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Human immunoglobulin G (hIgG) is a major immunoglobulin in serum that plays a central role in immune response. There is great interest in the use of IgGs in basic biomedical research and for the diagnosis and treatment of various diseases. Immunoglobulins (Ig) are usually purified by Protein A affinity chromatography. However, a high level of purification, including the removal of all contamination by endotoxins and by leaching Protein A, is required for pharmaceutical use of Igs.

The development of new purification procedures for antibodies that do not present these inconveniences is much needed. We investigated the use of antibody-binding peptides to build a new IgG purification system by screening a random peptide library constructed in the bacteriophage T7 display system (Yuji et al. 2008).

The ProteOn XPR36 protein interaction array system is a surface plasmon resonance (SPR) device that can measure 36 interactions between 6 ligands and 6 analytes in a single injection. This system is amenable to high-throughput screening since the measurement of 36 interactions can be completed within 30 minutes.

Here we report the screening of active phage clones obtained by phage panning from random peptide libraries constructed using a T7 phage display system. We subsequently analyzed the interaction of synthetic peptides with antibodies using the ProteOn XPR36 system (Sakamoto et al. 2009). We also describe a convenient method for the immobilization of human IgG on the ProteOn sensor chip.

Experiment 1: Screening of human IgG antibody-binding T7 phage clones from a T7 phage peptide library

To identify peptides with high binding affinity to hIgG we constructed a random peptide library, cyclized via cys-cys disulfide bridges — using the T7Select 10-3 vector (Novagen), and isolated several hIgG-binding phages by biopanning the library. Based on the peptide sequence obtained from the initially isolated clones, secondary and tertiary libraries for affinity maturation were constructed, and the clones with the highest binding affinity were isolated. Instead of using ELISA, we employed the ProteOn XPR36 system to screen the phage-infected lysed bacteria.

ProteOn GLM sensor chips were activated by a combination of sulfo-NHS and EDAC in the vertical direction. Goat IgG, rabbit IgG, mouse IgG, and two hIgG1s were dissolved in 10 mM acetate buffer (pH 5.0) at a concentration of 5 µg/ml and immobilized using a five minute injection; one channel received only buffer. The two hIgG1s were tocilizumab (trade name MRA) and trastuzumab (trade name Herceptin) which is an anti-HER2 human IgG. Approximately 2,000 to 6,000 response units (RU) of ligand were coupled to the chip. The chip surface was blocked using 1 M ethanolamine (pH 8.5). The lyzed bacteria and phage solution were centrifuged, filtered through a 0.22 µm filter, and injected in the horizontal direction. For each clone, a report point was taken at 255 sec after injection. See Figure 2 for an outline of the experimental design.

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