Sanne Hoeks

Chapter 6 92 either coated to a solid carrier (bead), acting as the capture antibody and or, in the case of the second antibody, bound to a labeled reporter. In plate-based assays however, the capture antibodies are bound in distinct positions within the wells of 96 well plate. 57 Pro’s Con’s Bioassay Sensitive detection of bioactive molecules Semi-quantitative Narrow analytical range Low specificity Time consuming Large sample size Protein microarray Sensitive High through put Rapid Parallel measurement of multiple proteins Low Protein detection system stability (denaturation) Non-specific activity of capture protein Masking of low protein levels by higher protein levels Matrix/Heterophlic (auto-) antibody interference HPLC Relatively rapid Low false positives High cost and complexity Co-elution of compounds Irreversible absorption of compounds Low sensitivity ELISA High specificity High sensitivity Wide analytical range Reproducibility Unable to distinguish between bioactive and inactive molecules Varying binding affinity of antibodies Large sample volume High reagent costs Narrow dynamic range Only measure one protein at a time Matrix/Heterophlic (auto-) antibody interference MSD Quantitative and qualitative analysis High sensitivity Low background Detection of multiple cytokines Unable to distinguish between bioactive and inactive molecules Performance of assay in various matrices is unknown Bead based multiplex immunoassay High specificity High sensitivity Broad analytical and dynamic range Reproducibility Rapid Small sample volume Unable to distinguish between bioactive and inactive molecules Matrix/Heterophlic (auto-) antibody interference TABLE 1. Pro’s and con’s of various assays