein IIb/IIIa

1992 ◽  
Vol 284 (3) ◽  
pp. 711-715 ◽  
Author(s):  
G Piétu ◽  
A S Ribba ◽  
G Chérel ◽  
D Meyer
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Fusion Proteins ◽  
Solid Phase ◽  
Platelet Glycoprotein ◽  
Rgd Sequence ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Beta Galactosidase

In order to study the structure-function relationship of von Willebrand Factor (vWF), we have located the epitope of a well-characterized monoclonal antibody (MAb) to vWF (MAb 9). This MAb reacts with the C-terminal portion of the vWF subunit, SPII fragment [amino acids (aa) 1366-2050], which includes an Arg-Gly-Asp (RGD) sequence at positions 1744-1746, and totally inhibits vWF and SPII binding to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). A recombinant DNA library was constructed by cloning small (250-500 nucleotides) vWF cDNA fragments into the lambda gt11 vector and these inserts were expressed as fusion proteins with beta-galactosidase. Immunological screening of the library with 125I-MAb 9 identified three immunoreactive clones. vWF inserts were amplified by the PCR and their sequences demonstrated overlapping nucleotides from positions 7630 to 7855 of vWF cDNA, coding for aa residues 1698-1773 of the mature subunit, indicating that this is the epitope of MAb 9. vWF-beta-galactosidase fusion protein reacted with 125I-MAb 9 by Western blotting. In a solid-phase radioimmunoassay, the purified fusion proteins decreased the binding of vWF to 125I-MAb 9 by 50%, and this inhibition was dose-dependent between 3.5 and 120 nM. Therefore the epitope of MAb 9 is located within aa 1698-1773 of the vWF subunit, which includes the RGD sequence implicated in the binding of adhesive proteins of GPIIb/IIIa.

A BIOLOGICAL ASSAY FOR CALCITONIN

1965 ◽  
Vol 33 (3) ◽  
pp. 469-475 ◽  
Author(s):  
M. A. KUMAR ◽  
E. SLACK ◽  
A. EDWARDS ◽  
H. A. SOLIMAN ◽  
A. BAGHDIANTZ ◽  
...  
Keyword(s):  
Satisfactory Agreement ◽  
Intravenous Administration ◽  
Dose Range ◽  
Plasma Calcium ◽  
Dose Effect ◽  
Biological Assay ◽  
Potency Ratio ◽  
Standard Preparation ◽  
Assay Design ◽  
Routine Assay

SUMMARY (1) Calcitonin preparations from acetone-dried thyroid were administered to rats by various routes. (2) Intravenous administration, especially by infusion, produced a much greater fall in plasma calcium than s.c. or i.p. injection. (3) The log dose-effect curves after i.v. injection or infusion showed no evidence of non-linearity over a 100-fold dose range and had highly significant slopes. (4) The potency ratio of two preparations was estimated by means of a (2+2) assay design using both i.v. infusion and single i.v. injection. There was satisfactory agreement. (5) The i.v. injection method is recommended for the routine assay of calcitonin. A simple assay schedule is given in the Appendix. (6) A unit of calcitonin activity is defined in terms of a standard preparation.

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