) In Phase 1 an enzymatic assay for homocysteine was developed with sensitivity down to concentrations of 10-20 micromolar, well below the range of 50-500 micromolar that is indicative of homocystinuria. The feasibility of a test strip for homocysteine detection was demonstrated using an immobilized form of the enzyme and a colorimetric visualization method, providing results within 20 minutes. In Phase 2, the test strip assay will be perfected for use in the neonatal screening for homocystinuria to replace the flawed Guthrie test, which cannot detect homocysteine. Immobilization of the enzyme will be optimized for loading and activity, and the visualization chemistry will be refined. Stability and shelf life will be evaluated and improved as necessary. Expression of the methionine gamma-lyase gene will be increased to reduce the cost of enzyme. The solution assay will be further developed to make the assay viable for routine blood testing. Enzyme selectivity for homocysteine will be enhanced by directed evolution of existing clones. Sample preparation procedures will be optimized to remove potentially interfering substances in physiological fluids. Sensitivity will be improved 5 to 10 fold using fluorescent substrates, with the goal of precise, reproducible quantitation of homocysteine down to the range of 5-10 micromolar.
Neonatal screening assay for homocystinuria; home and doctor's office test strip for measurement of L-methionine and L-homocysteine concentrations in urine or blood.
