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 enzyme-immunometric 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 [¹²⁵I] 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®', as the separation medium. This immunoassay produced a similar 'blanket' response as produced in the enzyme-immunometric assay. The coated-tube 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)