Antibodies to expanded virus antigen panels show elevated diagnostic sensitivities in multiple sclerosis and optic neuritis.

An antigen panel consisting of Epstein-Barr, measles, mumps, varicella zoster and rubella viruses (EMMRZ) was recently presented, which may aid in the diagnosis of multiple sclerosis (MS). The aim of this study was to validate and extend the EMMRZ panel. Various candidates, such as Cytomegalovirus and John Cunningham virus were analysed in relapsing-remitting MS (RRMS) and optic neuritis (ON) samples by enzyme-linked immunosorbent assay. IgG levels were elevated in RRMS samples and correlations were found between serum and cerebrospinal fluid levels. Cohort-dependent optimized panels were obtained for RRMS and ON, which obtained the highest sensitivity when combined with the status of oligoclonal bands.

Design, Production, Characterization, and Use of Peptide Antibodies.

Antibodies are key reagents in diagnostics, therapeutics, and experimental biology, capable of detecting numerous targets [...].

Antibodies to Epstein-Barr virus and neurotropic viruses in multiple sclerosis and optic neuritis.

Antibody indices to Measles, Mumps, Varicella Zoster (MRZ) are of diagnostic value in multiple sclerosis (MS). Here, we have investigated, if this panel could be extended to increase diagnostic value. Samples from relapsing-remitting (RR) MS and optic neuritis (ON) patients were tested for reactivity to antigens from Epstein-Barr, Varicella Zoster, Measles, Mumps and Rubella (EMMRZ) viruses. Increased IgG levels in serum and cerebrospinal fluid (CSF) were found in RRMS patients, along with a significant correlation between serum and CSF. The sensitivity of the EMMRZ panel was increased approximately 40% compared to the MRZ panel, suggesting that the EMMRZ panel may be useful in MS and ON diagnostics.

Antibodies as Diagnostic Targets and as Reagents for Diagnostics.

Antibodies (Abs) were discovered around the turn of the 19th century and characterized in the following decades as an essential part of the human adaptive immune system [...].

Specificity of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. The majority of individuals with RA are positive for the disease-specific anti-citrullinated protein antibodies (ACPAs). These antibodies are primarily of cross-reactive nature, hence, the true autoantigen to ACPA remains unidentified. In this study, we analyzed the reactivity of RA sera to several post-translationally modified epitopes, in order to further characterize the specific nature of ACPAs by immunoassays. Substituting citrulline with other amino acids, e.g., D-citrulline, homo-citrulline and methyl-arginine illustrated that ACPAs are utmost specific for citrullinated targets. Collectively, these findings support that ACPAs and citrullinated targets are specific for RA, making citrulline-containing peptide targets the most effective assays for detection of ACPAs.

Comparison of immunological adjuvants.

In this study, several innate immunological adjuvants and related compounds were compared with respect to complement activation in serum and induction of cytokine release in whole blood samples using immunoassays. As found, simple lipids had no effect on the complement system or on cytokine release, whereas lipopolysaccharides induced prominent release of pro-inflammatory cytokines (IL1ß, TNF and IFNγ) without affecting the complement system, except for one, which activated the lectin pathway (LP). Moreover, saponin induced IL1ß and MCP1 release and did not affect the complement system. The polysaccharide inulin exhausted the alternative pathway (AP) completely without affecting the LP and the classical pathway (CP), whereas zymosan exhausted the AP and had a major effect on the LP and CP as well. Peptidoglycans mainly affected the LP. Inulin, agarose and cellulose induced IL1ß and MCP1 release, while dextran had no effect on cytokine secretion. Zymosan mainly induced IL1ß release. The inorganic compound aluminium hydroxide, Al(OH)3 , activated the complement system very efficiently (all three pathways) but only induced MCP1 release. Other compounds tested had minor/individual effects. Collectively, well-known adjuvants, such as aluminum hydroxide, activated the complement system and/or induced pro-inflammatory cytokine release. Since complement activation generates anaphylactic peptides, a simple definition of an (innate) immunological adjuvant can be inferred: it activates the (innate) immune system by complement activation and/or release of cytokines so as to attract cells of the adaptive immune system.

Peptide Antibodies in Clinical Laboratory Diagnostics.

Peptide antibodies, with their high specificities and affinities, are invaluable reagents for peptide and protein recognition in biological specimens. Depending on the application and the assay, in which the peptide antibody is to used, several factors influence successful antibody production, including peptide selection and antibody screening. Peptide antibodies have been used in clinical laboratory diagnostics with great success for decades, primarily because they can be produced to multiple targets, recognizing native wildtype proteins, denatured proteins, and newly generated epitopes. Especially mutation-specific peptide antibodies have become important as diagnostic tools in the detection of various cancers. In addition to their use as diagnostic tools in malignant and premalignant conditions, peptide antibodies are applied in all other areas of clinical laboratory diagnostics, including endocrinology, hematology, neurodegenerative diseases, cardiovascular diseases, infectious diseases, and amyloidoses.

Epitope Specificity of Anti-Citrullinated Protein Antibodies.

Anti-citrullinated protein antibodies are primarily associated with a progressive course in the autoimmune disease rheumatoid arthritis, a disease with a chronic and inflammatory nature. These antibodies do not appear to have any strict dependency for reactivity except from the presence of the non-genetically encoded amino acid citrulline, which is the result of a posttranslational modification, catalyzed by calcium-dependent peptidylarginine deiminase enzymes. Nevertheless, several amino acids surrounding the citrulline residue notably influence antibody reactivity, especially with a central-Cit-Gly-motif being essential for antibody reactivity. Most importantly, these antibodies have been proposed to be divided into two groups, based on their ability to recognize multiple citrullinated peptides. Thus, an "overlapping" antibody group, which appears to recognize several citrullinated peptides, and a "non-overlapping" antibody group, which only recognizes a limited number of citrullinated peptides, have been proposed. Based on these findings, we suggest that antibodies recognizing several citrullinated targets, also referred to as cross-reactive antibodies, primarily are backbone-dependent, whereas less cross-reactive antibodies primarily depend on the side chains of the amino acids comprising the epitopes for stable antibody-antigen interactions, which reduces the degree of cross-reactivity significantly. Clarifying the reactivity pattern of anti-citrullinated protein antibodies may contribute to determining their true nature of origin.

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.

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.

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.