Quantitative Analysis of Cytokine Receptors Using Single-Photon Counting

Abstract

Fluorescent molecules are widely used as labels or probes in biological research [1,2]. In particular, they can be chemically attached to antibodies which, by binding to their specific antigens, can identify the location of particular molecules of biological interest. We have used this technique to identify cell surface receptors to a cytokine, tumor necrosis factor-α (TNFα), that are produced as a result of an immune challenge [3]. This approach works well for qualitative studies, but hitherto it has been difficult to quantify the technique so that the numbers per cell of the molecules of interest can be assessed, mainly because of the sensitivity required. One of us has previously used the technique of single-photon counting to quantify fluorescent molecules [4] and showed it to be highly sensitive and reproducible. We report here the application of this technique to the quantitation of fluoresceinated bound antibodies in small biological samples. We have measured the numbers TNFα receptors appearing on the surface of adipocytes (fat cells) surrounding a rat lymph node at various times after an immune challenge to that node. The change in receptor number confirms and refines our earlier qualitative results and lends support to our hypothesis that adipocytes are locally specialized to modulate the mammalian immune response [5].