Determination of cell concentration of CD45+ leukocytes in blood with corresponding antibody – anti-CD45 antibody bound to the fluorochrome is a very perspective method. For this purpose, two conjugates were prepared anti-CD45 antibody-FITC and anti-CD45 antibody-ATTO465. The isothiocyanate group of FITC and NHS group of ATTO465 were bound with amino groups of the anti-CD45 antibody. Purification of obtained conjugates was carried out by gel filtration chromatography through Sephadex G-25 column. The fractions of 2.0 mL were collected. The fraction absorbance was measured with Jenway 6900 UV/Vis spectrophotometer. Anti-CD45 antibody-FITC conjugate was in fraction 4 and anti-CD45 antibody-ATTO in fraction 3. Emission characteristics of the obtained conjugates and fluorescent dyes, FITC and ATTO465, were compared. The activity of the conjugates was investigated. The dilute cell samples with total nucleated cell concentration of 1 × 106 cells/mL was mixed with both conjugates separately (anti-CD45 antibody-FITC and anti-CD45 antibody-ATTO) and incubated for 20 min. The stained cells were counted by new fluorescent image cytometry EasyCounter BC from Milkotronic. EasyCounter BC is based on a fluorescence microscopic cell counting technique. Due to the fluorescent dye, LED optics, and CCD capture technologies, cell analysis is accurate, reliable, and fast. The obtained results were compared with results from fluorescence microscope Olympus and proved ability of the conjugates for immunofluorescence microscopic cell counting with new EasyCounter BC.

PREPARATION OF CONJUGATE OF ANTI-CD45 ANTIBODY WITH FLUOROCHROMES FITC AND ATTO465


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