Binding of Fc receptors on blood T and B cells with IgG from healthy cows and cows with chronic lympholeukemia.

Pure population of T and B cells was obtained by a combined method consisting in lymphocyte fractionation through Nylon wool and precipitation of E-rosettes in 17% Verograffin density gradient. Scatchard analysis showed that T and B cells isolated from the blood of healthy cattle bind similar quantities of IgG molecules. Binding of IgG to Fc receptors of T and B cell isolated from animals with chronic lympholeukemia increases. The expression of Fc receptors for IgG is changed in cattle with chronic lympholeukemia.

Isolation and immunochemical characterization of immunoglobulin-binding factor from bovine peripheral blood lymphocytes.

The shedding of immunoglobulin-binding factor (IBF) has been studied in peripheral blood lymphocytes during 1 h incubation at 37 degrees C. IBF was detectable in the incubating medium on the basis of their ability to bind immunoglobulin G (IgG) specifically. IBF was affinity purified on Sepharose beads coated with bovine IgG, fluoresceinated and identified by their biological activities, i.e., for binding immunoglobulin-binding factor labeled with fluoresceinizothiocyanate (FITC-IBF) to erythrocytes coated with purified antibodies (EAIgG) by fluorometric binding assay. The effect of various pH, temperatures and proteolytic enzymes on the binding properties of FITC-IBF to EAIgG was also studied. We showed that IBF are sensitive to pronase E, higher temperature and pH.

[The effect of gamma-interferon and phorbol myristate acetate on the Fc gamma-receptor ligand-binding capacity and on the target-conjugating and antibody-dependent cytotoxic activity of blood lymphocytes]. / Vliianie gamma-interferona i forbolomeristicheskogo atsetata na sviazyvaiushchuiu sposobnost' Fc gamma-retseptora s ligandom, mishen'kon''iugiruiushchuiu i antitelozavisimuiu tsitotoksicheskuiu aktivnost' limfotsitov krovi.

We investigated the ability of gamma-interferon (IFN-gamma) and phorbol 12-myristate 13-acetate (PMA) to influence the ligand-binding of Fc-receptors for immunoglobulin G (Fc gamma R) (conjugation of IgG to fluorescein izothiocyanate--FITC) of bovine blood lymphocytes and the antibody-dependent cell-mediated cytotoxic activity (ADCC). The results demonstrate that the IFN-gamma or PMA induced augmentation of ligand-binding capacity of Fc gamma R of bovine lymphocytes, and correlated well with the enhanced capacity to lyse L cells coated with anti-L cell antibody. The number of conjugates effector--target, formed by bovine lymphocytes in the reaction of ADCC, correlates well with ligand-binding capacity of Fc gamma R. It can be suggested that IFN-gamma or PMA may control the ADCC caused by Fc gamma R regulating its expression.

Chemiluminescence ELISA for the detection of antibodies to bovine leukaemia virus antigens.

A chemiluminescence enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine leukaemia virus antigens (BLV) has been developed. The possibility of using an enhanced chemiluminescence reaction for the determination of adsorbed immunoperoxidase conjugates was studied in this work. The intensity of chemiluminescence depends on both the concentration of reagents and experimental conditions used. The efficiency of the assay is determined by the formation of an immobilized antigen monolayer. A relationship between the quantity of the protein added and adsorbed has been shown. The optimal time and temperature for the antigen-antibody incubation steps have been estimated for each system (3 h at 37 degrees C was chosen as a standard incubation time). A linear dependence of the chemiluminescence intensity and optical density on the concentration of antibodies to the BLV antigens was observed. The detection limit of antibodies in the chemiluminescence ELISA is 2-3 times lower than that in the spectrophotometric one. The results obtained indicate the possibility of using both methods.

[The determination of microalbuminuria by immunoenzyme analysis]. / Opredelenie mikroal'buminurii metodom immunofermentnogo analiza.

The authors have defined the optimal conditions for inhibitory type enzyme immunoassay of microalbuminuria. The range of albumin detection is 30 to 250 micrograms/ml. The optimal time of antigen binding with antibody is 45 min, the lower threshold level of albumin detection 0.6 +/- 0.02 microgram/ml. Albumin concentration is unchanged after urine storage for 8 weeks at -20 degrees C or for 1 week at 4 degrees C.

[The comparative evaluation of spectrophotometric and chemiluminescent detection in an immunoenzyme test of antibodies to the antigens of the bovine leukosis virus]. / Sravnitel'naia otsenka spektrofotometricheskogo i khemiliuminestsentnogo detektirovaniia v immunofermentnom teste antitel k antigenam virusa leikoza krupnogo rogatogo skota.

Comparative evaluation of the sensitivity limit in the detection of antibodies to bovine leukemia virus in the enzyme immunoassay with the use of chemiluminescent and spectrophotometric detection techniques was carried out. In this assay 3-amino-1,4-phthalazinedion was used as chemiluminescent substrate and ortho-phenylenediamine, as chromogenic substrate. The chemiluminescent signal was registered by means of a special luminometer designed at the Institute of Biochemistry (Lithuanian Acad. Sci.). The use of the chemiluminescent substrate permitted the detection of proteins in amounts 2-3 times lower than those detected by the spectrophotometric technique.

Quantitative binding of Fc-receptors, and antibody-dependent cell-mediated cytotoxicity of blood lymphocytes in healthy and chronic lymphocytic leukemic cattle.

The binding capacity of Fc-receptors for IgG of blood lymphocytes was studied in healthy cattle and cattle with chronic lymphocytic leukemia before and after incubation at 37 degrees C in basal Eagle's medium without serum. It was found that lymphocytes of leukemic cattle possess twice the amount of Fc-receptors found in normal lymphocytes. The changes in the binding capacity of Fc-receptors for IgG of normal and leukemic lymphocytes correlated with those of antibody-dependent cell-mediated cytotoxicity (ADCC) of the corresponding lymphocytes. The lower ADCC of leukemic lymphocytes in comparison with normal ones was accompanied by a lower association constant of the leukemic lymphocyte Fc-receptors towards IgG.

Quantitative studies of Fc receptors on bovine leukemic blood lymphocytes: characterization by binding of homologous IgG1 and IgG2.

Receptors for IgG1 and IgG2 (FcR) on peripheral blood lymphocytes from cows with chronic lymphocytic leukemia (CLL) were detected by their ability to bind homologous IgG1 and IgG2 in fluorometric binding assay. Scatchard plots at 4 degrees C demonstrated that IgG1 bound the same number of FcR per cell (3.12 +/- 0.69 X 10(5)) as IgG2 (2.89 +/- 0.69 X 10(5)). The receptors bound IgG2 with an affinity of 4.09 +/- 1.08 X 10(5) 1/M and IgG1 with an affinity of 2.73 +/- 0.55 X 10(5) 1/M, although the difference was not of statistical significance (0.1 greater than P greater than 0.05). Inhibition studies demonstrated that the two ligands could inhibit each other. It might be assumed that FcR for the two subclasses were identical.