Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies toToxoplasma gondii

Abstract

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen‐specific B cells induces enhanced CD4+ T helper type 1 subset differentiation

“…Chimeric antibodies composed of variable regions from mouse antibodies joined to human constant regions retain their biological activity. 44,45 Consequently, we predicted that the same would apply to a membrane-bound BCR. We therefore replaced the variable regions from cDNAs encoding a human IgG1/j, anti-TTCF BCR 25,33 with those isolated from a newly generated mouse monoclonal hybridoma (C7.1) secreting an IgG1/j, anti-aggrecan antibody.…”

Preparation of Recombinant Human Monoclonal Antibody Fab Fragments Specific forEntamoeba histolytica

“…A possible use of recombinant human MAbs may be as controls in serodiagnosis (12). To interpret serologic data, known positive controls must be used.…”

Development and application of monoclonal antibodies to Chikungunya virus proteins

“…Moreover, there are safety concerns of disease contraction, at both the manufacturer and consumer ends, when dealing with potentially infectious human sera. Finally, continuous standardization will be required for each finite batch of material collected due to variable antibody titres and sensitivity or specificity (Hackett et al, 1998). With every varying batch of reference material collected, assays will then be re-calibrated against an international, national or in-house standard prior to aliqouting and storage for routine use (OIE Terrestrial Manual 2014, http://www.oie.int/international-standard-setting/terrestrial-manual/access-online/).…”

“…Naturally, with the use of chimeric mAbs as alternative controls, ethical problems regarding the collection of human blood will therefore be no cause for concern. The successful use of chimeric mAbs as positive controls in an ELISA-based diagnostic was first described for the detection of the parasitic protozoan Toxoplasma gondii (Hackett et al, 1998). There have since been several other published uses of protein-specific chimeric mAbs as diagnostic control reagents (Schuurman et al, 1997;Ichikawa et al, 1999;Simon et al, 2001;Jones and Barnard, 2007).…”

“…Furthermore, there are safety concerns dealing with potentially infectious human sera, from both the manufacturer and consumer perspectives. Lastly, each finite batch of collected serum will require standardization due to the batch variation of antibody titre (Hackett et al, 1998). As an alternative, the generation of a non-serum-derived, recombinant positive control antibody that interacts specifically with a CHIKV protein and is recognized by an enzyme-conjugated anti-human antibody can be a solution to overcoming the disadvantages.…”