Determination of crucial epitopes in the sperm protein calsperin employing synthetic peptides and monoclonal antibodies.

The chaperone protein calsperin is exclusively expressed in the testes and is essential for sperm migration from the uterus into the oviduct. During spermatogenesis, calsperin interacts with ADAM3, a spermatozoon membrane protein required for fertilization. In this study, we characterized a calsperin epitope by using two monoclonal antibodies and resin-bound calsperin peptides, which were tested for reactivity using a modified enzyme-linked immunosorbent assay. An epitope located at the C-terminal end of calsperin corresponding to amino acids 228 WEKHFLDAS237 was identified. Three hot spot amino acids were essential for antibody binding whereas the remaining amino acids in the identified epitope appeared to be essential for bringing the critical contact residues into an α-helix structure. No notable sequence similarity was determined between the identified calsperin epitope and calreticulin, a chaperone homologue with sequence similarity, indicating that the identified epitope was specific for calsperin. Characterization of the calsperin epitope and of the two antibodies tested may be used in assays for further characterization of calsperin, where knowledge about the binding sites is necessary, for example, in sandwich assays. Moreover, studies like these may be used to study the function of calsperin during spermatogenesis and fertilization in detail and to develop new male contraception methods by targeting calsperin and mediating neutralization of its function.

Antibody Cross-Reactivity in Auto-Immune Diseases.

Autoimmunity is defined by the presence of antibodies and/or T cells directed against self-components. Although of unknown etiology, autoimmunity commonly is associated with environmental factors such as infections, which have been reported to increase the risk of developing autoimmune diseases. Occasionally, similarities between infectious non-self and self-tissue antigens may contribute to immunological cross-reactivity in autoimmune diseases. These reactions may be interpreted as molecular mimicry, which describes cross-reactivity between foreign pathogens and self-antigens that have been reported to cause tissue damage and to contribute to the development of autoimmunity. By focusing on the nature of antibodies, cross-reactivity in general, and antibody-antigen interactions, this review aims to characterize the nature of potential cross-reactive immune reactions between infectious non-self and self-tissue antigens which may be associated with autoimmunity but may not actually be the cause of disease onset.

Advances in Antibody Design and Antigenic Peptide Targeting 2.0.

Antibodies possess numerous important functions in diagnostics, both as therapeutics and as research tools [...].

Peptide Antibody Reactivity to Homologous Regions in Glutamate Decarboxylase Isoforms and Coxsackievirus B4 P2C.

Two isoforms of the glutamate decarboxylase (GAD) enzyme exist, GAD65 and GAD67, which are associated with type 1 diabetes (T1D) and stiff-person syndrome (SPS), respectively. Interestingly, it has been reported that T1D patients seldom develop SPS, whereas patients with SPS occasionally develop T1D. In addition, coxsackievirus B4 (CVB4) has previously been proposed to be involved in the onset of T1D through molecular mimicry. On this basis, we aimed to examine antibody cross-reactivity between a specific region of GAD65 and GAD67, which has high sequence homology to the nonstructural P2C protein of CVB4 to determine potential correlations at antibody level. Monoclonal peptide antibodies generated in mice specific for a region with high similarity in all three proteins were screened for reactivity along with human sera in immunoassays. In total, six antibodies were generated. Two of the antibodies reacted to both GAD isoforms. However, none of the antibodies were cross-reactive to CVB, suggesting that antibody cross-reactivity between GAD65 and CVB, and GAD67 and CVB may not contribute to the onset of T1D and SPS, respectively.

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Molecular Recognition and Advances in Antibody Design and Antigenic Peptide Targeting.

Molecular recognition, the specific interaction between molecules by a combination of physical forces, has been a subject of scientific investigation for decades [...].

Peptides, Antibodies, Peptide Antibodies and More.

The applications of peptides and antibodies to multiple targets have emerged as powerful tools in research, diagnostics, vaccine development, and therapeutics. Antibodies are unique since they, in theory, can be directed to any desired target, which illustrates their versatile nature and broad spectrum of use as illustrated by numerous applications of peptide antibodies. In recent years, due to the inherent limitations such as size and physical properties of antibodies, it has been attempted to generate new molecular compounds with equally high specificity and affinity, albeit with relatively low success. Based on this, peptides, antibodies, and peptide antibodies have established their importance and remain crucial reagents in molecular biology.

Fine Mapping of Glutamate Decarboxylase 65 Epitopes Reveals Dependency on Hydrophobic Amino Acids for Specific Interactions.

