Making and Screening mAbs
A hybridoma cell line was prepared by selecting and fusing splenocytes from one of two CD1 mice, each of which were immunized with a serine-phosphorylated synthetic peptide coupled to diphtheria toxoid as a carrier protein. The animal with the highest serum titre—determined by ELISA 10 days after each immunization—was selected for fusion eight weeks after immunization.
Fusions were automated using a suite of cell culture robotics and dispensed into 20 96-well tissue culture plates, following standard protocols. After two weeks of incubation at 37°C, 40 µL of culture supernatant from each well was transferred by robotic liquid handler from the 96-well plates into the 384-well plates intended as source plates for the Super-Marathon. An additional seven fusions were simultaneously carried out in an identical fashion, using different protein and peptide targets.
Glass microscope slides were coated with an aminosilane to enable the peptide antigens to bind to the slide surface. Three aminosilane-coated chips were then coated with a monolayer of each of the three peptides: one phosphorylated, one unphosphorylated, and one an irrelevant peptide containing a phospho-serine moiety. Protein and peptide chips from each of the additional seven fusions were similarly prepared. The peptide and protein chips were subsequently placed on the Super-Marathon and 40 x 384-well plates, with lids, placed in the Microplate Stacker.
A Multiple Print Run definition enabled the designated supernatants to be printed on the designated slides in a single, unattended overnight print run. Each slide was washed and incubated with a fluorophore-labeled antimouse antibody, washed again, and visualized using a fluorescence microarray scanner. The phospho-peptide, nonphospho-peptide, and irrelevant-phospho-peptide chips were analyzed using standard software and compared using a custom-made database to look for phospho-specific signals.