Journal of immunoassay最新文献

In the present study, a competitive ELISA technique was developed to specifically quantitate 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT) detected in serum from AZT-treated patients. In order to eliminate cross-reacting AZT, serum sample was extracted with ethylacetate and then AMT was acetylated (Ac-AMT). A 5'-hemisuccinate-AMT-horseradish peroxidase conjugate was used as a tracer in the presence of anti-AMT rabbit antibodies which were raised against a 5' hemisuccinate-AMT-bovine serum albumin immunogen. Bound/free separation was achieved with an anti-rabbit IgG mouse monoclonal antibody insolubilized onto a microtiter plate. The limit of quantification of Ac-AMT was as low as 0.4 ng/ml in serum samples. This ELISA technique was applied for monitoring AMT plasma levels in patients receiving AZT therapy. The intra and inter-individual variations of the AZT/AMT plasma concentration ratios underlined the need for such a specific test in studying the formation of this toxic catabolite.

A direct competitive heterologous enzyme immunoassay (EIA) for conjugated cholic acid (CA) was developed using horseradish peroxidase labeled antigen having a shorter bridge length than that of the immunogen. An appropriate dose-response curve for conjugated CA was obtained in the range of 0.05-50 ng/well. Specificity of the EIA proved satisfactory in terms of cross-reactivities to 23 kinds of related bile acids. The proposed method was evaluated to be useful for the determination of conjugated CA in urine with acceptable accuracy and inter- and intra-assay precision. The results of analysis showed a reverse relationship between age and urinary excretion ratio of conjugated 1 beta-hydroxy-CA to conjugated CA in the first 9 months after birth.

A monoclonal antibody against NCS-chr was prepared and characterized. Because of the instability of NCS-chr, chemically synthesized stable analog compound, termed PS, was used as a hapten of immunogen. The obtained antibody, termed APS, reacted with NCS-chr, but neither with NCS, NCS-polystyrene-maleic acid conjugate (SMANCS), nor UV-irradiated NCS-chr. Epitope analysis using the compounds that have a structure similar to PS showed that APS recognized the total structure, particularly cyclopenten moiety, of PS. These results suggest that APS recognizes the enediyne structure of NCS-chr. Next, the inhibition enzyme-linked immunosorbent assay (ELISA) for determination of NCS-chr was established. The standard curve showed that the microgram order of NCS-chr were accurately measurable by the established ELISA. Furthermore, it was revealed that the established ELISA was more sensitive than the antibiotic activity determination, termed Bio-assay. The established ELISA will be useful as a quantitative method of NCS-chr.

Quantitation of antibody coupled to a derivatized polystyrene bead through a bifunctional cross linker can be accomplished by a competitive enzyme-linked immunosorbent assay (ELISA) method. This sensitive method is less subject to interference than other protein assay methods such as bicinchoninic acid (BCA) or Lowry. The competitive ELISA method consists of incubating the coupled bead with a (20/80) weight ratio of goat anti mouse kappa alkaline phosphatase/goat anti mouse kappa (GAMKAP/GAMK) for 1.5 hours at 37 degrees C, washing, adding p-nitrophenyl phosphate (PNPP) substrate, and reading the absorbance at 405/450 nm. A standard curve is established with radiolabeled antibody beads for microgram quantitation.

An Immunoradiometric assay for thymosin alpha 1(1-28) was developed using a monoclonal and a polyclonal antiserum which were raised against synthetic thymosin alpha 1(1-28) and thymosin alpha 1(16-28), respectively. A monoclonal antibody, specific for the (acetylated ser1) 1-5 sequence of thymosin alpha 1, was immobilized on polystyrene beads for the solid phase, and a polyclonal antiserum specific for the 16-28 sequence was employed. This method relies on the formation of an immune complex consisting of a 125I-labelled anti-rabbit IgG goat antibody, polyclonal antiserum, thymosin alpha 1, and the solid phase monoclonal antibody. Radioactivity on the solid phase is directly proportional to the amount of thymosin alpha 1 present in the specimen. The minimal detection limit of this assay system was approximately 1.9 pg/ml. The mean values of thymosin alpha 1 in plasma of healthy subject, ranging in age from 0 to 3 years was approximately five fold higher than that of higher ages. HPLC analysis of plasma of a healthy subjects revealed a single immunoreactive form which eluted with the same retention time as that of synthetic thymosin alpha 1. This assay will be extremely useful for the measurement of thymosin alpha 1 in biologic fluids and tissues.

