Validating a custom multiplex ELISA against individual commercial immunoassays using clinical samples

Area of Science:

• Biochemistry
• Immunology
• Clinical Diagnostics

Background:

• Multiplexed antigen measurement presents significant clinical and methodological hurdles.
• Accurate simultaneous detection of multiple biomarkers is crucial for effective disease diagnosis and monitoring.

Purpose of the Study:

• To validate a multiplexed sandwich enzyme-linked immunosorbent assay (microELISA) for simultaneous detection of nine clinically relevant antigens.
• To compare the performance of the microELISA with established individual immunoassays and conventional ELISAs.

Main Methods:

• A microELISA array was developed and validated for nine antigens: AFP, PSA, CEA, CA 125, CA 15-3, CA 19-9, beta-hCG, LH, and FSH.
• Clinical samples (n≥44 per antigen) were analyzed using microELISA and compared against results from individual chemiluminescent immunoassays.
• Methodological comparisons were performed against conventional ELISAs using identical antibodies and reagents.

Main Results:

• The microELISA demonstrated good intra-assay (7.3% cv) and inter-assay (12.6% cv) variability.
• Correlation coefficients between microELISA and clinical laboratory results ranged from 0.76 to 0.99.
• Deming regression indicated four of nine microELISA assays were high-quality and statistically comparable to individual assays.
• Comparison with conventional ELISAs revealed five of eight assays were high-quality, suggesting antibody differences contribute to assay variations.

Conclusions:

• The validated microELISA offers a reliable method for multiplexed antigen detection.
• Assay performance is influenced by methodological factors, including antibody specificity and reagent choices.
• This multiplexing approach shows promise for improving efficiency in biomarker analysis.