基于流式细胞微球阵列的 BALB/c 小鼠 T 细胞依赖性抗体反应（TDAR）方法。

A T-cell-dependent antibody response (TDAR) method in BALB/c mice based on a cytometric bead array.

机构信息

Hainan Medical University, Research Center for Drug Safety Evaluation of Hainan Province, Haikou, China.

Haikou Municipal People's Hospital and Xiangya Medical College Affiliated Hospital, Nursing Department, Haikou, China.

出版信息

J Immunotoxicol. 2022 Dec;19(1):34-40. doi: 10.1080/1547691X.2022.2067273.

DOI:10.1080/1547691X.2022.2067273

PMID:35477374

Abstract

Most current methods to assess T-cell-dependent antibody responses (TDAR) are semi-quantitative and based on measures of antibody titer generated against a standard antigen like keyhole limpet hemocyanin (KLH). The precision, sensitivity, and convenience of TDAR assays might be improved by applying rapid, sensitive, specific cytometric bead assays (CBA). In the study here, KLH antigen was covalently coupled onto the surface of cytometric beads using immune microsphere technology, and IgM antibody capture spheres were prepared for use in pretreatment processing of samples. The working parameters associated with this novel TDAR-CBA system were optimized in orthogonal experiments. The optimal concentration of the KLH coating solution in this system was 160 μg/ml, that of the anti-KLH IgG capture spheres 6.0 × 10/ml, and the optimal dilution of fluorescein isothiocyanate (FITC)-conjugated Affini-Pure Goat Anti-Mouse IgG (H + L) was 60 μg/ml. Repeated tests indicated that this approach yielded good linearity ( = 0.9937) method, with a within-run precision of 3.1-4.9%, and a between-run precision of 4.4-4.9%. This new approach had a limit of detection of 113.43 ng/ml (linear range = 390.63-50 000), and an interference rate of just 0.04-3.51%. Based on these findings, it seems that a new mouse TDAR assay based on CBA can be developed that would appear to be more sensitive, accurate, and precise than the current TDAR assay approaches based on traditional ELISA.

摘要

目前评估 T 细胞依赖的抗体反应（TDAR）的大多数方法都是半定量的，并且基于针对标准抗原（如血蓝蛋白）产生的抗体滴度的测量。通过应用快速、灵敏、特异的细胞计数珠分析（CBA），可以提高 TDAR 测定的精度、灵敏度和便利性。在本研究中，使用免疫微球技术将 KLH 抗原共价偶联到细胞计数珠的表面，并制备 IgM 抗体捕获珠，用于样品预处理。在正交实验中优化了与这种新型 TDAR-CBA 系统相关的工作参数。该系统中 KLH 包被溶液的最佳浓度为 160μg/ml，抗 KLH IgG 捕获珠的最佳浓度为 6.0×10/ml，异硫氰酸荧光素（FITC）标记的 Affini-Pure 山羊抗小鼠 IgG（H+L）的最佳稀释度为 60μg/ml。重复测试表明，该方法具有良好的线性（r = 0.9937），批内精密度为 3.1-4.9%，批间精密度为 4.4-4.9%。该新方法的检测限为 113.43ng/ml（线性范围 = 390.63-50000），干扰率仅为 0.04-3.51%。基于这些发现，似乎可以开发一种基于 CBA 的新型小鼠 TDAR 测定方法，该方法似乎比基于传统 ELISA 的当前 TDAR 测定方法更灵敏、准确和精确。