The utility of 5-hydroxy-2, 3-dihydrophthalazine-1, 4-dione (HDP) as a co-substrate for the chemiluminescent detection of horseradish peroxidase was assessed. Several substituted aryl boronic acid derivatives (4-phenyl, 4-iodo) acted as potent enhancers of the peroxidase catalyzed reaction. Addition of chelating agents (EDTA) and surfactants (Tween-20 and [poly (vinylbenzyl)tributylphosphonium chloride-poly (vinylbenzyl) trioctylphosphonium chloride copolymer]) modulated background light emission and the intensity and duration of the signal from both HDP and luminol. However, HDP was found to be inferior to luminol in the peroxidase assay. Comparative studies revealed that at 500 amol of peroxidase the S/B was ten-fold higher using a commercial luminol-based signal reagent as compared with an HDP-EDTA-Tween-20 reagent (S/B t = 0 min 21.8 vs 1.7, S/B t = 10 min 17.8 vs 2.0).

A sensitive ELISA using monoclonal antibodies (mAb) reactive with surface molecules specific for various leukocytes was devised to measure the adhesion of these cells to cultured monolayers of human umbilical vein endothelial cells. Superantigens, staphylococcal enterotoxin B or toxic shock syndrome toxin 1 were used to activate human peripheral blood mononuclear cells. The extent of adhesion of these cells to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 antimouse Ig and monoclonal antibody specific for leukocytes on fixed leukocytic cells that had adhered to endothelial cells. This method was fast and sensitive, and because the detection is by a specific marker on the cell of interest, it can be used in preparations of unseparated mixtures of cells. An increase in adhesion of superantigen-activated CD4+ and CD8- T lymphocytes to endothelial cells may contribute to the pathologic mechanism of superantigens.

An original solid phase method for direct radioimmunoassay of the antipsychotic savoxepine (CGP 19,486 A) in plasma has been developed which does not require the extraction of the parent drug with organic solvents. The assay showed good reproducibility over the working concentration range 1.9-30.6 nmol/l with intra- and inter-assay coefficients of variation < or = 16%. The procedure, which requires only small volumes of plasma (10 microliters), is simple to handle and well suited for routine analysis. The method allowed to investigate the pharmacokinetics of savoxepine in schizophrenic patients given low oral doses of the drug.

An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.

In this work we studied the effects of the molecular weight (M.W.) of thyroxin (T4)-conjugates and the sample dilution factor on the binding potential (C) of serum T4-binding proteins for these T4-conjugates. We prepared six T4-conjugates with great difference in molecular weight with proteins such as IgG, apoferritin, ferritin, transferrin, and thyroglobulin using p-benzoquinone as bifunctional reagent. The conjugates prepared were characterized in terms of their M.W., the molar ratio of T4 to the carrier protein, and their affinity to bind with the anti-Tr antibody. The binding potential values of serum T4-binding proteins for the T4-conjugates were determined, following appropriate mathematical interpretation of the results, obtained through an assay system containing 125I-labeled conjugated tracers, anti-T4 antibody in great excess compared with the concentration of the tracers, and increasing concentration of T4-binding proteins. We conclude that the M.W. of the conjugates is a parameter which significantly influences the binding of a conjugate to the T4-binding proteins. Additionally, for conjugates of very high M.W. (> 650,000), zero C values were obtained using 20-fold sample dilution in the final incubation mixture.