Maleimide activated mcklh

Peptides corresponding to sequences of the epitopes identified from panning on the gene fragment library (3C9, from DBPII subdomain 3; 3D10, from DBPII subdomain 1) and mimotopes from panning on the random peptide (3D10-M1, 3D10-M2, and 3D10-M3) (Table 1) were commercially synthesized (Pacific Immunology) and conjugated to keyhole limpet hemocyanin (KLH) using maleimide-activated mcKLH (ThermoScientific), following the manufacturer’s specifications. The conjugated peptides were used to immunize 6- to 8-week-old BALB/c mice (Harlan) to raise immune sera. All animals were handled in compliance with good animal practice, and all experimental procedures were performed in accordance with IACUC protocols approved by the Division of Research Integrity and Compliance, University of South Florida. Briefly, mice in all groups (n = 10) were bled for preimmune sera, and each mouse was immunized three times at 3-week intervals with 5 μg of KLH-conjugated peptide emulsified in Titermax Gold adjuvant. Each animal received a 50-μl antigen-adjuvant mix administered subcutaneously at the base of the tail. Mice immunized with KLH and adjuvant alone served as control. All mice were bled for final serum 4 weeks after the second boost.