## 3 Results
### Assay Specificity  
The ability of the LIPS-\(G_{A}\) and LIPS-\(G_{B}\) assays to detect anti-RSV-G IgG specific binding was evaluated using murine monoclonal antibodies (Figure 2). RSV anti-G MAbs 1227, 1197, 104-5, and 109 bound strongly in the LIPS-\(G_{A}\) assay only and did not bind to Ruc-\(G_{B}\) antigen. RSV anti-G MAb 131-2G, known to be reactive with both RSV-\(G_{A}\) and -\(G_{B}\) proteins, bound in both assays. MAbs directed against other viral antigens, including RSV-N (130-12H), RSV-F (1243), PIV3-F (108), and PIV-HN (170), were negative in both the LIPS-\(G_{A}\) and LIPS-\(G_{B}\) assays, while the anti-FLAG antibody bound both antigens. Cutoff values were determined using the mean value obtained for all four negative antibodies (130-12H, 1243, b108, and 170) and were set at 2.29 log\({}_{10}\) RLU (red line) and 2.16 log\({}_{10}\) RLU (blue line) for Ruc-\(G_{A}\) and Ruc-\(G_{B}\), respectively. All MAbs were negative when tested against Ruc only antigen without insert.  
Antisera from rabbits immunized parenterally with RSV-A or -B viruses or antigens were tested in the LIPS-\(G_{A}\) and -\(G_{B}\) assays to determine if binding was subtype specific or cross-reactive with  
Figure 1: Expression of Ruc-G antigens. Ruc-tagged antigens were immunoprecipitated using beads coated with anti-FLAG IgG and evaluated using Western blot assay as described in materials and methods to confirm expression of Ruc (no insert) (lane A), Ruc-\(G_{A}\) (lane B), and Ruc-\(G_{B}\) (lane C) antigens in COS-1 lysates using rabbit antisera for detection. Molecular weight markers are noted.  
the heterologous protein (Figure 3). Rabbit anti-RSV-A2 and anti-RSV-G\({}_{\rm A}\) glycoprotein were strongly positive when tested against \(\rm{Ruc\text{-}G_{A}}\) antigen with a lower but positive signal when tested against \(\rm{Ruc\text{-}G_{B}}\) antigen. In contrast, antisera from rabbits immunized with RSV-18537 and boosted with purified G\({}_{\rm B}\) protein were only positive in LIPS-G\({}_{\rm B}\) assay (cutoff = 2.47 log\({}_{10}\) RLU). Interestingly, subtype specific binding was noted using antisera from rabbits immunized with synthetic RSV-G\({}_{\rm A}\) peptides representing amino acids 231-250 or 262-291 since these antisera were positive against \(\rm{Ruc\text{-}G_{A}}\) antigen but negative when tested using the heterologous \(\rm{Ruc\text{-}G_{B}}\) antigen. The amino acids within the central region of the RSV-G primary sequence are highly-conserved and are thought to mediate an important first step in RSV binding to ciliated lung epithelial cells. Accordingly, we expected to see cross-reactivity in antibody responses raised against peptides that represent the RSV-G\({}_{\rm A}\) versus RSV-G\({}_{\rm B}\) central conserved domains. Anti-LHWT-G\({}_{\rm A171\text{-}202}\) was clearly positive against \(\rm{Ruc\text{-}G_{A}}\) antigen, and also bound to \(\rm{Ruc\text{-}G_{B}}\) antigen, albeit less well. Surprisingly, reciprocal cross-reactivity was not observed with anti-LHWT-G\({}_{\rm B171\text{-}202}\) since it was reactive only against \(\rm{Ruc\text{-}G_{B}}\) antigen and did not bind \(\rm{Ruc\text{-}G_{A}}\) antigen. Pre-immune serum did not react with either \(\rm{Ruc\text{-}G_{A}}\) or \(\rm{Ruc\text{-}G_{B}}\) antigens while rabbit anti-FLAG reacted positively in both assays. Rabbit antisera to MV or hMPV were negative against all antigens tested. All rabbit anti-sera were negative when tested against the \(\rm{Ruc}\) only antigen while the anti-FLAG antibody gave positive results as expected.