scholarly journals Evaluation of the discriminatory potential of antibodies created from synthetic peptides derived from wheat, barley, rye and oat gluten

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257466
Author(s):  
David Poirier ◽  
Jérémie Théolier ◽  
Riccardo Marega ◽  
Philippe Delahaut ◽  
Nathalie Gillard ◽  
...  
Keyword(s):  
Synthetic Peptides ◽  
Indirect Elisa ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Gluten Free ◽  
Immunization Strategy ◽  
Gluten Content ◽  
Concholepas Concholepas ◽  
Prepackaged Foods ◽  
The One

Celiac disease (CD) is triggered by ingestion of gluten-containing cereals such as wheat, barley, rye and in some cases oat. The only way for affected individuals to avoid symptoms of this condition is to adopt a gluten-free diet. Thus, gluten-free foodstuffs need to be monitored in order to ensure their innocuity. For this purpose, commercial immunoassays based on recognition of defined linear gluten sequences are currently used. These immunoassays are designed to detect or quantify total gluten regardless of the cereal, and often result in over or underestimation of the exact gluten content. In addition, Canadian regulations require a declaration of the source of gluten on the label of prepackaged foods, which cannot be done due to the limitations of existing methods. In this study, the development of new antibodies targeting discrimination of gluten sources was conducted using synthetic peptides as immunization strategy. Fourteen synthetic peptides selected from unique linear amino acid sequences of gluten were bioconjugated to Concholepas concholepas hemocyanin (CCH) as protein carrier, to elicit antibodies in rabbit. The resulting polyclonal antibodies (pAbs) successfully discriminated wheat, barley and oat prolamins during indirect ELISA assessments. pAbs raised against rye synthetic peptides cross-reacted evenly with wheat and rye prolamins but could still be useful to successfully discriminate gluten sources in combination with the other pAbs. Discrimination of gluten sources can be further refined and enhanced by raising monoclonal antibodies using a similar immunization strategy. A methodology capable of discriminating gluten sources, such as the one proposed in this study, could facilitate compliance with Canadian regulations on this matter. This type of discrimination could also complement current immunoassays by settling the issue of over and underestimation of gluten content, thus improving the safety of food intended to CD and wheat-allergic patients.

scholarly journals A peptide isolated from phage display libraries is a structural and functional mimic of an RGD-binding site on integrins.

1995 ◽  
Vol 130 (5) ◽  
pp. 1189-1196 ◽  
Author(s):  
R Pasqualini ◽  
E Koivunen ◽  
E Ruoslahti
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Synthetic Peptides ◽  
Cell Attachment ◽  
Polyclonal Antibodies ◽  
Divalent Cations ◽  
Amino Acid Sequences ◽  
Beta Subunit ◽  
Affinity Column ◽  
Fibronectin Fragments

Many integrins recognize short RGD-containing amino acid sequences and such peptide sequences can be identified from phage libraries by panning with an integrin. Here, in a reverse strategy, we have used such libraries to isolate minimal receptor sequences that bind to fibronectin and RGD-containing fibronectin fragments in affinity panning. A predominant cyclic motif, *CWDDG/LWLC*, was obtained (the asterisks denote a potential disulfide bond). Studies using the purified phage and the corresponding synthetic cyclic peptides showed that *CWDDGWLC*-expressing phage binds specifically to fibronectin and to fibronectin fragments containing the RGD sequence. The binding did not require divalent cations and was inhibited by both RGD and *CWDDGWLC*-containing synthetic peptides. Conversely, RGD-expressing phage attached specifically to immobilized *CWDDGWLC*-peptide and the binding could be blocked by the respective synthetic peptides in solution. Moreover, fibronectin bound to a *CWDDGWLC*-peptide affinity column, and could be eluted with an RGD-containing peptide. The *CWDDGWLC*-peptide inhibited RGD-dependent cell attachment to fibronectin and vitronectin, but not to collagen. A region of the beta subunit of RGD-binding integrins that has been previously demonstrated to be involved in ligand binding includes a polypeptide stretch, KDDLW (in beta 3) similar to WDDG/LWL. Synthetic peptides corresponding to this region in beta 3 were found to bind RGD-displaying phage and conversion of its two aspartic residues into alanines greatly reduced the RGD binding. Polyclonal antibodies raised against the *CWDDGWLC*-peptide recognized beta 1 and beta 3 in immunoblots. These data indicate that the *CWDDGWLC*-peptide is a functional mimic of ligand binding sites of RGD-directed integrins, and that the structurally similar site in the integrin beta subunit is a binding site for RGD.

