应用于RPA nfo反应产物结果可视化的胶体金试纸条制备与评价

Preparation and evaluation of colloidal gold strips for visualization of RPA nfo reaction product

旨在利用6-羧基荧光素(6-FAM)与6-FAM单克隆抗体、生物素与链霉亲和素之间的特异性结合,制备可用于nfo酶切割探针的重组酶聚合酶(RPA)反应产物结果可视化的胶体金侧向免疫层析试纸条(GICS)。通过杂交瘤法和诱生腹水法制备了高亲和力的6-FAM单克隆抗体(亲和力常数为5.38×10~9 L/mol),再运用高浓度NaCl破坏试验,确定了胶体金与6-FAM抗体的最佳标记pH值为8.0,最佳抗体标记量为12μg/mL,并使用模拟样本确定了最佳检测(T)线包被缓冲液为0.2 mol/L(pH值5.0)。然后使用RPA nfo反应产物作为实际样本,观察条带的深浅,最终确定在层析液为0.1 mol/L PB(pH值7.4)、T线包被1 mg/mL SA、反应液用量5μL及金标抗体用量10μL条件下,能实现试纸条最佳检测性能。自主研发的胶体金侧向免疫层析试纸条在加样5 min后能出现清晰的条带,与目前商品化试纸条的可视化判定结果一致,能替代现有的商品化试纸条,降低检测成本、缩短检测时间,对建立RPA nfo-GICS及RPA-CRISPR Cas12a-GICS检测方法奠定了基础。

Based on the specific binding between 6-FAM and anti-(6-FAM) monoclonal antibodies,biotin and streptavidin,colloidal gold lateral immunochromatographic strips were prepared,in this study,that can be used to visualize the results of recombinase polymerase amplification(RPA) reaction product of the probes cleaved by nfo enzyme.Firstly,6-FAM monoclonal antibodies with high affinity were prepared using the hybridoma and induced ascites methods(the affinity constant was 5.38×10^(9) L/mol).The labeling conditions of 6-FAM and colloidal gold were optimized by high concentration NaCl destruction test,the optimal labeling pH was 8.0 and the optimal labeling amount of colloidal gold was 12 μg/mL colloidal gold.Then,the optimal T-line coated buffer was determined to be 0.2 mol/L HAc-NaOAc(pH=5.0) using simulated samples.RPA nfo reaction products were used as actual samples to observe the depth of the strips.Finally,the optimal detection performance of the strips could be achieved under the conditions of 0.1 mol/L PB chromatography solution(pH=7.4),1 mg/mL SA coating,5 μL reaction solution and 10 μL gold labeled antibody dosage.The results was that the colloidal gold lateral immunochromatography strip we developed independently was able to show clear bands 5 min after sample loading,which was 100%(9/9) consistent with the results of the current commercial strips.This strip could replace the existing commercial strips,reducing the detection cost and shortening the detection time.The present result also laid a foundation for establishment of RPA nfo-GICS and RPA-CRISPR Cas12a-GICS detection methods.