Antibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis

doi:10.1006/meth.1996.0055

Methods

Volume 9, Issue 3, June 1996, Pages 473-481

https://doi.org/10.1006/meth.1996.0055 Get rights and content

Abstract

The multipin method for peptide synthesis was developed for antibody epitope mapping. From its initial invention as a method for testing many short peptides on the solid surface used for their synthesis, it has evolved into a method for producing solutions of peptides of a wide range of lengths in many “formats,” such as tagged (biotinylated) peptides for binding studies or affinity chromatography. The power of the multipin technique lies in the large number and variety of peptides that can be rapidly made and efficiently tested.

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Cited by (25)

Primary biliary cirrhosis following lactobacillus vaccination for recurrent vaginitis
2008, Journal of Hepatology
Citation Excerpt :
We present the case of a woman with an 8-year history of lactobacillus vaccination for recurrent vaginitis who developed characteristic clinical, biochemical and histological features of primary biliary cirrhosis. In view of known immunological cross-reactivity between a lactobacillus sequence and the major autoepitope of antimitochondrial antibody [11] we characterized autoreactivity and possible cross-reactivity [11,18,19,21,22,24–27,30] in this patient finding that her serum contains a high-titer AMA, which belongs mainly to IgG3 subclass and targets the immunodominant PDC-E2212–226 autoepitope. This serum also reacts with the Lactobacillus delbrueckii subs.
Antimitochondrial antibodies directed against the E2 subunit of the pyruvate dehydrogenase complex, PDC-E2, and other mitochondrial 2-oxoacid dehydrogenases (AMA-M2) are the hallmark for diagnosis of primary biliary cirrhosis (PBC). AMA-M2 formation as an early step in the pathogenesis of PBC has recently been assumed to be triggered by bacterial mimics of the E2 subunit and certain reactant xenobiotics. We report a case of symptomatic PBC diagnosed after sequential immunization with a lactobacillus vaccine for recurrent vaginitis over years.
Serum AMA-M2 specificity of the patient was evaluated by indirect immunofluorescence, immunoblotting and ELISA. Serum antibody responses against pyruvate dehydrogenase complex-E2 subunit (PDC-E2_212–226), the major PBC-specific mitochondrial autoepitope, and microbial mimics revealed cross-reactivity with beta-galactosidase of Lactobacillus delbrueckii (LACDE BGAL_266–280) which shows a high local homology with that of Lactobacillus species administered via the vaccine. The relative affinity of antibody reactivity to LACDE BGAL_266–280 was significantly higher than that against human PDC-E2_212–226.
We conclude that lactobacillus vaccination therapy may be another culprit for the development of PBC in genetically susceptible women.
Does Cross-Reactivity Between Mycobacterium avium paratuberculosis and Human Intestinal Antigens Characterize Crohn's Disease?
2006, Gastroenterology
Background & Aims: Most Crohn’s disease (CD) patients show seroreactivity against Mycobacterium avium paratuberculosis (MAP), suggesting a pathogenic role for this organism. Our aim was to seek amino acid similarities between MAP and intestinal proteins that, through molecular mimicry, could serve as targets for cross-reactive immunity in CD.
Methods: Fifty-three peptides comprising 23 sets of MAP/human intestinal peptidyl mimics chosen for maximal homology were constructed and tested for immunologic cross-reactivity by enzyme-linked immunosorbent assay in 50 patients with CD, 50 with ulcerative colitis, and 38 healthy controls.
Results: Antibody reactivity was present in only 7 of 23 peptide sets. MAP/self-reactivity in at least 1 of the 7 reactive sets was present in 21 (42%) CD patients but was virtually absent in the controls. Significant double-reactivity was found against MAP glycosyl transferase d (gsd)_230–244/human gastrointestinal glutathione peroxidase (GPg)_111–125 homologues in 15 of 50 (30%) CD patients; MAP alkylohydroperoxidase C (ahpC)_20–34/human tumor overexpressed protein (TOG)_637–651 double-reactivity was present in 10 (20%) CD patients, but in none of the controls. Inhibition studies confirmed that simultaneous reactivity to mimics was caused by cross-reactivity. Three-dimensional modeling predicts GPg_111–125 will be exposed in a solvent-accessible surface region of the protein compatible with antibody recognition. Antibody affinity was greater for the MAP mimics than for the self-sequences, suggesting that reactivity to the mycobacterial sequences precedes that against self-sequences.
Conclusions: We describe MAP/self-mimics as targets of cross-reactive antibody responses characterizing patients with CD. Our findings indicate gastrointestinal glutathione peroxidase as a novel autoantigen in CD.
Mapping of FVIII inhibitor epitopes using cellulose-bound synthetic peptide arrays
2006, Journal of Immunological Methods
Epitope mapping using antibodies against factor VIII (FVIII) has been performed using blotting techniques with truncated and/or digested FVIII molecules. Here, we focused on the precise mapping of affinity purified IgG from patients with an immune response against blood clotting FVIII using synthetic peptide arrays on cellulose membranes comprising the entire sequence of FVIII. The aim was to elucidate the epitope profile from different inhibitors and possibly detect new epitopes, which have not been described before. The epitope patterns from five patients showed reactivity with all domains in the FVIII molecule, but were different between various patients. These results included epitopes usually buried within the folded protein. However, in competition assays using FVIII as competitive agent in a mixture with inhibitor IgG, the most immunogenic regions were located in the FVIII light chain. Our results show that the C1 domain was the region with highest immunogenicity in all patients. Here, we demonstrate that the SPOT method is very well suited for the precise location of epitopes in the core of the protein, which usually cannot be detected by other methods.
