E. Gavrilova

Evaluation of dissociation constants of antigen-antibody complexes by ELISA.

In this communication some of the advantages and constraints in the use of ELISA (enzyme-linked immunosorbent assay) procedures to evaluate antigen-antibody dissociation constants (Kd) are discussed and experimental conditions under which the effective Kd is close to the true value are proposed. Interactions between horseradish peroxidase (POD), human myoglobin and insulin with mono- and polyclonal antibodies (McAb and PcAb) were used to demonstrate that ELISA can be used to determine the average Kd, characterizing the interaction between antigens and PcAb. The Kd values obtained by ELISA were similar to those determined by luminescent immuno-cofactor analysis (LICA).

Bioluminescent analysis in a biomembrane-like medium consisting of surfactant, hydrocarbon solvent, and water: ATP determination using firefly luciferase

ATP is the most important coupling agent between exergonic and endergonic processes in living organisms. It is either consumed or regenerated in every metabolic sequence. Hence, accurate, sensitive, and facile methods are required for ATP determination. The most promising (specific) assay involves the firefly luciferase-catalyzed reaction in which a single enzyme uses ATP as a substrate and produces light as a At present,6 aqueous solution is the most widely used medium for carrying out the bioluminescent assay. Colloidal solutions of water in organic solvents have been recently demonstrated to be more promising media for some enzymatic reactions.’-’* In the colloidal case, enzyme molecules are entrapped in the inner cavities of hydrated surfactant reversed micelles. Such a microheterogeneous solution has proven13 to be quite a favorable environment for firefly luciferase as well. We now report a striking enhancement of the sensitivity of the bioluminescent response to ATP concentration in the Brij 96-cyclohexane-water system, as compared to that in aqueous solution. Under the conditions of our experiments, this ternary system exists in the form of a cubic lattice composed of reversed micelles of surfactant.14 A similarity between some of the structural and environmental aspects of such systems and those of biomembranes has been recently discussed.” Firefly luciferase was isolated from Luciola mingrelica and was purified using ion-exchange chromatography on DEAE-Sephadex.I6 The protein concentration in aqueous solution was determined spectrophotometrically a t 280 nm. Stock solutions of the purified enzyme and substrates (luciferin and ATP) were prepared in 0.05 M Tris-acetate buffer, pH 7.8, containing 2 m M EDTA and 10 mM MgS04. A typical

Study of the monoclonal antibody - insulin interaction

We generated four stable hybridoma lines producing monoclonal antibodies. All antibodies were as reactive with insulin from other species as with swine insulin. The apparent affinity of the monoclonal antibodies for insulin varied in the range from 2 X 10(8) M-1 to 2 X 10(10) M-1. According to their affinity constants some antibodies could be used for the preparation of immunosorbent, and others for the development of immunoassay methods. Bioluminescent cofactor immunoassay is a method of the choice for the detection of insulin concentration in physiological fluids due to its extreme sensitivity and reproducibility. High affinity antibodies of clones I and II may serve as immunospecific constituents for immunoassay method. Epitope specificity of all monoclonal antibodies has not been studied yet in detail and will be the subject of further investigation.

Enzyme-linked immunosorbent assay for determination of cyclosporin a in the whole blood

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range of 60–1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radioimmunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.984, respectively. The developed test system is stable for at least 9 months when stored at 4°C and can be used in clinical practice.