ESR spectroscopy in the study of antigen processing--uptake of spin-labelled antigens by macrophages

Macrophages were briefly pulsed with a spin-labelled synthetic polypeptide, poly(L-tyrosine:L-glutamic acid) poly DL-alanine:poly L-lysine (n-TGAL) in the presence and absence of anti-TGAL-antibody, and the electron spin resonance (ESR) spectra of the cell suspension compared with the spectrum of free n-TGAL in solution. Spectral analysis indicated two cell-associated n-TGAL pools, one composed of freely rotating label held in an aqueous environment, susceptible to protease digestion and ascorbate reduction, and a second highly concentrated pool, sequestered intracellularly, and held within a highly ordered, polar microenvironment. The ESR analyses were completed within minutes of antigen pulsing, employed very small numbers of live cells, and did not damage the cells being tested. The utility of the technique in screening fatty acid-antigen conjugates for macrophage uptake was demonstrated.