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Immunological Tests for Diagnosis of Disease and Identification of Molecules
Published in Julius P. Kreier, Infection, Resistance, and Immunity, 2022
Such antibody-sandwich ELISAs may be more useful than the direct sandwich ELlSAs for detecting antibody because they are frequently between two and five times more sensitive than ELlSAs in which antigen is directly bound to the solid phase. Antibody-sandwich ELlSAs are especially useful when screening for specific antibodies in cases when a small amount of specific antibody is available and purified antigen is unavailable. The antibody mediated binding of the antigen of interest assures that only the antigen of interest is bound. This method can be used for epitope mapping of monoclonal antibodies that are directed against the antigen. Figure 20.10 is a diagram of this type of test.
Mite allergens
Published in Richard F. Lockey, Dennis K. Ledford, Allergens and Allergen Immunotherapy, 2020
Enrique Fernández-Caldas, Leonardo Puerta, Luis Caraballo, Victor Iraola, Richard F. Lockey
Cross-reactivity is a common feature among mite allergens, especially in those from taxonomically related species. This phenomenon may be the cause of polysensitization occurring in some mite-allergic individuals, although species-specific reactions do occur and should not be neglected. Originally, cross-reactivity was studied using whole extracts and radioallergosorbent test (RAST) inhibition techniques. In recent years, new tools, such as purified native or recombinant allergens, epitope mapping, T-cell proliferation techniques, and bioinformatics prediction have been applied [198]. When serum pools are used in cross-reactivity studies, the result depends to a great extent on the characteristics of the individual sera in the serum pool.
Hypersensitivity and Allergic Fungal Manifestations: Diagnostic Approaches
Published in Johan A. Maertens, Kieren A. Marr, Diagnosis of Fungal Infections, 2007
Application of recombinant DNA technology to fungal allergens provided scope for better understanding on molecular nature of the fungal allergens and identification of immunodominant epitopes. The knowledge of IgE-binding epitopes of the major fungal allergens may be of importance for increasing the specificity and sensitivity of diagnostic tests for designing model allergens representing such epitopes for in vitro tests. Advances made in epitope mapping of major antigens of pathogenic microbes resulted in the rapid developmentof diagnostic technologies and products. Availability of the deduced amino acid sequences of a few major allergens/antigens and partial sequencing of several other allergens/antigens have now provided opportunity to analyze regions both by algorithms and by experiment (by chemical/enzymatic cleavage of allergenic/antigenic proteins). Identified peptide fragments can be synthesized and evaluated for their potential in immunodiagnosis. Synthesis of overlapping peptides spanning the whole protein is one of the approaches for development of peptide-based diagnostics for infectious diseases. Another approach is based on the recombinant expression of the epitopic sequences.
Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and AlphaFold
Published in mAbs, 2023
Tiphanie Pruvost, Magali Mathieu, Steven Dubois, Bernard Maillère, Emmanuelle Vigne, Hervé Nozach
All methods for epitope mapping have limitations. X-ray crystallography or cryoEM can reveal simultaneously both the epitope and paratope of a mAb/antigen complex. However, they are dependent on the quality of the complex and its capacity to crystallize at high enough resolution or generate high-quality images, respectively. Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a fast and cost-effective alternative approach enabling parallelized epitope mapping. However, its accuracy and precision can be compromised by insufficient peptide coverage for large complexes or highly glycosylated antigens, or the inability to discriminate between direct binding interface and allosteric conformational change.32 The Ala mutagenesis technique can provide some answers on the areas of the antigen involved in the interaction, but is far less precise than DMS, which scans the 20 proteinogenic amino acids. In our dataset, we observe that Ala substitutions would not have identified some important positions, such as K151 for mAb B or L310 for mAb A, and of course not A108, which is already an alanine residue.
Epitope mapping of anti-drug antibodies to a clinical candidate bispecific antibody
Published in mAbs, 2022
Arthur J. Schick, Victor Lundin, Justin Low, Kun Peng, Richard Vandlen, Aaron T. Wecksler
There are many technologies available for epitope mapping, including peptide array, electron microscopy, crystallography, and mutagenesis (reviewed by Nilvebrant et al.10). However, each of these technologies has disadvantages, such as cost, throughput, protein amount/purity requirements, and ability to detect conformational vs. linear epitopes. Bottom-up mass spectrometry (MS) technologies using covalent and non-covalent labeling have shown promise for deciphering protein–protein interactions and are able to circumvent some of the inherent challenges described by other epitope mapping technologies.11–13 One of the emerging MS-based technologies for epitope mapping is hydroxyl radical footprinting (HRF)-MS, a technology that uses hydroxyl radicals to label the side chains of solvent-exposed amino acids.14 The high affinity, specificity, and large surface area of antibody-antigen complexes are ideal for epitope mapping using HRF.13,15,16 However, identifying the difference between a binding site and a conformational change is difficult for all bottom-up MS technologies.
Reverse engineering approach: a step towards a new era of vaccinology with special reference to Salmonella
Published in Expert Review of Vaccines, 2022
Shania Vij, Reena Thakur, Praveen Rishi
The advancements in structural biology, B-cell technologies, and knowledge of pangenome have not only improved the assessment and prioritization of bacterial antigens as vaccine candidates but also enabled the better characterization of the immunogenicity of antigens that are identified using in-silico approaches. These advances have led to a new model for rational vaccine design, which can be termed ‘Reverse vaccinology 2.0’ (Figure 3). In this approach, genomics is not only limited to antigen discovery but is also exploited for antigen expression, its conservation, and to have a better understanding of epidemiology. Human monoclonals are employed for the identification of epitopes or protective antigens. Antigen design is instructed after structural characterization of the antigen–antibody complex by conformational epitope mapping studies so as to come up with the atomic details of the immunogenic and protective epitopes, which can be easily recognized by broadly neutralizing antibodies. The incorporation of advanced approaches into the traditional RV scheme has led to reverse vaccinology 2.0 [181]. A schematic illustration of the reverse vaccinology 2.0 approach has been provided in Figure 4.