Abstract
Multiplex serological immunoassays, such as implemented on microarray or microsphere-based platforms, provide greater information content and higher throughput, while lowering the cost and blood volume required. These features are particularly attractive in pediatric food allergy testing to facilitate high throughput multi-allergen analysis from finger- or heel-stick collected blood. However, the miniaturization and microfluidics necessary for creating multiplex assays make them highly susceptible to the “matrix effect” caused by interference from non-target agents in serum and other biofluids. Such interference can result in lower sensitivity, specificity, reproducibility and quantitative accuracy. These problems have in large part prevented wide-spread implementation of multiplex immunoassays in clinical laboratories. We report the development of a novel method to eliminate the matrix effect by utilizing photocleavable capture antibodies to purify and concentrate blood-based biomarkers (a process termed PC-PURE) prior to detection in a multiplex immunoassay. To evaluate this approach, it was applied to blood-based allergy testing. Patient total IgE was purified and enriched using PC-PURE followed by multiplex microsphere-based detection of allergen-specific IgEs (termed the AllerBead assay). AllerBead was formatted to detect the eight most common pediatric food allergens: milk, soy, wheat, egg, peanuts, tree nuts, fin fish and shellfish, which account for >90% of all pediatric food allergies. 205 serum samples obtained from Boston Children’s Hospital were evaluated. When PC-PURE was employed with AllerBead, excellent agreement was obtained with the standard, non-multiplex, ImmunoCAP® assay (average sensitivity above published negative predictive cutoffs = 96% and average Pearson r = 0.90; average specificity = 97%). In contrast, poor ImmunoCAP®-correlation was observed when PC-PURE was not utilized (average sensitivity above published negative predictive cutoffs = 59% and average Pearson r = 0.61; average specificity = 97%). This approach should be adaptable to improve a wide range of multiplex immunoassays such as in cancer, infectious disease and autoimmune disease.
Highlights
IntroductionA major problem for biomarker-based diagnostic assays is the low sensitivity and specificity provided by a single biomarker
Multiplex assays and the problem of the matrix effectA major problem for biomarker-based diagnostic assays is the low sensitivity and specificity provided by a single biomarker
The digoxigenin moiety conjugated to the IgE provided a convenient affinity tag to allow quantification of the digoxigenin labeled IgE (Dig-IgE) using a Luminex1 microsphere-based sandwich immunoassay, where an anti-digoxigenin antibody-coated microsphere captures the Dig-IgE which is detected using a fluorescently labeled anti-IgE detection antibody
Summary
A major problem for biomarker-based diagnostic assays is the low sensitivity and specificity provided by a single biomarker. To overcome this problem, researchers have focused on developing effective multi-biomarker panels. A high priority is to transition these multi-biomarker panels to multiplex assay formats both for large-scale biomarker validation and for clinical assay. In this regard, the utilization of multiplex platforms will be critical for providing lower cost and higher throughput, especially necessary for population-wide screening. A second example is blood drawn using finger-stick and heel-stick methods for pediatric diagnostic testing where only ~100–200 μL of serum is typically available [13, 14]
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