The identification of immunodominant linear epitopes of dengue type 2 virus capsid and NS4a proteins using pin-bound peptides
2005, Virus Research
We have used multi-pin peptide synthesis strategy to identify B-cell epitopes on two small dengue virus proteins, capsid and NS4a. We have identified several linear, immunodominant epitopes on both these proteins. Almost all these epitopes mapped to regions predicted to be hydrophilic based on Kyte and Doolittle profiles. Of the capsid epitopes identified in this study, the most immunogenic ones mapped to the C-terminal α4 helix, which lies on the solvent-exposed surface of the capsid dimer. The capsid epitopes were dengue-specific in that they could recognize antibodies in dengue virus-, but not yellow fever virus (YFV)- or Japanese encephalitis virus (JEV)-immune sera. This study has demonstrated the presence of anti-NS4a antibodies in dengue-patient sera definitively, for the first time, using authentic NS4a-derived pin-bound peptides as capture antigens. All the NS4a epitopes mapped to the amino-terminal third of the NS4a molecule. Our study suggests that the immunodominant epitopes of these two dengue proteins might have the potential to be used as a part of a recombinant multi-epitope protein containing carefully chosen E and NS1 epitopes for the detection of dengue infections with a high degree of sensitivity and specificity.
Susceptibility to thyroid disorders in hepatitis C
2005, Clinical Gastroenterology and Hepatology
Background & Aims: Autoimmune thyroid disorders (AITDs) are reported, especially during interferon treatment, in chronic HCV infection, in which non-organ-specific autoantibodies (NOSAs) are common. We wondered whether seropositivity for NOSA is associated with susceptibility to AITDs. Methods: We evaluated thyroid function and antithyroglobulin and antithyroperoxidase antibodies in 348 Italian patients with chronic hepatitis C (34% NOSA-positive), 196 patients (33% NOSA-positive) of whom received interferon treatment. Results: At baseline, thyroid disorders were significantly more frequent in liver/kidney microsomal antibody type 1 (LKM1)–positive patients (29% vs 9%, P < .005). Similarly, on interferon therapy de novo autoimmune thyroid markers and/or symptomatic thyroid disorders appeared more often in LKM1-positive patients (50% vs 3%, P < .0001). Both female sex and LKM1 positivity were predictors of AITD, but only the latter remained significant after logistic regression analysis. Cross-reactivity to all 7 linear epitopes encoding homologous amino acid sequences shared by the HCV polyprotein, CYP2D6 (the LKM1 autoantigen), and thyroperoxidase was detected in 86% LKM1-positive HCV patients with clinical thyroid disorders, but in none of the LKM1-positive or negative HCV patients without thyroid disease, and none of an HCV-negative control group comprising subjects with LKM1-positive autoimmune hepatitis or AITD without liver disease (P < .0001). Conclusions: Patients receiving interferon therapy for hepatitis C seropositive for LKM1 are susceptible to develop AITDs, in association with treatment. Molecular mimicry and epitope spreading are potential pathogenic mechanisms.
Biosensor Characterization of Structure-Function Relationships in Viral Proteins
2004, Methods in Microbiology
Citation Excerpt :
In rare instances will a linear peptide fragment contain a sufficient number of residues of the original discontinuous epitope to enable it to bind to antibodies raised against the intact protein. Nowadays, continuous epitopes of proteins are frequently identified by analysing the antigenic reactivity of sets of overlapping synthetic peptides encompassing the entire sequence of the protein antigen (Rodda and Tribbick, 1996). Another approach consists in testing the antigenic reactivity of sets of peptides obtained from a combinatorial library.
This chapter discusses the biosensor characterization of structure–function relationships in viral proteins. The most widely used biosensor instrument is the BIACORE. The chapter discusses the mapping of viral epitopes. The regions of the viral proteins recognized by the antibody are usually described in terms of amino-acid residues although it is at the level of atomic interactions that the recognition takes place. As antigenic reactivity is usually ascribed to particular residues in a protein—this has led to the classification of epitopes as either continuous or discontinuous—depending on whether the residues involved in the epitopes are contiguous in the polypeptide chain or not. The mapping of epitopes on the surface of viral proteins is commonly done by double-sandwich enzyme-linked immunosorbent assays (ELISA) using all possible pairwise combinations of a panel of monoclonal antibodies (Mabs). Biosensors are useful for selecting diagnostic reagents suitable for viral diagnosis by high throughput immunoassays.

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Regular ArticleAntibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis

Abstract

Regular Article
Antibody-Defined Epitope Mapping Using the Multipin Method of Peptide Synthesis