Development of fully automated determination of marker-specific immunoglobulin G (IgG) avidity based on the avidity competition assay format: application for Abbott Architect cytomegalovirus and Toxo IgG Avidity assays

Abstract

Determination of the avidity of immunoglobulin G (IgG) directed against a specific marker has become an established diagnostic tool for identifying or excluding acute infections with pathogens. A novel assay format termed AVIcomp (avidity competition based on mass action) circumventing the conventional chaotropic format has been developed for determination of the avidity of marker-specific IgG in patient specimens. Its applications for cytomegalovirus (CMV) and Toxoplasma gondii are presented. Specific high-avidity IgG from the patient specimen is selectively blocked using a soluble antigen in a sample pretreatment reagent, and the amount of remaining specific low-avidity IgG is determined relative to that in an untreated control. The comparison of the conventional chaotropic format, represented by the Radim CMV IgG Avidity assay, and the newly developed AVIcomp method, as exemplified by the Architect CMV IgG Avidity assay, on blood drawn within 4 months after seroconversion revealed a sensitivity of 100% (97.3% by an alternative calculation) for the AVIcomp format versus 87.5% (75.7% by an alternative calculation) for the chaotropic avidity assay. The specificity on 312 CMV IgG reactive and CMV IgM nonreactive specimens from pregnant women was 100% for the AVIcomp assay and 99.7% for the conventional avidity assay. The Architect Toxo IgG Avidity assay showed an agreement of 97.2% with the bioMérieux Vidas Toxo IgG Avidity Assay employing chaotropic reagents. These performance data suggest that the AVIcomp format shows superior sensitivity and equivalent specificity for the determination of IgG avidity to assays based on the chaotropic method and that the AVIcomp format may also be applicable to other disease states.

FIG. 1.

Comparison of conventional (chaotropic) and AVIcomp avidity assays. (Top) Principle of conventional (denaturing) avidity assays; (bottom) principle of the AVIcomp avidity assays.

FIG. 2.

CMV IgG avidity as a function of CMV antigen concentration. Three samples precharacterized as low, gray zone, or high avidity by the Radim CMV IgG Avidity assay were tested using seven different antigen concentrations in the neutralizing pretreatment solution. The error bars indicate 1 standard deviation. The gray field between 50 and 60% avidity represents the gray zone of the Architect CMV IgG Avidity assay.

FIG. 3.

Impact of the CMV IgG concentration on the CMV IgG Avidity assay result. Four samples (Smp) precharacterized by the Radim CMV IgG Avidity assay as low or high avidity (two samples at each level) were diluted to six concentrations of specific IgG before testing. % Avi, percent avidity. The error bars represent 1 standard deviation; the horizontal gray field represents the gray zone of the assay; and the vertical gray field represents the target dilution range, into which the final assay dilutes every sample automatically before the avidity of the specific IgG is determined.

FIG. 4.

Impact of the T. gondii IgG concentration on the Toxo IgG Avidity assay result. Three samples precharacterized by the Biomerieux Vidas Toxo IgG Avidity assay as low (one sample) or high (two samples) avidity were diluted to five concentrations of specific IgG before testing using the Architect Toxo IgG Avidity assay. % Avi, percent avidity.

FIG. 5.

Manual versus automated sample dilution in Architect wash buffer exemplified for the Architect CMV IgG Avidity assay. Shown is a comparison of automatically performed dilution of samples to the optimal testing range versus manual endpoint dilution to a fixed concentration of CMV-specific IgG (10 AU/ml). % Avi, percent avidity.

FIG. 6.

IgG avidity by the Architect and Radim CMV IgG Avidity assays. Histograms show data from ROC analysis. Light shaded bars, specimens tested for specificity; dark shaded bars, specimens tested for seroconversion sensitivity. % Avi, percent avidity. Numbers of specimens, given below the histograms, include all specimens from specificity and sensitivity testing with a valid avidity result (IgG reactivity for the Architect CMV IgG Avidity assay; an OD of >0.300 for the Radim CMV IgG Avidity assay).

FIG. 7.

Agreement between the Architect and Vidas Toxo IgG Avidity assays on specimens from pregnant females (n = 104) and on seroconversion specimens (n = 51). The gray zones of both assays are indicated by dashed lines (avidity index, 0.2 to 0.3; percent avidity, 50 to 60%). The relative agreement between the Architect and Vidas Toxo IgG Avidity assays was 97.2% (140/144 specimens). % Avi, percent avidity.