Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.
Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.
复制标题
地高辛-氨苄青霉素缀合物，用于通过化学发光检测青霉素结合蛋白。
DOI：
10.1128/aac.38.2.330
复制
发表时间：
1994
影响因子：
4.9
通讯作者：
Norgard,MV
中科院分区：
文献类型：
--
作者：
Weigel,LM;Belisle,JT;Radolf,JD;Norgard,MV
This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled beta-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 micrograms/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 microgram of conjugated ampicillin per ml) and of free ampicillin (0.5 microgram/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 micrograms of conjugated ampicillin per ml) and of free ampicillin (8 micrograms/ml) were widely disparate, suggesting that the DIG-AMP conjugate was too large and/or hydrophobic to traverse the E. coli outer membrane via porins. DIG-AMP binding assays with E. coli and S. aureus cell envelopes revealed profiles of PBPs similar to those detected with 125I-ampicillin or [3H]penicillin. DIG-AMP binding to PBPs was completely inhibited in competition experiments with free ampicillin or penicillin, supporting the specificity of the DIG-AMP conjugate for PBPs. DIG-AMP thus represents an advantageous alternative to radioactive beta-lactams for the identification and analysis of PBPs.
