## 3 Results
### Sensitivity of LIPS-G Assays in Detecting Anti-RSV-G IgG Antibodies Compared to PRN  
Human immune globulin (hulgG, BEI NR-21973) was tested over a wide range of IgG concentrations in the LIPS-G\({}_{\rm A}\), LIPS-G\({}_{\rm B}\), and PRN assays to compare sensitivities in detecting anti-RSV antibodies. Anti-RSV-G specific IgG binding was detected at concentrations as low as 0.1% (1.0 mg/mL total IgG) in LIPS-G\({}_{\rm A}\) (Figure 4A) and 0.01% (0.1 mg/mL total IgG) in LIPS-G\({}_{\rm B}\) (Figure 4B) assays. PRN antibodies capable of inhibiting RSV infectivity by 50% or more were detected at concentrations as low as 0.01% (0.1 mg/mL total IgG) for both RSV-A2 and RSV-B1 viruses. No binding was seen at any dilution when hulgG was tested against \(\rm{Ruc\text{ antigen alone}}\) and \(\rm{IgA}\) did not inhibit RSV in the PRN assay. Cutoffs for a positive response in the LIPS-G\({}_{\rm A}\) and -G\({}_{\rm B}\) assays were determined using the mean values obtained for the tests on IgA in each assay (3.46 log\({}_{10}\) RLU and 2.86 log\({}_{10}\) RLU for \(\rm{Ruc\text{-}G_{A}}\) and \(\rm{Ruc\text{-}G_{B}}\), respectively).  
Figure 2: Specificity of the LIPS-G assays. Specificity of respiratory syncytial virus (RSV) LIPS-G assays in detecting anti-RSV-G IgG antibodies was shown using murine monoclonal antibodies. The mean reactivities against \(\rm{Ruc\text{-}G_{A}}\) and \(\rm{Ruc\text{-}G_{B}}\) antigens are shown in red and blue bars, respectively, and error bars represent standard deviations. Data were normalized against murine anti-FLAG antibody. Cutoff values for a positive signal were five standard deviations above the mean value of all four negative controls and are shown as horizontal red (\(\rm{Ruc\text{-}G_{A}}\)) and blue lines (\(\rm{Ruc\text{-}G_{B}}\)). * Represents a negative result.  
Figure 4: Detection of anti-RSV antibodies using LIPS-G\({}_{\rm A}\) and -G\({}_{\rm B}\) assays versus plaque reduction neutralization (PRN) testing. Human immune globulin (hulgG, BEI NR-21973) was used neat or serially diluted 10-fold into Ig\(\Delta\) from 10% to 0.00001% IgG. Samples were tested in triplicate in immunoprecipitation assays and in duplicate in plaque reduction neutralization assays against Ruc-G\({}_{\rm A}\) antigen and RSV-A2, respectively (panel **A**) or against Ruc-G\({}_{\rm B}\) antigen and RSV-B1, respectively (panel **B**). The results of testing in the immunoprecipitation assay are shown by the black lines in each figure. Cutoff values for a positive result in the immunoprecipitation assays were five standard deviations above the mean value of the negative control (IgGA) and are shown as horizontal red (Ruc-G\({}_{\rm A}\)) and blue lines (Ruc-G\({}_{\rm B}\)). In the PRN test, a reduction in the mean plaque count of \(\geq\)50% (in wells containing hulgG) relative to the mean plaque count seen in virus control wells (in the absence of hulgG) was considered significant.  
Figure 3: Specificity of the LIPS-G assays. Specificity of RSV LIPS-G assays was also shown using rabbit antisera. Mean reactivities against Ruc-G\({}_{\rm A}\) and Ruc-G\({}_{\rm B}\) antigens are shown in red and blue bars, respectively, and error bars represent standard deviations. Data were normalized against rabbit anti-FLAG antibody. Cutoff values for a positive signal were five standard deviations above the mean value of the negative control and are shown as horizontal red (Ruc-G\({}_{\rm A}\)) and blue lines (Ruc-G\({}_{\rm B}\)).