Immunological detection of in vitro formed phosphatidylethanol--an alcohol biomarker--with monoclonal antibodies
| Autor: | Markku J. Savolainen, Marja K. Liisanantti, Antti E. Nissinen, Sohvi Hörkkö, Sanna Mäkelä, Minna L. Hannuksela, Jussi Vuoristo |
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| Rok vydání: | 2008 |
| Předmět: |
Male
medicine.drug_class Serum albumin Immunoglobulin Variable Region Medicine (miscellaneous) Fluorescent Antibody Technique Glycerophospholipids Biology Toxicology Monoclonal antibody Cell Line chemistry.chemical_compound Mice Antibody Specificity Phosphatidylcholine medicine Animals Humans RNA Messenger Immunoassay Hybridomas medicine.diagnostic_test Base Sequence Sequence Analysis RNA Antibodies Monoclonal Flow Cytometry Molecular biology Lipoproteins LDL Mice Inbred C57BL Psychiatry and Mental health Red blood cell Alcoholism medicine.anatomical_structure chemistry Biochemistry Immunoglobulin M Monoclonal biology.protein Phosphatidylethanol Female Immunization Antibody Lipoproteins HDL Biomarkers |
| Zdroj: | Alcoholism, clinical and experimental research. 32(6) |
| ISSN: | 1530-0277 |
| Popis: | Background: Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography–mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. Methods: C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. Results: We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. Conclusions: The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes. |
| Databáze: | OpenAIRE |
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