DETECTION OF AND DISCRIMINATION BETWEEN TOTAL AND FREE HUMAN INTERLEUKIN-4 AND FREE SOLUBLE INTERLEUKIN-4 RECEPTOR BY ELISA

Interleukin-4 (IL-4) signaling is initiated by binding of IL-4 to the high-affinity IL-4 receptor alpha-chain and subsequent interaction wit h the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluid s and, dependent on the concentration, enhance or inhibit IL-4 activit y by forming IL-4/sIL-4R complexes. To discriminate between free and p otentially active IL-4 from the inactive and complexed form, we have e stablished a set of new ELISA systems for the measurement of human IL- 4 in its distinct forms. To select suitable pairs of anti-IL-4 antibod ies, a chequerboard interference analysis with six highly-selective hu man IL-4 specific monoclonal antibodies was performed. For the determi nation of total IL-4, a monoclonal capture antibody was used that bind s IL-4 outside the binding site of the IL-4R alpha-chain. Another anti body recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4, The binding of this antibody was inhi bited in a dose-dependent fashion by recombinant sIL-4R. Assays for bo th total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynami c range up to 1000 pg/ml, Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibit ed by recombinant IL-4 suggesting the measurement of predominantly fre e sIL-4R, Complexes between soluble IL-4R and IL-4 were detected by a monoclonal anti-sIL-4R antibody in combination with an anti-IL-4 antib ody. When supernatants of activated T cells were analyzed, the majorit y of the IL-4 was in free form. The amount of complexed IL-4 was low a s indicated by the fact that most of total IL-4 could be detected as f ree IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not b e determined, presumably due to the dynamic nature of the complex betw een the two proteins. We suggest that the ability to quantitate total and free IL-4 in combination with sIL-4R may provide a new insight of the role that IL-4 plays in different pathophysiological conditions. ( C) 1998 Elsevier Science B.V. All rights reserved.