nonlinear least-squares regression model  (OriginLab corp)

Journal: Nature medicine

Article Title: Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model

doi: 10.1038/s41591-020-0937-x

Figure Lengend Snippet: Stalk-only constructs with or without the A388V mutation (See Extended Data Fig. 7b for the amino acid sequence) were produced to measure the level of antibodies recognizing the mutant stalk in human serum while excluding head-binding antibodies (Fig. 3j). To confirm that the stalk-only construct with A388V mutation appropriately represents the natural A388V stalk structure, the level of decrease in broadly neutralizing monoclonal antibodies (bNAbs) binding was compared between the stalk-only construct and the full-length HA. ELISA was performed using serially diluted (a,b) CR6261, (c,d) CR9114, (e,f) FI6V3, (g,h) 70–1F02, (l,j) C179, and (k,l) CT149. (m,n) Anti-StrepTag II antibody was used to show that equal amounts of wild-type and mutant antigen were used for the analysis. The AUC was calculated using GraphPad Prism8 (v.8.3.0). The AUC for the full-length HAs (b,d,f,h,j,l) was calculated using the data from Fig. 3. Graphs show mean and standard deviations from three independent measurements. The summary table shows the mean OD492 values and standard error of mean (s.e.m.) of the three independent measurements. The comparison result (summarized in the table) shows that the A388V stalk-only construct closely represents the natural stalk structure of the full-length A388V HA.

Article Snippet: The percentage of infection was normalized to the lowest antibody concentration, and the IC 50 values were determined using a nonlinear least square regression model (variable slope, four parameters) using Prism8 software v.8.3.0 (GraphPad Software).

Techniques: Construct, Mutagenesis, Sequencing, Produced, Binding Assay, Bioprocessing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature medicine

Article Title: Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model

doi: 10.1038/s41591-020-0937-x

Figure Lengend Snippet: Raw ELISA data used to generate Fig. 3j are shown. Stalk-only constructs with or without the A388V mutation (See Extended Data Fig. 7b for the amino acid sequence) were used to measure changes in the level of antibodies recognizing the wild-type (A388) of mutant (V388) stalk in human serum. twenty-nine pre-challenge serum samples from the influenza human challenge study participants were serially diluted and used for the ELISA. the AUC was calculated using graphPad Prism8 (v.8.3.0) with the baseline value of 0.1 (approximately 2 times the OD492 value from the control wells). Blue lines and numbers show the ELISA data and the AUC, respectively, obtained using the wild-type (A388) stalk construct. Red lines and numbers show the ELISA data and the AUC, respectively, obtained using the mutant (V388) stalk construct. graphs show mean and standard deviations from three independent measurements.

Article Snippet: The percentage of infection was normalized to the lowest antibody concentration, and the IC 50 values were determined using a nonlinear least square regression model (variable slope, four parameters) using Prism8 software v.8.3.0 (GraphPad Software).

Techniques: Enzyme-linked Immunosorbent Assay, Construct, Mutagenesis, Sequencing, Control

Journal: Nature medicine

Article Title: Pre-existing immunity to influenza virus hemagglutinin stalk might drive selection for antibody-escape mutant viruses in a human challenge model

doi: 10.1038/s41591-020-0937-x

Figure Lengend Snippet: Selection pressure placed by individual human sera was measured using pre-challenge serum from the influenza human challenge study participants. a, An equal mixture of the wild-type (A388) and mutant (V388) virus was cultured with 1:50 diluted serum samples. Sera from Q1, Q2, Q3 and Q4 from Fig. 1 (n = 7 per quartile) were used. Culture supernatants were collected at 48 and 72 hours after infection followed by viral RNA extraction. The selection dynamics were measured using a Single Nucleotide Polymorphism (SNP) assay utilizing a set of Minor Groove Binder (MGB)-based TaqMan probes; VIC-labeled probe detects the wild-type (A388); FAM-labeled probe detects the mutant (V388). Data are presented as the threshold cycle (Ct) value from the SNP assay. Dashed lines show the Ct value limit (Ct 40) of the SNP assay. A Ct value of 41 was given to undetected signals to generate graphs. Error bars represent standard deviations from three independent experiments. The final dilution is noted on the individual graph if higher than 1:50. b, Mutant selection index was calculated based on data from (a) by ΔΔCt method using controls cultured without serum (see Methods). A mutant selection index higher than 0 (pink area) indicates a serum sample selected for the mutant virus. An index lower than 0 (blue area) indicates a serum sample selected for the wild-type virus. Horizontal lines show median values and error bars represent 95% CI. The indexes between samples from different quartiles were compared using nonparametric one-way analysis of variance (Kruskal-Wallis test) and Dunn’s test as a post-hoc test. c, Correlation between the anti-stalk serum IgG titer and the selection index of 29 sera samples were analyzed by calculating two-tailed Spearman’s rank correlation coefficient (Spearman r). The best-fit line was plotted using simple linear regression analysis. Statistical analyses were performed using GraphPad Prism8 (v.8.3.0).

Article Snippet: The percentage of infection was normalized to the lowest antibody concentration, and the IC 50 values were determined using a nonlinear least square regression model (variable slope, four parameters) using Prism8 software v.8.3.0 (GraphPad Software).

Techniques: Selection, Mutagenesis, Virus, Cell Culture, Infection, RNA Extraction, Labeling, Two Tailed Test