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Abstract: Introduction: In the Luminex multiplexed immunoassay system, complex samples such as human serum are diluted to minimize disturbing matrix effects with a specific diluent. This diluent has to imitate the sample matrix to allow interpolation and has to provide optimal cytokine–antibody binding for all cytokines. Because diluents influence multiplex immunoassay results, this paper explores several methods to determine the quality of a chosen diluent. Materials and methods: Two commercially available diluents, DY997 and RD6 from R&D Systems, were compared in a 19-plex immunoassay setup from Luminex. Results: Using diluent DY997, multiplex signal intensity was reduced by 55% when spiked samples (chemokines and cytokines at 100 pg/mL) contained 50% v/v human serum, compared to samples containing 25% v/v. When using diluent RD6, signal intensity was reduced by 20% when samples contained 50% v/v human serum, compared to 25% v/v human serum. Diluent DY997 showed decreasing multiplex assay sensitivity with increasing protein concentrations, but not as low as in the presence of 50% v/v human serum. Conclusions: In a 19-plex setup, this paper describes signal intensity, assay sensitivity and background signal levels in relation to the total volume-fraction of serum and protein concentration. For the determination of cytokines in serum samples with the multiplexed system Luminex the diluent RD6 seems more appropriate than the diluent DY997. [Copyright &y& Elsevier]

Patients treated with interferons, other cytokines, or various biologically active proteins may form neutralizing antibodies, which can adversely affect clinical outcome. It is therefore important to understand how antibodies neutralize such soluble protein antigens and how best to quantitate such antibodies. By applying the mass action law to antigen–antibody reactions, we previously developed a mathematical model applicable in two situations: first, for antibodies having low affinity for the antigen concerned (the Constant Proportion (CP) case), and, second, for antibodies having high affinity (the Fixed Amount (FA) case). The results allowed calculation of neutralization titers which were independent of the particular assay method used. Neutralization by antibodies of intermediate affinity, however, requires different mathematical treatment because the mode of neutralization does not fit the two cases mentioned above.In this paper, theoretical neutralization curves were derived, based on the same mathematical model, for antibodies of intermediate affinity. We show that the slope of the neutralization curve relating residual active antigen to the concentration of antibodies is determined by the antibody association constant and the molar concentration of the effector antigen. It is therefore possible to infer the magnitude of the association constant from the observed neutralization curve. We show that values obtained for the neutralization titer of antibodies of intermediate affinity by the use of the formula previously described for the Fixed Amount and Constant Proportion cases may deviate from the theoretically sound values; the magnitude of the deviation can be estimated by applying the formulas described herein. These relationships should apply generally to antibody neutralization reactions with all biologically active soluble protein effector molecules that have a single and nonrepetitive epitope. [Copyright &y& Elsevier]