Quantitative immunofluorescence

Summary Instrumental requirements for quantitative immunofluorescence were analyzed. Commercially available equipment was found to be sufficiently sensitive and light sources such as a halogen lamp and the 75 W xenon high pressure arc lamp were found not to fluctuate more than one percent if operated on a stabilized power supply. Only minor modifications of the instrumentation were needed to obtain readings with a reproducibility within one percent, provided the environmental temperature was kept constant. A uranyl glass slide proved to be a suitable calibration standard, to be used for each series of measurements. As standard fluorescein unit (SFU) 10^–3 of the fluorescence of 1 picoliter of a 50 M fluorescein solution pH 8.5 was chosen. The pH of fluorescein dyes had to be about 8.5 since the fluorescence was then maximal and the influence of small pH variations neglectable. The SFU was measured either from microdroplets of fluorescein solution or in microcapillaries containing this solution. The microcapillary system is to be preferred since it is easier to handle, measurements are less time consuming and fading is less disturbing the measurements.From the Study Group on Quantitative Immunofluorescence.