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The electrode was calibrated with dilute standard acid and alkali solutions, thus defining pH=−log[H+] (p KW=14.28).
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After a washing with PBS Tween 20 (0.05%), a solution made of TBS BSA 0.1%)–Tween 20 (0.05%) was used to dilute standards (rhIL-18, R&D Systems) and sera.
After the plate was washed, an identical set of samples and serially diluted standard serum (1 500 to serial 1 5 dilutions) were added to the left and right halves (control for reactivity against mouse IgG) of the plate.
All samples were tested against a serially diluted standard reference serum (supplied by NIH as was previously reported12) with a known antibody titer.
The percent-injected activity per gram (%IA/g) was calculated by comparison to a weighed, diluted standard.
A stock solution of 1000 ppm of each dye was prepared and diluted with distilled water to prepare diluted standard solutions.
LODs and LOQs were determined using diluted standard solution when the signal-to-noise ratios (S/N) of analytes were about 3 and 10, respectively.
The reported peak areas and peak heights were compared to those obtained injecting diluted standard solutions of the same perchlorate concentrations using identical conditions.
Absolute quantification of splice variants was performed using a serially diluted standard containing the same sequence as the target amplicon.
Concentrations of mRNA were calculated from serially diluted standard curves simultaneously amplified with unknown samples and normalized with respect to β-actin mRNA levels.
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