Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity

Stok, Jeanette E. and Baldwin, Jack E. (2006) Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity. Analytica Chimica Acta, 577 2: 153-162. doi:10.1016/j.aca.2006.06.040


Author Stok, Jeanette E.
Baldwin, Jack E.
Title Development of enzyme-linked immunosorbent assays for the detection of deacetoxycephalosporin C and isopenicillin N synthase activity
Journal name Analytica Chimica Acta   Check publisher's open access policy
ISSN 0003-2670
1873-4324
Publication date 2006-09-08
Year available 2006
Sub-type Article (original research)
DOI 10.1016/j.aca.2006.06.040
Volume 577
Issue 2
Start page 153
End page 162
Total pages 10
Place of publication Amsterdam, The Netherlands
Publisher Elsevier
Language eng
Subject 0304 Medicinal and Biomolecular Chemistry
030405 Molecular Medicine
0399 Other Chemical Sciences
Abstract Although there are a number of existing assays for monitoring the activity of both isopenicillin N synthase (IPNS) and deacetoxycephalosporin C synthase (DAOCS), none have demonstrated the qualities required for screening a mutant library. Hence, enzyme-linked immunosorbent assays (ELISAs) for IPNS and DAOCS were developed based on the detection of the catalytic turnover products isopenicillin N and cephalexin/phenylacetyl-7-aminodeacetoxycephalosporanic acid (G-7-ADCA), respectively. These assays are relatively fast compared to existing assays, such as the hole-plate bioassay, and are amenable with high-throughput screening. Both the IPNS and DAOCS-ELISAs were optimised for use with crude protein extracts rather than purified protein, thereby eliminating any additional time required for purification. The ELISA developed for the detection of cephalexin had an IC50 value of 154±9 ngmL−1 and LOD of 7.2±2.2 ngmL−1 under conditions required for the assay. Good recoveries and correlation was observed for spiked samples when the concentration of crude protein was kept below 1 mgmL−1. The DAOCS-ELISAwas found to have increased sensitivity compared to the hole-plate bioassay (10.3 µgmL−1). The IPNS-ELISA did not significantly increase the sensitivity (approximately 5 µgmL−1) compared to that of the hole-plate bioassay (16 µgmL−1) for isopenicillin N. The minimum amount of crude protein extract required for producing detectable amounts of product for both assays was below 0.5% of the maximum amount of protein that the assay could contain without any effect on the ELISA. This suggests that when screening a mutant library, mutants producing low amounts of the product could still be detected using these assays.
Keyword Enzyme-linked immunosorbent assay
Deacetoxycephalosporin C synthase
Isopenicillin N synthase
Cephalexin
Ampicillin
Isopenicillin N
Penicillin G
High-throughput
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Chemistry and Molecular Biosciences
 
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Created: Thu, 17 Dec 2009, 11:57:49 EST by Macushla Boyle on behalf of Faculty of Science