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Validating a custom multiplex ELISA against individual commercial immunoassays using clinical samples
Michael Liew1, Matthew C. Groll2, James E. Thompson2, Sara L. Call2, Joann E. Moser2, Justin D. Hoopes2, Karl Voelkerding1,3, Carl Wittwer1,3, and Rex S. Spendlove2
1ARUP Laboratories, Salt Lake City
2Spendlove Research Foundation, Logan
3University of Utah School of Medicine, Salt Lake City, UT, USA
BioTechniques, Vol. 42, No. 3, March 2007, pp. 327–333
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The measurement of multiple antigens in a single sample poses clinical and methodological challenges. Here we describe the validation of a multiplexed sandwich enzyme-linked immunosorbent assay (ELISA) array (microELISA) of nine antigens. The antigens tested simultaneously were: α-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), CA 15-3, CA 19-9, β-human chorionic gonadotropin (β-hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). At least 44 clinical samples were tested for each antigen. microELISA results for the nine antigens were then compared with clinical laboratory results obtained for the same antigens in individual chemiluminescent immunoassays. The microELISA had a coefficient of variation (CV) of 7.3% within an assay and 12.6% for assays run at different times. A statistical comparison of results from the microELISA with results from the clinical laboratory showed that the assays had correlation coefficients ranging from 0.99 to 0.76, and Deming regression demonstrated that four of the nine assays were high-quality assays and not statistically different to the individual assays. To determine if the differences in the assays were due to methodology, the microELISA was also compared with conventional ELISAs using identical antibodies and reagents. Deming regression demonstrated that five of the eight assays were high-quality, indicating that a poor correlation between a microELISA and an individual immunoassay are partly due to antibody differences.


Identifying multiple proteins in a single sample has advantages. In cancer diagnostics, specificity can be improved with more than one marker (1,2). For example, cancer antigen 125 (CA 125) is elevated in different types of cancer such as endometrial cancer (3), renal cell carcinoma (4), and ovarian cancer (5). In addition, if different diseases have overlapping clinical symptoms, the qualitative and quantitative assessment of multiple antigens may provide diagnosis. So, it has been reported that the same CA 125 antigen is also elevated in noncancerous conditions, such as endometriosis (6), mitral valve stenosis (7), and hypothyroidism (8,9). Therefore, if other markers can be identified to categorize the diseases, the potential benefits of measuring multiple antigens simultaneously from the same sample are obvious. Currently, technology is available for measuring multiple antigens, but the clinical interpretation is not available.

There are a variety of different methods available for identifying multiple antigens in the same sample simultaneously. The most common method for measuring multiple antigens from a complex mixture of proteins has been by two-dimensional gel electrophoresis (2-DE) (10,11), sometimes followed by identification by tandem mass spectrometry. Also available are protein arrays that require antibodies of known specificity and affinity (10,12) that are immobilized on a surface. Time-of-flight mass spectrometer techniques like matrix-assisted laser desorption/ionization (MALDI) and surface-enhanced laser desorption/ionization (SELDI) have also been used for identifying multiple antigens in complex mixtures (13,14).

Many methods allow parallel protein identification, but measurement of their concentrations is an important indicator used in both life science research and clinical practice (15). Arrays of antibodies for simultaneous antigen quantification are considered the most accurate (10,12). Enzyme-linked immunosorbent assay (ELISA) microarrays were first reported as printed arrays on glass (16). These arrays had 144 spots each that corresponded to the location of a well in a microtiter plate. A 16-spot array printed onto nitrocellulose attached to a microscope slide has also been reported (17). Microarrays can now be printed directly onto the bottom of a 96-well plate and have been used by different investigators and companies (15,18). For example, SearchLight™ arrays (Pierce Biotechnology, Rockford, IL, USA) have up to 16 assays per well in a sandwich format similar to our multiplexed sandwich ELISA array (microELISA) (15). When multicytokine assays based upon microELISA technology (SearchLight and FAST® Quant systems; Whatman Schleicher & Schuell, Keene, NH, USA) were compared with a bead-based array (Beadlyte® Luminex human multicytokine detection system; Upstate, Charlottesville, VA, USA) (19), each assay system had its own merits. For example, the FAST Quant assay tested had the broadest dynamic range and lowest sample volume requirements compared with the other assays, and the SearchLight assay had the best reported detection limits.

A rigorous comparison of microELISAs to identical assays performed as individual conventional ELISAs has not been performed. Our microELISA uses up to 25 antibodies bound to the bottom of a microtiter plate well in an array format. The microELISA was compared with routine clinical testing and identical conventional ELISAs using six tumor markers [α-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), CA 125, CA 15-3, and CA 19-9] and three hormones [β-human chorionic gonadotropin (β-hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH)].

Materials and Methods


All samples were de-identified according to a global Associated Regional and University Pathologists (ARUP) Laboratories protocol under International Review Board (IRB) no. 7275. The samples used in this study had been submitted to ARUP for the quantification of AFP, total PSA, CEA, CA 125, CA 15-3, CA 19-9, β-hCG, LH, or FSH antigen levels. AFP, total PSA, CA 15-3, and CA 125 were measured on the Modular E170 analyzer (Roche Diagnostics, Indianapolis, IN, USA). CEA, CA 19-9, and β-hCG were measured on the Immulite® 2000 (Diagnostic Products Corporation, Los Angeles, CA, USA). LH and FSH were measured on the ADVIA Centaur® Immunoassay system (Bayer Healthcare, Tarrytown, NY, USA). Samples were either human serum or plasma (heparin or EDTA). Three hundred and sixty-four samples were tested by ARUP for one or two of the nine antigens. The only antigens that were measured on the same sample were LH and FSH; all other antigens were measured separately on different samples. Forty-four samples were tested for both FSH and LH, with three of those having LH results below the detection limit of the assay. Forty-six samples were tested for CA 125, 61 samples for CA 19-9, 19 samples for β-hCG, 55 samples for CEA, 37 samples for CA 15-3, 50 samples for PSA, and 52 samples for AFP. Upon completion of testing, the samples were stored at −80°C until transfer to the Spendlove Research Foundation (Logan, UT, USA) where the microELISA analysis was performed.

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