A polyclonal antibody was generated by immunizing New Zealand rabbits with the synthesized HR2 peptide mariculture keyhole limpet hemocyanin (mcKLH) conjugate. The maleimide-activated mcKLH carrier protein (Pierce) was reconstituted by adding dH2O to Hypo-vial to yield a solution of 10 mg/ml. A mixture of 2 mg of peptide and 2 mg of activated mcKLH was used, and the mcKLH formed a suspension, not a solution, that was typically opaque to whitish blue in color. The suspension was not solubilized by vortexing or heating, as these treatments would cause mcKLH to further precipitate from solution. Next, 2 mg of sulfhydryl-containing hapten was dissolved in 200 μl of dH2O. The peptide and activated mcKLH solutions were then immediately mixed, and the mixture was allowed to react for 2 hours at room temperature. The conjugate was purified by dialysis against PBS buffer. For the first immunization, the purified peptide-mcKLH conjugate (100 μg) in PBS (1 ml) was mixed with an equal volume of Freund’s complete adjuvant (Bio Basic Inc.) to form a stable emulsion. One rabbit was subcutaneously injected at two to four different sites. Three booster injections were administered at 20-day intervals using 100 μg of the purified peptide-mcKLH conjugate with incomplete Freund’s adjuvant. Blood samples (0.5 to 1 ml) were collected before each injection, and bleeding (30 ml) was performed 10 days after the last booster to evaluate the immune response. Polyclonal antibodies (IgGs) were purified from rabbit serum by affinity chromatography on a 5-ml HiTrap protein A column (GE Healthcare) according to the manufacturer’s instructions. Binding was performed in 0.02 M sodium phosphate (pH 7.0), and elution was performed in 0.1 M citric acid (pH 3.0). The eluted IgGs were collected, immediately neutralized to physiological pH by adding 1 M tris-HCl buffer (pH 9.0), and then concentrated to 1 mg/ml by using 30-kDa molecular weight cutoff concentrators. Next, an N-hydroxysuccinimide (NHS)–activated Sepharose column (GE Healthcare) was used for affinity purification of the HR2-specific IgGs from the total rabbit antibodies. Concentrated HR2 peptide (20 mg) in coupling buffer [0.2 M NaHCO3 and 0.5 M NaCl (pH 8.3)] was applied to the NHS-activated column and allowed to bind for 2 hours at room temperature. Excess uncoupled, activated NHS groups were deactivated by sequential washing with two buffers: 0.5 M ethanolamine and 0.5 M NaCl (pH 8.3), followed by 0.1 M sodium acetate and 0.5 M NaCl (pH 4.0). The IgGs purified from rabbit serum using protein A were passed through the HR2 column. Unbound IgGs were removed by wash buffer [0.05 M sodium phosphate and 0.15 M NaCl (pH 7.0)] to obtain pure HR2-specific IgGs, which were eluted with three- to five-column volumes of 0.1 M citric acid buffer and then neutralized with 1 M tris-HCl buffer (pH 9.0). Purified HR2-specific IgG samples were buffer exchanged with 0.05 M sodium phosphate buffer (pH 7.0) and then concentrated to 1 mg/ml. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Peking University.

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