Antibody‐affinity blotting, a sensitive technique for the detection of α‐fetoprotein separated by lectin affinity electrophoresis in agarose gels
Antibody‐affinity blotting, a sensitive technique for the detection of α‐fetoprotein separated by lectin affinity electrophoresis in agarose gels
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抗体亲和印迹，一种用于检测通过凝集素亲和电泳在琼脂糖凝胶中分离的甲胎蛋白的灵敏技术
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发表时间：
1985
期刊：
影响因子：
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通讯作者：
H. Hirai
中科院分区：
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作者：
K. Taketa;E. Ichikawa;H. Taga;H. Hirai
开发了一种灵敏的抗体亲和印迹技术，用于在琼脂糖凝胶亲和电泳中在高背景凝集素和其他血清蛋白存在的情况下检测少量人甲胎蛋白（AFP）。通过印迹将 AFP 特异性转移到硝酸纤维素膜上，该膜预涂有亲和纯化的马或山羊抗人 AFP 多克隆抗体。 AFP 转移物用兔免疫球蛋白处理至人 AFP，然后用亲和纯化的山羊抗兔 IgG(H+L)-辣根过氧化物酶缀合物与 3,3'-二氨基联苯胺进行显色。该方法使我们能够检测低至 4 pg/mm2 的 AFP（或 4 ng/ml AFP，将 3 µl 样品体积施加到 0.5 × 6 × 1 mm 槽中）。在凝集素亲和电泳中，将 5 μl 125-500 ng/ml AFP 加入 1 × 5 × 1 mm 槽中，定量检测占总 AFP 10% 以上的 AFP 条带。当使用 AFP 或其免疫球蛋白组分的马抗血清预包被硝酸纤维素膜时，琼脂糖凝胶中伴刀豆球蛋白 A (Con-A) 的背景染色变得明显。在随后的抗体处理之前，通过用 0.2 M α-甲基-D-甘露糖苷清洗转移物可以消除这种情况。用 α-甲基-D-甘露糖苷洗涤还可以抵消 Con-A 分离的 AFP 条带染色不均匀的情况。抗体亲和印迹最适合在存在不同量的其他背景蛋白质的情况下将特定蛋白质定量转移到硝酸纤维素膜上。
A sensitive technique of antibody‐affinity blotting was developed for the detection of small amounts of human α‐fetoprotein (AFP) in the presence of high background lectins and other serum proteins in agarose‐gel affinity electrophoresis. AFP was specifically transferred by blotting to nitrocellulose membranes which were precoated with affinity‐purified horse or goat polyclonal antibodies to human AFP. The AFP transfers were treated with rabbit immunoglobulins to human AFP, followed by affinity‐purified goat anti‐rabbit IgG(H+L)‐horseradish peroxidase conjugate for color development with 3,3′‐diaminobenzidine. The method allowed us to detect as low as 4 pg/mm2 AFP (or 4 ng/ml AFP with a 3 μl sample volume applied to a 0.5 × 6 × 1 mm trough). In lectin affinity electrophoresis, AFP bands constituting more than 10% of the total AFP were quantitatively detected with 5 μl of 125–500 ng/ml AFP applied to a 1 × 5 × 1 mm trough. When horse antiserum to AFP or its immunoglobulin fraction was used to precoat nitrocellulose membranes, the background stain due to concanavalin A(Con‐A) in agarose gel became marked. This was eliminated by washing the transfers with 0.2 M α‐methyl‐D‐mannoside before subsequent antibody treatments. The washing with α‐methyl‐D‐mannoside also counteracted the uneven staining of separated AFP bands by Con‐A. The antibody‐affinity blotting suits best for the quantitative transfer of a specific protein to nitrocellulose membrane in the presence of varying amounts of other background proteins.