26 Chapter 2 using top-down (intact), middle-down (semi-intact) and bottom-up (signature peptide) strategies are briefly explored. Finally, the most widely used bottom-up quantification strategy via signature peptide is chronologically discussed in a general workflow where strengths and weaknesses of each step are extensively explained. Ligand binding assay Enzyme-linked immunosorbent assay (ELISA) is arguably the most widely used form of ligand binding assay (LBA), because of its high sensitivity, ease of use, and low instrumental costs [22, 23]. Moreover, due to the spectrophotometric detection principle, ELISA offers a safer alternative with a high ease of use compared to historically used radio immunoassays which require special facilities and operators trained to handle radioactive material [24]. In general, ELISA methods are based on the quantification of the target protein (antigen) using strategies, such as direct, indirect or sandwich type ELISA (Figure 1) [25-27]. In the final step, quantification is performed by adding a substrate solution, often tetramethylbenzidine (TMB), which is gradually oxidized by horseradish peroxidase (HRP) to a colored product. To improve sensitivity the use of a polyclonal secondary antibody in indirect or in sandwich type ELISA can help boost the signal intensity [28]. Alternatively, polymerized HRP can be used to increase sensitivity. Background staining is a very common challenge in these assays, and is often caused by non-specific interactions of either the primary or the secondary polyclonal antibody used [29]. The choice of binding strategy can help minimize the risk of non-specific interactions. For example, in sandwich type ELISA’s, an additional anti-idiotypic antibody binding the antigen at a different epitope provides higher specificity [30]. The reliance of ELISA methods on these specific anti-idiotypic antibodies or ligands can lead to lengthy method development times [31] and higher consumables costs compared to LC-MS/MS methods. Furthermore, ELISA’s allow for one component analysis in one specific bio-matrix, while LC-MS/MS methods allows for multiplexed measurement of multiple therapeutic and endogenous proteins in various bio-matrices [32-34] and due to the narrow linear dynamic range in ELISA, accurate quantification may require multiple sample dilutions thus limiting sample throughput. Importantly, the validation acceptance criteria for ELISAs are less stringent in comparison to LC-MS/MS methods [35, 36], mainly because ELISA methods cannot incorporate internal standards to correct for binding efficiencies influenced by sample matrix or component loss due to binding and washing steps. Finally, different results can be obtained with different ELISA assays as was demonstrated by Vande Casteele by comparison of three commercially available ELISA kits for infliximab and anti-infliximab quantification, stating that comparison of drug levels and ADA monitoring is hampered by lack of standardization [37]. Inter-assay variability makes it difficult for clinicians to compare results from other centers and invalid measurements affects decisions made in patient’s diagnosis and treatment.
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