Molecular calibration in flow cytometry with sub-attogram detection limit.
A monoclonal antibody to complement receptor type 1 (CR1) has been double labelled with 125iodine and fluorescein isothiocyanate (FITC) and used for parallel radioimmune assay (RIA) and flow cytometric studies. Serial dilutions of the reagent with unlabelled antibody were used to obtain a linear correlation between radioactivity counts and fluorescence over the whole range of labelled antibody molecules bound per cell, namely 700-25,000. This has enabled us to calibrate our flow cytometer in terms of numbers of molecules per channel as opposed to the customary arbitrary fluorescence units. Normal human granulocytes from a single donor were found to have a mean of 25,000 CR1 molecules per cell with a normal distribution and standard deviation of 4775 molecules. The detection limit of the flow cytometer was 730 CR1 molecules per cell which is equivalent to between 125 and 150 molecules of free fluorescein. This represents less than 0.1 attogram (ag) of fluorescein per cell, where 1 ag = 10(-18) g. The resolution was 44 CR1 molecules per digitization step of the analogue-to-digital converters (ADC) which corresponds to the fluorescence from less than ten molecules of free fluorescein.
D002138:Calibration; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D006801:Humans; D007457:Iodine Radioisotopes; D011863:Radioimmunoassay; D011951:Receptors, Complement / Q000032:analysis* / Q000276:immunology; D013861:Thiocyanates
D005452:Fluoresceins; D007457:Iodine Radioisotopes; D011951:Receptors, Complement; D013861:Thiocyanates; D016650:Fluorescein-5-isothiocyanate