Use of Chimeric Antibodies as Positive Controls in an Enzyme-Linked Immunosorbent Assay for Diagnosis of Scrub Typhus (Infection by Orientia tsutsugamushi)
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| Published in | Clinical and Vaccine Immunology Vol. 14; no. 10; pp. 1307 - 1310 |
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| Main Authors | , |
| Format | Journal Article |
| Language | English |
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American Society for Microbiology
01.10.2007
American Society for Microbiology (ASM) |
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| AbstractList | The use of human sera collected from individuals of known infected and noninfected status is necessary for the validation of diagnostic assays and for the determination of cutoff values. However, the routine inclusion of pooled human sera from infected individuals for use as positive controls in commercial assay kits has many disadvantages. Sufficient quantities of sera can be difficult to obtain, and there are ethical and safety issues to be considered. Additionally, each batch of control material requires standardization, as each will differ in antibody titer. We have genetically engineered chimeric immunoglobulin G (IgG), IgM, and IgA antibodies consisting of mouse-derived variable regions and human constant regions derived from peripheral blood lymphocytes. The chimeric nature of these antibodies allows the desired antigen specificity created through mouse immunization and hybridoma technology while retaining a human constant region required for recognition by the enzyme-conjugated antihuman signal antibody. We have investigated the potential use of chimeric IgG with specificity for the major surface antigen of
Orientia tsutsugamushi
as an alternative positive control for inclusion in a commercial enzyme-linked immunosorbent assay kit for the diagnosis of rickettsia scrub typhus (caused by infection with
O. tsutsugamushi
). Chimeric IgG was expressed in stably transfected CHO cells, allowing production of unlimited quantities. The purified protein was found to have a much greater specificity for the scrub typhus antigen than the serum-derived controls. The methods described could be applied to other assay kits for the detection of antibodies against infectious agents. The use of human sera collected from individuals of known infected and noninfected status is necessary for the validation of diagnostic assays and for the determination of cutoff values. However, the routine inclusion of pooled human sera from infected individuals for use as positive controls in commercial assay kits has many disadvantages. Sufficient quantities of sera can be difficult to obtain, and there are ethical and safety issues to be considered. Additionally, each batch of control material requires standardization, as each will differ in antibody titer. We have genetically engineered chimeric immunoglobulin G (IgG), IgM, and IgA antibodies consisting of mouse-derived variable regions and human constant regions derived from peripheral blood lymphocytes. The chimeric nature of these antibodies allows the desired antigen specificity created through mouse immunization and hybridoma technology while retaining a human constant region required for recognition by the enzyme-conjugated antihuman signal antibody. We have investigated the potential use of chimeric IgG with specificity for the major surface antigen of Orientia tsutsugamushi as an alternative positive control for inclusion in a commercial enzyme-linked immunosorbent assay kit for the diagnosis of rickettsia scrub typhus (caused by infection with O. tsutsugamushi). Chimeric IgG was expressed in stably transfected CHO cells, allowing production of unlimited quantities. The purified protein was found to have a much greater specificity for the scrub typhus antigen than the serum-derived controls. The methods described could be applied to other assay kits for the detection of antibodies against infectious agents. Classifications Services CVI Citing Articles Google Scholar PubMed Related Content Social Bookmarking CiteULike Delicious Digg Facebook Google+ Mendeley Reddit StumbleUpon Twitter current issue CVI About CVI Subscribers Authors Reviewers Advertisers Inquiries from the Press Permissions & Commercial Reprints ASM Journals Public Access Policy CVI RSS Feeds 1752 N Street N.W. • Washington DC 20036 202.737.3600 • 202.942.9355 fax • journals@asmusa.org Print ISSN: 1556-6811 Online ISSN: 1556-679X Copyright © 2014 by the American Society for Microbiology. For an alternate route to CVI .asm.org, visit: CVI |
| Author | Martina L. Jones Ross T. Barnard |
| AuthorAffiliation | School of Molecular and Microbial Sciences, University of Queensland, St. Lucia 4072, QLD, Australia |
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| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/17687111$$D View this record in MEDLINE/PubMed |
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| Cites_doi | 10.1002/jcla.20016 10.1016/S1286-4579(00)01352-6 10.1093/clinids/18.4.624 10.20506/rst.17.2.1119 10.1128/JCM.36.5.1277-1284.1998 10.1128/CDLI.5.4.519-526.1998 10.2144/05382BM01 |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Corresponding author. Mailing address: School of Molecular and Microbial Sciences, University of Queensland, St. Lucia 4072, QLD, Australia. Phone: 61 7 3365 4635. Fax: 61 7 3365 4699. E-mail: martina.jones@uq.edu.au |
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| References | 9665960 - Clin Diagn Lab Immunol. 1998 Jul;5(4):519-26 8038320 - Clin Infect Dis. 1994 Apr;18(4):624-6 9574691 - J Clin Microbiol. 1998 May;36(5):1277-84 15103679 - J Clin Lab Anal. 2004;18(3):159-64 9713892 - Rev Sci Tech. 1998 Aug;17(2):469-526 11226850 - Microbes Infect. 2001 Jan;3(1):11-21 15727121 - Biotechniques. 2005 Feb;38(2):181-2 (e_1_3_2_8_2) 1994; 18 (e_1_3_2_4_2) 1998; 17 (e_1_3_2_7_2) 2001; 3 (e_1_3_2_5_2) 2005; 38 (e_1_3_2_6_2) 2004; 18 e_1_3_2_3_2 e_1_3_2_2_2 |
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Reddit... The use of human sera collected from individuals of known infected and noninfected status is necessary for the validation of diagnostic assays and for the... |
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| SubjectTerms | Animals Antibodies, Bacterial - genetics Antibodies, Bacterial - isolation & purification Clinical and Diagnostic Laboratory Immunology Cross Reactions Enzyme-Linked Immunosorbent Assay - methods Enzyme-Linked Immunosorbent Assay - standards Humans Immunoglobulin G - biosynthesis Immunoglobulin G - genetics Immunoglobulin G - isolation & purification Mice Mutant Chimeric Proteins - genetics Mutant Chimeric Proteins - isolation & purification Mutant Chimeric Proteins - standards Orientia tsutsugamushi Orientia tsutsugamushi - immunology Rickettsia Scrub Typhus - diagnosis Scrub Typhus - immunology Scrub Typhus - microbiology |
| Title | Use of Chimeric Antibodies as Positive Controls in an Enzyme-Linked Immunosorbent Assay for Diagnosis of Scrub Typhus (Infection by Orientia tsutsugamushi) |
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