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 with the common gamma-chain. Soluble forms of the extracellular domain of the alpha-chain (sIL-4R) were shown to be present in biological fluids and, dependent on the concentration, enhance or inhibit IL-4 activity by forming IL-4/sIL-4R complexes. To discriminate between free and potentially active IL-4 from the inactive and complexed form, we have established 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 antibodies, a chequerboard interference analysis with six highly-selective human IL-4 specific monoclonal antibodies was performed. For the determination of total IL-4, a monoclonal capture antibody was used that binds IL-4 outside the binding site of the IL-4R alpha-chain. Another antibody recognizing an epitope of the alpha-chain binding site was chosen for the detection of free IL-4. The binding of this antibody was inhibited in a dose-dependent fashion by recombinant sIL-4R. Assays for both total and free IL-4 exhibited a sensitivity of 8 pg/ml and a dynamic range up to 1000 pg/ml. Human sIL-4R was detected by two monoclonal antibodies directed against different epitopes. This ELISA was inhibited by recombinant IL-4 suggesting the measurement of predominantly free 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 antibody. When supernatants of activated T cells were analyzed, the majority of the IL-4 was in free form. The amount of complexed IL-4 was low as indicated by the fact that most of total IL-4 could be detected as free IL-4. Although values obtained for complexed IL-4 correlated with the difference between total and free IL-4, precise values could not be determined, presumably due to the dynamic nature of the complex between 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.
Department of Dermatology, University Göttingen, Germany.
T
T
Jung
J P
JP
Bews
K H
KH
Enssle
K
K
Wagner
C
C
Neumann
C H
CH
Heusser
D000911:Antibodies, Monoclonal / Q000276:immunology; D017077:Culture Media, Conditioned; D003876:Dermatitis, Atopic / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D006801:Humans; D015847:Interleukin-4 / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation; D016410:Lymphoma, T-Cell, Cutaneous / Q000473:pathology; D011485:Protein Binding; D019948:Receptors, Interleukin-4 / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D012680:Sensitivity and Specificity; D012878:Skin Neoplasms / Q000473:pathology; D012995:Solubility
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D000939:Epitopes; D019948:Receptors, Interleukin-4; D015847:Interleukin-4