Hybridoma analysis by ELISA.

Mouse sera, polyclonal supernatants from each hybridoma library, and monoclonal hybridoma supernatants after single-cell cloning were analyzed using a coated antigen, anti-IgG reporter ELISA. EIA/RIA 96-well plates (Corning) were coated overnight (4°C) with either fixed concentrations or serial dilutions of RLN1 (R&D Systems) or RLN2 (Phoenix Peptide) diluted in 1× PBS. After blocking with PBS/casein, plates were washed and incubated with serum (1/1000 dilution) or hybridoma supernatant (undiluted), followed by 1/5000 dilution of goat anti-mouse IgG-HRP (Jackson ImmunoResearch), washed, and incubated with TMB Substrate Solution (Moss Substrates) for 30 minutes. ELISA Stop Solution (1M H3PO4) was added to the wells, and absorbance was measured at 450 nm on an EnSpire microplate reader (PerkinElmer). Incubations were for 1.5 to 2 hours at ambient temperature. To isolate clones from the RLN2Am34 library, viable hybridoma cells were sorted via a BD FACS Aria III, 1 cell per well, into 96-well plates (10–20 plates from each library). After 12 days in culture, monoclonal hybridoma–conditioned supernatants from each 96-well plate were sampled for ELISA screening against RLN2. From ten 96-well plates from the RLN2Am34 library, 41 RLN2-binding clones were identified, and 25 were selected for expansion and cryopreservation.