Antibodies, Fluorescent Markers and Their Conjugates. Preparation and Purification of Fluorescently Labeled Antibodies

Abstract

Immunoassays are highly selective bioanalytical methods that detect or quantify analytes using antibodies or antigens as biorecognition elements. In fluorescent immunoassays, fluorescent probes serve as sensitive and versatile detectable signals, enabling quantitative and multiplexed analysis. The performance of these assays critically depends on the properties of the antibodies, fluorescent markers, and their conjugates. This review summarizes both classical fluorescent dyes, derived from cyanine, fluorescein, rhodamine, and coumarin, which established the foundations of immunofluorescence, and modern fluorophores developed over the last decade with improved photostability, brightness, and spectral definition. Particular emphasis is placed on chemical strategies for the preparation of antibody-fluorophore conjugates, as well as on purification and validation methods that ensure conjugate stability and preservation of antigen-binding activity. Recent advances in fluorophore development and conjugation approaches relevant to diagnostic and biomedical applications are also discussed, highlighting the transition from traditional random labeling methods to more controlled and reproducible practices.