基于可控相转变温度热敏高分子的人IgG酶联荧光免疫分析

以温度敏感高分子聚N－异丙基丙烯酰胺－丙烯酰胺P(NIP-AA)]作为载体，建立了酶联荧光免疫分析人IgG的新方法。AA摩尔含量为8％的高分子P(NIP-AA)其临界溶解温度为37 °C。竞争型免疫测定中，被固定的IgG和标准溶液（或样品）在33 °C均相条件下竞争性地与辣根过氧化物酶标记抗体反应，升高温度分离出高分子免疫复合物，沉淀重新溶解后通过偶联过氧化氢与对羟基苯乙酸的荧光反应进行定量，线性范围为100-1000 ng/mL，检测限为2.0 ng/mL。方法灵敏、快速操作简便，且提高了免疫反应效率。此外，灵敏度与用传统微孔板做载体相似，但测定时间更快（从100-120分钟减少到30分钟）。该法用于测定人血清中IgG的含量，结果满意。

Competitive Enzymatic Fluorescence Immunoassay for Human IgG by Using a Temperature‐Sensitive Phase Separating Polymer with Regulated Phase Transition Temperature

A new enzymatic fluorescence immunoassay for human IgG was developed using a temperature‐sensitive polymer, poly(N‐isopropylacrylamide‐co‐acrylamide) P(NIP‐AA)], as a carrier. The lower critical solution temperature of the P(NIP‐AA) containing molar fraction of 8% for AA was 37 °C. In a competitive immunoassay, immobilized IgG and the standard IgG (or sample) competed for binding to a horseradish peroxidase labeled antibody at 33 °C in homogeneous format. After changing the temperature to separate the polymer‐immune complex, the complex precipitate was re‐dissolved and determined by coupling with the fluorescence reaction of hydrogen peroxide and p‐hydroxyphenylacetic acid. The calibration graph for human IgG was linear over the range of 100–1000 ng/mL with a detection limit of 2.0 ng/mL. The method is rapid, sensitive and simple. The immune reaction efficiency was improved. In addition, the sensitivity of this method was close to that using traditional microtitration plates as carriers. However, the assay was much faster (the assay time decreased from 100–120 to 30 min). The method has been applied to the determination of the human IgG levels in human sera with satisfactory results.