Step by step with ELISA: Mechanism of operation, crucial elements, different protocols, and insights on immobilization and detection of various biomolecular entities
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© The Author(s) 2018. Current chapter describes the essential components of ELISA including the solid phase, the adsorbents (different types of target biomolecules), and the washing and blocking agents used in assay procedure. The chapter also r,!iews widely applied enzymes and substrates with their specific characteristics. To complete the assay, the chapter offers information regarding the stopping procedure and readout techniques such as colorimetric, fluorescence and luminescence, along with their reading instruments. To secure a high specificity, the chapter describes protocols for conducting different types of controls in the assay procedure. These controls are namely: positive, endogenous, negative, standard, and spike controls. The chapter subsequently describes available ELISA protocols including direct, indirect, sandwich, double sandwich, and competitive assays. Finally, this chapter is dedicated to reviewing immobilization techniques including physical, covalent, oriented strategies as well as immobilization via entrapment. In the case of covalent immobilization of the biomolecules, protein attachment via zero-length cross linkers and spacers (linear or branched) are described.