Allergy Testing
Specific · Sensitive · Multiplexed

Introduction

Luminex has developed an allergy test that measures specific IgE antibodies for sixteen grass allergens simultaneously in a multiplexed format. Each assay consists of sixteen FlowMetrix microsphere sets, each coated with a different allergen (target) that binds allergen-specific antibodies. Detection of allergen-specific IgE is achieved with goat anti-IgE (affinity purified) followed by green fluorescence-labeled anti-goat IgG (reporter). In parallel, the allergen-specific IgG in each test serum was measured using an anti-IgG reporter. Results indicated in Figure 1 show that the FlowMetrix System provides a uniquely powerful platform for allergy screening that provides sensitivity and specificity in a true multiplexed format.

Materials

Grass allergen extracts, canine sera and affinity purified goat anti-canine IgE were kindly provided by Dr. Bill Mandy, BioMedical Services, Austin, TX. Rabbit anti-Goat IgG-FITC and Goat anti-canine IgG-FITC were purchased from Sigma Chemical Co., Inc., St. Louis, MO. EDC (1-ethyl-3-3 [3-dimethylaminopropyl] carbodiimide hydrochloride), and Sulfo-NHS (N-hydroxysulfosuccinimide) were purchased from Pierce Chemicals, Rockford, IL. RIA grade Bovine Serum Albumin (BSA) was purchased from Sigma Chemical Co. St. Louis, MO.

Figure 1

Methods

Allergen Conjugation to Microspheres: Sixteen grass allergens were conjugated to sixteen distinct FlowMetrix microsphere sets with a two-step EDC coupling. Twenty microliters of each microsphere set was activated, washed and suspended in fifty microliters of diluted allergen (1:100). After two hours, the microspheres were washed and stored in 0.02% Tween 20, 1 mg/ml BSA in PBS, pH 7.4 (PBSTB).

Multiplexed Grass Allergen IgE assay: Equivalent amounts of each of the sixteen grass allergen loaded microspheres were mixed. Twenty microliters of the microsphere mixture was reacted with sixty microliters of a 1:3 dilution of canine serum in PBSTB and the mixture incubated for fifteen minutes. Microspheres were washed in PBSTB by centrifugation at 13,400 x g for thirty seconds and suspended in forty microliters of a 5 m g/ml solution of goat anti-canine IgE. After incubation for fifteen minutes, microspheres were washed in PBSTB and treated with forty microliters of rabbit anti-goat IgG-FITC at 20 m g/ml. After fifteen minutes, the assay was read using the FlowMetrix System. Negative controls included the canine serum with the rabbit anti-goat IgG-FITC. Allergen-specific canine IgE was determined by subtraction of the mean intensity of fluorescence (MFI) of the green channel (FL1) for the negative controls for each grass allergen from the MFI of FL1 for the tubes including the goat anti-canine IgE.

Multiplexed Grass Allergen IgG assay: Equivalent amounts of each of the sixteen grass allergen loaded microspheres were mixed. Twenty microliters of the microsphere mixture was reacted with sixty microliters of a 1:3 dilution of canine serum in PBSTB and the mixture incubated for fifteen minutes Microspheres were washed in PBSTB and suspended in twenty-five microliters of a 50 m g/ml solution of goat anti-canine IgG-FITC. After incubation for fifteen minutes, the assay was read using the FlowMetrix System. Negative controls included the microspheres without canine serum. Allergen-specific canine IgG was determined by subtraction of the MFI of FL1 for the negative control for each grass allergen from the MFI of FL1 for the tubes including canine serum.

Results

Multiplexed Assay for Grass Allergen Specific IgE and IgG:

Six canine sera were assayed for IgE or IgG reactivities to sixteen different grass allergens simultaneously. The results are shown in Figure 1. Two canines, Rex and Ace demonstrated relatively low IgE reactivity to most of the grass allergens with the exception of Wheat grass and several others. The other four canines demonstrated significant IgE responses to the majority of the sixteen grass allergens. These results agreed with the ELISA results provided by BioMedical Services. There was no apparent correlation between IgG and IgE response to grass allergens in the six canines. Some canines were low responders for both IgE and IgG, some were reactive with both immunoglobulin subclasses, and some demonstrated IgE reactivity in a low background of IgG specific for the grass allergens.

As seen in Figure 1, one hundred ninety two data points were obtained from twelve samples representing the sixteen assays multiplexed in each tube. This is a clear demonstration of the power of FlowMetrix technology.

Conclusions

Two multiplexed assays for serum IgG and IgE specific for sixteen grass allergens have been developed. The FlowMetrix System and an Elisa system obtained comparable results with six canine sera. This assay was developed at Luminex Corporation for the purpose of demonstrating the power of FlowMetrix technology.

Luminex Corporation provides the FlowMetrix 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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