|
 |
|||||||||||||||||||
|
|||||||||||||||||||
|
|||||||||||||||||||
 |
 |
|
|
a R&D Diasorin SpA, Saluggia, Vercelli, Italy b Centro Ricerche Fiat, Strada Torino, Orbassano, Torino, Italy c Servizio di Reumatologia ed Immunologia Clinica Spedali Civili, Brescia, Italy
Key Words: anticardiolipin • antibodies • immunoassay • LIAISON  2-glycoprotein I purification • antiphospholipid syndrome • SEM
Address for correspondence: Fabrizio Bonelli, R&D Diasorin SpA, via Crescentino s.n.c., 13040 Saluggia (VC), Italy. Voice: +39-0161-487514; fax: +39-0161-487628. fabrizio.bonelli{at}diasorin.it
The lack of reliable standardization tools as well as the poorly defined nature of the "Cardiolipin antigen" makes the development of the anticardiolipin antibody (ACA) assays (for anti-IgG and IgM detection) highly challenging. This article describes how several issues have been solved during the development of an automated ACA immunoassays, based on a technology that includes paramagnetic microbeads as solid-phase reagents and chemiluminescence as a signal. The technology is adapted to an automatic immunoanalyzer, called LIAISON, which performs, in an automatic manner, the whole assay, starting from the primary tube of the bleeding to the display of the assay result. Briefly, the magnetic microbeads were coated with an ethanolic solution of cardiolipin (CL) followed by an affinity-purified, cross-linked human
2-glycoprotein I. CL-coated paramagnetic microbeads, after incubation with an ACA-positive sera plus addition of immunogold-protein A, were visualized by SEM, showing the presence of well-defined protein clusters on the microbeads surface as an indication of the successful occurrence of the "antigen" coating. The assay standardization was achieved on the basis of human samples containing various amount of ACA, which were previously classified according to consensus doses. The evaluation of the optimized LIAISON Cardiolipin assays (IgG and IgM) was conducted by using clinically characterized APS sera. The results of the evaluation showed that the LIAISON assays perform at least similar to certain well-established ACA enzyme-linked immunosorbent assay (ELISA) products.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
