UC Santa Cruz

Abstract

A Surrogate Based Characterization Technique

For Antibody Coupling Efficiency

by

Mustafa Mutlu

The immunoprecipitation protocol is of key importance to antigen purification, to downstream protein analysis, and to clinical research. The success of an immunoprecipitation experiment depends on many variables of which antibody immobilization is the most important. Antibodies must be coupled with specific solid surfaces, such as agarose beads or superparamagnetic beads, during immunoprecipitation. Although antibody coupling is crucial for the protocol, there is not an easy-to-apply, low cost, or fast method to maximize and characterize the immuno-binding capacity. In this work we propose a characterization method which gives a clear result of the coupling efficiency, which does not require complicated experimental steps or expensive laboratory equipment. By forming a two-particle complex that sandwiches an antibody, the total amount of antibody coupled to the given number of magnetic beads can be readily translated into the number of surrogate beads that are attached to the magnetic beads through the antibodies. This method doesn’t require additional biological reagents. The surrogate bead alone suffices to quantify immobilization efficiency. This method requires only fluorescence imaging, which can be done with any portable fluorescence platform. It has multiplexing capability, provides high-throughput and high-yield, is cost-effective and time-efficient. Researchers can easily track the shelf-life, quality, degradation, and deterioration of the immobilized beads. This research has characterized a couple of variables, as well as tested automating the protocol. Our design also allows other researchers to explore more variables and automate their protocols.