链格孢霉毒素细交链格孢菌酮酸人工抗原的合成及其多克隆抗体制备

Synthesis of Tenuazonic Acid Artificial Antigens and Preparation of an Anti-tenuazonic Acid Polyclonal Antibody

摘要:

本研究采用肟化法用羧甲基羟胺半盐酸盐对链格孢霉毒素细交链格孢菌酮酸（Tenuaconic acid, TA）进行衍生后引入连接臂，得到了半抗原TAO。通过活性酯法将半抗原TAO分别与牛血清蛋白（Bovine serum albumin，BSA）偶联制备得到免疫原TAO-BSA、与血蓝蛋白（Keyhole Limpet Hemocyanin，KLH ）偶联制备得到包被原TAO-KLH。经透析纯化后分别采用紫外光谱、红外光谱扫描法对合成的人工抗原进行初步鉴定，制备得到的人工抗原的偶联比分别为18.3:1、38.7:1。此人工抗原免疫Balb/c小鼠后获得的鼠源抗血清进一步验证了人工抗原合成成功，制备得到特异性识别TA的多克隆抗体。通过ELISA棋盘法确定了最佳包被原浓度以及最佳抗体稀释度并建立了TA间接竞争ELISA检测方法，其灵敏度以半抑制浓度IC50表示为335.55 ng/mL，最低检出限（LOD）为19.00 ng/mL，定量线性检测范围为200.00 ~1563.00 ng/mL。

Abstract:

The oximation reaction was used to derivatize tenuazonic acid (TA), and a connecting arm was introduced to synthesize the hapten, TAO. The TAO-BSA immunogen and TAO-KLH coating antigen conjugates were prepared by the active ester method. After dialysis purification, UV spectrum scanning, Fourier transform infrared (FT-IR) spectroscopy, and animal immunization experiments were used to test and verify the synthesis products. The coupling ratios of the two conjugates were 18.3:1 and 38.7:1. Balb/c mice were immunized by TAO-BSA and a polyclonal TA antibody was obtained from the antiserum. The sensitivity of the mouse antiserum was measured by indirect competitive ELISA. The optimum concentrations of the coating antigen and antibody dilutions were determined by the chessboard method. The sensitivity, as measured by IC50, was 335.55 ng/mL. The detection limit (LOD) was 19.00 ng/mL, and the linearity range was 200.00~1563.00 ng/mL.