Monoplex and multiplex immunoassays: approval, advancements, and alternatives.

Immunoassays are a powerful diagnostic tool and are widely used for the quantification of proteins and biomolecules in medical diagnosis and research. Enzyme-linked immunosorbent assay (ELISA) is the most commonly used immunoassay format and allows the detection of biomarkers at a very low concentration. The diagnostic platforms such as enzyme immunoassay (EIA), chemiluminescence (CL) assay, polymerase chain reaction (PCR), flow cytometry (FC), and mass spectrometry (MS) have been used to identify molecular biomarkers. However, these diagnostic tools requiring expensive equipment, long testing time, and qualified personnel that is not always available in small local hospitals with limited resources. The lateral flow immunoassay (LFIA) platform was developed for rapidly obtaining laboratory results and to make urgent decisions in emergency medicine, as well as the recently introduced concept of testing at the site of care (point-of-care, POC). The simultaneous measurement of different substances from a single sample called multiplex assays have become increasingly significant for in vitro quantification of multiple analytes in a single sample, thereby minimising cost, time, and volume. In multiplex immunoassays, the ligands are immobilized either in planar format (flat surface) or on microspheres in suspension that binds to target analytes in sample. The multiplex technology has established itself in proteomic networks and pathways, validation of genomic discoveries, and in the development of clinical biomarkers. In the present review article, various types of monoplex/simplex and complex/multiplex immunoassays have been analysed that are increasingly being applied in laboratory medicine. Also, some advantages and disadvantages of these multiplex assays have also been included such as experimental animals, in vitro tests using cell lines and tissue samples, 3-dimensional modelling and bioprinting, in silico tests, organ-on-chip, and computer modelling.