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Clinical and Experimental Immunology Mar 1990Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo... (Review)
Review
Antibodies with two distinct binding specificities have great potential for a wide range of clinical applications as targeting agents for in vitro and in vivo immunodiagnosis and therapy, and for improving immunoassays. They have shown great promise for targeting cytotoxic effector cells, delivering radionuclides, toxins or cytotoxic drugs to specific targets, particularly tumour cells. We discuss potential applications of bispecific antibodies, the theoretical basis and problems associated with their production and purification, cell fusion and chemical conjugation techniques, and propose a new manufacturing strategy by genetic engineering. This approach has enormous potential applications for producing tailor-made bispecific antibodies, and will enable widespread clinical uses of these antibodies both for diagnostic purposes and therapy.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Humans
PubMed: 2180597
DOI: 10.1111/j.1365-2249.1990.tb08089.x -
Archives of Pathology & Laboratory... Sep 2016-There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant... (Review)
Review
CONTEXT
-There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant about, their influence.
OBJECTIVE
-To highlight 3 arenas in which errors may be introduced.
DATA SOURCES
-For choosing the correct primary antibody, selection of the most appropriate antibodies for a given clinical application can be aided by obtaining information from the vendor; however, this can yield incomplete information. There are a number of online databases that have comparisons of antibodies from different vendors, particularly with respect to their use and properties. Reading the published literature can assist in this process, particularly with respect to determining antibody sensitivity and specificity, but it is a daunting task to keep up with all of the immunohistochemistry-related papers published. Finally, Web sites of a number of quality assurance organizations are accessible and can provide a wealth of information comparing the "real world" performance characteristics of different antibodies to the same target protein. False-positive signals can result from a number of factors, including the use of inappropriately high antibody concentration, and "pseudospecific" signal that is in the wrong compartment of the cell. False-negative signal can result from factors such as use of a nonoptimized epitope retrieval method. It is critical that epitope retrieval methods be optimized for each antibody employed in the laboratory.
CONCLUSIONS
-By paying attention to these potential problems, the "black box" of diagnostic immunohistochemistry can be made more transparent.
Topics: Antibodies; Antibody Specificity; Diagnostic Tests, Routine; Epitopes; Humans; Immunohistochemistry; Pathology, Clinical; Reproducibility of Results; Sensitivity and Specificity
PubMed: 27575264
DOI: 10.5858/arpa.2016-0119-RA -
New Biotechnology Jun 2012The reversible phosphorylation of tyrosine residues is one of the most frequent post-translational modifications regulating enzymatic activities and protein-protein...
The reversible phosphorylation of tyrosine residues is one of the most frequent post-translational modifications regulating enzymatic activities and protein-protein interactions in eukaryotic cells. Cells responding to internal or external regulatory inputs modify their phosphorylation status and diseased cells can often be diagnosed by observing alterations in their qualitative or quantitative phosphorylation profile. As a consequence the ability to describe the phosphorylation profile of a cell is central to many approaches aiming at the characterisation of signalling pathways. Anti-phosphotyrosine (pY) antibodies are widely used as experimental tools to monitor the phosphorylation status of a cell. By using peptide microarray technology we have characterised the substrate specificity of three widely used pY antibodies. We report that they are more sensitive to sequence context than is generally assumed and that their sequence preferences differ.
Topics: Amino Acid Sequence; Antibodies, Phospho-Specific; Antibody Specificity; HEK293 Cells; Humans; Molecular Sequence Data; Phosphopeptides; Phosphorylation; Protein Array Analysis; Proteomics; Reproducibility of Results; Signal Processing, Computer-Assisted
PubMed: 22178400
DOI: 10.1016/j.nbt.2011.12.001 -
Current Opinion in Organ Transplantation Aug 2014Human leukocyte antigen (HLA) antibodies are now recognized as being specific for epitopes which can be defined structurally with amino acid differences between HLA... (Review)
Review
PURPOSE OF REVIEW
Human leukocyte antigen (HLA) antibodies are now recognized as being specific for epitopes which can be defined structurally with amino acid differences between HLA alleles. This article addresses two general perspectives of HLA epitopes namely antigenicity, that is their reactivity with antibody and immunogenicity, that is their ability of eliciting an antibody response.
RECENT FINDINGS
Single-antigen bead assays have shown that HLA antibodies recognize epitopes that are equivalent to eplets or corresponding to eplets paired with other residue configurations. There is now a website-based Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br). Residue differences within eplet-defined structural epitopes may also explain technique-dependent variations in antibody reactivity determined in Ig-binding, C1q-binding and lymphocytotoxicity assays.HLA antibody responses correlate with the numbers of eplets on mismatched HLA antigens, and the recently proposed nonself-self paradigm of epitope immunogenicity may explain the production of epitope-specific antibodies.
SUMMARY
These findings support the usefulness of HLA matching at the epitope level, including the identification of acceptable mismatches for sensitized patients and permissible mismatching for nonsensitized patients aimed to reduce HLA antibody responses.
Topics: Animals; Antibodies; Antibody Formation; Antibody Specificity; Epitopes; HLA Antigens; Histocompatibility Testing; Humans
PubMed: 25010064
DOI: 10.1097/MOT.0000000000000100 -
Human Antibodies 2005Antibody cross-reactivity can compromise interpretation of experiments and derail therapeutic antibody development. Standard techniques such as immunohistochemistry or... (Review)
Review
Antibody cross-reactivity can compromise interpretation of experiments and derail therapeutic antibody development. Standard techniques such as immunohistochemistry or Western analysis provide important but often inadequate approaches to assess antibody specificity. Protein microarrays are providing a new approach to rapidly characterize antibody cross-reactivity against 1,000s of proteins simultaneously. This review will focus on reported examples of antibody cross-reactivity, methods used to characterize them, and the recent development and use of protein microarrays for assessing antibody specificity.
