Strategies for immunoassay

Abstract

The scope and diversity of immunoassay technology has shown phenomenal growth. Requirements for convenience (whole blood), reliability, simplicity (nonseparation), multiple simultaneous assays, and extreme sensitivity (>zeptomole detection limits) are increasingly demanding. Various strategies have been devised to address these requirements. Many new labels have been tested including laccase, acetate kinase, Vargula luciferase, rare earth cryptates, and Pd-coproporphyrin. Whole blood nonseparation immunoassays have been devised based on porous antibody-coated immuno-electrodes. Fusion conjugates provide a reproducible source of bioluminescent conjugates (e.g., firefly luciferase-Protein A). New nonseparation assay strategies use singlet oxygen channeling, phase modulation fluorescence, or dyesensitized photobleaching principles. Multiple simultaneous assays provide a means of consolidating analytical workload and devising screening tests (eg, strategies based on combinations of labels and spatially separated tests zones). Ultrasensitive chemiluminescent detection reactions for amplifying labels, such as alkaline phosphatase (adamantyl 1,2-dioxetane aryl phosphate substrates) and peroxidase (HRP) (luminol or pyridopyridazine - enhancer (substituted phenol or boronic acid) type detection reagents) have produced significant improvements in sensitivity in sandwich-type assays. Other amplification techniques replicate a bound DNA label directly using the polymerase chain reaction, or replicate a bound peroxidase label indirectly via a catalyzed deposition procedure.