Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

L. M. Weigel, J. T. Belisle, J. D. Radolf, M. V. Norgard

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Abstract

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 β- 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 μg/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 μg of conjugated ampicillin per ml) and of free ampicillin (0.5 μg/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 μg of conjugated ampicillin per ml) and of free ampicillin (8 μg/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 β- lactams for the identification and analysis of PBPs.

Original languageEnglish (US)
Pages (from-to)330-336
Number of pages7
JournalAntimicrobial Agents and Chemotherapy
Volume38
Issue number2
StatePublished - 1994

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Penicillin-Binding Proteins
Digoxigenin
Ampicillin
Luminescence
Adenosine Monophosphate
Escherichia coli
Staphylococcus aureus
Lactams
Penicillins
Porins
X-Ray Film
Immunoblotting

ASJC Scopus subject areas

  • Pharmacology (medical)

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Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence. / Weigel, L. M.; Belisle, J. T.; Radolf, J. D.; Norgard, M. V.

In: Antimicrobial Agents and Chemotherapy, Vol. 38, No. 2, 1994, p. 330-336.

Research output: Contribution to journalArticle

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