A method of quantitatively determining an analyte in a fluid sample by an immunoassay comprises binding of the analyte to a ligand capable of specifically binding to the analyte, wherein at least part of the analyte is present as an analyte complex is disclosed. The method comprises the steps of: a) subjecting the sample to a first acidic pH to at least substantially dissociate any analyte complex present and provide substantially all analyte in free form, b) raising the first acidic pH to a second acidic pH where re-formation of complexes is prevented but where binding of analyte to the ligand is permitted, and c) determining the binding of analyte to the ligand to quantitatively determine the analyte in the sample.
