Sanne Hoeks

Chapter 6 94 Multiplex immunoassays In bead-based multiplex immunoarrays, identifiable bead sets are stably coated with desired and specific capture antibodies. These beads are then incubated with a small sample volume allowing the capture of the analyte that binds specifically to the capture antibody. Following this, labeled detection antibodies bind to the analyte- capture antibody-bead complex to make a four member solid phase sandwich that when passed through the detection system allows the identification and quantification of the desired compound. In comparison to ELISA, multiplex assays in general are more sensitive, show a broad analytical and dynamic range – measuring a few pg/ml, are highly specific, are rapid, require smaller sample volumes 58 and allow the simultaneous measurement of up to 500 different proteins (xMAP technology, Luminex, Austin Texs, USA). Even in the presence of all these advantageous features however, these multiplex assays, similar to other antibody based technologies, are affected by the presence of heterophilic and auto-antibodies. These antibodies cause false positive and false negative signals by binding to either the capture antibody, detection antibody or to the antigen. 35 In order to combat this phenomenon three methods can be undertaken. The first involves heterophilic/auto-antibody blockage with animal serum (ineffective when antibody titers are high as seen in a diseased state). 58 The second is through the use of internal assay markers that allow the interfering antibodies to be monitored. These internal markers can clearly indicate when heterophilic or auto-antibodies are influencing data and can thus allow the exclusion of unfit samples during assay analysis. 30, 61 The third and most preferable method is the removal of these antibodies by incubating samples with either protein-L, 30 or with antibodies cocktail such as Hetero-Block. 62