Antibody-Based Protein Multiplex Platforms: Technical and Operational Challenges

AbstractBackground: The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen.Content: We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed.Summary: Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.

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Clinical laboratory medicine. In vitro diagnostic medical devices. Validation of user quality control procedures by the manufacturer

10.3403/03084253u ◽

2015 ◽

Keyword(s):

Quality Control ◽

Medical Devices ◽

Clinical Laboratory ◽

Laboratory Medicine ◽

In Vitro Diagnostic ◽

Control Procedures

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Quality Control of Microarray Assays for Toxicogenomic and In Vitro Diagnostic Applications

Essential Concepts in Toxicogenomics - Methods in Molecular Biology™ ◽

10.1007/978-1-60327-048-9_3 ◽

2008 ◽

pp. 45-68 ◽

Cited By ~ 6

Author(s):

Karol L. Thompson ◽

Joseph Hackett

Keyword(s):

Quality Control ◽

In Vitro Diagnostic ◽

Diagnostic Applications

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Going beyond clinical routine in SARS-CoV-2 antibody testing - A multiplex corona virus antibody test for the evaluation of cross-reactivity to endemic coronavirus antigens

10.1101/2020.07.17.20156000 ◽

2020 ◽

Cited By ~ 3

Author(s):

Matthias Becker ◽

Monika Strengert ◽

Daniel Junker ◽

Tobias Kerrinnes ◽

Philipp D. Kaiser ◽

...

Keyword(s):

Vaccine Development ◽

Antibody Test ◽

Cross Reactivity ◽

Antibody Testing ◽

N Protein ◽

Humoral Immune ◽

In Vitro Diagnostic ◽

Human Coronaviruses ◽

Multiplexed Immunoassay

Given the importance of the humoral immune response to SARS-CoV-2 as a global benchmark for immunity, a detailed analysis is needed to (i) monitor seroconversion in the general population, (ii) understand manifestation and progression of the disease, and (iii) predict the outcome of vaccine development. Currently available serological assays utilize single analyte technologies such as ELISA to measure antibodies against SARS-CoV-2 antigens including spike (S) or nucleocapsid (N) protein. To measure individual antibody (IgG and IgA) responses against SARS-CoV-2 and the endemic human coronaviruses (hCoVs) NL63, 229E, OC43, and HKU1, we developed a multiplexed immunoassay (CoVi-plex), for which we included S and N proteins of these coronaviruses in an expanded antigen panel. Compared to commercial in vitro diagnostic (IVD) tests our CoVi-plex achieved the highest sensitivity and specificity when analyzing 310 SARS-CoV-2 infected and 866 uninfected individuals. Simultaneously we see high IgG responses against hCoVs throughout all samples, whereas no consistent cross reactive IgG response patterns can be defined. In summary, our CoVi-plex is highly suited to monitor vaccination studies and will facilitate epidemiologic screenings for the humoral immunity toward pandemic as well as endemic coronaviruses.

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Clinical laboratory testing and in vitro diagnostic test systems. Reference method for testing the in vitro activity of antimicrobial agents against yeast of fungi involved in infectious diseases

10.3403/30237472 ◽

2012 ◽

Keyword(s):

Infectious Diseases ◽

Diagnostic Test ◽

Laboratory Testing ◽

Antimicrobial Agents ◽

Clinical Laboratory ◽

Reference Method ◽

In Vitro Activity ◽

In Vitro Diagnostic ◽

Clinical Laboratory Testing

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A Reference Method Laboratory Network for Cholesterol: A Model for Standardization and Improvement of Clinical Laboratory Measurements

Clinical Chemistry ◽

10.1093/clinchem/46.11.1762 ◽

2000 ◽

Vol 46 (11) ◽

pp. 1762-1772 ◽

Cited By ~ 102

Author(s):

Gary L Myers ◽

Mary M Kimberly ◽

Parvin P Waymack ◽

S Jay Smith ◽

Gerald R Cooper ◽

...

Keyword(s):

Reference Materials ◽

Clinical Laboratory ◽

Matrix Effects ◽

Reference Method ◽

Performance Criteria ◽

Blood Cholesterol ◽

Field Methods ◽

Laboratory Network ◽

In Vitro Diagnostic

Abstract Background: Accurate and precise measurement of blood cholesterol plays a central role in the National Cholesterol Education Program’s strategy to reduce the morbidity and mortality attributable to coronary heart disease. Matrix effects hamper the ability of manufacturers to adequately calibrate and validate traceability to the National Reference System for Cholesterol (NRS/CHOL). CDC created the Cholesterol Reference Method Laboratory Network (CRMLN) to improve cholesterol measurement by assisting manufacturers of in vitro diagnostic products with validation of the traceability of their assays to the NRS/CHOL. Methods: CRMLN laboratories established the CDC cholesterol reference method (modification of the Abell-Levy-Brodie-Kendall chemical method) and are standardized using CDC frozen serum reference materials. CRMLN laboratories use common quality-control materials and participate in monthly external performance evaluations conducted by CDC. The CRMLN performance criteria require member laboratories to agree with CDC within ± 1.0% and maintain a CV ≤2.0%. Results: From 1995 to 2000, the CRMLN laboratories met the accuracy criterion 97% of the time and the precision criterion 99% of the time. During this time period, the CRMLN maintained an average bias to CDC of 0.01% and an average collective CV of 0.33%. Conclusions: CDC established the CRMLN as the first international reference method laboratory network. The CRMLN assists manufacturers in the validation of the calibration of their diagnostic products so that clinical laboratories can measure blood cholesterol more reliably. The CRMLN can serve as a model for other clinical analytes where traceability to a hierarchy of methods is needed and matrix effects of the field methods with processed calibrators or reference materials are present.

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