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Allergology International : Official... Jan 2016Prompt diagnosis of allergic bronchopulmonary mycosis (ABPM) is an important clinical issue in preventing irreversible lung damage. Therefore, a good serological marker... (Review)
Review
Prompt diagnosis of allergic bronchopulmonary mycosis (ABPM) is an important clinical issue in preventing irreversible lung damage. Therefore, a good serological marker for the diagnosis of ABPM is desired in clinical practice. The measurement of IgE antibody to crude Aspergillus fumigatus allergen is considered the first step in screening asthmatic patients for allergic bronchopulmonary aspergillosis (ABPA). However, presence of IgE to A. fumigatus does not always indicate genuine sensitization to A. fumigatus because of cross-reactivity between crude extracts from different fungal sources. The application of molecular-based allergy diagnosis can solve this problem. The specificity of testing can be greatly improved by measuring the IgE antibody to Asp f 1 and f 2, specific allergen components for genuine A. fumigatus allergy. The problem of cross-reactivity between crude fungal extracts is also true for the identification of genuine causal fungi in each ABPM patient. Some patients with ABPM induced by fungi other than Aspergillus may be consistent with ABPA diagnostic criteria because current criteria depend on IgE/IgG reactivity to crude extracts. Accurate identification of genuine causal fungi for ABPM is of clinical importance, considering that clinical presentation, anti-fungal treatment strategies and disease prognosis can be influenced by different causal fungi. The diagnosis of causal fungi can be robustly validated by the confirmation of genuine sensitization to fungi after measuring IgE to specific allergen components, as well as repeated microbiological isolation of the fungi from their airway.
Topics: Allergens; Antibodies, Fungal; Antibody Specificity; Antigens, Fungal; Aspergillosis, Allergic Bronchopulmonary; Aspergillus fumigatus; Cross Reactions; Humans; Immunoglobulin E; Immunoglobulin G; Molecular Diagnostic Techniques; Serologic Tests
PubMed: 26740298
DOI: 10.1016/j.alit.2015.08.004 -
Science Signaling Jan 2020Monoclonal antibodies recognize epitopes so specifically that altering a single residue can disrupt binding. In this issue of , Schüchner and Frohner report that... (Review)
Review
Monoclonal antibodies recognize epitopes so specifically that altering a single residue can disrupt binding. In this issue of , Schüchner and Frohner report that flanking amino acids and an underappreciated posttranslational modification perturb epitope affinity for two groups of widely used monoclonal antibodies.
Topics: Animals; Antibodies, Monoclonal; Antibody Affinity; Antibody Specificity; Epitopes; Humans
PubMed: 31992582
DOI: 10.1126/scisignal.aaz8130 -
Protein Engineering, Design & Selection... Nov 2018Specificity is one of the most important and complex properties that is central to both natural antibody function and therapeutic antibody efficacy. However, it has...
Specificity is one of the most important and complex properties that is central to both natural antibody function and therapeutic antibody efficacy. However, it has proven extremely challenging to define robust guidelines for predicting antibody specificity. Here we evaluated the physicochemical determinants of antibody specificity for multiple panels of antibodies, including >100 clinical-stage antibodies. Surprisingly, we find that the theoretical net charge of the complementarity-determining regions (CDRs) is a strong predictor of antibody specificity. Antibodies with positively charged CDRs have a much higher risk of low specificity than antibodies with negatively charged CDRs. Moreover, the charge of the entire set of six CDRs is a much better predictor of antibody specificity than the charge of individual CDRs, variable domains (VH or VL) or the entire variable fragment (Fv). The best indicators of antibody specificity in terms of CDR amino acid composition are reduced levels of arginine and lysine and increased levels of aspartic and glutamic acid. Interestingly, clinical-stage antibodies with negatively charged CDRs also have a lower risk for poor biophysical properties in general, including a reduced risk for high levels of self-association. These findings provide powerful guidelines for predicting antibody specificity and for identifying safe and potent antibody therapeutics.
Topics: Amino Acid Sequence; Antibodies; Antibody Specificity; Complementarity Determining Regions; Humans
PubMed: 30770934
DOI: 10.1093/protein/gzz002 -
Proceedings of the National Academy of... Mar 2022The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a...
