For the anti-MPER response ELISA plates (Nunc Maxisorp, Rochester, NY) were coated with 10 mM of the MPER peptide (GenScript Co, Piscataway, NJ) in 100 ml of 50 mM carbonate (pH 9.6) per well, according to the method we described previously [29] . Plates were washed and then blocked with 3% BSA/PBS. After washing, 60 ml of 1:20 diluted sera was added. After incubation for at least 2 hr at RT, the plates were extensively washed and blocked with 3% BSA/PBS. The plates were then washed with HRP-conjugated goat anti-human antibody (Southern Biotech, Birmingham, AL). ELISAs were developed with TMB substrate. In order to determine MPER isotype-specific reactivity, ELISA plates were coated with 10 mM of the MPER peptide in 100 ml of 50 mM carbonate (pH 9.6) per well, according to the method we described previously [29] . Plates were washed and then blocked with 3% BSA/PBS. After washing, 60 ml of 1:20 diluted sera was added. After incubation for at least 2 hr at RT, the plates were extensively washed and blocked with 3% BSA/PBS. Isotype-specific mouse antibody (Sigma-Aldrich, St. Louis, MO) was then bound to ELISA plates. Plates were then washed and HRP-conjugated anti-mouse antibody (Dako Denmak, Denmark). ELISAs were developed with TMB substrate (Sigma-Aldrich, St. Louis, MO). OD 450 nm was measured on an Emax microplate reader.