Immunological detection of in vitro formed phosphatidylethanol--an alcohol biomarker--with monoclonal antibodies.
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.
Nissinen
Antti E
AE
Institute of Clinical Medicine, Department of Internal Medicine, University of Oulu, Clinical Research Center, Oulu University Hospital, Oulu, Finland.
Mäkelä
Sanna M
SM
Vuoristo
Jussi T
JT
Liisanantti
Marja K
MK
Hannuksela
Minna L
ML
Hörkkö
Sohvi
S
Savolainen
Markku J
MJ
D000437:Alcoholism / Q000175:diagnosis*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis*; D002460:Cell Line; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020404:Glycerophospholipids / Q000032:analysis* / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D007118:Immunoassay; D007075:Immunoglobulin M / Q000096:biosynthesis; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000737:chemistry; D017423:Sequence Analysis, RNA
D000911:Antibodies, Monoclonal; D015415:Biomarkers; D020404:Glycerophospholipids; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D012333:RNA, Messenger; C051521:phosphatidylethanol