Pestka J J
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824-1224.
J Immunol Methods. 1991 Feb 15;136(2):177-83. doi: 10.1016/0022-1759(91)90004-y.
A new immunoblot approach called ELISAGRAM was devised that combines the sensitivity and selectivity of competitive ELISA with the capacity of high performance thin layer chromatography (HPTLC) to separate structurally related haptens. The procedure involved (1) separation of haptens by monoclonal antibody, (3) incubation of NC with hapten-enzyme conjugate to identify unreacted antibody binding sites. (4) detection of bound enzyme conjugate with a precipitating substrate and (5) visual or densitometric assessment of inhibition bands indicative of a cross-reacting hapten. The technique was applied to two major mycotoxin families, the zearalenones and aflatoxins (AFs), which are to toxicological significance. Detection limit for zearalenone and alpha-zearalenol in the method was 300 pg/assay. AFB1, AFB2, and AFG1 were detectable at 380 pg/assay and AFG2 was detectable at 1500 pg/assay. Multiple standard curves for the zearalenones and AFs could be constructed using scanning densitometry. Cross-reactivity in ELISAGRAM curves was analogous to that found in competitive ELISA. This procedure should be widely applicable to the simultaneous quantitation and confirmation of multiple haptens with a single cross-reactive antibody.
一种名为酶联免疫吸附色谱图（ELISAGRAM）的新型免疫印迹方法被设计出来，它将竞争性酶联免疫吸附测定（ELISA）的灵敏度和选择性与高效薄层色谱法（HPTLC）分离结构相关半抗原的能力相结合。该程序包括：（1）通过单克隆抗体分离半抗原；（3）将硝酸纤维素膜（NC）与半抗原 - 酶偶联物孵育，以识别未反应的抗体结合位点；（4）用沉淀底物检测结合的酶偶联物；（5）对指示交叉反应性半抗原的抑制带进行视觉或光密度评估。该技术应用于两个主要的霉菌毒素家族，即玉米赤霉烯酮和黄曲霉毒素（AFs），它们具有毒理学意义。该方法中玉米赤霉烯酮和α - 玉米赤霉醇的检测限为300 pg/测定。黄曲霉毒素B1（AFB1）、黄曲霉毒素B2（AFB2）和黄曲霉毒素G1（AFG1）在380 pg/测定时可检测到，黄曲霉毒素G2（AFG2）在1500 pg/测定时可检测到。使用扫描光密度计可以构建玉米赤霉烯酮和黄曲霉毒素的多条标准曲线。酶联免疫吸附色谱图曲线中的交叉反应性与竞争性酶联免疫吸附测定中发现的类似。该程序应广泛适用于用单一交叉反应抗体同时定量和确证多种半抗原。
