METHOD FOR NEONATAL SCREENING FOR HOMOCYSTINURIA

描述：（申请人的描述）在第1阶段， 高半胱氨酸的灵敏度低至10-20 微摩尔，远低于50-500微摩尔的范围，这表明 同型胱氨酸尿症。对同型半胱氨酸检测试纸条的可行性进行了研究。 使用固定化形式的酶和比色法证明了这一点。 可视化方法，在20分钟内提供结果。在第二阶段，测试 试纸条检测将被完善用于新生儿筛查， 高胱氨酸尿症，以取代有缺陷的古特里测试，它不能检测 同型半胱氨酸酶的固定化将被优化用于装载和 活动，可视化化学将得到完善。稳定性和保质 生活将得到评估和必要的改善。蛋氨酸的表达 增加γ-裂解酶基因，降低酶的成本。溶液 将进一步开发检测方法，使其适用于常规血液检测 试验.酶对同型半胱氨酸的选择性将通过定向 现有克隆的进化。样本制备程序将得到优化 以去除生理流体中的潜在干扰物质。 使用荧光底物，灵敏度将提高5至10倍， 高半胱氨酸精确、可重复定量的目标 范围为5-10微摩尔。 拟定商业应用： 高胱氨酸尿症的新生儿筛查测定;家庭和医生办公室试纸条 测量尿液或血液中L-蛋氨酸和L-高半胱氨酸的浓度。

DESCRIPTION: (Applicant's description) 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. PROPOSED COMMERCIAL APPLICATION: 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.