Biomarker Testing Assay By LC-MS/MS, ELISA, MSD QuickPlex, And Luminex Multiplex

The ultimate goal of a biomarker is to have a reliable, yet powerful detection tool for early diagnosis and monitoring of diseases. Biomarker detection technologies have experienced enormous progress in the past decade. Research has developed several detection methods that include LC-MS/MS, enzyme-linked immunosorbent assay (ELISA), multiplex assays, fluorescence methods, and electrochemical assays. Today, biomarker assay services and biomarker validation services extensively use LC-MS/MS strategies and capture-based strategies in biomarker analysis.

LC-MS/MS biomarker testing

Several biomarker assay services use LC-MS/MS to detect biomarkers in biological samples. The basic LC-MS/MS setup uses the same principle of tandem mass spectrometry. Biomarker assay services introduce protease-digested mixture onto the liquid chromatography column. A technician elutes this mixture over time using a solvent gradient. Peptides after elution enter a tandem mass spectrometer, where they are ionized, separated, and detected.

Depending on the application, we can broadly divide LC-MS/MS into three techniques. In a data-dependent approach, biomarker assay services use the first detection “MS1” to quantify the biomarker. They may seldom subject a subset of ions to another round of mass spectrometry and get MS2 fragments. Technicians use targeted LC-MS/MS approaches when they already know the target of interest. These approaches quantity biomarkers at the MS2 level and therefore are more precise than data-dependent approaches. The third approach is called a data-independent acquisition. Data-independent acquisition is an untargeted approach where the data is obtained out of the tiled fragment scans. 

Capture-based strategies in biomarker testing

Antibodies have been the foundation of biomarker assays for a very long time. They provide a flexible yet specific biomarker testing tool that can be conjugated to several reporter proteins and used in biomarker assays.

ELISA tests are the most fundamental capture-based biomarker assays. ELISA biomarker assays use enzyme-linked antibodies that are targeted against specific biomarkers. The assay begins with the binding of antigens to a solid surface. Then a researcher applies the enzyme-linked antibody over the surface so that it binds the antigen in a sandwich-like complex. This antigen/antibody binding activates the enzyme, which changes the color of the substrate. The researcher measures the color change and quantifies the biomarker in biological fluids.

Although ELISA tests remain the primary method for biomarker assays, the emergence of new technology, electrochemiluminescent immunoassays such as MSD QuickPlex, and Luminex Multiplex are now the new standards in biomarker testing. In MSD QuickPlex biomarker assays, researchers coat the carbon electrodes with antibodies so that they can multiplex up to 10 targets. They then introduce ECL-conjugated secondary antibodies, which when supplied with electricity emits light. MSD QuickPlex biomarker assays have high sensitivity and can measure even low pg/ml levels. 

Luminex Multiplex biomarker assays use matrix-bound color-coded beads to capture the antibodies. Technicians bind a biotinylated-detection antibody to a streptavidin-conjugated fluorescent dye for detecting biomarkers in samples. They then subject this complex through two lasers, where the first one decodes the beads while the second one quantifies the color intensity. 

Future directions

Recent advancements in the sensitivity and reproducibility of LC-MS/MS instrumentation coupled with improvements in the immunoassay-based single-molecule and multiplex biomarker assays offer a spectrum of tools for bioanalytical testingor bioanalytical method development. These tools will aid biomarker validation services and biomarker assay services to carry out hypothesis-free target bioanalysis with accurate and sensitive quantification of small target molecules.

About This Author

Maria Brown Joined: April 1st, 2020