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      Detection of cynomolgus monkey anti-protein XYZ antibody using immunocapture-LC/MS

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      Journal of Applied Bioanalysis

      Betasciencepress Publishing

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          Abstract

          Although enzyme-linked immunosorbent assays and electrochemiluminescence (ECL) immunoassays are the most widely used platform for ADA detection, they may be compromised by drug interference. We describe here an alternate, free of drug interference, immunocapture-LC/MS methodology for detecting anti-protein XYZ antibody in cynomolgus monkey plasma. In our approach, ADA-protein XYZ complexes are captured by a mouse monoclonal anti-drug antibody on streptavidin magnetic beads and separated from monkey plasma by a magnet. After elution, ADA are digested with trypsin and detected by LC/MS using a surrogate peptide common to monkey IgG subclasses 1-4. The immunocapture-LC/MS assay was applied to support a toxicology study and results were in close agreement with those from a modified ECL immunoassay. To our knowledge, this is the first application of using LC/MS for monkey ADA detection.

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          Most cited references 18

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          Bioequivalence and the immunogenicity of biopharmaceuticals.

          The expiry of the first patents for recombinant-DNA-derived biopharmaceuticals will open the possibility of marketing generics, if they can be shown to be essentially similar to the innovator product. However, as shown by the problem of immunogenicity, the properties of biopharmaceuticals are dependent on many factors, including downstream processing and formulation. Products from different sources cannot be assumed to be bioequivalent, even if identical genes are expressed in the same host cells and similar production methods are used. Some of the influencing factors are still unknown, which makes it impossible to completely predict biological behaviour, such as immunogenicity, which can sometimes lead to serious side effects.
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            Mass spectrometry based targeted protein quantification: methods and applications.

            The recent advance in technology for mass spectrometry-based targeted protein quantification has opened new avenues for a broad range of proteomic applications in clinical research. The major breakthroughs are highlighted by the capability of using a "universal" approach to perform quantitative assays for a wide spectrum of proteins with minimum restrictions and the ease of assembling multiplex detections in a single measurement. The quantitative approach relies on the use of synthetic stable isotope labeled peptides or proteins, which precisely mimic their endogenous counterparts and act as internal standards to quantify the corresponding candidate proteins. This report reviews recently developed platform technologies for emerging applications of clinical proteomics and biomarker development.
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              Quantification of thyroglobulin, a low-abundance serum protein, by immunoaffinity peptide enrichment and tandem mass spectrometry.

              Quantification of serum tumor markers plays an important role in determining whether patients treated for cancer require further therapy. Whereas large-scale proteomic efforts aim to identify novel tumor markers to facilitate early detection, optimization of methods for quantifying known tumor markers offers another approach to improving management of malignancies. For example, immunoassays used in clinical practice to measure established tumor markers suffer from potential interference from endogenous immunoglobulins and imperfect concordance across platforms-problems that also plague many other immunoassays. To address these important limitations, this study used peptide immunoaffinity enrichment in concert with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify thyroglobulin, a well-characterized tumor marker. We identified 3 peptides in tryptic digests of thyroglobulin that were detected at low concentrations by tandem mass spectrometry, raised polyclonal antibodies to those peptides, and used the antibodies to extract the 3 corresponding peptides from tryptic digests of human serum. We quantified each endogenous peptide using LC-MS/MS and multiple reaction monitoring with external calibrators. The detection limit for endogenous thyroglobulin in serum was 2.6 microg/L (4 pmol/L). Direct comparison with immunoassay revealed good correlation (r(2) = 0.81). Immunoaffinity peptide enrichment-tandem mass spectrometry can detect tryptic peptides of thyroglobulin at picomolar concentrations while also digesting the endogenous immunoglobulins that can potentially interfere with traditional immunoassays. Our observations suggest a general analytical strategy for using immunoaffinity isolation together with tandem mass spectrometry to quantify tumor antigens and other low-abundance proteins in human serum.
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                Author and article information

                Journal
                Journal of Applied Bioanalysis
                J Appl Bioanal
                Betasciencepress Publishing
                2405710X
                October 13 2016
                October 13 2016
                : 2
                : 4
                : 117-128
                Article
                10.17145/jab.16.016
                © 2016

                This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

                Product
                Self URI (journal-page): https://jab.scholasticahq.com/

                General life sciences, Chemistry, Analytical chemistry, Life sciences

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