Effects of antibody labeling chemistry on assays developed for the Gyrolab immunoassay platform

University essay from Uppsala universitet/Molekylär systembiologi

Abstract: The aim of this project was to make a comparison of the effects of antibody labeling chemistries on assays developed for the Gyrolab immunoassay platform. One of the labeling techniques was a heterogenous labeling technique targeting amino groups on the antibody. The other labeling technique was a site-specific labeling technique targeting the conserved Fc-glycan at the aspargine 297 residue on the IgG molecule. The site-specific labeling was performed using a kit from Genovis called GlyCLICK. The two labeling techniques were compared on four different assays developed for the Gyrolab platform. The assays tested in this project were two anti-drug antibody assays, a pharmacokinetics assay, a polyclonal antibody assay, and a monoclonal antibody assay. The drug tolerance was tested for the anti-drug antibody assays, resulting in better drug tolerance for reagents labeled with amino conjugation for the Humira assay with incubation overnight. A confirmatory analysis, testing the inhibition of negative control with addition of unlabeled drug in the Master Mix, was performed. This resulted in small differences in the inhibition between the different reagents, except for Keytruda on Gyrolab Bioaffy 200, for which the GlyCLICK labeled reagents led to a lower inhibition of the negative control. For all the assays the effects on signal to background ratio and limit of detection was investigated. The greatest advantages of GlyCLICK on the signal to background was observed for anti-drug antibody Keytruda assay and polyclonal antibody assay. For the polyclonal antibody assay, the results indicated potentially reduced need for the polishing step and for two wash solutions after addition of the detect reagent.  

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