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Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency.
Anal Chem. 2006 Sep 15; 78(18):6601-7.AC

Abstract

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.

Authors+Show Affiliations

Gehring AG
Agricultural Research Service, United States Department of Agriculture, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania 19038, USA.
Albin DM
No affiliation info available
Bhunia AK
No affiliation info available
Reed SA
No affiliation info available
Tu SI
No affiliation info available
Uknalis J
No affiliation info available

MeSH

Antibodies, BacterialColony Count, MicrobialEscherichia coli O157Food MicrobiologyMicroscopy, FluorescenceProtein Array AnalysisSensitivity and Specificity

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

16970339

Citation

Gehring, Andrew G., et al. "Antibody Microarray Detection of Escherichia Coli O157:H7: Quantification, Assay Limitations, and Capture Efficiency." Analytical Chemistry, vol. 78, no. 18, 2006, pp. 6601-7.
Gehring AG, Albin DM, Bhunia AK, et al. Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency. Anal Chem. 2006;78(18):6601-7.
Gehring, A. G., Albin, D. M., Bhunia, A. K., Reed, S. A., Tu, S. I., & Uknalis, J. (2006). Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency. Analytical Chemistry, 78(18), 6601-7.
Gehring AG, et al. Antibody Microarray Detection of Escherichia Coli O157:H7: Quantification, Assay Limitations, and Capture Efficiency. Anal Chem. 2006 Sep 15;78(18):6601-7. PubMed PMID: 16970339.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency. AU - Gehring,Andrew G, AU - Albin,David M, AU - Bhunia,Arun K, AU - Reed,Sue A, AU - Tu,Shu-I, AU - Uknalis,Joseph, PY - 2006/9/15/pubmed PY - 2007/4/27/medline PY - 2006/9/15/entrez SP - 6601 EP - 7 JF - Analytical chemistry JO - Anal Chem VL - 78 IS - 18 N2 - A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7. SN - 0003-2700 UR - https://www.unboundmedicine.com/medline/citation/16970339/Antibody_microarray_detection_of_Escherichia_coli_O157:H7:_Quantification_assay_limitations_and_capture_efficiency_ L2 - https://doi.org/10.1021/ac0608467 DB - PRIME DP - Unbound Medicine ER -