[Enzyme immunoassay of (24R)-brassinosteroids].

Brassinosteroids are a new group of phytohormones that are widely distributed in plants and play an important role in the processes of plant growth and development. Physiological concentrations of brassinosteroids in plants are extremely low, and their analysis in organs and tissues is very difficult. This study is devoted to the chemical aspects of elaboration and to bioanalytical parameters of an immunoenzymatic system for quantitative determination of the phytohormones 24-epicastasterone and 24-epibrassinolide.

[Characteristics of immunoaffinity chromatography and a new method for isolation of human thyroid peroxidase from subcellular fractions of thyroid gland].

A system for quantitative determinations of human thyroid peroxidase (TPO) in biological fluids has been obtained, based on the use of enzyme-linked immunosorbent assay. Immunochemical properties of TPO were studied under variable conditions, and a new method for isolating the protein from microsomes, mitochondria, and cytosol of thyroid glands of patients with diverse thyroid diseases was developed. The procedure involves solubilization of subcellular fractions with detergents, their sonication, two sequential runs of chromatography (on sorbents with immolbilized monoclonal antibodies against TPO and goat anti-human immunoglobulin antibodies), treatment with ribonuclease, and dialysis. Highly purified preparations of intact TPO and a product of its limited trypsinolysis are expected to be used as research tools and components of high-sensitivity immunoassays.

[Characteristics of monoclonal antibodies against human thyroid peroxidase for use in immunoaffinity chromatography and immunoassays].

Two types of monoclonal antibodies (MABs) against human thyroid peroxidase (TPO) have been obtained, which interact with spatially separated conformational epitopes of the antigen (Ka values are in the range 10(8)-10(9) M(-1)). The binding site of MAB F8 is in the immunodominant region of the TPO molecule, in the vicinity of the autoantigenic determinants, whereas the epitope specific for MAB A1 lies outside this location. Both MABs retain the ability to form immune complexes after solid-phase immobilization and chemical modification with a biotin derivative. The above properties suggest that MABs A1 and F8 may be used in immunoaffinity chromatography (isolation and purification of TPO from natural sources) and immunoassays for determinations of TPO (in biological fluids) and TPO autoantibodies (in human blood serum).

[Qualitative characteristics of Soviet kits for the radioimmunological determination of cortisol and progesterone in human blood serum]. / Kharakteristika kachestva otechestvennykh naborov dlia radioimmunologicheskogo opredeleniia kortizola i progesterona v syvorotke krovi cheloveka.

Radioimmunoassays and kits of reagents based on them CORT-I-3H,STERON-K-125I, STERON-P-3H and STERON-P-125I intended for medical microanalysis, were studied. Their development and serial production were undertaken in the Institute of Bioorganic Chemistry, AS BSSR. The data obtained characterized the quality of the radioimmunoassays under study, long-term stability and reliability of the kits of reagents. A high specificity of antisera and correlation between the results of measurements and a degree of native blood serum dilution as well as the accuracy of the results obtained and sensitivity of the assays as a whole were demonstrated. The above properties of the radioimmunoassays made it possible to develop serial kits of reagents for determination of cortisol and progesterone concentration in the human blood serum, employed effectively in medical diagnosis of endocrine and other diseases. A long-term control over the reproducibility of parameters of calibration charts and the results of determination of the level of cortisol and progesterone in the pool blood serum of women showed a high reliability of the kits of reagents of different production lots within the time of their application observing instructions for use.

[Radioimmunological system for the quantitative determination of progesterone in human blood serum]. / Radioimmunologicheskaia sistema dlia kolichestvennogo opredeleniia progesterona v syvorotke krovi cheloveka.

A methodical approach to development of a radioimmune system for quantitative assay of progesterone in the blood serum of man is described. It implies investigation of every component in the system and conformity of these components by the physico-chemical properties and concentrations. An antiserum to the progesterone conjugate with bovine serum albumin was prepared. The "fitting density" of the heptaene was 35 mol/mol of the protein. The linkage with the carrier protein in it was performed by the functional group at C(ii) of the steroid molecule. The antiserum had a high titre (28000), high specificity and affinity to progesterone (Ka (3.5 +/- 0.4) X 10(9) M-1). The antiserum characteristics did not depend on the hydrogen ion concentration in the solution at pH 3.5-10.0 and the ionic strength of the buffer solutions (0.05 to 1.0 M). This allowed performing the radioimmunological assay at pH 3.5. Under such conditions complete inhibition of the endogenic binding proteins of the blood serum was possible. 125I preparations of progesterone made in the USSR were used in the study. The progesterones were prepared by adding the molecule of histamine containing the 125I atom to the molecule of progesterone modified at C(3). The quality of the 125I preparations of progesterone was estimated by their immune reactivity. The standard solutions of progesterone were prepared with the donor blood serum. It was shown that quantitative determination of progesterone in the blood serum at clinically significant ranges of the hormone concentration was possible at the final antiserum dilution of 1:28000, the label activity of 20000-30000 imp/min per a sample and the content of the blood serum in the analyzed sample equal to at least 10 per cent. A simple and reliable procedure for the assay of progesterone in the blood serum of man was developed.