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Rapid Screening of Fab Phage Display Libraries Using the ProteOn™ XPR36 Protein Interaction Array System and One-shot Kinetics™ Technique
Sponsored,vendor-submitted protocol   Published in November 2009 (p.63) DOI: 10.2144/000113025

During the development and optimization of antibody reagents for therapeutics, the key is to identify and characterize antibodies with very high affinity and specificity for the intended target, as well as those with low cross-reactivity. The use of fragment antigen binding (Fab) phage display libraries for panning potential Fab candidates, though powerful, is both time-consuming and costly. The conventional Fab library screening workflow usually consists of four phases: Fab panning, assembly of the Fab and Fc, affinity maturation, and Fc mutation analysis. The ProteOn XPR36 system replaces the first two phases with a single method, eliminating multiple steps and analyses (such as purification, ELISA, and FACS). The system also provides the advantage of collecting kinetic data on 36 interactions simultaneously, in the same step.

Protocols for Fab Panning and IgG Screening Fab Panning of 62 Fab Supernatants

  1. Fab anti-human Fab is covalently immobilized on all six horizontal channels of a GLM sensor chip using amine-coupling chemistry (Figure 1A).

  2. Six different Fab supernatants, flown in the vertical direction, are captured by the antihuman Fab.

  3. Five antigen concentrations are injected simultaneously in the horizontal channels. Running buffer is injected in the sixth channel to correct for any decay of the captured Fab from the Fab anti-human Fab.

  4. The captured Fab-antigen complexes are removed in a regeneration step, allowing the analysis of six new Fab supernatants.



Screening of 20 IgG Complexes

  1. Protein A is covalently immobilized on two horizontal channels of a GLM sensor chip using amine-coupling chemistry (Figure 1B).

  2. Four different assembled IgGs, a control IgG, and running buffer are injected into the six vertical channels.

  3. The specific antigen and a nonspecific antigen (negative control) are injected into the horizontal channels.

  4. A regeneration step is carried out to remove the captured IgG-antigen complexes, allowing the analysis of four new assembled IgGs.

Summary

The multiplexing and one-shot kinetics capabilities of the ProteOn XPR36 protein interaction array system from Bio-Rad provide high-throughput, efficient screening and characterization of Fabs without the need for purification.

Correspondence

Address correspondence to Bio-Rad Laboratories, Inc., Life Science Group, 2000 Alfred Nobel Drive, Hercules, CA 94547, 1-800-4BIORAD (1-800-424-6723), www.discover.bio-rad.com

For Product Technical Support: [email protected]

For Literature Request: [email protected]

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