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Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

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.

SUBMITTER: Wan Z

PROVIDER: S-EPMC5794080 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

Wan Zhi Z Ostendorff Heather P HP Liu Ziying Z Schneider Lynda C LC Rothschild Kenneth J KJ Lim Mark J MJ

PloS one 20180201 2

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 susceptib ...[more]

PMID: 29389948

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Project description:BackgroundApproximately 50% of uveal melanoma (UM) patients develop metastases preferentially in the liver leading to death within 15 months. Currently, there is no effective treatment for metastatic UM, in part because the tumor burden is typically high when liver metastases are detected through abnormal liver function tests (LFTs) or imaging studies. The use of LFTs results followed by diagnostic tests has high specificity and predictive values but low sensitivity, and better tests are needed for early diagnosis of the primary tumor as well as its metastatic spread. To evaluate serum biomarkers for the early detection of UM, multiplex immunoassays were developed.MethodsMagnetic bead-based multiplex immunoassays were developed for the selected serum biomarkers using a Bio-Plex 200 system. The dynamic ranges, lower limits of detection and quantification, cross-reactivity, and intra- and inter-assay precision were assessed. All proteins were analyzed in sera of 48 patients diagnosed with UM (14 metastatic, 9 disease-free (DF) ≥ 5 years, 25 unknown) and 36 healthy controls. The performance of the biomarkers was evaluated individually and in combination for their ability to detect UM.ResultsA 7-plex immunoassay of OPN, MIA, CEACAM-1, MIC-1, SPON1, POSTN and HSP27 was developed with negligible cross-reactivity, recovery of 84-105%, and intra-assay and inter-assay precision of 2.3-7.5% or 2.8-20.8%, respectively. Logistic regression identified a two-marker panel of HSP27 and OPN that significantly improved the individual biomarker performance in discriminating UM from healthy controls. The improved discrimination of a two-marker panel of MIA and MIC-1 was also observed between metastatic UM and DF, however not statistically significant due to the small sample size.ConclusionsThe multiplex immunoassay provides sufficient analytical performance to evaluate serum biomarkers that complement each other in detection of UM, and warrants further validation with a larger number of patient samples.

Dataset Information

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

Publications

Photocleavage-based affinity purification of biomarkers from serum: Application to multiplex allergy testing.

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