Service Communautaire d'Information sur la Recherche et le Développement - CORDIS

Platelet Immuno Assay using TIRF

DiaMed / ADS in collaboration with Leuze electronic has developed a new technology best described as real-time Fluorescence Immuno-Assay also known as TIRF. This basic technology has been exploited to transform our 40 minute HPA-1a ELISA to a 5 min real-time FIA. The TIRF Assay is completed in 5 minutes with just two pipetting steps and no washing step. A field trial indicated good performance of the real-time FIA with ELISA and genotyping.

For antibody assays we developed a MAIPA / real-time FIA procedure to be performed in 1 h. Basically we incubate platelets and human plasma to be examined in the same way as is done for a conventional MAIPA and then follow the MAIPA wash procedures. The major difference is the detection of the complex monoclonal antibody / platelet glycoprotein / human immune globulin where our real-time FIA uses a one-step binding / detection step and gives a result in ten minutes. We have proven the feasibility of the new fast MAIPA with gpIIbIIIa, an optimisation procedure and the application to the other immunologically relevant platelet glycoproteins gpIaIIa, gpIbIX, CD109 and HLA will be the necessary. A development in 2005 for a one-hour MAIPA test is foreseen using the real-time fluorescence immuno assay.

To expand the HPA typing range further than the HPA-1a typing an approach to HPA genotyping has been evaluated in a feasibility study. Using the real-time fluorescence immuno assay platform and HPA-1a oligonucleotides as analyte we can in 15minute hybridisation and detection determine the oligonucleotide concentration with an analytical sensitivity of 5 pmol/L


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