Antiserum from mice immunized against GP<sub>1,2</sub> or sGP display different reactivity patterns.
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Abstract
<p>(A) Detection by Western blot of antibodies against GP<sub>1,2</sub> and sGP from immunized mice. 50 ng of purified His-sGP and His-GP<sub>1,2</sub> were run by SDS-PAGE under denaturing conditions and probed with 1∶1000 pooled GP<sub>1,2</sub>Edit or sGPEdit antisera followed by blotting with HRP-conjugated goat anti-mouse IgG. (B) Schematic of competition ELISA. Wells were coated with GP<sub>1,2</sub> and incubated with pooled antisera as well as increasing concentrations of competing antigen (sGP or GP<sub>1,2</sub>) to compete for antibodies. After two hours, plates were washed and then incubated with HRP-conjugated secondary antibody followed by addition of substrate to develop color. (C, D) Competition ELISA. Antisera from mice immunized with sGPEdit, GP-7A, GP-8A, and GP<sub>1,2</sub>Edit were diluted to give similar anti-GP<sub>1,2</sub> signal. Diluted antiserum was mixed with increasing quantities of purified His-sGP (C) or His-GP<sub>1,2</sub> (D) and incubated in His-GP<sub>1,2</sub> coated wells and developed as described above. Experiments were performed in duplicate and repeated at least three times, with representative results shown. (E, F) Competition Immunoprecipitation. Pooled antisera from GP<sub>1,2</sub>Edit-immunized mice (E) or sGP-immunized mice (F) were incubated with no GP, purified sGP or GP<sub>1,2</sub> alone, or with fixed GP<sub>1,2</sub> and increasing concentrations of sGP to compete for anti-GP<sub>1,2</sub> antibodies. GP<sub>1,2</sub> was incubated with recombinant HA as a negative control. The upper panel for the sGPEdit antisera shows the GP<sub>1,2</sub> portion of the blot at a longer exposure time to show the attenuation of signal with increasing sGP concentration. Results are representative of three independent experiments.</p