The antigen specificity and long serum half-life of monoclonal antibodies have made them a critical part of modern therapeutics. These properties have been coopted in a number of synthetic formats, such as antibody-drug conjugates, bispecific antibodies, or Fc-fusion proteins to generate novel biologic drug modalities. Historically, these new therapies have been generated by covalently linking multiple molecular moieties through chemical or genetic methods. This irreversible fusion of different components means that the function of the molecule is static, as determined by the structure. Here, we report the development of a technology for switchable assembly of functional antibody complexes using chemically induced dimerization domains. This approach enables control of the antibody's intended function in vivo by modulating the dose of a small molecule. We demonstrate this switchable assembly across three therapeutically relevant functionalities in vivo, including localization of a radionuclide-conjugated antibody to an antigen-positive tumor, extension of a cytokine's half-life, and activation of bispecific, T cell-engaging antibodies.
Topics: Antibodies; Antibody Specificity; Humans; Immunoconjugates; Small Molecule Libraries
PubMed: 35210365
DOI: 10.1073/pnas.2117402119 -
Clinical and Experimental Immunology Dec 2019This review takes the reader through 45 years of islet autoantibody research, from the discovery of islet-cell antibodies in 1974 to today's population-based screening... (Review)
Review
This review takes the reader through 45 years of islet autoantibody research, from the discovery of islet-cell antibodies in 1974 to today's population-based screening for presymptomatic early-stage type 1 diabetes. The review emphasizes the current practical value of, and factors to be considered in, the measurement of islet autoantibodies.
Topics: Antibody Specificity; Autoantibodies; Autoimmunity; Diabetes Mellitus, Type 1; Glutamate Decarboxylase; Humans; Islets of Langerhans; Mass Screening; Population Surveillance
PubMed: 31397889
DOI: 10.1111/cei.13360 -
PloS One 2019The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather...
The commercially available D-dimer assays used in the clinical practice often show differences in the results, and their specificity and sensitivity are rather unsatisfactory. Our aim was to develop a new monoclonal antibody against D-dimer with a proper specificity, and estimating its suitability using in a latex agglutination diagnostic test. Monoclonal antibodies were generated using hybridoma technology. Their titer was determined by a self-developed ELISA method. The cross-reactions of the antibodies were tested. Characterization of the epitope specificity of a selected antibody was performed through digestion of D-dimer followed by Western blotting. The amino acid sequences of the active antigen fragments were determined. According to the ELISA results, 38 cell groups were constated as antibody-producing hybridomas, among them 7 gave raised titer of antibody and were cloned. Based on the cross-reaction analysis, none of the antibodies gave cross-reaction with fibrin-E and fibrinogen-E fragments but reacted with fibrin D and fibrinogen D fragments. A low cross-reaction was showed with fibrinogen and fibrin X and Y. Contrary to the others, antibody 2B9 gave no cross-reaction with fibrinogen and reacted weakly with fibrin X and Y fragments. According to the epitope analysis the antibody 2B9 binds to amino acids 94-99 and to amino acids 140-147 on the beta chain and it recognizes the amino acids 23-32 and 93-98 on the gamma chain of D-dimer. Considering the characteristics of the above mentioned monoclonal antibody 2B9, we found that it is suitable to be a basis for a D-dimer diagnostic test with proper specificity.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibody Specificity; Female; Fibrin Fibrinogen Degradation Products; Humans; Immunization; Latex Fixation Tests; Mice
PubMed: 30763351
DOI: 10.1371/journal.pone.0212104 -
Frontiers in Immunology 2019Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology...
Dengue Virus (DENV) associated disease is a major public health problem. Assessment of HLA class II restricted DENV-specific responses is relevant for immunopathology and definition of correlates of protection. While previous studies characterized responses restricted by the HLA-DRB1 locus, the responses associated with other class II loci have not been characterized to date. Accordingly, we mapped HLA-DP, DQ, and DRB3/4/5 restricted DENV-specific CD4 T cell epitopes in PBMCs derived from the DENV endemic region Sri Lanka. We studied 12 DP, DQ, and DRB3/4/5 alleles that are commonly expressed and provide worldwide coverage >82% for each of the loci analyzed and >99% when combined. CD4+ T cells purified by negative selection were stimulated with pools of HLA-predicted binders for 2 weeks with autologous APC. Epitope reactive T cells were enumerated using IFNγ ELISPOT assay. This strategy was previously applied to identify DRB1 restricted epitopes. In parallel, membrane expression levels of HLA-DR, DP, and DQ proteins was assessed using flow cytometry. Epitopes were identified for all DP, DQ, and DRB3/4/5 allelic variants albeit with magnitudes significantly lower than the ones previously observed for the DRB1 locus. This was in line with lower membrane expression of HLA-DP and DQ molecules on the PBMCs tested, as compared to HLA-DR. Significant differences between loci were observed in antigen immunodominance. Capsid responses were dominant for DRB1/3/4/5 and DP alleles but negligible for the DQ alleles. NS3 responses were dominant in the case of DRB1/3/4/5 and DQ but absent in the case of DP. NS1 responses were prominent in the case of the DP alleles, but negligible in the case of DR and DQ. In terms of epitope specificity, repertoire was largely overlapping between DRB1 and DRB3/4/5, while DP and DQ loci recognized largely distinct epitope sets. The HLA-DP, DQ, and DRB3/4/5 loci mediate DENV-CD4 specific immune responses of lower magnitude as compared to HLA-DRB1, consistent with their lower levels of expression. The responses are associated with distinct and characteristic patterns of immunodominance, and variable epitope overlap across loci.
