The analytical approach is referred to as “middle-up” if protein subunit masses are determined. The latter usually involves digestion with the IdeS protease, which has become a valuable tool for the characterization of human therapeutic mAbs 10, 11, 12, 13. Alternatively, mAb subunits obtained by reduction of disulphide bonds or limited proteolysis may be analysed. With protein variants being highly relevant in the context of biopharmaceuticals, numerous methods for the characterization of intact therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins have been described 2, 3, 4, 5, 6, 7, 8, 9. If combined with gas-phase fragmentation in a so-called “top-down” approach, information on the amino acid sequence may be obtained. In contrast to conventional peptide analysis, referred to as “bottom-up” approach, intact mass determination provides information on protein integrity and on the coexistence of protein variants arising from glycosylation and other post-translational modifications (PTMs) 1. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.Įmergence of novel analytical technologies has driven the development of high-performance liquid chromatography (HPLC) and mass spectrometry (MS) strategies for intact protein characterization. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. The diagnosis depends upon clinical features combined with laboratory testing.In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. No objective tests for Lyme borreliosis are both 100% sensitive and specific. Diagnosis of Lyme Disease, however, can only be guided and not fully established by laboratory results. These include syphilis, subacute bacterial endocarditis (SBE), peridontitis, HIV infection, and hypergammaglobulinemia. If the Western Blot is negative, causes of false positive immunoassays should be considered. All specimens found to be positive or equivocal by the EIA screening test(s) should be tested by the Western Blot procedure. In early suspected disease, tests for both IgM and IgG antibodies are recommended. Testing for Lyme Disease should follow a two-test protocol: The first step employs a sensitive polyvalent screening test enzyme immunoassay (EIA). Serologic diagnosis is often not established until later stages of the disease. However, many patients with early disease are seronegative by immunoassay (up to 40%) at the time they present with Erythema migrans. burgdorferiis not practical in the microbiology laboratory because of its special growth requirements and very slow growth. In most cases, Lyme Disease is easily treated and does not progress to the chronic stage.Ĭulture of B. The varying clinical manifestations, coupled with the difficulty in confirming the diagnosis in many patients, have led to some misconceptions about Lyme Disease. It is a systemic disease with variable manifestations, including dermatologic, rheumatologic, neurologic, and cardiac abnormalities. Lyme Disease is caused by infection with the spirochete Borrelia burgdorferiand is transmitted by the bite of an infected tick of the Ixodid species. Positive results will reflex to the following tests: Specimen not centrifuged within 2 hours of collection Frozen: Aliquot serum for longer storage.Immediately after collection, gently invert tube 5-10 times.Lyme Disease Serology Lyme Disease Antibody
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