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

Abstract

The diagnosis and monitoring of cancer have been facilitated by discovering tumor "biomarkers" and methods to detect their presence. Yet, for certain cancers, we still lack sensitive and specific biomarkers or the means to quantify subtle concentration changes successfully. The identification of new biomarkers of disease and improving the sensitivity of detection will remain key to changing clinical outcomes. Patient liquid biopsies (serum and plasma) are the most easily obtained sources for noninvasive analysis of proteins that tumor cells release directly and via extracellular microvesicles and tumor shedding. Therefore, an emphasis on creating reliable assays using serum/plasma and "direct, in-solution" ELISA approaches has built an industry centered on patient protein biomarker analysis. A need for improved dynamic range and automation has resulted in the application of ELISA principles to paramagnetic beads with chemiluminescent or fluorescent detection. In the clinical testing lab, chemiluminescent paramagnetic assays are run on automated machines that test a single analyte, minimize technical variation, and are not limited by serum sample volumes. This differs slightly from the R&D setting, where serum samples are often limiting; therefore, multiplexing antibodies to test multiple biomarkers in low serum volumes may be preferred. This review summarizes the development of historical biomarker "standards", paramagnetic particle assay principles, chemiluminescent or fluorescent biomarker detection advancements, and multiplexing for sensitive detection of novel serum biomarkers.

Figure 1

Minimum detectable concentration versus actual point of detection. The minimum detectable concentration (MDC) is the earliest detection point for a biomarker that exceeds its normal level of expression. As biomarkers are secreted by both healthy and tumor cells, the average biomarker level can be above the assay's detection limit before tumor initiation. Therefore, a cutoff value is required to differentiate between tumor-bearing and unaffected or benign conditions in serum assays. As tumor size increases, biomarker expression increases (blue circles). Although a biomarker is detected at or below the cutoff value, samples are not considered diseased until they exceed this cutoff value (the actual point of detection).

Figure 2

Types of ELISA. (a) Direct: an antigen is immobilized on the surface of a multiwell plate. A labeled primary antibody binds to the target antigen and is detected using an enzymatic substrate. (b) Indirect: an indirect ELISA consists of an unconjugated antibody binding to the target antigen, followed by a conjugated antibody. (c) Competitive: this assay is also known as an inhibition assay. The target antigen is precoated on a multiwell plate. An enzyme-labeled antibody is preincubated with the sample and may form antibody-antigen complexes with the inhibitor antigen before being added to the multiwell plate. The free antibody binds to the target antigen immobilized on the surface of a multiwell plate. A lower signal corresponds to a higher amount of antigen. (d) Sandwich: the sandwich “capture” assay is the most complex but provides sensitive and highly specific detection using two antibodies that preferably bind to two different epitopes. The antigen binds to the capture antibody and is detected using a second “detection” antibody. A labeled secondary antibody is then used to produce a measurable signal.

Figure 3

Magnetic antibody-microparticle conjugation. A strategy to prepare a universal magnetic microparticle that can be conjugated to any biotinylated capture antibody has been devised following the methodology set forth in Beckman Coulter literature.

Figure 4

Antibody conjugation to the surface of the paramagnetic particle: (a) antibody conjugation using conventional antibody methods; (b) “orientation-specific” antibody conjugation using Beckman Coulter's 3-layer antibody coupling approach.

Figure 5

Biomarker detection using flow cytometry. The Luminex strategy detects multiplexed biomarkers using dual wavelengths. The two fluorescent dyes are excited at a wavelength of 635 nm, and the fluorophore is excited at 532 nm. The fluorescent light emitted from the bead is detected at two separate wavelengths, corresponding to a specific biomarker, while the signal produced from each fluorophore is detected at another wavelength.