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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:…
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  • 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 .
  • 4. Advantages Part 1: general principle <ul><li>Advantages of measuring n analytes within the same reaction compartment : </li></ul><ul><li>- The sample history is exactly the same for all analytes (freeze/thaw, etc…) </li></ul><ul><li>- Reduced sample manipulation (1x taking out from freezer instead of n x) </li></ul><ul><li>- Reduced sample volume needed to perform all analysis ( 1/n X) </li></ul><ul><li>- Absence of any sample pipetting error on ratio determination </li></ul><ul><li>- Reduced personnel and material cost (one performance for n analytes) </li></ul><ul><li>Advantages of using a common capture antibody </li></ul><ul><li>- The inter assay variation of capture antibody freshness, coating efficiency and </li></ul><ul><li>capture step incubation conditions is the same for all analytes, and thus has no </li></ul><ul><li>influence on the determination of the analyte’s ratios. </li></ul><ul><li>- The capture antibody cost is reduced (1 capture antibody for n analytes) </li></ul><ul><li>- The solid phase can be maximally saturated with one capture antibody (no </li></ul><ul><li>concurrence between capture antibodies) </li></ul>
  • 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
  • 6. The PI/C ratio e Convertases Part 2: application <ul><li>dynamic marker for functional state of β cells (K. Hostens 1999) </li></ul><ul><li>independent predictive marker of T1DM (I. Truyen 2005) </li></ul><ul><li>simple: - requires only a (random) blood prelevation </li></ul><ul><li> - relatively easy to analyze </li></ul><ul><ul><li> - can be analyzed on large scale </li></ul></ul>Beta cell Cell membrane Golgi apparatus Secretory granules PI/C ratio
  • 7. State of the art Part 2: application <ul><li>Advantages: </li></ul><ul><li>low background </li></ul><ul><li>broad dynamic range </li></ul><ul><li>Disadvantage: </li></ul><ul><li>cumulative imprecision </li></ul><ul><li>Solution: </li></ul><ul><li>Trefoil-type </li></ul><ul><li>simultaneous assay </li></ul>Individual Time –Resolved Fluorescence Assays C-peptide TRFIA PI/C ratio proinsulin TRFIA
  • 8. PI/C ratio = PI/(Cp T – PI)*100 Part 2: application Trefoil-type simultaneous assay Proinsulin: PEP-001 Tb Tb Tb Tb Tb Tb Tb Tb Tb HUI-001 <ul><li>three monoclonal antibodies </li></ul><ul><li>with no mutual steric hindrance </li></ul><ul><li>amplification of the lowest </li></ul><ul><li>signal (proinsulin) with the </li></ul><ul><li>biotin/ streptavidin system </li></ul><ul><li>two independently measurable </li></ul><ul><li>Labels (Eu and Tb) </li></ul>Eu CPT3F11 Eu Eu Insulin C-peptide Tb Tb Eu
  • 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?
  • 12. Objectives Part 3: objectives <ul><ul><li>Aim : </li></ul></ul><ul><ul><li>We want to implement the protected general principle of trefoil-type assay in existing analytical methods. </li></ul></ul><ul><ul><ul><ul><li> </li></ul></ul></ul></ul><ul><ul><ul><ul><li>Application field : </li></ul></ul></ul></ul><ul><ul><ul><ul><li>I n the niche where ratios between a limited number of molecules of the same family have to be determined with the highest possible accuracy and precision, i.e. conditions where different molecules with shared epitopes need to be simultaneously quantified. </li></ul></ul></ul></ul>
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