Development of a Chemiluminescent Assay for Autoantibodies to Thyroid Peroxidase

Abstract

The presence of antibodies to thyroid peroxidase (i.e. TPOAb) in human serum has been measured using an immunoradiometric assay (IRMA), an enzymeimmunometric assay, and three other immunoassay systems which incorporated the chemiluminescent label acridinium ester (AE). The IRMA successfully utilised a commercial preparation of TPO labelled with [ 125 I] and magnetic particles coupled to Protein A (Staphylococcus aureus) (MPA), with the latter separation medium used in an enzyme-immunometric assay which incorporated TPO labelled with horse-radish peroxidase (HRP-TPO). TPO was also labelled with AE and used in a chemiluminometric assay with magnetic particles coupled to human anti-IgG (MAb-Anti-IgG). The latter assay exhibited an improved response, as compared with the non-viable enzyme-immunometric assay, but was inferior to the IRMA lacking the required sensitivity and precision for a viable, clinical assay. A competitive assay system was also investigated which utilised human anti-TPO (obtained from a purified preparation of IgG) labelled with acridinium ester (Acrid- IgG), TPO labelled with biotin (Biotin-TPO) and magnetic streptavidin-labelled 'Dynabeads®1 , as the separation medium. This immunoassay produced a similar 'blanket' response as produced in the enzyme-immunometric assay. The coatedtube assay involved the immobilisation of TPO onto plastic tubes and the use of a commercial preparation of sheep anti-human IgG labelled with AE (Acrid-Anti-IgG). This system proved to be the most viable of all the non-radioactive assays, with the results comparing favourably with the established ELISA with a correlation coefficient (r) of 0.96 and P = <0.001, (using the 'least squares linear regression after logarithmic conversion of the data). A good correlation was also demonstrated in the Deming and the Passing & Bablok plots. The coated-tube assay also compared favourably with indirect agglutination (r) = 0.80, P = <0.001. The results indicated that the less pure source of TPO used in the non-radioactive assays, could only be successfully applied to the measurement of TPOAb, when immobilised onto a solid-phase support such as the ELISA micro-titre plate or onto plastic tubes, but not in the more random, assay systems which utilised magnetic particles as the separation phase. The solid-phase, coated-tube chemiluminometric assay was comparable with ELISA for the measurement of TPOAb in human serum.

Date of Award	Jul 2000
Original language	English
Supervisor	Stuart Hogg (Supervisor)