Simplified Assay for Measuring Toxoplasma gondii Immunoglobulin G Avidity

Abstract

A Toxoplasma gondii immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) was developed that combines the accuracy of assays based on end point titers and the relative ease of assays based on optical density values. Like published procedures, the new assay's avidity index (AI) was based on differential T. gondii-specific IgG reactivity in serum-treated wells washed with urea buffer versus that in wells washed with control buffer; unlike previous assays, however, the IgG reactivity was measured quantitatively using a standard curve. The assay was evaluated using 24 IgG-positive and IgM-positive sera collected within 5 months of the onset of symptoms (recent-infection group) and 25 IgG-positive and IgM-negative sera (past-infection group). All sera in the recent-infection group exhibited AI values of <0.18, whereas all sera in the past-infection group exhibited AI values of >0.27. The AI values of the recent-infection group showed significant correlation with the number of days after the onset of symptoms. A subset of 16 sera (8 recent and 8 past) was tested using a commercially available T. gondii IgG avidity ELISA based on end point titration; the results of the two assays showed highly significant correlation (R 2 ‫؍‬ 0.9125). In addition, we confirmed and extended the findings of other investigators, showing that AI values calculated using optical density values, but not AI values calculated using quantitative IgG values, varied significantly depending on the serum dilution used. This new assay should facilitate the accurate measurement of T. gondii IgG avidity in a reference laboratory setting.Measurement of Toxoplasma gondii immunoglobulin G (IgG) avidity (binding strength) is a powerful tool for distinguishing recent from past T. gondii infection. Detection of low-avidity IgG is a reliable indicator of infection within the previous 8 months, whereas detection of high-avidity IgG essentially excludes the possibility that infection occurred within the previous 5 months (2, 4, 7). T. gondii IgG avidity measurement is particularly valuable for approximating the time of infection in pregnant women found to be positive for T. gondii IgG and IgM at the time of their first prenatal care visit (4,7,8,12).IgG avidity measurement is based on the differential elution of antigen-bound IgG using urea-containing wash buffer; lowavidity IgG dissociates from antigen in the presence of urea, whereas high-avidity IgG remains bound to antigen (2). Results for avidity assays are generally expressed as an avidity index (AI), which reflects the relative amount of T. gondiispecific IgG detected following washes with urea buffer versus that found following washes with control buffer. Published methods differ, however, in how these IgG levels are expressed. Using the end point titration method, multiple dilutions of patient sera are tested, and titers are defined in relation to a cutoff optical density (OD) value (2, 4-7). Drawbacks to this method include the requirement for four or more dilutions of a specimen, t...

Spiramycin Treatment of Toxoplasma gondii Infection in Pregnant Women Impairs the Production and the Avidity Maturation of T. gondii -Specific Immunoglobulin G Antibodies

“…One study evaluated the IgG AI using single-dilution assays with and without urea and showed that because the signal obtained in an enzyme immunoassay system is not linear, it makes a difference whether the Toxoplasma IgG AI is calculated from the optical density values or from the activity of Toxoplasma-specific IgG antibodies measured in international units per ml (23).…”

European Multicenter Study of the LIAISON Automated Diagnostic System for Determination of Toxoplasma gondii -Specific Immunoglobulin G (IgG) and IgM and the IgG Avidity Index

“…This process introduces uncertainty, although experiments with only two dilutions showed excellent agreement with IgG avidity measurements obtained with four serial dilutions (13). Testing of a single dilution with and without urea was further evaluated by Prince and Wilson (21), who showed that because the signal obtained in an enzyme immunoassay system is not linear, it makes a difference whether the Toxoplasma-specific IgG avidity index is calculated from optical densities or activities measured in international units of Toxoplasma-specific IgG antibodies per milliliter (21).…”

High-avidity IgG Autoantibodies against DFS70/LEDGF in Atopic Dermatitis

“…Antibody avidities are used widely for discrimination between the acute and chronic phases of infectious diseases [13,14]. High-avidity autoantibodies against double-strand DNA are associated with renal damage in systemic lupus erythematosus [15], and high-affinity immune responses to insulin are associated with high risk for developing type 1 diabetes [16,17].…”

“…The ELISAs were performed as previously described [13][14][15][16], with modifications. Microtiter wells (Medisorp; Nunc, Roskilde, Denmark) were coated with recombinant DFS70 [9] or diphtheria toxoid (DT; List Biological Laboratories, Inc., Campbell, CA) in PBS (1 and 2 µg/ ml, respectively).…”