ELISA microplate Research Article
form has a limited capture capability and a confined assay linear range versus magnetic beads because of the fixed plate surface area. This limitation can be minimized by sample dilution. The nature of capture protein, such as its size, hydrophobicity, polarity, etc. should be considered when the type of an unmodi- fied microplate is selected in order to have optimal capacity.
Future perspective An emerging LC–MS-based approach for quantita- tion of protein therapeutics has been growing quickly to support the shift in drug discovery and develop- ment from traditional small-molecule drugs toward biotherapeutics. One of the biggest challenges for this approach versus LBA is perhaps the assay sensitivity. Immunoaffinity has been incorporated into an LC– MS-based approach (i.e., Immunoaffinity-LC–MS/ MS) for sample enrichment to enhance assay sensi- tivity. The common platform for sample preparation for this approach is currently magnetic beads. This is
likely attributed to the following considerations
besides its ability for sample enrichment: a uniform surface could be provided to all samples by using pre- treated pooled magnetic beads solution. This reduces sample-to-sample variation of the capture antibody capacity; by altering the amount of the beads, it pro- vides flexibility in adjusting capture capacity to meet the specifications of an assay; there are various com- mercialized precoated surfaces of magnetic beads
Executive summary
• Magnetic beads are commonly used as an immunocapture platform with pros and cons for immunoaffinity- LC–MS/MS quantitation of protein therapeutics.
• The comparable performance from ELISA microplates suggests they are a viable alternative platform to magnetic beads.
• The performance of chemically preactivated microplates depends on the surface chemistry. Project-specific investigation should be conducted for this type of plate.
References Papers of special note have been highlighted as: • of interest; •• of considerable interest
1 Timmerman P, Lausecker B, Barosso B et al. Large meets small: connecting the bioanalytical community around peptide and protein bioanalysis with LC–MS(MS). Bioanalysis 4(6), 627–631 (2012).
• Presents a good overview of scientific progress in the bioanalysis of peptides and proteins with MS-based techniques.
2
Li F, Fast D, Michael S. Absolute quantitation of protein therapeutics in biological matrices by enzyme digestion and LC–MS. Bioanalysis 39(2), 2459–2480 (2011).
• Presents a good review of LC–MS- based technology for absolute quantitation of protein therapeutics.
3 Ouyang Z, Furlong MT, Wu S, Sleczka B et al. Pellet digestion: a simple and efficient sample preparation technique for LC–MS/MS quantification of large therapeutic proteins in plasma. Bioanalysis 4(24), 17–28 (2012).
4
Liua G, Ji Q, Dodgea R et al. Liquid chromatography coupled with tandem mass spectrometry for the bioanalysis of proteins in drug development: practical considerations in assay development and validation. J. Chromatogr. A 1284(1), 155–162 (2013).
5 Hagman C, Ricke D, Ewert S et al. Absolute quantitation of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry. Anal. Chem. 80(4), 1290–1296 (2008).
6 Anderson L, Hunter CL. Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins. Mol. Cell. Proteomics 5(4), 573–588 (2006).
available for diverse assay requirements. However, its high cost (Table 2) and potentially troublesome bead wash steps are drawbacks to compete with LBA. Throughout this case study, the outstanding per-
formance of the ELISA microplate platform combined with LC–MS/MS was evident based on low cost, ease of operation and uncompromised results. This suggested an ELISA microplate is a viable alternative to magnetic beads as a solid support for bound affinity capture reagents in biological sample pretreatment for LC–MS-based quan- titation of protein therapeutics, while more applications will be preferred to further support this observation.
Financial & competing interests disclosure The authors have no relevant affiliations or financial involve- ment with any organization or entity with a financial inter- est in or financial conflict with the subject matter or mate- rials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this
manuscript.
Ethical conduct of research The authors state that they have obtained appropriate institu- tional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations in- volving human subjects, informed consent has been obtained from the participants involved.
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