Characterization of multiple antibody epitopes has revealed the necessity of specific groups of amino acid residues for reactivity. This applies to the majority of antibody-antigen interactions, where especially charged and hydrophilic amino acids have been reported to be essential for antibody reactivity. This study describes thorough characterization of glutamic acid decarboxylase (GAD) 65 antigenic epitopes, an immunodominant autoantigen in type 1 diabetes (T1D). As linear epitopes are sparsely described for GAD65 in T1D, we aimed to identify and thoroughly characterize two GAD65 antibodies using immunoassays. A monoclonal antibody recognized an epitope in the N-terminal domain of GAD65, 8FWSFGSE14, whereas a polyclonal antibody recognized two continuous epitopes in the C-terminal domain, corresponding to amino acids 514RTLED518 and 549PLGDKVNF556. Hydrophobic amino acids were essential for antibody reactivity, which was verified by competitive inhibition assays. Moreover, the epitopes were located in flexible linker regions and turn structures. These findings confirm the versatile nature of antibody-antigen interactions and describe potential continuous epitopes related to T1D, which predominantly have been proposed to be of discontinuous nature.

Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans.

Graves' hyperthyroidism, a common autoimmune disease caused by pathogenic autoantibodies to the thyrotropin (TSH) receptor (TSHR), can be treated but not cured. This single autoantigenic target makes Graves' disease a prime candidate for Ag-specific immunotherapy. Previously, in an induced mouse model, injecting TSHR A-subunit protein attenuated hyperthyroidism by diverting pathogenic TSHR Abs to a nonfunctional variety. In this study, we explored the possibility of a similar diversion in a mouse model that spontaneously develops pathogenic TSHR autoantibodies, NOD.H2h4 mice with the human (h) TSHR (hTSHR) A-subunit transgene expressed in the thyroid and (shown in this article) the thymus. We hypothesized that such diversion would occur after injection of "inactive" hTSHR A-subunit protein recognized only by nonpathogenic (not pathogenic) TSHR Abs. Surprisingly, rather than attenuating the pre-existing pathogenic TSHR level, in TSHR/NOD.H2h4 mice inactive hTSHR Ag injected without adjuvant enhanced the levels of pathogenic TSH-binding inhibition and thyroid-stimulating Abs, as well as nonpathogenic Abs detected by ELISA. This effect was TSHR specific because spontaneously occurring autoantibodies to thyroglobulin and thyroid peroxidase were unaffected. As controls, nontransgenic NOD.H2h4 mice similarly injected with inactive hTSHR A-subunit protein unexpectedly developed TSHR Abs, but only of the nonpathogenic variety detected by ELISA. Our observations highlight critical differences between induced and spontaneous mouse models of Graves' disease with implications for potential immunotherapy in humans. In hTSHR/NOD.H2h4 mice with ongoing disease, injecting inactive hTSHR A-subunit protein fails to divert the autoantibody response to a nonpathogenic form. Indeed, such therapy is likely to enhance pathogenic Ab production and exacerbate Graves' disease in humans.

Human MHC-II with Shared Epitope Motifs Are Optimal Epstein-Barr Virus Glycoprotein 42 Ligands-Relation to Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder of unknown etiology, which is characterized by inflammation in the synovium and joint damage. Although the pathogenesis of RA remains to be determined, a combination of environmental (e.g., viral infections) and genetic factors influence disease onset. Especially genetic factors play a vital role in the onset of disease, as the heritability of RA is 50-60%, with the human leukocyte antigen (HLA) alleles accounting for at least 30% of the overall genetic risk. Some HLA-DR alleles encode a conserved sequence of amino acids, referred to as the shared epitope (SE) structure. By analyzing the structure of a HLA-DR molecule in complex with Epstein-Barr virus (EBV), the SE motif is suggested to play a vital role in the interaction of MHC II with the viral glycoprotein (gp) 42, an essential entry factor for EBV. EBV has been repeatedly linked to RA by several lines of evidence and, based on several findings, we suggest that EBV is able to induce the onset of RA in predisposed SE-positive individuals, by promoting entry of B-cells through direct contact between SE and gp42 in the entry complex.

Characterization of continuous monoclonal antibody epitopes in the N-terminus of Ro60.

