## 4 Discussion  
We have described the development of an immunoprecipitation assay using luciferase-tagged RSV-G\({}_{\rm A}\) and -G\({}_{\rm B}\) proteins to detect IgG antibodies in serum. Although RT-PCR is routinely used for RSV diagnosis, and this testing is very sensitive for detecting RSV genome, the logistics of sample collection in a timely manner may be complicated. A strategy that relies only upon RT-PCR to  
Figure 9: Reactivity of adolescent sera using native and mutated \(\rm{Ruc}\)-G\({}_{\rm A}\) and -G\({}_{\rm B}\) antigens. Binding to native versus mutated antigen was compared using the adolescent serum panel. Data were normalized against rabbit anti-FLAG antibody for each antigen comparison; (**A**) \(\rm{Ruc}\)-G\({}_{\rm A}\) versus \(\rm{Ruc}\)-mG\({}_{\rm A}\) or (**B**) \(\rm{Ruc}\)-G\({}_{\rm B}\) versus \(\rm{Ruc}\)-mG\({}_{\rm B}\). Immunoglobulin depleted serum (IgG\(\Delta\)) was used as the negative control and cutoff values are shown as either solid (native) or dashed (mutated) horizontal red (\(\rm{Ruc}\)-G\({}_{\rm A}\), \(\rm{Ruc}\)-mG\({}_{\rm A}\)) or blue lines (\(\rm{Ruc}\)-G\({}_{\rm B}\), \(\rm{Ruc}\)-mG\({}_{\rm B}\)). Results are ranked from low to high based on testing against the \(\rm{Ruc}\)-G\({}_{\rm A}\) or \(\rm{Ruc}\)-G\({}_{\rm B}\) antigens and paired with the corresponding result for each subject against the \(\rm{Ruc}\)-mG\({}_{\rm A}\) or \(\rm{Ruc}\)-mG\({}_{\rm B}\) antigens, respectively. (**C**) Group RLU responses were plotted for all antigens tested. * indicates significant difference between groups (\(p\) = 0.0013). identify cases may underestimate the number infected with RSV. When RT-PCR is combined with a serological assay, RSV incidence rates are increased [17]. Commercially available enzyme-linked immunosorbent assay (ELISA) kits that detect anti-RSV-IgG are easy to use and provide rapid results. However, these kits typically use whole virions or lysates from RSV-infected cells and may not be able to discriminate between antibodies elicited by natural infection from those resulting from immunization or immune-prophylaxis. As such, serological assays to detect antibodies against RSV-specific, non-vaccine antigens are needed to support surveillance efforts and help identify RSV-exposed subjects following immunization or immune-prophylaxis. Electrochemiluminescent serological assays using non-vaccine target antigens successfully identified subjects exposed to RSV in the post-immunization period, thus providing support for the practical use of these types of assays [18]. However, reagents for these assays are not commercially available. Similarly, immune profiling by immunoprecipitation of luciferase-tagged proteins (LIPS) allows detection of protein-specific IgG antibodies. Additionally, the use of luciferase-tagged antigens from crude cell lysates is advantageous since protein purification can be time consuming and often results in low yields. Assays that distinguish between the two RSV antigenic subtypes (A and B) may also contribute to our understanding of the role these IgG antibodies may play in determining subsequent risk of RSV re-infection. Lastly, detection of anti-RSV-G IgG antibodies among subjects given RSV-F-only vaccines may help to confirm RSV exposures that support safety and efficacy assessments. The LIPS-G assays described herein specifically detected IgG antibodies directed against RSV-G\({}_{\text{A}}\) and/or -G\({}_{\text{B}}\) proteins, as shown by murine MAbs, while MAbs directed against RSV-N, RSV-F, PIV3-F, and PIV3-HN failed to bind in either assay. Specificity was further confirmed using mono-specific rabbit antisera induced by immunization with purified RSV-G\({}_{\text{A}}\) protein, or synthetic linear RSV-G\({}_{\text{A}}\) and -G\({}_{\text{B}}\) peptides while rabbit antisera induced by immunization with either purified RSV-F protein, UV-inactivated MV, or UV-inactivated hMPV failed to bind in either assay.