In direct comparison of both real and simulated immunoblot data, the 1-step method consistently exhibited smaller errors than the conventional “2-step” method. It is less sensitive to measurement error on standards and enables use of some batches that do not include standards. It returns the most probable values for the sample compositions under the assumptions of a statistical model, making them the maximum likelihood predictors. The 1-step method computes all calibration results iteratively from all measurements. Here we show that a “1-step calibration method” reduces these problems for the common situation in which samples are measured in batches, where a batch could be an immunoblot (Western blot), an enzyme-linked immunosorbent assay (ELISA), a sequence of spectra, or a microarray, provided that some sample measurements are replicated across multiple batches. It also implies that any data collected without reliable standards must be discarded. The deduction of the conversion function from only the standard measurements causes the results to be quite sensitive to experimental noise. The conventional approach has two steps: the experimenter deduces a conversion function using measurements on standards and then calibrates (or normalizes) measurements on unknown samples with this function. ![]() Experimental measurements require calibration to transform measured signals into physically meaningful values.
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