Peter J, Unverzagt C, Lenz H, Hoesel W
Technische Universität München, Lichtenbergstrasse 4, Garching, 85748, Germany.
Anal Biochem. 1999 Aug 15;273(1):98-104. doi: 10.1006/abio.1999.4215.
When isolating proteins from complex biological material by immunosorption, the nonspecific binding and elution of other proteins present in much larger concentrations than the target protein are often a general problem, preventing the isolation of a pure protein. To improve this situation we have developed a new indirect immunosorption method, which makes use of a digoxigenylated anti-analyte antibody. This antibody is linked to streptavidin-coated magnetic beads via a biotinylated anti-digoxigenin antibody. After binding of the analyte to the affinity matrix, the complex composed of analyte and digoxigenylated anti-analyte antibody is specifically eluted with a solution of digoxigenin-lysine at pH 7.3. Coelution of nonspecifically bound proteins was highly reduced as revealed by SDS-PAGE when compared to the acidic eluates of the direct immunosorption. The efficiency of the indirect immunosorption method was demonstrated with the isolation of free prostate-specific antigen (PSA) and PSA/alpha(1)-antichymotrypsin complex from human serum and subsequent analysis of the intact proteins by SDS-PAGE and MALDI-TOF-mass spectrometry.
当通过免疫吸附从复杂生物材料中分离蛋白质时，存在比目标蛋白浓度高得多的其他蛋白质的非特异性结合和洗脱通常是一个普遍问题，这会妨碍纯蛋白的分离。为改善这种情况，我们开发了一种新的间接免疫吸附方法，该方法利用了地高辛配基化的抗分析物抗体。这种抗体通过生物素化的抗地高辛配基抗体与链霉亲和素包被的磁珠相连。分析物与亲和基质结合后，由分析物和地高辛配基化的抗分析物抗体组成的复合物用pH 7.3的地高辛配基赖氨酸溶液特异性洗脱。与直接免疫吸附的酸性洗脱液相比，SDS-PAGE显示非特异性结合蛋白的共洗脱显著减少。通过从人血清中分离游离前列腺特异性抗原（PSA）和PSA/α(1)-抗糜蛋白酶复合物，并随后通过SDS-PAGE和MALDI-TOF质谱对完整蛋白进行分析，证明了间接免疫吸附方法的效率。
