Tumor Biomarker In-Solution Quantification, Standard Production, and Multiplex Detection

Table 1

Advantages and limitations of current immunoassay methods.

Method	Advantages	Limitations
ELISA (colorimetric)	(i) Simple and easy to implement; (ii) Cost-effective; (iii) High sensitivity and specificity; (iv) Wide variety of applications; (v) Protocol conditions can be applied to bead-based assay development	(i) Immobilization on solid surface; (ii) Sample volumes of (50-100 μL per well); (iii) Protocols range from a few hours to days; (iv) Narrow dynamic range (2-3 orders of magnitude); (v) High variability; (vi) High nonspecific absorption; (vii) Unable to multiplex (singleplex assay format)
CLIA (bead-based, paramagnetic, luminescence)	(i) Short protocols; (ii) High sensitivity and robustness; (iii) Wide dynamic range (6-7 orders of magnitude); (iv) High stability of reagents; (v) In-solution detection	(i) Sample volumes of (50-100 μL per well); (ii) High cost of equipment (luminometer); (iii) Complex reaction kinetics (nonlinear response); (iv) Emission intensity dependent on time; (v) Influenced by environmental factors (e.g., temp., light, and pH); (iv) Unable to multiplex (singleplex assay format)
Bead Array Kits (BD™ CBA assay)	(i) Large surface area-to-volume ratio; (ii) Small sample volumes (12-25 μL); (iii) Medium to high throughput (multiplex); (iv) High signal-to-noise ratio; (v) High sensitivity/low variability	(i) Stability and emittance of fluorophore dependent on pH; (ii) Potential for antibody cross-reactivity in multiplex assays; (iii) High cost of instrumentation (dedicated system or flow cytometer); (iv) Antibody clones unknown in commercial kits; (v) Autofluorescence from interfering components in serum
Bead-based multiplexing technologies (Luminex, Quanterix/SiMoA, Millipore SMC™)	(i) Minimize nonspecific binding; (ii) Medium to high throughput (multiplex); (iii) High sensitivity (low limit of detection); (iv) Wide dynamic range (4-6 orders of magnitude)	(i) Unique equipment for each type of analysis platform; (ii) Tradeoffs between number of possible multiplexed; (iii) Limited applications (proteins, nucleic acids, and small molecules); (iv) Fluorophores may interfere with antibody-antigen binding