Detection of very low receptor numbers on cells by flow cytometry using a sensitive staining method

Abstract

We describe a staining method for flow cytometry that resolves with a high degree of sensitivity very low numbers of cell surface molecules, which are normally too few to detect using the conventional fluorescein-conjugated reagents. We took advantage of the fact that liposomes can be constructed to contain hundreds of thousands of fluorochrome molecules per vesicle; antigen specificity can be conferred by covalently conjugating them to antibodies or protein A. Unlike fluorochromes such as fluorescein isothiocyanate (FITC) that are directly conjugated to protein ligands with a fluorochrome to protein ratio of about 2 to 1 on the average, their large encapsulating capacity gives liposomes a tremendous potential for signal amplification.In an indirect immunofluorescence study using liposomes that contained the fluorochrome carboxyfluorescein (CF) and that were covalently conjugated to protein A, we were able to obtain up to 50 times the fluorescence signal over background that could be detected with FITC-conjugated protein A. Scatchard analysis showed that the thymoma cell line RDM4 expresses 23,000 and 2,600 binding sites for monoclonal antibodies ( d b ) against H-2K and H-ZD, respectively. When RDM4 cells were treated with anti-H-ZK mAb followed by FITC-conjugated protein A, at best we were able to obtain a fluorescence signal that was only 7 times above background. However, when these cells were treated with the same antibody followed by protein A conjugated to small unilamellar liposomes or large unilamellar liposomes, the fluorescence signals were 110 and 335 times above background, respectively. Using the liposome conjugates, we were also able to detect with ease the 2,600 binding sites for the anti-H-2D mAb, whereas the FITC conjugate failed completely to resolve specific binding from background. We estimate conservatively that by using this methodology, it will be possible to detect as few as 800 binding sites per cell.

Quantitative Prediction of Human Pharmacokinetics for mAbs Exhibiting Target-Mediated Disposition

“…For some targets expressed in soluble form or solely on circulating blood cells, it may be possible to measure baseline levels or turnover rates in human whole blood or purified PBMCs using techniques such as ELISA or flow cytometry (27)(28)(29)(30). On the other hand, for targets expressed mainly in tissues, there are experimental limitations to accurately determine the total target abundance in tissues.…”

Labelling of liposomes with intercalating perylene fluorescent dyes

“…Truneh et al [1,2] have avoided this fundamental disadvantage of the prevailing immunofluorescence technique by coupling monoclonai antibodies to liposomes which have several hundred molecules of a fluorescent dye encapsulated in their aqueous inner volume. With the help of these fluorescent immunoliposomes even such antigens can be detected which cannot be visualized by fluorescent antibodies but only by an expensive fluorescence multiplication technique.…”

Enzymatic amplification staining for flow cytometric analysis of cell surface molecules

“…We have directly compared our enzymatic amplification technology with this procedure and have found that enzymatic amplification still gives a 10 -100-fold enhancement in the fluorescent signal (data not shown). Another approach to enhance the fluorescent signal for flow cytometry has involved the use of fluorescent liposomes (14,15). Although excellent results have been obtained by a few groups, the difficulties of working with liposomes have prevented this technology from being widely used.…”