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.

Partial characterization of bovine leukemic cell populations.

Peripheral blood lymphocytes (LL) from cows with chronic lymphocytic leukemia (CLL) have been studied using a number of surface markers. Cell populations were obtained by partial enrichment and depletion using Sephadex G-200-anti-F(ab')2 and Sephadex G-200-anti-LL-Ig immunoadsorbent columns. It was shown that cell populations having different markers, i.e., surface immunoglobulins (sIg) and leukemia-associated antigens (LAA), are characterized by similar parameters. Thus the adherent cells obtained by both fractionation methods formed 1.1-4.0% of rosettes with sheep red blood cells, while 90.6-94.3% were sIg positive cells. The cytotoxicity test (CTT) performed with anti-B and anti-LL sera indicated that a vast majority of adherent cells reacted to the sera. Thus the adherent cells represent a population with a high percentage of B-cells.

Immunochemical and biochemical study of an abnormal heavy chain of bovine IgG1.

An abnormal heavy chain (HC) protein of IgG1 was isolated from the serum of a leukemic cow by gel filtration on a Sephadex G-200 column with the following ion-exchange chromatography. The HC protein migrated electrophoretically in the anodic region. The molecular weight of the untreated HC protein was about 48,000. Enzymatic treatment with papain and reduction with beta-mercaptoethanol showed no effect on the protein. Antigenic analysis of the HC protein using a specific rabbit antiserum against bovine IgG1 showed a complete identity with the Fc fragment and a partial identity with the intact bovine IgG1. Antigenic determinants of the light-chain were not found. The HC protein consisted of only one polypeptide chain.