A highly specific two-site ELISA for pneumococcal C-polysaccharide using monoclonal and affinity-purified polyclonal antibodies |
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Authors: | A M Sj?gren T Holme |
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Affiliation: | 1. Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China;2. Department of Microbiology, School of Basic Medicine, Fourth Military Medical University, Xi’an, Shaanxi 710032, China;5. Wellcome Trust, United Kingdom;6. KEMRI-Wellcome, Kilifi County Hospital, Kenya;7. Federal Agency for Medicines and Health Products (FAMHP), Belgium;8. University of Oxford, United Kingdom;9. hVIVO, United Kingdom;10. KEMRI-Wellcome Trust Research Programme, Kenya;11. Centre for Infectious Disease Research in Zambia, Roma, Zambia;12. University of Bergen, Norway;13. University of Oxford, United Kingdom;14. Johns Hopkins Bloomberg School of Public Health, USA;15. University of Oxford, United Kingdom;p. McMaster University, Canada;q. University of Cincinnati, USA;r. Monash University, Australia;s. University of North Carolina, USA;t. Christian Medical College, Vellore, India;u. University of York, United Kingdom;v. St. George''s, University of London, United Kingdom and mrc/uvri @lshtm, Uganda;w. University of Maryland School of Medicine, USA;x. University of Oxford, United Kingdom;y. Imperial College, United Kingdom;z. Murdoch Children''s Research Institute (MCRI), Australia;11. University of Oxford, United Kingdom;12. Hospices Civils de Lyon, France;13. Naval Medical Research Center, USA;14. Leiden University Medical Center, the Netherlands;15. Monash University, Australia;16. SGS LIfe Sciences, Belgium;1. Department of Paediatrics, University of Oxford, United Kingdom;2. Radboud University Medical Center, the Netherlands;3. P95 Epidemiology & Pharmacovigilance, Leuven, Belgium;4. International Alliance for Biological Standardization, Belgium;1. Division of Infectious Diseases, Department of Internal Medicine, Korea University College of Medicine, Seoul, Republic of Korea;2. Division of Infectious Diseases, Chungbuk National University College of Medicine, Cheongju, Chungcheongbuk-do, Republic of Korea;3. Division of Nephrology, Department of Internal Medicine, Hallym University College of Medicine, Chuncheon, Gangwon-do, Republic of Korea;4. Division of Infectious Disease, Department of Internal Medicine, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Seoul, Republic of Korea;5. Division of Infectious Diseases, Department of Internal Medicine, Dongtan Sacred Heart Hospital , Hallym University College of Medicine, Hwasung, Republic of Korea;6. Department of Microbiology, Institute for Viral Diseases, Korea University College of Medicine, Seoul, Republic of Korea;1. Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand;2. Research Institute for Health Sciences, Chiang Mai University, Chiang Mai, Thailand;3. Clinical and Molecular Epidemiology of Emerging and Re-emerging Infectious Diseases Cluster, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand |
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Abstract: | A two-site ELISA for the detection of pneumococcal C-polysaccharide (PnC) has been developed. A monoclonal antibody directed against the phosphorylcholine residue of the PnC was used as catcher and an affinity-purified polyclonal anti-PnC rabbit antiserum for detection. Polyclonal antibodies against the PnC as well as capsular antigens were obtained by immunizing rabbits with type 1 pneumococci. Antibodies against the phosphorylcholine determinant of PnC could be removed by affinity purification. Remaining antibodies reacted in an ELISA with type 1 capsular polysaccharide as well as with PnC. Only in the fraction with the highest antibody activity against PnC, phosphorylcholine exhibited a slight inhibitory action. It is concluded that the purified antibody preparation reacted with an antigenic determinant shared by the two polysaccharides, in all probability a determinant associated with 2-acetamido-4-amino-2,4,6-trideoxygalactose which is the only monosaccharide component in common between PnC and the type 1 capsular polysaccharide. By the use of this affinity-purified antibody preparation, reactions with alpha-streptococci, occurring with non-purified serum, were abolished. The sensitivity and specificity of the test was determined using capsulated and non-capsulated pneumococci and alpha-streptococci known to cross-react with unpurified serum against the pneumococcal C-polysaccharide. |
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