以pH敏感相分离高分子为载体的酶联荧光免疫分析法测定人IgG

Enzyme-linked Fluorescence Immunoassay for Human IgG by Using a pH-Sensitive Phase Separating Polymer as Carrier

pH敏感高分子因其独特的pH敏感性质而在药物的控制释放[1,2]、分子分离[3]以及生物传感器[4]等方面得到应用. 应用于免疫分析时, 要求pH敏感高分子在溶液pH 7.4左右波动时做出响应, 过多偏离生理pH会对免疫反应生成的抗原-抗体复合物造成不同程度的破坏. 目前, pH敏感高分子并未在免疫分析中广泛应用, 这主要是由于高分子的相转变pH大多在4或10左右[5,6]. 我们[7]曾合成了37 ℃下相转变pH在5.6左右的pH敏感高分子, 并将其作为免疫反应载体, 建立了乙肝表面抗原的分析系统, 虽然其相转变pH比以往的高分子更接近于中性[5], 但并没有达到生理pH值7.4范围, 分离时仍可能对免疫复合物造成一定的损害.

Poly(N-isopropylacrylamide-N,N-dimethyl aminopropyl methacrylamide) [P(NIP-DMAPM)], a novel linear water-soluble pH-sensitive polymer whose phase transition pH is about 7.4 at 37 ℃, was synthesized. P(NIP-DMAPM) polymer precipitated out of water above a critical pH 7.4 at 37 ℃ and redissolved at pH below 7.4. P(NIP-DMAPM) was used as a novel separation carrier for the reactants in immunoassay and a enzyme-linked fluorescence immunoassay for human IgG was built. The linear range of determination was 0~1 700 ng/mL and the limit of detection was 2.2 ng/mL. The sensitivity of this method was close to that of traditional ELISA by using same reactants. However, the assay was much faster. This method has been applied to the determination of the human IgG levels in human serum with satisfactory results.