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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

Hapten evaluation. a Ponceau S staining indicates protein presence in the coupled haptens. b Immunoblotting indicates corresponding antibiotic presence in both coupled haptens specifically
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Fig1: Hapten evaluation. a Ponceau S staining indicates protein presence in the coupled haptens. b Immunoblotting indicates corresponding antibiotic presence in both coupled haptens specifically

Mentions: The development of these ELISAs required the coating of gentamicin or vancomycin to standard polystyrene ELISA wells. As the antibiotics themselves cannot be coated directly to well plates, we generated BSA haptens of each individual antibiotic by EDC-mediated coupling between a BSA carrier and the antibiotic. To acquire evidence of successful hapten generation we separated freshly prepared BSA-gentamicin and BSA-vancomycin haptens by SDS-PAGE, transferred the separated proteins to nitrocellulose membranes and performed anti-gentamicin or anti-vancomycin immunoblotting. To first visualize the total amount of SDS-PAGE-loaded hapten/protein, membranes were stained with Ponceau S after electro-blotting of SDS-PAGE gels. BSA runs at 66 kDa in SDS-PAGE and besides a major 66 kDa band, we were able to confirm the presence of ~140 kDa (BSA dimer) and ~210 kDa (BSA trimer) main bands, as well as a smear of higher molecular weight BSA species which were generated as a result of the EDC cross-linking (Fig. 1a). A band running at the size for BSA was found in the BSA-vancomycin haptene preparation, but no other higher molecular weight BSA species were detected by Ponceau S staining (Fig. 1a). To confirm the coupling of each individual antibiotic to BSA, antibiotic-specific immunodetection was performed. As presented in Fig. 1b, immuno-blotting against gentamicin specifically generated a signal in the lane loaded with the BSA-gentamicin hapten and not in the BSA-only lane. Similarly, a specific signal was detected in the BSA-vancomycin lane as compared to the BSA-only lane after anti-vancomycin immuno-blotting. These data show that we were successful in covalently coupling gentamicin or vancomycin to BSA for use as a hapten in the ELISA.Fig. 1


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)

Hapten evaluation. a Ponceau S staining indicates protein presence in the coupled haptens. b Immunoblotting indicates corresponding antibiotic presence in both coupled haptens specifically
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Hapten evaluation. a Ponceau S staining indicates protein presence in the coupled haptens. b Immunoblotting indicates corresponding antibiotic presence in both coupled haptens specifically
Mentions: The development of these ELISAs required the coating of gentamicin or vancomycin to standard polystyrene ELISA wells. As the antibiotics themselves cannot be coated directly to well plates, we generated BSA haptens of each individual antibiotic by EDC-mediated coupling between a BSA carrier and the antibiotic. To acquire evidence of successful hapten generation we separated freshly prepared BSA-gentamicin and BSA-vancomycin haptens by SDS-PAGE, transferred the separated proteins to nitrocellulose membranes and performed anti-gentamicin or anti-vancomycin immunoblotting. To first visualize the total amount of SDS-PAGE-loaded hapten/protein, membranes were stained with Ponceau S after electro-blotting of SDS-PAGE gels. BSA runs at 66 kDa in SDS-PAGE and besides a major 66 kDa band, we were able to confirm the presence of ~140 kDa (BSA dimer) and ~210 kDa (BSA trimer) main bands, as well as a smear of higher molecular weight BSA species which were generated as a result of the EDC cross-linking (Fig. 1a). A band running at the size for BSA was found in the BSA-vancomycin haptene preparation, but no other higher molecular weight BSA species were detected by Ponceau S staining (Fig. 1a). To confirm the coupling of each individual antibiotic to BSA, antibiotic-specific immunodetection was performed. As presented in Fig. 1b, immuno-blotting against gentamicin specifically generated a signal in the lane loaded with the BSA-gentamicin hapten and not in the BSA-only lane. Similarly, a specific signal was detected in the BSA-vancomycin lane as compared to the BSA-only lane after anti-vancomycin immuno-blotting. These data show that we were successful in covalently coupling gentamicin or vancomycin to BSA for use as a hapten in the ELISA.Fig. 1

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