Evaluation of two International Reference Standards for antibodies to Trypanosoma cruzi in a WHO collaborative study
Expert Committee on biological standardization, Geneva, 17 to 21 October 2011
Overview
Two freeze dried preparations of defibrinated human plasma containing anti-Trypanosoma cruzi antibodies, coded 09/186 and 09/188, were assessed for their suitability as International Reference Standards (IS) for the serodiagnosis of Chagas disease. A WHO collaborative study was undertaken by 24 laboratories from 16 countries. Preparations 09/186 and 09/188 were tested in 30 commercially available assays: enzyme immunoassays (EIA, n=16), one chemiluminescent immunoassay, indirect immunofluorescence assays (IFA, n=4), indirect hemagglutination assays (IHA, n=4), one particle agglutination assay and rapid immunochromatographic assays (RIC, n=4). In addition 10 in-house assays were used: EIA (n=2), IFA (n=5) and western blots (n=3). A radioimmunoprecipitation assay (RIPA) was used to characterize prototypes of 09/186 and 09/188 in a pilot study.
Both preparations 09/186 and 09/188 were identified as sero-positive in all assays except for one laboratory. Based on the results of the collaborative study, the reactivity in EIA and ChLIA was used as a guide to establish the unitage mL-1. The geometric mean endpoint titre estimated ranged from 2 to 37 for preparation 09/186, and from 3 to 59 for preparation 09/188. For 09/186, the overall Geometric mean (GM) was calculated as: 11 (Geometric Coefficients of variation [GCV] 104.7%; n=29) for assays using native antigen and 9 (GCV 89.4%; n=25) for assays using recombinant antigen. For 09/188, the overall GM was calculated as: 17 (GCV 101.3%; n=29) for assays using native antigen and 16 (GCV 125.1%; n=25) for assays using recombinant antigen.
It is proposed that preparations 09/186 and 09/188 are established as the 1st WHO IS for anti-Trypanosoma cruzi antibodies:
- Preparation 09/186, defined as the anti-T.cruzi antibody Standard representative of the region where T.cruzi II is predominant, with an arbitrary unitage of 1 TcII International Unit mL-1 for the undiluted concentration after reconstitution, according to the instructions for use.
- Preparation 09/188, defined as the anti-T.cruzi antibody Standard representative of the region where T.cruzi I is predominant, with an arbitrary unitage of 1 TcI International Unit mL-1 for the undiluted concentration after reconstitution, according to the instructions for use.
These standards , containing 0.5 mL freeze dried plasma, will be distributed together, with an assigned unitage of 0.5 IU per ampoule. The unitage to be associated with the endpoint titre can thus be taken as the reciprocal value of the titre, and this will inform the user of the relative strength of 09/186 and 09/188 in the assay of use.