Use of chimeric antibodies as positive controls in an enzyme-linked immunosorbent assay for diagnosis of scrub typhus (infection by Orientia tsutsugamushi).
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.
Jones
Martina L
ML
School of Molecular and Microbial Sciences, University of Queensland, St. Lucia 4072, QLD, Australia. martina.jones@uq.edu.au
Barnard
Ross T
RT
D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D051379:Mice; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000592:standards; D012285:Orientia tsutsugamushi / Q000276:immunology*; D012612:Scrub Typhus / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology
D000907:Antibodies, Bacterial; D007074:Immunoglobulin G; D050506:Mutant Chimeric Proteins