ToRCH Assay
Multiplexed · Sensitive · Specific

Introduction

Luminex Corporation has developed a ToRCH assay by coupling purified antigens of T. gondii, Rubella, CMV and HSV Type 1 and Type 2 to five FlowMetrix microsphere sets. Screening serum for antibodies specific for infectious pathogens is used to determine if a patient has been exposed to the agent in question. ToRCH assays detect serum IgG and IgM responses to Toxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes Simplex Virus Types 1 and 2. These infectious agents are capable of crossing the placental barrier and entering the immunologically naive fetus where they can cause severe fetal damage. FlowMetrix technology provides for a multiplexed format that simultaneously quantitates antibodies specific for each of these pathogens. The multiplexed assay was optimized using human serum calibrators certified as either highly positive, slightly positive or negative for IgG reactivity to all five agents. The assay was developed using a reporter of fluorescent goat anti-human IgG.

Figure 1

Methods

Antigen Conjugation to Microspheres: Six FlowMetrix microsphere sets were conjugated separately to the five ToRCH antigens and human serum albumin (HSA). Fifty microliters (5 x10⁶ microspheres) of each microsphere type was activated using a two-step EDC/Sulfo-NHS procedure. Activated microspheres were suspended in each antigen at empirically determined concentrations. Antigen coated microspheres were washed and stored in 0.02% Tweenâ 20, 1 mg/ml BSA in PBS, pH 7.4 (PBSTB).

Multiplex assay for IgG activities: Equivalent amounts of the six antigen-loaded microspheres were combined to produce a ToRCH microsphere mixture. ten microliters of the mixture was reacted with ten microliters of a 1:400 dilution of ToRCH calibrator serum for thirty minutes. The four tube assay used the three serum calibrators (high, low and negative) and one with no human serum as a negative control. Microspheres were washed in PBSTB, then suspended in twenty microliters of a 40 m g/ml solution of fluorescent anti-human IgG. This mixture was incubated for thirty minutes and assayed in fifteen to twenty seconds using the FlowMetrix System.

Results

Results are shown in Figure 1. A serum calibrator with high IgG reactivity to all five pathogens was uniformly positive (red bars). Similarly, a serum calibrator with no IgG reactivity to all five was uniformly negative (green bars). Specificity for each individual assay was demonstrated by reaction of the microsphere set with the strongly positive calibrator in the presence of each soluble antigen. For example, soluble Rubella antigen in this reaction strongly inhibited the Rubella microsphere signal specifically and not the other four pathogen microsphere reactions (data not shown). The slightly positive serum calibrator (yellow bars) was demonstrated to have an IgG reactivity to the five pathogen antigens ranging from 1.1 to 2.7 times the limit of detection for each assay in two commonly used diagnostic instruments. Even at a 1:400 dilution, the IgG reactivity of this control serum to the five antigens was three to six times the limit of detection in the multiplexed assay. (Figure 2)

Figure 2

For IgM reactivity, a parallel assay was performed and developed with a reporter of fluorescent Goat anti-human IgM. It should be noted that this ToRCH assay can also be performed in its multiplexed format with whole blood as well as serum or plasma.

Conclusions

These results demonstrate that a multiplexed ToRCH assay developed for the FlowMetrix System provides a uniquely powerful technology for identifying recent or remote seroconversion. The FlowMetrix ToRCH assay is multiplexed, specific and sensitive. This assay was developed at Luminex Corporation for the purpose of demonstrating the power of FlowMetrix technology. In addition to antibody detection immunoassay, this exciting technology can be applied to immunometric assays, enzyme assays and nucleic acid based genetic analyses.

Luminex Corporation provides the system including microspheres in a format ready to apply to your specific application. Using this Assay Development Kit, your research group can develop multiplexed assays of interest in a matter of days. For further information in the US call toll free 1-888-219-8020, outside the US call 1-512-219-8020 or email scombs@luminexcorp.com.

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