Topics: Alleles; Antibody Specificity; CD4-Positive T-Lymphocytes; Dengue; Dengue Virus; Epitopes, T-Lymphocyte; HLA-DP Antigens; HLA-DRB1 Chains; Humans; Interferon-gamma
PubMed: 31333679
DOI: 10.3389/fimmu.2019.01568 -
Communications Biology Feb 2024Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic...
Anti-DNA antibodies (Abs), serological hallmarks of systemic lupus erythematosus (SLE) and markers for diagnosis and disease activity, show a specificity for non-nucleic acid molecules, such as N-pyrrolated proteins (pyrP) containing N-pyrrole-L-lysine (pyrK) residues. However, the detailed mechanism for the binding of anti-DNA Abs to pyrP remains unknown. In the present study, to gain structural insights into the dual-specificity of anti-DNA Abs, we used phage display to obtain DNA-binding, single-chain variable fragments (scFvs) from SLE-prone mice and found that they also cross-reacted with pyrP. It was revealed that a variable heavy chain (VH) domain is sufficient for the recognition of DNA/pyrP. Identification of an antigenic sequence containing pyrK in pyrP suggested that the presence of both pyrK and multiple acidic amino acid residues plays important roles in the electrostatic interactions with the Abs. X-ray crystallography and computer-predicted simulations of the pyrK-containing peptide-scFv complexes identified key residues of Abs involved in the interaction with the antigens. These data provide a mechanistic insight into the molecular basis of the dual-specificity of the anti-DNA Abs and provide a basis for therapeutic intervention against SLE.
Topics: Mice; Animals; Antibodies, Antinuclear; Antibody Specificity; Lupus Erythematosus, Systemic; Single-Chain Antibodies; DNA
PubMed: 38310133
DOI: 10.1038/s42003-024-05851-0 -
Protein Science : a Publication of the... Nov 2022Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant...
Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant pathogens. Regulatory agencies cannot do the rigorous, expensive testing required to keep up with the volume of seafood shipments. Current rapid test kits for these drugs enable the increase in testing needed for adequate monitoring of food supply chains, but they lack a high degree of accuracy. To combat this, we set out to discover and engineer single-domain antibodies (VHHs) that bind to small molecule antibiotics, and that can be used in rapid test kits. The small size, solubility, and stability of VHHs are useful properties that can improve the reliability and shelf-life of test kits for these adulterants. Here, we report a novel anti-chloramphenicol VHH (Chl-VHH) with a disassociation constant of 57 nM. This was achieved by immunizing a llama against a chloramphenicol-keyhole limpet hemocyanin (KLH) conjugate and screening for high affinity binders through phage display. The crystal structure of the bound-VHH to chloramphenicol was key to identifying a mutation in the binding pocket that resulted in a 16-fold improvement in binding affinity. In addition, the structure provides new insights into VHH-hapten interactions that can guide future engineering of VHHs against additional targets.
Topics: Animals; Single-Domain Antibodies; Chloramphenicol; Reproducibility of Results; Camelids, New World; Anti-Bacterial Agents; Antibody Specificity
PubMed: 36153664
DOI: 10.1002/pro.4457 -
Molecular Neurodegeneration Nov 2017Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation,...
BACKGROUND
Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer's disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear.
METHODS
In this study, we first designed an online tool called 'TauPTM', which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies.
RESULTS
We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains.
CONCLUSION
This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at http://www.tauptm.org .
Topics: Acetylation; Animals; Antibody Specificity; Brain; Humans; Methylation; Mice; Mice, Transgenic; Phosphorylation; Protein Processing, Post-Translational; tau Proteins
PubMed: 29157277
DOI: 10.1186/s13024-017-0229-1