Human thyroglobulin reference material (CRM 457). 1st Part: Assessment of homogeneity, stability and immunoreactivity.

This paper describes the assessment of the homogeneity and stability of a purified and lyophilized human thyroglobulin (Tg), and characterizes its immunoreactivity. The purified and lyophilized Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The homogeneity of the content of the ampoules was considered acceptable (< 9%). The stability was tested by accelerated temperature degradation showing predicted annual relative losses of 0.01% at -70 degrees C and 1.04% at -20 degrees C. The immunoreactivity of the Tg material as measured in different laboratories varied mostly according to the method used rather than the laboratory. The interlaboratory variability showed that the two commercial methods used in several laboratories (kit 1 and 2) had an interlaboratory variation (CV) of 15.9% (N = 5) and 7.1% (N = 3), respectively, whereas the total interlaboratory CV was 64.3% (N = 18). The immunoreactive Tg had dilution curves parallel with other Tg calibrators (those of the methods). Dilution curves of the Tg material after storage at various temperatures and time were parallel in both RIA and IRMA. In conclusion, we have prepared a Tg reference material which in extensive studies in several participating laboratories has demonstrated a sufficient homogeneity and stability as well as dilution curves parallel to the calibrators of all the immunoassays tested in the study. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg immunoassays.

Human thyroglobulin reference material (CRM 457). 2nd Part: Physicochemical characterization and certification.

This report describes the characterization of a purified human thyroglobulin (Tg) reference material, and details the procedures used in its certification. The purified Tg is intended to be used as a primary reference material to establish calibration of working serum based reference material for immunoassay procedures. The programme involved the participation of 15 European laboratories and one laboratory from the United States. The physicochemical characterization showed by polyacrylamide gel electrophoresis and immunoblotting that the purified Tg had for the major part the expected molecular size of 660 kDa with traces of lower molecular forms. The amino acid composition was close to that demonstrated for the cDNA and the content of iodine was in keeping with a moderately to highly iodinated Tg. The mass concentration in reference material RM 457 is certified to be (0.324 +/- 0.018) g/L on the basis of protein determined by the Lowry method and supported by nitrogen determination, absorbance measurement, and amino acid analysis. This reference material is considered the first step towards decreasing the interlaboratory variability between Tg methods of measurement.