Application of the Checkerboard Immunoblotting Technique to the Quantification of Host Biomarkers in Gingival Crevicular Fluid
Background: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high‐throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples. Methods: Monoclonal antibodies were used to bind...
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| Vydáno v: | Journal of periodontology (1970) Ročník 80; číslo 3; s. 447 - 456 |
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| Hlavní autoři: | , , , , |
| Médium: | Journal Article |
| Jazyk: | angličtina |
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Chicago, IL
American Academy of Periodontology
01.03.2009
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| Témata: | |
| ISSN: | 0022-3492, 1943-3670 |
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| Abstract | Background: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high‐throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples.
Methods: Monoclonal antibodies were used to bind GCF interleukin (IL)‐1β and −8 and matrix metalloproteinase (MMP)‐8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross‐reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme‐linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty‐one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples.
Results: The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL‐1β and −8 compared to healthy subjects.
Conclusion: The CBIB technique is a sensitive and specific assay for the high‐throughput quantification of MMP‐8 and IL‐8 and −1β in GCF. |
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| AbstractList | Background: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high‐throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples.
Methods: Monoclonal antibodies were used to bind GCF interleukin (IL)‐1β and −8 and matrix metalloproteinase (MMP)‐8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross‐reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme‐linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty‐one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples.
Results: The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant ( P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL‐1β and −8 compared to healthy subjects.
Conclusion: The CBIB technique is a sensitive and specific assay for the high‐throughput quantification of MMP‐8 and IL‐8 and −1β in GCF. The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples. Monoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples. The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects. The CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF. The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples.BACKGROUNDThe aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification of multiple inflammatory mediators in gingival crevicular fluid (GCF) samples.Monoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples.METHODSMonoclonal antibodies were used to bind GCF interleukin (IL)-1beta and -8 and matrix metalloproteinase (MMP)-8 to the surface of membranes. Biotinylated antibodies were used to detect bound antigens in a checkerboard format. Signals were developed using chemiluminescence, captured on film, and quantified using software for array analysis. The assay was tested for potential cross-reactions among the three pairs of antibodies. Eleven CBIBs were processed to determine the analytical sensitivity of the assay. Forty GCF samples were analyzed using CBIB and enzyme-linked immunosorbent assay (ELISA) in parallel, and the significance of the correlations among the results was tested using the Pearson correlation coefficient. Nine hundred thirty-one GCF samples were collected from 20 periodontally healthy subjects and 20 periodontitis subjects and analyzed using CBIB to test the assay's sensitivity and dynamic ranges using clinical samples.The CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects.RESULTSThe CBIB was capable of distinguishing among the three analytes. The sensitivity and dynamic ranges of the assay were suitable for the detection of the three targets in the majority of GCF samples. There were highly statistically significant (P <0.0001) positive correlations between CBIB and ELISA data for all three biomarkers. The periodontitis subjects had statistically significantly higher mean levels of IL-1beta and -8 compared to healthy subjects.The CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF.CONCLUSIONThe CBIB technique is a sensitive and specific assay for the high-throughput quantification of MMP-8 and IL-8 and -1beta in GCF. |
| Author | Sakellari, Dimitra Teles, Ricardo P. Socransky, Sigmund S. Konstantinidis, Antonis Haffajee, Anne D. |
| AuthorAffiliation | Department of Periodontology, The Forsyth Institute, Boston, MA, USA Department of Periodontology, Dental School, Aristotle University of Thessaloniki, Greece |
| AuthorAffiliation_xml | – name: Department of Periodontology, The Forsyth Institute, Boston, MA, USA – name: Department of Periodontology, Dental School, Aristotle University of Thessaloniki, Greece |
| Author_xml | – sequence: 1 givenname: Ricardo P. surname: Teles fullname: Teles, Ricardo P. email: rTeles@forsyth.org – sequence: 2 givenname: Dimitra surname: Sakellari fullname: Sakellari, Dimitra – sequence: 3 givenname: Antonis surname: Konstantinidis fullname: Konstantinidis, Antonis – sequence: 4 givenname: Sigmund S. surname: Socransky fullname: Socransky, Sigmund S. – sequence: 5 givenname: Anne D. surname: Haffajee fullname: Haffajee, Anne D. |
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| CitedBy_id | crossref_primary_10_1016_j_trac_2011_06_016 crossref_primary_10_1902_jop_2010_090643 crossref_primary_10_1097_MD_0000000000006932 crossref_primary_10_1111_j_1600_0757_2009_00316_x crossref_primary_10_1111_jre_12244 crossref_primary_10_1902_jop_2009_090397 crossref_primary_10_1007_s00784_013_0972_9 crossref_primary_10_5402_2012_581207 crossref_primary_10_1111_j_1600_0757_2009_00344_x crossref_primary_10_1111_jcpe_12194 crossref_primary_10_1177_0022034511400078 |
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| Keywords | Stomatology Crevicular fluid Cytokine Biological marker Dentistry Chemokine Periodontal disease gingival crevicular fluid cytokines Technique periodontal diseases Application matrix metalloproteinases Chemokines Gingival fluid |
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| Snippet | Background: The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high‐throughput... The aim of this study was to describe the development and validation of the checkerboard immunoblotting (CBIB) technique for the high-throughput quantification... |
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| SubjectTerms | Adult Antibodies, Immobilized Biological and medical sciences Biomarkers - analysis Chemokines Chronic Periodontitis - metabolism Cross Reactions cytokines Dental Plaque - metabolism Enzyme-Linked Immunosorbent Assay - methods Facial bones, jaws, teeth, parodontium: diseases, semeiology Female gingival crevicular fluid Gingival Crevicular Fluid - chemistry Gingival Hemorrhage - metabolism Gingival Recession - metabolism Gingivitis - metabolism Humans Immunoblotting - methods Interleukin-1beta - analysis Interleukin-8 - analysis Luminescence Male Matrix Metalloproteinase 8 - analysis matrix metalloproteinases Medical sciences Membranes, Artificial Middle Aged Non tumoral diseases Otorhinolaryngology. Stomatology periodontal diseases Periodontal Pocket - metabolism Periodontium - metabolism Polyvinyls Sensitivity and Specificity Software |
| Title | Application of the Checkerboard Immunoblotting Technique to the Quantification of Host Biomarkers in Gingival Crevicular Fluid |
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