Conjugação e validação de controle isotípico IgG1-FITC para uso em citometria de fluxo

Abstract

It was during the 1950's that the development of flow cytometry started, technology that allow to measure physiochemical characteristics of cells or suspended particles in fluid. This technology uses monoclonal antibodies labeled by fluorochromes as investigation tool in several analysis and needs isotype controls to define the negative region (background). These controls are constituted by immunoglobulins of the same isotype and fluorochrome from test antibodies, being fluorescein isothiocyanate (FITC) the most fluorescent marker used in antibody conjugations. The isotype controls have the function to define the unspecific fluorescence (negative cells) and the fluorescent regions (positive cells). In this study was selected monoclonal antibody (mAb) against canine erythrocyte antigen, produced in the Monoclonal Antibodies Laboratory - Blood Center of Botucatu, which reacts positively with dog red blood cells, but never with human leukocytes, having therefore, utility potential as negative control in flow cytometry. The purification mAb of IgG1 subclass was made by affinity chromatography in Sepharose Protein-A and the purification control was performed by electrophoresis in ágarose and polyacrylamide gels (SDS-PAGE). The purified immunoglobulin was conjugated to FITC and after was filtered in Sephadex G25 column to separation of labeled and unlabeled proteins. The conjugated mAb was tested against dog red blood cells and the conjugation success was verified by fluorescence tests, being the median positivity of 94.70. To the human leucocytes the positivity median was 0.03 against 0.50 of the commercial reagents. The nonparametric statistical tests of Wilcoxon and the correlation Spearman showed the efficiency and validate the isotype control produced in relation to the tested commercial reagents.