One of the major targets of the autoimmune response in the rheumatic autoimmune diseases, Systemic Lupus Erythematosus and Sjögrens Syndrome, is the protein Ro60. Ro60 is known to associate with small misfolded RNAs, and is involved in RNA quality control and in enhancing cell survival during cellular stress, e.g. after ultaviolet irradiation. In this study, six monoclonal antibodies to Ro60 were analyzed in order to identify antigenic regions and the nature of these. Preliminary analyses revealed that two of the antibodies recognized continuous epitopes, while the remaining antibodies most likely recognized conformational epitopes. The continuous epitopes of Ro60 were characterised by modified immunoassays employing resin-bound peptides and free peptides. Peptide screenings located the epitopes to the N-terminus of Ro60, and further analyses indicated that the epitopes of the monoclonal antibodies TROVE2 and SSI-HYB 358-02 were located to amino acids 8-17 and 34-49, respectively. Moreover, charged amino acids were found to be especially important for antibody reactivity, although antibody reactivity of the monoclonal antibody TROVE2 primarily was found to be epitope backbone-dependent.

The dependency on neighboring amino acids for reactivity of anti-citrullinated protein antibodies to citrullinated proteins.

Rheumatoid arthritis (RA) is an autoimmune connective tissue disease, associated with the presence of anti-citrullinated protein antibodies (ACPA). These antibodies have been found in approximately 70% of patients suffering from RA and they are currently used for diagnosis of RA. Although they exhibit an absolute need for citrulline for antibody reactivity, no precise cognate antigen for these antibodies has been determined. In this study, we analyzed the reactivity of ACPA to various citrullinated peptides by modified enzyme-linked immunosorbent assays, in order to determine the dependency of specific amino acids for antibody reactivity. A non-human protein (ovalbumin) and antigens directly related to RA were used as templates for synthesis of non-modified and citrullinated peptides, becoming potential target epitopes. Mainly peptides containing a Cit-Gly motif were recognized by ACPAs, while no particular amino acids N-terminal of citrulline were found to be essential for antibody reactivity. Moreover, ACPA reactivity was not restricted to antigens known to be associated with ACPA-positive RA alone, but also to proteins without relation to RA, primarily illustrating that any protein in theory can be turned into an RA autoantigen, by introducing Cit-Gly motifs. Knowledge about the interaction between ACPAs and their citrullinated targets is important for understanding autoimmune ACPA responses in RA, which are known to contribute to the pathophysiology.

Species cross-reactivity of rheumatoid factors and implications for immunoassays.

Rheumatoid factors (RFs) are antibodies recognizing other antibodies usually by binding to the Fc part, while heterophilic antibodies (HAbs) are antibodies reacting with immunoglobulins (Igs) from other species. In particular, RFs have been found to cause false positive results in sandwich immunoassays. In this work, we analyzed RF-positive and RF-negative sera for content of cytokines and for heterophilic reactions by enzyme-linked immunosorbent assay and bead-based sandwich immunoassays. All sera, including those with RFs, contained insignificant amounts of cytokines and chemokines, but RF-positive sera showed large false positive values for several cytokines when analyzed by fluorescent bead-based multiplex immunoassays. This non-specific binding could be minimized by reagents designed to block HAbs, i.e. by selected animal IgGs. Furthermore, sera positive for RFs reacted with several animal IgGs, when these were immobilized on beads or coated on the polystyrene surface in enzyme-linked immunosorbent assays. This reaction could be inhibited by human IgG and by agents designed to inhibit heterophilic reactions (i.e. mixtures of IgGs from different species). In conclusion, RFs and HAbs represent an identical/overlapping set of antibodies, causing false positive reactions in sandwich and other immunoassays. Such assays must be conducted in the presence of appropriate blocking agents, e.g. HBR+, and must be carefully controlled.

Characterization of continuous B-cell epitopes in the N-terminus of glutamate decarboxylase67 using monoclonal antibodies.

Glutamate decarboxylase (GAD) is an autoantigen associated with the autoimmune disorders Type-1 diabetes (T1D) and stiff-person syndrome (SPS). The protein, being an essential enzyme involved in the production of the inhibitory neurotransmitter γ-aminobutyric acid, exists in two isoforms, GAD67 and GAD65. Both isoforms may be targeted by autoantibodies in SPS and T1D patients, although SPS primarily is associated with the presence of GAD67 autoantibodies, whereas T1D mainly is associated with the presence of GAD65 autoantibodies. In this study, we describe antibody reactivity to overlapping GAD67 peptides covering the complete protein sequence by modified peptide enzyme-linked immunosorbent assay in order to identify potential GAD67 epitopes using two monoclonal antibodies (mAbs). Both GAD67 mAbs showed reactivity to linear epitopes located at the N-terminal end of GAD67. The epitopes of GAD mAb 1 and 2 were identified as the amino acid sequences NAGADPNTTN and TETDFSNLF, respectively, corresponding to amino acids 14-23 and 91-99. Fine mapping of the epitopes revealed that antibody reactivity was related to amino acid side-chain functionality, rather than amino acid side-chain specificity. Additionally, results suggested that non-contact amino acids in the epitope structure were essential for antibody reactivity. The exact role of these amino acids remains to be determined, but they are thought to be involved in backbone hydrogen bonds or stabilization of the epitope structure. As only limited knowledge is available in relation to antigenic regions of GAD67, this study contributes to characterization of GAD67 epitopes and may be a first step in the development of peptide-based therapeutics against SPS.

