Calibration standards immunoassay development

An immunoassay was developed to determine the penicillinase stable isoxazolyl penicillins cloxacillin and dicloxacillin in milk by Usleber et alJ The assay detected lOpgkg" of cloxacillin and 30pgkg of dicloxacillin with recoveries of 102% and 84%, respectively. The calibration curve was prepared by fortifying skimmed milk powder (lOOgL ) with standards. Fortified samples were prepared in pasteurized milk and analyzed directly after decreaming by centrifugation. This immunoassay was performed with minimal sample preparation, probably because the extensive water solubility of the penicillins prevents problems associated with more lipid-soluble analytes. 

As for immunoassays for pharmaceutical proteins, in-study validation of biomarker assays should include one set of calibrators to monitor the standard curve as well as a set of QC samples at three concentrations analyzed in duplicate for the decision to accept or reject a specific run. Recommended acceptance criterion is the 6-4-30 rule, but even more lenient acceptance criteria may be justified based on statistical rationale developed from experimental data [14]. 

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