Patent information for investors

1. Trefoil type multiplex immunoassay (P.A. n° PCT/EP2005/000785, filed on January 27, 2005) Part 1: general principle Part 2: application on the PI/C ratio Part 3: objectives of the patent claim Inventor: Pieter De Pauw

2. Aims 1 ° To quantify different peptide molecules of a same family in one assay 2 ° Determine their mutual concentration ratios with high precision (applications: LDH isoforms, pituitary hormones (= α , ≠ β subunits) , PI/C, etc…) Part 1: general principle common (“constant”) part specific (“variable”) parts

3. Method One reaction compartment for the whole multiple assay Part 1: general principle When for each variable part a specific detecting antibody is available, the “total” is not necessary, but can be used as supplementary control One common capture antibody L1 L1 L1 L1 One common detecting antibody (for “Total”) L3 L2 One antibody with specific label for each variable part (for differentiation) L4 In the case where a variable part is lacking, this analyte can still be measured by subtraction of the others from the total .

5. PART II: APPLICATION Determination of the PI/C ratio with a Dual Time-Resolved Fluorescence Immunoassay (TRFIA) of circulating Proinsulin and C-peptide Part 2: application

9. Limits of quantitation Criterion*: RE ≤ 25 % with RE = bias ( ▲ ) ± 95% CI of total imprecision (■) *Findlay J.W.A., 2000 Part 2 : application C-peptide in simultest Proinsulin in simultest Range = 43 – 8200 pmol Cp / L Range = 2.7 – > 260 pmol PI / L

10. Limits of quantitation of PI/C *Findlay J.W.A., 2000 Part 2 : application PI/C unrelated Criterion*: RE ≤ 25 % with RE = bias ( ▲ ) ± 95% CI of total imprecision (■) Unsatisfactory range Range: 72 – 7900 pmol Cp/L PI/C related

11. *International reference standards in plasma with 1480 pmol Cp / L and 31.2 pmol P I/ L Recovery Spiked IRR* Further data are in “Clinical Chemistry” article < 0.01% cross ~100% recovery 7200 pM Cp < 0.01% cross < 0.01% cross 2000 pM INS ~100% recovery < 0.02% cross 1024 pM PI Proinsulin C-peptide Part 2: application crossreaction Cp/INS/PI?