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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: 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.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.

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

Specificity and sensitivity of the antibiotic ELISAs. a The influence of vancomycin on the gentamicin ELISA. b The influence of gentamicin on the vancomycin ELISA. c The validation of the calibration curve of the gentamicin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. d The validation of the calibration curve of the vancomycin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. Error bars indicate standard deviation
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Fig3: Specificity and sensitivity of the antibiotic ELISAs. a The influence of vancomycin on the gentamicin ELISA. b The influence of gentamicin on the vancomycin ELISA. c The validation of the calibration curve of the gentamicin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. d The validation of the calibration curve of the vancomycin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. Error bars indicate standard deviation

Mentions: The specificity of the gentamicin ELISA was assessed by performing the gentamicin ELISA using a concentration series of either gentamicin or vancomycin. If the gentamicin ELISA would be aspecific for distinguishing gentamicin from vancomycin, it is expected that with increasing vancomycin concentrations a vancomycin-induced A450 shift would take place. The same principle was used for the vice versa situation where we determined the specificity of the vancomycin ELISA with a concentration series of gentamicin. The presence of vancomycin in the gentamicin ELISA did not result in a change of A450 absorbance in any of the tested concentrations, indicating that this ELISA setup is highly specific for gentamicin (Fig. 3a). These data also show that with this high specificity, the gentamicin ELISA is very sensitive and allows reliable detection of gentamicin in a range between 2 and 500 ng/ml. Gentamicin did not influence the vancomycin ELISA at any of the concentrations that were tested, indicating that also the vancomycin ELISA set-up is very specific for vancomycin (Fig. 3b). The detection range of the vancomycin ELISA was determined to be reliable between 20 and 5000 ng/ml vancomycin. To determine how the sensitivity of the ELISAs might depend on the antibiotic concentration in the sample, we used two separately prepared antibiotic concentration series. One series was used to generate a calibration curve and the other concentration series (“validation series” in the Figure) was subsequently measured and the antibiotic concentration in the series was estimated by using the calibration curve. This was done for both ELISAs separately. As shown in Fig. 3c, d we found that for both ELISAs the samples in the validation series generated absorbances that were within the 10 % deviation range of the measured absorbance of the calibration curve (often used in commercial kits, to compensate for potential pipetting errors, differences between wells and the standard deviation of the measurements) (Fig. 3c, d).Fig. 3


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)

Specificity and sensitivity of the antibiotic ELISAs. a The influence of vancomycin on the gentamicin ELISA. b The influence of gentamicin on the vancomycin ELISA. c The validation of the calibration curve of the gentamicin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. d The validation of the calibration curve of the vancomycin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. Error bars indicate standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Specificity and sensitivity of the antibiotic ELISAs. a The influence of vancomycin on the gentamicin ELISA. b The influence of gentamicin on the vancomycin ELISA. c The validation of the calibration curve of the gentamicin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. d The validation of the calibration curve of the vancomycin ELISA. Lines indicate upper and lower 10 % range of the calibration curve. Error bars indicate standard deviation
Mentions: The specificity of the gentamicin ELISA was assessed by performing the gentamicin ELISA using a concentration series of either gentamicin or vancomycin. If the gentamicin ELISA would be aspecific for distinguishing gentamicin from vancomycin, it is expected that with increasing vancomycin concentrations a vancomycin-induced A450 shift would take place. The same principle was used for the vice versa situation where we determined the specificity of the vancomycin ELISA with a concentration series of gentamicin. The presence of vancomycin in the gentamicin ELISA did not result in a change of A450 absorbance in any of the tested concentrations, indicating that this ELISA setup is highly specific for gentamicin (Fig. 3a). These data also show that with this high specificity, the gentamicin ELISA is very sensitive and allows reliable detection of gentamicin in a range between 2 and 500 ng/ml. Gentamicin did not influence the vancomycin ELISA at any of the concentrations that were tested, indicating that also the vancomycin ELISA set-up is very specific for vancomycin (Fig. 3b). The detection range of the vancomycin ELISA was determined to be reliable between 20 and 5000 ng/ml vancomycin. To determine how the sensitivity of the ELISAs might depend on the antibiotic concentration in the sample, we used two separately prepared antibiotic concentration series. One series was used to generate a calibration curve and the other concentration series (“validation series” in the Figure) was subsequently measured and the antibiotic concentration in the series was estimated by using the calibration curve. This was done for both ELISAs separately. As shown in Fig. 3c, d we found that for both ELISAs the samples in the validation series generated absorbances that were within the 10 % deviation range of the measured absorbance of the calibration curve (often used in commercial kits, to compensate for potential pipetting errors, differences between wells and the standard deviation of the measurements) (Fig. 3c, d).Fig. 3

Bottom Line: 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.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.

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