Journal of immunoassay最新文献

Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis as an alternative when doing T-cell blots.

An indirect enzyme-linked assay was developed for quantifying biotin concentrations in human sera. Biotin standard solutions or unknown samples are preincubated with streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and added to plates coated with biotinylated bovine IgG (B-IgGb). The concentration of the streptavidin-HRP is such that the streptavidin binding sites are sufficient to bind apparently all the biotin present in samples, whereas, the remaining sites are inversely proportional to the amount of biotin in analysed sample. These sites could subsequently interact with the immobilized B-IgGb providing signal. The assay demonstrated dynamic range 5 to 640 ng/L, detection limit 2 ng/L, intra- and interassay C.V., 1.6-3.9% and 3.7-7.2% respectively, recovery 100-114% and linear recovery 90-117%. Serum biotin determined: healthy individuals 66 to 600 ng/L, pregnant women (> or = 36 weeks) 60 to 360 ng/L, and patients under chronic haemodialysis 0.56 to 1.62 micrograms/L. The method described is among those few which have been experimentally evaluated for their capabilitity of assessing biotin in human sera.

Mathematical models of competitive ELISAs with labelled antibody and with labelled antigen taking into account bivalent interactions between antibodies and hapten-protein conjugates were developed and analyzed. It was shown that in the kinetic model of the immunochemical reaction the conjugate composition influenced the amplitude of detected signal but not ELISA sensitivity. In the equilibrium model decreased sensitivity correlated with bivalent complexes formation. The predictions were tested experimentally using 2,4-dichlorophenoxyacetic acid (2,4-D) and testosterone as haptens. It was confirmed that increasing of the hapten : protein ratio resulted in formation of bivalent complexes with antibodies. The equilibrium binding constants for these complexes were two orders of magnitude higher than for monovalent ones. Optimal conjugate compositions have been chosen for ELISA of these haptens.

A microparticle-enhanced nephelometric immunoassay was developed for alpha-lactalbumin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-alpha-lactalbumin conjugate with an anti-alpha-lactalbumin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 1.5 micrograms/L) and could be performed in high dilution of milk, excluding any interference or sample pretreatment. It allowed the quantification of alpha-lactalbumin on a large range of concentrations (0.5-16.9 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 1 to 7%). Changes in the alpha-lactalbumin concentration of human milk during lactation were determined in 162 samples. The concentration and ratio of alpha-lactalbumin total protein were found to be significantly lower in colostrum (4.9 g/l, 27%) than in transitional milk (5.2 g/L, 40%), then decreased in mature milk (3.4 g/L, 31%).

A descriptive mathematical model was chosen to fit the antigen-antibody association kinetics of a new homogeneous immunometric assay for prolactin, involving time-resolved fluorescence detection (TRACE technology, Time Resolved Amplified Cryptate Emission). We paid special attention to the methodology and criteria applied, to yield a convenient and statistically valid model, designed to allow potential exploitation of kinetic information in the data processing of the assay. We compared specific parameterizations of an hyperbolic model, the Gompertz, and the monomolecular models on the basis of morphological considerations, a statistical analysis of fit, and an assessment of the parameters estimation quality, over a wide range of antigen concentrations. The monomolecular model gave the best fit, and the most precise and stable estimation of its parameters. The study of parameter properties confirmed this choice.

Bovine serum albumin (BSA), which has a free thiol, was blotted onto a polyvinylidene difluoride membrane. The membrane was reacted with a sulfhydryl-reactive (maleimide-containing) biotin derivative, 1-biotinamido-4-[4'-(maleimidomethyl) cyclohexanecarboxamido] butane (Biotin-BMCC), and then probed. BSA on membranes was detected semi-quantitatively at 50 ng of protein (0.76 pmol of free thiol) and among range of higher extent. BSA on membranes was less efficiently biotinylated compared with biotinylation in solution. Regardless, these results suggested that sulfhydryl-containing proteins were specifically and semi-quantitatively identified by membrane biotinylation with Biotin-BMCC.

We have developed a Threshold System immunoligand assay for the quantitation of residual, process-specific, Escherichia coli host cell contaminant proteins (HCP) in somavubove (a normal sequence recombinant bovine somatotropin). The assay has a dynamic range of 2 to 160 ng/mL, with a limit of quantitation of 2 ng/mL. Daily analytical precision (CV) for six replicates of the designated somavubove laboratory standard is 3.0%. Cumulative analytical precision for multiple assay runs of this standard (a total of 69 laboratory standard replicates) is 8.4%. A conservative alert limit of 100 ng/mL has been assigned in order to assure analytical precision for each assay sample under conditions of stoichiometric antibody excess. Although the assay was designed for use as a profile-release specification assay, it has also been used to validate removal of HCP by the proprietary somavubove purification process. This use is consistent with regulatory guidelines related to "well characterized" recombinant biopharmaceutical proteins.

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid (GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.

MAb against delta 1-THCA was produced by fusing hybridoma with splenocytes immunized with delta 1-THCA-BSA conjugate and hypoxanthine, aminopterine, thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. The cross-reaction of anti-delta 1-THCA antibody against other cannabinoids was very wide, thus many cannabinoids and a spiro-compound were reactive suggesting that 2'-hydroxyl, 6'-hydroxyl or 6'-O-alkyl, 4'-alkylbenzene ring moiety is necessary for its reactivity. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. The metabolites of delta 6-THC such as two major metabolites, 7-oxo-delta 6-THC and 7-hydroxyl-delta 6-THC were also detectable by this ELISA.

The first step of sandwich ELISA, namely adsorption of antibodies to plastic microtiter plates, was studied as a function of the pH of the coating buffer. Coating efficiency was assessed in terms of maximum signal (absorbance) observed in ELISA and also estimated by measuring the amount of functional antibodies adsorbed to the plate. While goat antibodies displayed better results after coating with acetate pH 5 buffer, rabbit IgGs generally worked well at pH 7.4. On average, the classical carbonate pH 9.6 buffer was only 50% as efficient.