Antibodies are quintessential affinity reagents for the investigation and determination of a protein's expression patterns, localization, quantitation, modifications, purification, and functional understanding. Antibodies are typically used in techniques such as Western blot, immunohistochemistry (IHC), and enzyme-linked immunosorbent assays (ELISA), among others. The methods employed to generate antibodies can have a profound impact on their success in any of these applications. We raised antibodies against 10 serum proteins using 3 immunization methods: peptide antigens (3 per protein), DNA prime/protein fragment-boost (“DNA immunization”; 3 per protein), and full length protein. Antibodies thus generated were systematically evaluated using several different assay technologies (ELISA, IHC, and Western blot). Antibodies raised against peptides worked predominantly in applications where the target protein was denatured (57% success in Western blot, 66% success in immunohistochemistry), although 37% of the antibodies thus generated did not work in any of these applications. In contrast, antibodies produced by DNA immunization performed well against both denatured and native targets with a high level of success: 93% success in Western blots, 100% success in immunohistochemistry, and 79% success in ELISA. Importantly, success in one assay method was not predictive of success in another. Immunization with full length protein consistently yielded the best results; however, this method is not typically available for new targets, due to the difficulty of generating full length protein. We conclude that DNA immunization strategies which are not encumbered by the limitations of efficacy (peptides) or requirements for full length proteins can be quite successful, particularly when multiple constructs for each protein are used.
Immunological methods such as ELISA have been traditionally employed to quantify protein levels in plants improved through modern biotechnology. Combined trait products (i.e., plants producing multiple recombinant proteins) created by introducing multiple genetic traits by transformation or traditional breeding methods have prompted the need for the development of analytical assay technologies capable of detecting and quantifying multiple proteins in a single assay. The development of a two-site, sandwich, dual-label, time-resolved fluorometry-based immunoassay (TRFIA) capable of simultaneously quantitating two recombinant proteins (CP4 EPSPS and Cry3A) in plant sample extracts of genetically improved potato cultivars is reported here. The performance characteristics of TRFIA were similar to or exceeded those of current ELISA methods used to detect and quantitate these proteins. TRFIA is a practical and reliable assay for the quantitation of proteins in genetically improved potato plants and offers an alternative approach to conventional ELISA methods with the added benefit of multiple analyte detection.
This study agrees with the existing literature demonstrating that the LE test strips are among the lowest sensitivity tests for PJI. The urinary LE tests strips should not be used to rule-out PJI, as they often fail to detect abundant levels of LE in synovial fluid. Instead, it is more appropriate to use the (++) LE test strip result as a secondary confirmatory rule-in test for PJI because of its high specificity.
Tags are widely used to monitor a protein’s expression level, interactions, protein trafficking, and localization. Membrane proteins are often tagged in their extracellular domains to allow discrimination between protein in the plasma membrane from that in internal pools. Multipass membrane proteins offer special challenges for inserting a tag since the extracellular regions are often composed of small loops and thus inserting an epitope tag risks perturbing the structure, function, or location of the membrane protein. We have developed a novel tagging system called snorkel where a transmembrane domain followed by a tag is appended to the cytoplasmic C-terminus of the membrane protein. In this way the tag is displayed extracellularly, but structurally separate from the membrane protein. We have tested the snorkel tag system on a diverse panel of membrane proteins including GPCRs and ion channels and demonstrated that it reliably allows for monitoring of the surface expression.
Transmembrane proteins, including multipass transmembrane proteins like GPCRs and ion channels, are important targets for therapeutic monoclonal antibody (mab) discovery. Therapeutic antibodies to this class of proteins are generally targeted to extracellular domains displayed on the surfaces of living cells. Challenges associated with developing antibodies to this class of targets are small numbers of extracellular amino acids, membrane-dependent protein conformation, difficulty in expression at high levels, high amino acid sequence homology of human and mouse proteins, and post-translational modifications. DNA immunization strategies with full-length constructs and high throughput flow cytometry screening of mab binding to transfected and control cells was used to generate and identify large numbers of mabs to CXCR4 and ADORA2A (GPCRs) and CD20 (a tetraspan membrane protein). Panels of mabs were generated for all 3 targets with low numbers of hybridoma fusions. For each target the mab gene sequences were shown to be unique and contain levels of somatic hypermutation comparable to existing benchmark therapeutic antibodies. Epitope mapping with mutant proteins identified diverse patterns of reactivity including known and novel specificities. Functional assays including apoptosis and receptor modulation (calcium flux and cAMP modulation) further demonstrated that the technical approach generated diverse panels of antibodies that exhibit functional activity as good or better than existing benchmark therapeutic antibodies Citation Format: Michael C. Brown, Ross Chambers, Dale V. Onisk, Tony R. Joaquim, Lewis J. Stafford, Klaus Lindpaintner, Daniel Keter, James W. Stave. Monoclonal antibodies to transmembrane proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2013-4325
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