基于独特型-抗独特型玉米赤霉烯酮间接竞争ELISA检测方法的建立

Development of indirect competitive inhibition ELISA method for zearalenone based on idiotype-anti-idiotype reaction

目的建立基于独特型．抗独特型玉米赤霉烯酮（zearalenone，ZEN）间接竞争ELISA检测方法，并进行验证。方法采用ProteinG亲和层析法纯化ZEN的p型抗独特型单抗1D5（Ab213-1D5）腹水；以ZEN单抗Fab片段包被酶标板，Ab2β-1D5为一抗，建立ZEN的竞争ELISA检测方法，绘制标准抑制曲线，根据回归方程计算半数抑制浓度（IC50），并确定检出限（IC10）及线性范围。以ZEN类似物呕吐毒素、伏马毒素、赭曲霉素A代替ZEN作为竞争抗原，用建立的方法进行检测，验证方法的特异性；配制6个浓度的ZEN溶液（20、30、40、50、60和75ng／m1），用建立的方法进行检测，计算试验内及试验间回收率和变异系数（CV），验证方法的准确性和精密性。结果Ab2β-1D5单抗的纯化效果较好，浓度为4mg／ml，效价为1：6×104；ZEN单抗Fab片段的最佳包被浓度为1μg／ml。建立的ZEN竞争EHSA检测方法的线性回归方程为：y=-34．099x＋81．857，R2=0．9944，IC50为8．60ng／ml，IC10为0．58ng／ml，线性范围为0．58-128．03ng／ml；该方法检测ZEN类似物伏马毒素、赭曲霉素A和呕吐毒素的交叉反应率均小于0．01％；检测不同浓度ZEN的试验内平均回收率在98．65％-113．45％之间，试验间平均回收率在82．96％-98．00％之间；试验内cy值在0．48％。6．40％之间，试验间cy值在0．94％-8．27％之间。结论应用ZEN单抗的Fab片段和ZEN的B型抗独特型单抗建立了ZEN的间接竞争ELISA检测方法，该方法特异性较强，准确性和精密性良好，且成本较低，快速简便，易于标准化，适用于样品的大量筛选。

Objective To develop and verify an indirect competitive inhibition ELISA method for zearalenone （ZEN） based on idiotype-anti-idiotype reaction. Methods The ascites of β-type monoclonal anti-idiotype antibody （Ab2β-1D5） against ZEN was purified by Protein G affinity chromatography. Microtiter plate was coated with monoclonal antibody against ZEN, based on which a competitive ELISA method for ZEN was developed using Ab2β-1D5 as the first antibody. The standard curve of the developed method was plotted, and the median inhibitory concentration （ICs0）, detection limit （IC10） and linear range were obtained according to the regression equatiorr The developed method was verified for specificity by substitution of ZEN with its analogues vomitoxin, Fumonisin and ochratoxin A. The ZEN solution at six concentrations （20, 30, 40, 50, 60 and 75 ng/ml） was determined by the developed method, and the recovery rates and coefficients of variation （CVs） in intra- and inter-assays were calculated to verify the accuracy and precision of the method. Results The McAb against Ab2β-1D5 was effectively purified, of which the concentration was 4 mg / ml and the titer was 1 ： 6 x 104. The optimal concentration of Fab fragment of McAb against ZEN was 1 μg/ml. The linear regression equation of developed ELISA method was： y = -34. 099 x ＋ 81. 857, Rz = 0. 994 4, while the IC50 was 8. 60 ng/ml, the IC10 was O. 58 ng/ml, and the linear range was O. 58 - 128. 03 ng/m]. All the cross reaction rates with vomitoxin, Fumonisin and ochratoxin A were less than 0. 01%. The mean recovery rates of ZEN at various concentrations in intra- and inter-assays were 98. 65% - 113. 45% and 82. 96% -98. 00%, while the CVs were 0. 48% N 6. 40% and 0. 94% N 8. 27%, respectively. Conclusion The indirect com- petitive inhibition ELISA method for ZEN was developed by using the Fab fragment of McAb and D-type monoclonal anti- idiotype antibody against ZEN, which showed high specificity, accuracy and pr6cision, and was low-cost, rapid, simple, easy to be standardized and suitable for large screening of samples.