Journal: Infection

Article Title: Development and validation of a respiratory syncytial virus multiplex immunoassay

doi: 10.1007/s15010-024-02180-6

Figure Lengend Snippet: Technical validation of RSV multiplex immunoassay. Several parameters were assessed for technical validation of the RSV multiplex immunoassay. a Dilution linearity was assessed in five validation samples from DF200-3276800. Linearity within the assay corresponded to a range of 250–25,000 MFI. Linearity for other antigens within the assay are included as Supplementary Fig. . Inter-assay variation and Intra-assay variation ( b , c ) were assessed in four validation samples at DF3200. For inter-assay variation, samples were measured in triplicate across five independent experiments, means of the triplicate for each sample is shown. %CV was < 5.7% for all samples. For intra-assay variation, samples were measured in triplicate (technical replicates), 12 × on a single plate. The mean of the technical triplicate for each of the 12 biological replicates is shown. %CV was < 4%. CVs for Inter-assay variation and Intra-assay variation for all other antigens are included as Supplementary Table . To assess prozone effects ( d ), a monoclonal post-F antibody was spiked at various concentrations (DF32000-6.5 × 10 7 ) into either eight negative serum samples or assay buffer with percentage recovery calculated. Mean %recovery for the eight samples is shown with 100% indicating no difference between serum and assay buffer. Standard bioanalytical margins for successful recovery (85% and 115%) are indicated on the panel. To confirm antigen specificity, binding titer for the post-F, N, and G was assessed for the same antibody. Binding responses were found only on the post-F antigen bead (orange) and not on any others (G rsb1734 shown in blue as an example, N is included as Supplementary Fig. ). Binding specificities for the N, GA, and GB are included as Supplementary Fig. . Lastly, the RSV multiplex immunoassay performance was compared to a commercial RSV ELISA (see methods for details), with linear regression and Spearman r used to calculate agreement between the two assays. The correlation was highly significant ( p < 0.0001)

Article Snippet: G , A-rsb1734 , 11070-V08H2 , Sino Biological , Anteo , 25.

Techniques: Multiplex Assay, Inter Assay, Intra Assay, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Infection

Article Title: Development and validation of a respiratory syncytial virus multiplex immunoassay

doi: 10.1007/s15010-024-02180-6

Figure Lengend Snippet: Antigens used during assay development including antigen, RSV subtype, strain (if known), the manufacturer and catalog number as well as the final coupling method and concentration

Article Snippet: G , A-rsb1734 , 11070-V08H2 , Sino Biological , Anteo , 25.

Techniques: Concentration Assay, Virus

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IM immunization with MF59/preF vaccine induced systemic humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intramuscularly with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and ILN were harvested on day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intramuscularly immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–G) The representative images and quantitative analysis of preF‐specific IgG + ASCs in bone marrow (E), spleen (F), and lung (G). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–G. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–G. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: IN immunization with MF59/preF vaccine induced local humoral and B‐cell immune responses. (A) The schematic representation of the mouse immunization and sample collection protocol. Mice were immunized intranasally with PBS, MF59, preF, MF59/preF‐low, or MF59/preF‐high on Days 0, 21, and 42. Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (B) Endpoint titers of anti‐preF/postF IgG in sera from mice intranasally immunized with adjuvanted preF on Days 14, 35, and 56. (C) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (D) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (E–J) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (E, H), spleen (F, I), and lung (G, J). Data are presented as geometric mean values ± SD in B–D. The middle line indicates the median while the whisker shows the data range in E–J. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in B, and D–J. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay

Journal: MedComm

Article Title: Combining Intramuscular and Intranasal Immunization With the MF59‐Adjuvanted Respiratory Syncytial Virus Pre‐Fusion Protein Subunit Vaccine Induces Potent Humoral and Cellular Immune Responses in Mice

doi: 10.1002/mco2.70301

Figure Lengend Snippet: Combination of IM and IN immunization using MF59/preF vaccine elicited both local and systemic humoral and B‐cell immune responses. (A–B) The schematic representation of the mouse immunization and sample collection protocol. BALB/c mice received two intramuscular doses of the MF59/preF vaccine followed by a single intranasal dose (IM‐IM‐IN), or one intramuscular dose followed by two intranasal doses (IM‐IN‐IN). Sera were collected on Days 14, 35, and 56, and BALF, spleen, lung, and mLN were harvested on Day 72. (C) Endpoint titers of anti‐preF/postF IgG in sera on Day 56. (D) Titers of virus neutralizing antibody (VNA) against RSV A2 and RSV B in sera collected on Day 56. (E) Endpoint titers of anti‐preF/postF IgG and IgA in BALF collected on Day 72. (F‐K) The representative images and quantitative analysis of preF‐specific IgG + (left) and IgA + (right) ASCs in bone marrow (F, I), spleen (G, J), and lung (H, K). Data are presented as geometric mean values ± SD in C–E. The middle line indicates the median while the whisker shows the data range in F–K. n = 6 mice per group. p values were conducted by One‐way ANOVA analysis followed by Tukey's multiple comparisons test in C–K. **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant.

Article Snippet: Briefly, 96‐well ELISA plates (NUNC‐MaxiSorp, Thermo Fisher Scientific) were coated overnight at 4°C with either 1 μg/mL of RSV preF or postF protein (Sino Biological, 11049‐V49H5‐B).

Techniques: Virus, Whisker Assay