Enable Biosciences Inc., South San Francisco, CA, USA.
Department of Clinical Sciences, Lund University CRC, Skåne University Hospital, Malmö, Sweden.
J Immunol Methods. 2022 Jul;506:113265. doi: 10.1016/j.jim.2022.113265. Epub 2022 Mar 28.
Multiplex Antibody-Detection by Agglutination-PCR (ADAP) assay was compared to singleplex standard radiobinding assays (RBA) to detect autoantibodies against insulin (IAA), GAD65 (GADA), islet antigen-2 (IA-2A), ZnT8 (ZnT8A) and tissue transglutaminase (TGA). Serum samples from 273 (114F/158M), 15-73 years of age healthy controls and 227 (109F/118M) newly diagnosed type 1 diabetes children, 1-11 years of age, were analyzed in both assay systems.The original WHO standard 97/550 and in-house reference standards for RBA were compared to ADAP. The ADAP and RBA generated parallel reference standards in all assays except TGA. Lower detection limits were observed in the ADAP assay for GADA,IAA and ZnT8A, markedly for TGA, but not for IA-2A. The Receiver Operating Characteristics (ROC) curve AUC analyses for pairwise comparison of ADAP with RBA showed no difference for GADA (n.s.), ADAP greater AUC for IAA (p = 0.005), RBA greater AUC for IA-2A (p = 0.0004) and ZnT8A (p < 0.0001) while ADAP TGA had a greater AUC compared to both RBA TGA-IgG (p < 0.0001) and TGA-IgA (p < 0.0001). These data suggest that the ADAP and RBA assays are comparable with equal performance for GADA, better ADAP performance for IAA while the RBA showed better performance in both IA-2A and ZnT8A associated with greater heterogeneity in autoantibody levels. The simultaneous analysis of 5 different autoantibodies by ADAP in sample volume reduced to only 4 μL and at an increased lower detection limit in all assays except IA-2A makes the ADAP automated autoantibody assay a distinct advantage for high throughput screening.
多重抗体检测凝集-PCR（ADAP）检测与单重标准放射结合检测（RBA）相比较，以检测针对胰岛素（IAA）、GAD65（GADA）、胰岛抗原-2（IA-2A）、锌转运体 8（ZnT8A）和组织转谷氨酰胺酶（TGA）的自身抗体。在这两个检测系统中分析了 273 例（114 例女性/158 例男性）年龄 15-73 岁的健康对照者和 227 例（109 例女性/118 例男性）年龄 1-11 岁的新诊断 1 型糖尿病患儿的血清样本。原始的世界卫生组织 97/550 标准和 RBA 的内部参考标准与 ADAP 进行了比较。除 TGA 外，ADAP 和 RBA 在所有检测中生成了平行的参考标准。在 ADAP 检测中，GADA、IAA 和 ZnT8A 的检测下限较低，TGA 的检测下限明显较低，但 IA-2A 则不然。ADAP 与 RBA 之间的两两比较的接收者操作特征（ROC）曲线 AUC 分析显示，GADA（无统计学意义）、IAA 的 ADAP AUC 更大（p=0.005）、IA-2A 的 RBA AUC 更大（p=0.0004）和 ZnT8A（p<0.0001），而 ADAP TGA 的 AUC 与 RBA TGA-IgG（p<0.0001）和 TGA-IgA（p<0.0001）相比均更大。这些数据表明，ADAP 和 RBA 检测具有可比性，GADA 具有相同的性能，IAA 的 ADAP 性能更好，而 RBA 在 IA-2A 和 ZnT8A 方面表现更好，与自身抗体水平的更大异质性有关。ADAP 可在仅 4μL 样本量的情况下同时分析 5 种不同的自身抗体，且除 IA-2A 外，所有检测的检测下限均增加，这使得 ADAP 自动化自身抗体检测成为高通量筛选的显著优势。
