Abstract：
OBJECTIVE: Neutrophil gelatinase-associated lipocalin (NGAL) has been identified by the International Nephrology Association (INA) as a promising biomarker for the early evaluation of renal injury. This study aimed to develop and evaluate NGAL test strips as a rapid, simple, and economical method for the early diagnosis of acute kidney injury (AKI).
METHODS: Recombinant prokaryotic expression vectors, purified NGAL protein, and anti-NGAL monoclonal antibodies were prepared. NGAL test strips were developed, and serum samples were collected from healthy individuals and patients with early-stage kidney injury at the Third Affiliated Hospital of Sun Yat-sen University between January 2023 and May 2024. Samples were tested using both the self-made strips and commercially available reagents.
RESULTS: The NGAL test strip comprised a conjugate pad containing 0.2 μL of fluorescent microspheres conjugated with anti-NGAL monoclonal antibody (McAb7#), a test line containing 1 mg/mL of a different anti-NGAL monoclonal antibody (McAb3#), and a control line containing 0.5 mg/mL of goat anti-mouse IgG. The test utilized 60 μL of sample (30 μL serum diluted with 30 μL of sample diluent) and was completed within 15 min at 25 °C and 35 %-85 % relative humidity. The developed strip accurately detected NGAL, demonstrating good linearity within the range of 0-160 ng/mL (R2 = 0.9943). The sensitivity and specificity of the NGAL strip for AKI diagnosis were 86.1 % and 78.8 %, respectively, comparable to the performance of commercially available testing reagents.
CONCLUSIONS: The developed test strip, utilizing anti-NGAL antibodies coupled with fluorescent microspheres, effectively detected trace amounts of NGAL protein in serum samples.
METHODS: Recombinant prokaryotic expression vectors, purified NGAL protein, and anti-NGAL monoclonal antibodies were prepared. NGAL test strips were developed, and serum samples were collected from healthy individuals and patients with early-stage kidney injury at the Third Affiliated Hospital of Sun Yat-sen University between January 2023 and May 2024. Samples were tested using both the self-made strips and commercially available reagents.
RESULTS: The NGAL test strip comprised a conjugate pad containing 0.2 μL of fluorescent microspheres conjugated with anti-NGAL monoclonal antibody (McAb7#), a test line containing 1 mg/mL of a different anti-NGAL monoclonal antibody (McAb3#), and a control line containing 0.5 mg/mL of goat anti-mouse IgG. The test utilized 60 μL of sample (30 μL serum diluted with 30 μL of sample diluent) and was completed within 15 min at 25 °C and 35 %-85 % relative humidity. The developed strip accurately detected NGAL, demonstrating good linearity within the range of 0-160 ng/mL (R2 = 0.9943). The sensitivity and specificity of the NGAL strip for AKI diagnosis were 86.1 % and 78.8 %, respectively, comparable to the performance of commercially available testing reagents.
CONCLUSIONS: The developed test strip, utilizing anti-NGAL antibodies coupled with fluorescent microspheres, effectively detected trace amounts of NGAL protein in serum samples.
摘要：
目的：中性粒细胞明胶酶相关脂质运载蛋白（NGAL）已被国际肾脏病学协会（INA）确定为早期评估肾损伤的有希望的生物标志物。本研究旨在开发和评估NGAL测试条作为一种快速，简单,急性肾损伤(AKI)早期诊断的经济方法。
方法：重组原核表达载体，纯化的NGAL蛋白，制备抗NGAL单克隆抗体。开发了NGAL测试条，和血清样本收集自2023年1月至2024年5月在中山大学附属第三医院的健康个体和早期肾损伤患者。使用自制条带和市售试剂测试样品。
结果：NGAL测试条包含含有0.2μL与抗NGAL单克隆抗体（McAb7＃）偶联的荧光微球的偶联垫，含有1mg/mL不同抗NGAL单克隆抗体（McAb3#）的测试线，和含有0.5mg/mL山羊抗小鼠IgG的对照品系。该测试使用60μL样品(用30μL样品稀释剂稀释的30μL血清),并在25°C和35%-85%相对湿度下在15分钟内完成。开发的试纸准确检测NGAL，在0-160ng/mL范围内表现出良好的线性（R2=0.9943）。NGAL试纸条诊断AKI的敏感性和特异性分别为86.1%和78.8%,分别,与市售测试试剂的性能相当。
结论：开发的测试条，利用抗NGAL抗体与荧光微球偶联，有效检测血清样品中的微量NGAL蛋白。
方法：重组原核表达载体，纯化的NGAL蛋白，制备抗NGAL单克隆抗体。开发了NGAL测试条，和血清样本收集自2023年1月至2024年5月在中山大学附属第三医院的健康个体和早期肾损伤患者。使用自制条带和市售试剂测试样品。
结果：NGAL测试条包含含有0.2μL与抗NGAL单克隆抗体（McAb7＃）偶联的荧光微球的偶联垫，含有1mg/mL不同抗NGAL单克隆抗体（McAb3#）的测试线，和含有0.5mg/mL山羊抗小鼠IgG的对照品系。该测试使用60μL样品(用30μL样品稀释剂稀释的30μL血清),并在25°C和35%-85%相对湿度下在15分钟内完成。开发的试纸准确检测NGAL，在0-160ng/mL范围内表现出良好的线性（R2=0.9943）。NGAL试纸条诊断AKI的敏感性和特异性分别为86.1%和78.8%,分别,与市售测试试剂的性能相当。
结论：开发的测试条，利用抗NGAL抗体与荧光微球偶联，有效检测血清样品中的微量NGAL蛋白。
