METHOD FOR NEONATAL SCREENING FOR HOMOCYSTINURIA

描述：(申请人的描述)在第一阶段的酶分析中 同型半胱氨酸被开发出来，其灵敏度可降至10-20浓度 微摩尔，远低于50-500微摩尔的范围 同型半胱氨酸尿。建立同型半胱氨酸检测试纸的可行性 使用固定化形式的酶和比色法进行演示 可视化方法，在20分钟内提供结果。在第二阶段，测试 试条试验将完善用于新生儿筛查 同型半胱氨酸尿症取代有缺陷的格思里试验，后者无法检测到 同型半胱氨酸。酶的固定化将进行优化，以加载和 活跃性，可视化化学将被提炼。稳定性和货架 生活将根据需要进行评估和改善。甲硫氨酸的表达 将增加伽马裂解酶基因，以降低酶的成本。解决方案 将进一步开发化验方法，使其适用于常规血液 测试。同型半胱氨酸的酶选择性将通过定向提高 现有克隆的进化。将优化样品制备程序 去除生理液体中潜在的干扰物质。 使用荧光衬底，灵敏度将提高5到10倍， 同型半胱氨酸精确、可重复性定量的目标 5-10微摩尔的范围。 建议的商业应用： 新生儿同型半胱氨酸尿症的筛查试验.家庭和医生办公室的试纸 尿或血中L-蛋氨酸和L-同型半胱氨酸浓度的测定。

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.