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ProteOn™ XPR36 Protein Interaction Array System: High-Throughput Antibody Screening and Epitope Mapping
Sponsored,vendor-submitted protocol   Sponsored by Bio-Rad Laboratories    Published in BioTechniques Protocol Guide 2008 (p.71)

Monoclonal antibodies are of particular interest in basic biomedical research and for the diagnosis and treatment of various diseases. Screening, profiling, and production of antibodies involve tedious and time-consuming procedures. Bio-Rad now offers the ProteOn XPR36 protein interaction array system, a label-free, multiplex system for rapid screening and comprehensive kinetic profiling of antibody interactions.

The ProteOn XPR36 instrument is based on surface plasmon resonance (SPR) technology and meets the challenges of high-throughput processing by bringing a novel one-shot kinetics approach to the study of protein interactions. The system's parallel and real-time sample processing capabilities enable simultaneous acquisition of comprehensive data for 36 molecular interactions — without the need for regeneration.

Protocol for Selection and Ranking of 250 Mouse Monoclonal Antibody Supernatants against Human Interleukin 12 (IL-12)

  1. Anti-mouse IgG is immobilized covalently on six microfluidic channels using a ProteOn GLM sensor chip (see Figure 1 for protocol workflow).

  2. Six different unpurified mouse anti-human IL-12 supernatant solutions are injected through six orthogonal channels in parallel. Specific mouse antibodies are captured by the immobilized anti-mouse IgG capture agent.

  3. Five concentrations of purified IL-12 antigen are applied simultaneously in the orientation of the original anti-mouse IgG capture antibodies. Running buffer is injected in the sixth channel as a double reference to correct for any decay of the captured anti-IL-12 from the anti-mouse IgG. Thirty-six sensorgrams (six sets of six) are generated simultaneously, providing the association and dissociation kinetics of all six antibodies.

  4. IL-12 antigen-antibody pairs are removed using diluted phosphoric acid, allowing capture and analysis of a new panel of 6 hybridoma supernatants.


Fig 1. (Click to enlarge)


Repeating the cycle 42 times on the same chip allows determination of a comprehensive kinetic profile for all 250 supernatants in less than 13 hours, enabling rapid and high-throughput screening for high-affinity cell lines (see Figure 2).


Fig 2. (Click to enlarge)


Summary

The multiplexing and one-shot kinetics capabilities of the Bio-Rad ProteOn XPR36 system provide a powerful approach for the rapid screening of large numbers of hybridoma supernatants, a previously time-consuming and laborious component in the area of antibody research.

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