Topics: Animals; Antibody Specificity; Blotting, Western; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Humans; Immunohistochemistry; Protein Array Analysis
PubMed: 16424595
DOI: No ID Found -
Monoclonal Antibodies in... Apr 2016
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; HEK293 Cells; Heparin-binding EGF-like Growth Factor; Humans; Immunohistochemistry; Mice
PubMed: 27097072
DOI: 10.1089/mab.2016.0011 -
Journal of Neurochemistry Feb 2008High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease.... (Review)
Review
High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease. The efficacy of anti-amyloid immunization strategies has been demonstrated preclinically, prompting the design of clinical studies. However, the detailed mechanisms of action of therapeutic antibodies, especially their influence on the complex amyloid beta peptide (Abeta) metabolism and various Abeta-equilibria present both within and outside the CNS, are far from being clear. Furthermore, physiological Abeta metabolism is poorly understood and the analytical tools to characterize and quantify treatment effects on Abeta metabolism are suboptimal. Thus, the design of immunization strategies with optimized benefit-to-risk ratios for patients is subjected to significant obstacles. Indeed, an active immunization trial with Abeta was discontinued because of severe adverse effects. Anti-Abeta immunization protocols designed to attain high blood levels of antibodies bear the potential to induce brain inflammation and/or hemorrhage, thus directing the biomedical research towards development of more predictable therapies for minimizing the risk of adverse effects. The focus of this review is to summarize current knowledge of Abeta metabolism under physiological and antibody-based therapeutic conditions and to introduce a promising approach, namely the passive immunization using antibody fragments, which are characterized by entirely different pharmacokinetic and pharmacodynamic properties compared with conventional monoclonal antibodies.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antibodies; Antibody Specificity; Humans; Immunization, Passive
PubMed: 17986215
DOI: 10.1111/j.1471-4159.2007.05064.x -
Proteins 1990The mouse hybridoma cell line 40-150 secretes antibodies with high affinity toward the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40-150 A2.4,...
The mouse hybridoma cell line 40-150 secretes antibodies with high affinity toward the cardiac glycosides digoxin and digitoxin. A spontaneous mutant, 40-150 A2.4, produces an antibody which carries a single residue mutation, Ser----Arg, in its heavy chain (H94) and has an altered specificity. A second-order mutant, 40-150 A2.4 P.10, produces two antibody molecules, one the same as 40-150 A2.4, the other lacking two residues at the N-terminus of its H chain, and having a specificity profile approaching that of 40-150 antibody. The N-terminus and the position H94 are distant from the antigen-binding site of the antibody; thus, the structural basis of the specificity changes was not immediately clear. Approximate structures of the 40-150 antibody and its mutants were constructed in the computer, based on atomic coordinates of the homologous mouse antibody McPC 603. Using the program CONGEN, the torsional space of the polypeptide backbone and side chains around position H94 was uniformly sampled, and the lowest energy conformations were analyzed in detail. The results indicate that when Arg-H94 is substituted for Ser, Arg-H94 can hydrogen bond to side chains of Asp-H101, Arg-L46, and Asp-L55. This results in a change in the surface of the combining site which may account for the affinity changes. Deletion of the two N-terminal residues increases solvent accessibility of Arg-H94. The solvation may cause a hydrogen bond between Arg-H94 and Asp-H101 to be lost, restoring the structure to one similar to that of 40-150.
Topics: Animals; Antibody Specificity; Cell Line; Electronic Data Processing; Humans; Hybridomas; Hydrogen Bonding; Immunoglobulins; Mice; Mutation; Protein Conformation
PubMed: 2330371
DOI: 10.1002/prot.340070109 -
Analytical Biochemistry Dec 2008Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g.,...
Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.
Topics: Animals; Antibody Affinity; Antibody Specificity; Antigen-Antibody Reactions; Biuret Reaction; Copper; Cross Reactions; Cross-Linking Reagents; Humans; Mice; Peptides
PubMed: 18801330
DOI: 10.1016/j.ab.2008.08.024 -
Hybridoma and Hybridomics Feb 2003Human IgG is comprised of four subclasses (IgG(1), IgG(2), IgG(3), and IgG(4)). Each subclass possesses different biological properties. One of the differential...
Human IgG is comprised of four subclasses (IgG(1), IgG(2), IgG(3), and IgG(4)). Each subclass possesses different biological properties. One of the differential specificities of human IgG subclasses is binding of Fc fragment of IgG(1), 2, and 4 but, not IgG(3) to staphylococcal protein A (SPA). This study was conducted to produce, select and characterize a monoclonal antibody (MAb) recognizing human IgG subclasses with specificity similar to SPA. Splenocytes from Balb/c mice immunized with Fc fraction of a human IgG(1) myeloma protein were fused with Sp2/0 myeloma cells. Fused cells were grown in hypoxanthine, aminopterine, and thymidine (HAT) selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAb was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. A murine hybridoma designated 6F11E1 was obtained that secretes an MAb specific for the Fc fragment of the immunizing protein. This MAb reacts with isotypic epitope common to IgG(1), 2 and 4 subclasses. An allelic epitope linked to IgG(3) molecules is also recognized by 6F11E1. This pattern of reactivity was found to be highly similar to that of SPA. Our findings imply that similar or overlapping epitopes are recognized by 6F11E1 and SPA.
Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Epitopes; Humans; Mice; Mice, Inbred BALB C; Staphylococcal Protein A
PubMed: 12713688
DOI: 10.1089/153685903321538062