COMPARISON OF 2 METHODS OF PREPARING ENZYME-ANTIBODY CONJUGATES - APPLICATION OF THESE CONJUGATES FOR ENZYME IMMUNOASSAY
COMPARISON OF 2 METHODS OF PREPARING ENZYME-ANTIBODY CONJUGATES - APPLICATION OF THESE CONJUGATES FOR ENZYME IMMUNOASSAY
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DOI：
10.1016/0003-2697(79)90117-9
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发表时间：
1979-01-01
影响因子：
2.9
通讯作者：
MARKS, V
中科院分区：
文献类型：
--
作者：
OSULLIVAN, MJ;GNEMMI, E;MARKS, V
对两种制备酶抗体偶联物的方法进行了评价。两种方法均获得了较高的共轭物收率。第一种方法使用了均双功能交联试剂N，N' -邻苯二马来酰亚胺。巯基残基通过与甲基巯基丁胺酸反应引入第二抗体。修饰抗体与N，N′-邻苯二马来酰亚胺反应，通过凝胶过滤去除多余的试剂，活化抗体与- β -半乳糖苷酶交联。高达80%的酶与免疫活性抗体结合，酶活性保留约90%。第二种方法采用异双官能团间马来酰亚苯甲酰- n -羟基琥珀酰亚胺酯（MBS）。抗体与MBS反应，凝胶过滤去除多余的试剂，活化抗体与- β -半乳糖苷酶交联。大约80%的酶与免疫活性抗体结合，大约90%的酶和抗体活性保持不变。用这两种方法制备的偶联物在免疫分析系统中用作标记物，能够检测大约5-10 ng的第一抗体。MBS操作更简单，更容易适应大规模工作，结果可重复性更高，所产生的偶联物能够检测到较低浓度的第一抗体。
Two methods of preparing enzyme-antibody conjugates were evaluated. High yields of conjugate were obtained with both methods. The 1st procedure utilized the homobifunctional crosslinking reagent N,N''-o-phenylenedimaleimide. Sulfydryl residues were introduced into 2nd antibodies by reaction with methyl-mercaptobutyrimidate. Modified antibodies were reacted with N,N''-o-phenylenedimaleimide, excess reagent was removed by gel filtration and activated antibodies were cross-linked to .beta.-galactosidase. Up to 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of enzyme activity. The 2nd method utilized the heterobifunctional m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). The antibodies were reacted with MBS, excess reagent was removed by gel filtration and activated antibodies were crosslinked to .beta.-galactosidase. Approximately 80% of the enzyme was conjugated to immunologically active antibody with approximately 90% retention of both enzyme and antibody activity. Conjugates prepared using these 2 procedures were used as labels in an immunoassay system and were able to detect approximately 5-10 ng of 1st antibodies. The MBS procedure was simpler to perform could more easily be adapted to large-scale work, gave more reproducible results and conjugates produced were able to detect slightly lower concentrations of 1st antibody.