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ELISA-based detection of gentamicin and vancomycin in protein-containing samples.

Odekerken JC, Logister DM, Assabre L, Arts JJ, Walenkamp GH, Welting TJ - Springerplus (2015)

Bottom Line: Both methods are heavily influenced due to proteins in the samples.Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples.An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Orthopaedics, Department of Orthopaedic Surgery, Research School CAPHRI, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.

ABSTRACT

Background: Orthopaedic implant infections are treated by surgical debridement, systematic antibiotic treatment or local antibiotic treatment with antibiotic-loaded beads. Currently antibiotic concentrations in wound exudate, serum, urine or tissue samples are determined with HPLC or fluorescent spectrometric assays. Both methods are heavily influenced due to proteins in the samples.

Questions/purposes: Is ELISA capable to detect gentamicin and vancomycin in protein-containing samples like serum and wound exudate.

Methods: Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples. An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection.

Results: The developed ELISAs perform at a detection range of 2-500 ng/ml gentamycin and 20-5000 ng/ml vancomycin. Both ELISAs were capable of detecting these antibiotics in human serum and wound exudate without being compromised by the presence of proteins. We did not detect cross-reactivity for gentamicin in the vancomycin ELISA or vice versa.

Conclusions: The antibiotic ELISAs detect gentamicin and vancomycin at low concentrations in protein-rich samples and they can be used as a high throughput and cost-effective alternative for chromatographic or fluorescent methods.

Clinical relevance: These ELISAs can be used to detect very low gentamicin or vancomycin concentrations in clinical samples or assess novel orthopaedic antibiotic release systems in in vitro and in vivo studies.

No MeSH data available.


Related in: MedlinePlus

ELISA for gentamicin and vancomycin. A combined hapten of both gentamicin and vancomycin was used to coat wells for the detection of either gentamicin (black circles) or vancomycin (black squares) in a calibrations series of the respective antibiotics. Error bars indicate standard deviation
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Fig4: ELISA for gentamicin and vancomycin. A combined hapten of both gentamicin and vancomycin was used to coat wells for the detection of either gentamicin (black circles) or vancomycin (black squares) in a calibrations series of the respective antibiotics. Error bars indicate standard deviation

Mentions: By using BSA as a coatable protein for generating the gentamicin and vancomycin haptens, we explored the possibility of combining different antibiotics in one hapten coupling reaction. This potentially offers the opportunity to generate a combined assay for the detection of both antibiotics by using the same hapten in separate dedicated ELISAs. A combined hapten of both gentamicin and vancomycin cross-linked to BSA indeed allowed the detection of either gentamicin or vancomycin by their respective antibodies, in a comparable range as the individual ELISA protocols (Fig. 4). Furthermore, when this hapten was used for the ELISA detection of gentamicin, it was not influenced by vancomycin presence in the sample and vice versa.Fig. 4


ELISA-based detection of gentamicin and vancomycin in protein-containing samples.

Odekerken JC, Logister DM, Assabre L, Arts JJ, Walenkamp GH, Welting TJ - Springerplus (2015)

ELISA for gentamicin and vancomycin. A combined hapten of both gentamicin and vancomycin was used to coat wells for the detection of either gentamicin (black circles) or vancomycin (black squares) in a calibrations series of the respective antibiotics. Error bars indicate standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4628023&req=5

Fig4: ELISA for gentamicin and vancomycin. A combined hapten of both gentamicin and vancomycin was used to coat wells for the detection of either gentamicin (black circles) or vancomycin (black squares) in a calibrations series of the respective antibiotics. Error bars indicate standard deviation
Mentions: By using BSA as a coatable protein for generating the gentamicin and vancomycin haptens, we explored the possibility of combining different antibiotics in one hapten coupling reaction. This potentially offers the opportunity to generate a combined assay for the detection of both antibiotics by using the same hapten in separate dedicated ELISAs. A combined hapten of both gentamicin and vancomycin cross-linked to BSA indeed allowed the detection of either gentamicin or vancomycin by their respective antibodies, in a comparable range as the individual ELISA protocols (Fig. 4). Furthermore, when this hapten was used for the ELISA detection of gentamicin, it was not influenced by vancomycin presence in the sample and vice versa.Fig. 4

Bottom Line: Both methods are heavily influenced due to proteins in the samples.Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples.An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Experimental Orthopaedics, Department of Orthopaedic Surgery, Research School CAPHRI, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands.

ABSTRACT

Background: Orthopaedic implant infections are treated by surgical debridement, systematic antibiotic treatment or local antibiotic treatment with antibiotic-loaded beads. Currently antibiotic concentrations in wound exudate, serum, urine or tissue samples are determined with HPLC or fluorescent spectrometric assays. Both methods are heavily influenced due to proteins in the samples.

Questions/purposes: Is ELISA capable to detect gentamicin and vancomycin in protein-containing samples like serum and wound exudate.

Methods: Two specific competitive ELISA-assays were set-up to detect either gentamicin or vancomycin in protein-rich samples. An antibiotic-BSA hapten was generated as a coatable antigen and commercially available antibodies were applied for downstream immunodetection.

Results: The developed ELISAs perform at a detection range of 2-500 ng/ml gentamycin and 20-5000 ng/ml vancomycin. Both ELISAs were capable of detecting these antibiotics in human serum and wound exudate without being compromised by the presence of proteins. We did not detect cross-reactivity for gentamicin in the vancomycin ELISA or vice versa.

Conclusions: The antibiotic ELISAs detect gentamicin and vancomycin at low concentrations in protein-rich samples and they can be used as a high throughput and cost-effective alternative for chromatographic or fluorescent methods.

Clinical relevance: These ELISAs can be used to detect very low gentamicin or vancomycin concentrations in clinical samples or assess novel orthopaedic antibiotic release systems in in vitro and in vivo studies.

No MeSH data available.


Related in: MedlinePlus