Conversion of mean fluorescent intensity to concentration

Preparations of standardized multiplex analyte titration series were manufactured using recombinant analytes diluted in buffer covering the range from 12 pg/mL to 81 ng/mL at 14 discrete points plus zero analyte buffer blanks. These titration points were distributed among the 16 available wells on three control slides. The standard titrations, designed to overlap the linear range of detection for each individual analyte, were used to generate standard curves from which sample analyte concentrations were determined. A four-point standard titration was run on every slide for normalization and quality control purposes. The four wells, designated "anchor point" controls, were derived from the standard 14-point titration series run on separate control slides to generate standard curves for each analyte. Anchor point controls contained a cocktail of all cytokines corresponding to the printed capture antibodies for a given array. The anchor points were prepared at four concentrations that fell within the linear range of detection for each analyte. Individual sample values were normalized using linear regression of the anchor points to reduce assay imprecision observed among replicates. Fluorescence intensities of the four spot replicates for each analyte within an anchor point well were averaged on a logarithmic (base two) scale to generate within-slide titration curves. Linear regression coefficients (slope and intercept) were calculated between individual titration curves from each slide to generate an "average" titration curve. Calculated slope and intercept were used to transform averaged analyte values for each sample well. Data normalization was performed on the data set after removal of outliers.

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