Thyroid hormone conjugates with rhodamine B as fluorescent ligands of human plasma transport proteins]

Conjugates of thyroxine (T4) and triiodothyronine (T3) with rhodamine B in which the hormone and the fluorescent dye are linked via a thiourea bond have been synthesized. These conjugates possess an ability to inhibit in a competitive manner the binding of [125I]T4 to three protein preparations: T4-binding globulin (TBG), apolipoprotein A-I (ApoA-I), and high density lipoprotein particles (ApoA-I-HDL) isolated from human serum by T4-Sepharose 4B chromatography and further purified. The following values of association constants have been estimated: for the T4 derivative-3 x 10(7) M-1 (TBG), 4.1 x 10(5) M-1 (ApoA-I), and 4.2 x 10(5) M-1 (ApoA-I-HDL); for the T3 derivative-1.6 x 10(7) M-1 (TBG), 5.3 x 10(5) M-1 (ApoA-I), and 5.4 x 10(5) M-1 (ApoA-I-HDL). The binding of rhodamine B-labeled thyroid hormones to TBG or ApoA-I do not alter significantly the parameters of rhodamine B chromophore absorption and fluorescence. The interaction of the conjugates with ApoI-HDL leads to a significant enhancement of the absorption intensity and a 3 nm blue shift in the absorption maximum as well as to a 1.5-fold increase in the fluorescence band amplitude at 586 nm. Biological and fluorescent properties of T4 and T3 derivatives suggest that these compounds may be a useful tool in fluorescence studies of plasma binding protein-driven transport of thyroid hormones in model biological systems.     

Interaction of triiodothyronine-biotin conjugate with binding proteins in immunoassay systems

The study presents a new design of a test system for the detection of a thyroid hormone, free 3,3??,5-triiodo-L-thyronine (free T₃), by enzyme-linked immunosorbent assay (ELISA) and immunoradiometric assay (IRMA) in human blood serum. For this purpose, a low-molecular-weight bifunctional conjugate of T₃ with biotin (Bt) was synthesized. The conjugate T₃-Bt can be bound to biotin-binding proteins on a solid support via its biotin residue, and T₃ portion of the conjugate can interact with a monoclonal antibody against T₃ (anti-T₃-MAb) that is labeled with horseradish peroxidase or iodine-125 in ELISA and IRMA, respectively. The immunochemical interaction is hindered if the T₃ residue is involved in a preformed complex of T₃-Bt with avidin in a solid phase. Computer simulations revealed that the iodothyronine residue is shielded by a glycan component of avidin in such a complex. Binding of T₃-Bt to both T₃-free sites of anti-T₃-MAb and to avidin on a solid support was achieved by delayed introduction of T₃-Bt into avidin-coated plates that were preliminarily co-incubated with labeled anti-T₃-MAb and the analyzed T₃ sample.     

IMMUNOHISTOCHEMICAL STUDIES OF S-100 PROTEIN DURING POSTNATAL ONTOGENESIS OF THE BRAIN OF TWO STRAINS OF RATS

Abstract— We have studied the dynamics of the appearance of cells reacting positively with anti-S-100 protein antiserum, during postnatal neurocytogenesis in the brain of rats of two strains differing in their susceptibility to sound stimuli. The postnatal time of appearance of cells reacting positively with anti-S-100 protein antiserum was somewhat later in rats susceptible to sound-induced seizures than in sound-resistant rats. These differences concerned mainly the cerebral cortex of 12-day-old rats. By day 21 of postnatal life these differences had disappeared. In subcortical structures of the brain, S-100 protein was first found on the 4th to the 5th day of life and the rate of appearance of cells containing this protein was similar in the two strains.     

Determination of Tetanus Toxin and Toxoid by ELISA Using Monoclonal Antibodies

The procedure for obtaining monoclonal antibodies TT-1, TT-2, and TT-3 against tetanus toxin/toxoid is described. It is shown that the commercial DTP vaccine and tetanus toxoid conjugated with a low-molecular-weight hapten can both be used as immunogens. Monoclonal antibodies TT-1 and TT-2 neutralized tetanus toxin in vivo. The monoclonal antibodies obtained were used to design and compare several schemes of quantitative determination of tetanus toxoid and toxin by ELISA. A more sensitive competitive ELISA allowed the detection of as much as 0.01 EC/ml toxoid and 50 LD₅₀/ml toxin.