Janus liquid handling stations

A library of peptide based epitope mimics was synthesized using solid-phase Fmoc synthesis. An amino functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and subsequent cleavage of the Boc-groups using trifluoroacetic acid (TFA). Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer). The binding of antibody to each of the synthesized peptides was tested in a pepscan-based ELISA. The peptide arrays were incubated with primary antibody solution (overnight at 4 °C). After washing, the peptide arrays were incubated with a 1:1000 dilution of anti-rabbit IgG HRP conjugate (DAKO) for 1 h at 25 °C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml of 3% H₂O₂ were added. After 1 h, the color development was measured with a charge coupled device (CCD) - camera and an image processing system. Epitope targets were read as the largest contiguous stretch of amino acids shared by all peptides recognized by the primary antibodies.

Affinity maturation by amino acid mutagenesis was performed by Pepscan (Lelystad, Netherlands). Briefly, a library of peptides was synthesized on an amino functionalized polypropylene support using solid-phase Fmoc synthesis. The library was constructed from single point substitutions and deletions of each position in the parent peptide sequences. Eighteen of the 20 natural amino acids, excluding methionine and cysteine, were used for substitutions. Synthesis protocols were performed by custom modified JANUS liquid handling stations (Perkin Elmer, Waltham MA).
Natalizumab binding to the library peptides was tested by array-based ELISA (Pepscan). Briefly, peptide arrays were incubated with natalizumab solution overnight at 4 °C. After washing, the arrays were incubated with a 1:1000 dilution of peroxidase-labeled anti-human IgG secondary antibody for 1 h at 25 °C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate (ABTS) and 20 μl/ml of 3% H₂O₂ were added for 1 h. Color development was measured and quantified by charge coupled device (CCD) camera and an image processing system. The values obtained from the CCD camera range from 0 to 3000 milli-Arbitrary Units (mAU).

To reconstruct epitopes of the EGFRvIII ECD, a library of 15-mer peptides was synthesized. In addition to peptides covering the native amino acid sequence of EGFRvIII, amino acid substitutions were introduced into all relevant positions to pinpoint crucial binding residues. An amino-functionalized polypropylene support was obtained by grafting with a proprietary hydrophilic polymer formulation, followed by reaction with t-butyloxycarbonyl-hexamethylenediamine using dicyclohexylcarbodiimide with N-hydroxybenzotriazole and subsequent cleavage of the Boc groups using trifluoroacetic acid. Standard Fmoc-peptide synthesis was used to synthesize peptides on the amino-functionalized solid support by custom modified JANUS liquid handling stations (Perkin Elmer). The binding of antibody to each of the synthesized peptides was tested by ELISA. The peptide arrays were incubated with primary antibody solution (overnight at 4°C). After washing, the peptide arrays were incubated with a 1:1,000 dilution of an appropriate antibody peroxidase conjugate for 1 h at 25°C. After washing, the peroxidase substrate 2,2′-azino-di-3-ethylbenzthiazoline sulfonate and 2 µL/mL of 3% H₂O₂ were added. After 1 h, the color development was measured. The color development was quantified with a charge-coupled device-camera and an image processing system.