Identification and fine mapping of a linear B cell epitope of human vimentin.

Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular, the two aspartate residues were found to be essential for antibody reactivity since these amino acids could not be substituted without a reduction in antibody reactivity. The majority of the remaining amino acids could be substituted without reducing antibody reactivity notably. These results confirm that charged amino acids are essential for antibody reactivity and that the vimentin antibody is dependent on side-chain interactions in combination with backbone interactions.

Characterization of Peptide Antibodies by Epitope Mapping Using Resin-Bound and Soluble Peptides.

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Sequential Double Immunoblotting with Peptide Antibodies.

Immunoblotting, also termed western blotting, is a powerful method for detection and characterization of proteins separated by various electrophoretic techniques. The combination of sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE), having high separating power, immunoblotting to synthetic membranes, and detection with highly specific peptide antibodies, is especially useful for studying individual proteins in relation to cellular processes, disease mechanisms, etc. Here, we describe a protocol for the sequential detection of various forms of an individual protein using peptide antibodies, exemplified by the characterization of antibody specificity for different forms of the protein calreticulin by double SDS-PAGE immunoblotting.

Correlation between centromere protein-F autoantibodies and cancer analyzed by enzyme-linked immunosorbent assay.

BACKGROUND: Centromere protein-F (CENP-F) is a large nuclear protein of 367 kDa, which is involved in multiple mitosis-related events such as proper assembly of the kinetochores, stabilization of heterochromatin, chromosome alignment and mitotic checkpoint signaling. Several studies have shown a correlation between CENP-F and cancer, e.g. the expression of CENP-F has been described to be upregulated in cancer cells. Furthermore, several studies have described a significant correlation between the expression of autoantibodies to CENP-F and cancer. METHODS: Autoantibodies to CENP-F were detected in a small number of samples during routine indirect immunofluorescence (IIF) analysis for anti-nuclear antibodies (ANA) using HEp-2 cells as substrate. Using overlapping synthetic peptides covering a predicted structural maintenance of chromosomes (SMC) domain, we developed an enzyme-linked immunosorbent assay (ELISA) for detection of CENP-F antibodies. RESULTS: Analyzing the reactivity of the sera positive in IIF for CENP-F antibodies to overlapping CENP-F peptides, we showed that autoantibodies to several peptides correlate with the presence of antibodies to CENP-F and a diagnosis of cancer, as increased CENP-F antibody expression specific for malignant cancer patients to five peptides was found (A9, A12, A14, A16, A27). These antibodies to CENP-F in clinical samples submitted for ANA analysis were found to have a positive predictive value for cancer of 50%. Furthermore, the expression of cancer-correlated CENP-F antibodies seemed to increase as a function of time from diagnosis. CONCLUSION: These results conform to previous findings that approximately 50% of those patients clinically tested for ANA analyses who express CENP-F antibodies are diagnosed with cancer, confirming that these antibodies may function as circulating tumor markers. Thus, a peptide-based CENP-F ELISA focused on the SMC domain may aid in identifying individuals with a potential cancer.

Identification and mapping of a linear epitope of centromere protein F using monoclonal antibodies.

Autoantibodies against centromere protein -F have been reported to be associated with various types of cancer with poor prognosis. The characterization of these autoantibody specificities is important in both diagnostics and basic research. In this study, we mapped the epitope (NELSRIRSEKA) of two monoclonal centromere protein F antibodies. The epitope was localized by screening of overlapping peptides followed by a fast and efficient estimation of the minimal peptide length required for antibody recognition, based on the screening of terminally truncated resin-bound peptide analogs. The epitope was determined through competitive inhibition assays of systematically truncated free peptides. In addition, the importance of the involved amino acid side chains of the identified epitope was determined through competitive inhibition assays using alanine-substituted analogs.