The creation of immunofluorescence probes based on monoclonal antibodies series ICO and phycoerythrin - fluorophore of phycobiliprotein group for analysis of cell populations by flow cytometry

Abstract

Introduction . Fluorescent probes based on monoclonal antibodies (MAb) are widely used in scientific and clinical research in the field of oncology, hematology, immunology, epidemiology. The unique spectral properties of the natural protein, phycoerythrin (PE), made it the dominant dye widely used for the preparation of fluorescent probes based on MAb. Objective . To create of fluorescent probes based on the MAb and the fluorescent dye PE by two alternative methods of protein synthesis. Materials and methods . MAb to T lymphocyte antigen (clone ICO-86, clone ICO-31), fluorescent dye PE and bifunctional agents SPDP and SMCC were used in the work. Liquid chromatography methods were used for isolation and purification of MAb: in the first step, affinity isolation of immunoglobulins on the immobilized protein G or anion exchange column chromatography. Gelfiltration on a PD-10 column was used to purify the conjugates (immunofluorescent probes, IFP) in the second step, the concentration and labeling density of the IFP were determined spectrophotometrically. Results . Conjugation of MAb with PE was carried out at a molar ratio of components of 1: 2. For this both components of the conjugation reaction (PE and MAb) were separately modified. For the creation of IFP on the basis of the MAb of the ICO series, both methods of conjugation of Mab with PE are applicable, but method II, in which the chemical modification by the bifunctional SMCC agent is exposed only to the PE molecule by its free amino groups, and the immunoglobulin molecule is conserved in the maximally native state is preferred. Conclusion . The methods described in the article allow to obtain immunofluorescent probes based on antibodies of the ICO series that are comparable in their analytical characteristics to foreign commercial analogs.