scholarly journals Molecular cloning of two human liver 3 α-hydroxysteroid/dihydrodiol dehydrogenase isoenzymes that are identical with chlordecone reductase and bile-acid binder

1994 ◽  
Vol 299 (2) ◽  
pp. 545-552 ◽  
Author(s):  
Y Deyashiki ◽  
A Ogasawara ◽  
T Nakayama ◽  
M Nakanishi ◽  
Y Miyabe ◽  
...  
Keyword(s):  
Bile Acid ◽  
Amino Acid ◽  
Human Liver ◽  
Polyclonal Antibodies ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Amino Acid Residues ◽  
Human Bile ◽  
Coding Regions ◽  
Computer Based

Human liver contains two dihydrodiol dehydrogenases, DD2 and DD4, associated with 3 alpha-hydroxysteroid dehydrogenase activity. We have raised polyclonal antibodies that cross-reacted with the two enzymes and isolated two 1.2 kb cDNA clones (C9 and C11) for the two enzymes from a human liver cDNA library using the antibodies. The clones of C9 and C11 contained coding sequences corresponding to 306 and 321 amino acid residues respectively, but lacked 5′-coding regions around the initiation codon. Sequence analyses of several peptides obtained by enzymic and chemical cleavages of the two purified enzymes verified that the C9 and C11 clones encoded DD2 and DD4 respectively, and further indicated that the sequence of DD2 had at least additional 16 residues upward from the N-terminal sequence deduced from the cDNA. There was 82% amino acid sequence identity between the two enzymes, indicating that the enzymes are genetic isoenzymes. A computer-based comparison of the cDNAs of the isoenzymes with the DNA sequence database revealed that the nucleotide and amino acid sequences of DD2 and DD4 are virtually identical with those of human bile-acid binder and human chlordecone reductase cDNAs respectively.

scholarly journals Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor

2001 ◽  
Vol 31 (2) ◽  
pp. 573-579 ◽  
Author(s):  
Pierre Eftekhari ◽  
Jean-Christophe Roegel ◽  
Frank Lezoualc'h ◽  
Rodolphe Fischmeister ◽  
Jean-Louis Imbs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
Amino Acid Sequences ◽  
Neonatal Lupus

scholarly journals Evaluation of a Handheld Gluten Detection Device

2018 ◽  
Vol 81 (10) ◽  
pp. 1723-1728 ◽  
Author(s):  
STEVE L. TAYLOR ◽  
JULIE A. NORDLEE ◽  
SHYAMALI JAYASENA ◽  
JOSEPH L. BAUMERT
Keyword(s):  
Ice Cream ◽  
Weight Basis ◽  
Processed Foods ◽  
Gluten Free ◽  
Specific Enzyme ◽  
Different Types ◽  
Food Matrices ◽  
The One ◽  
Addition Level ◽  
Positive Results

ABSTRACT A portable, handheld gluten detection device, the Nima sensor, is now available for consumers wishing to determine if gluten is present in food. By U.S. regulation, gluten-free foods should contain <20 ppm of gluten. Thirteen gluten-free foods (muffins, three different types of bread, three different types of pasta, puffed corn snack, ice cream, meatballs, vinegar and oil salad dressing, oatmeal, and dark chocolate) were prepared; each food was spiked on a weight to weight basis with gluten levels of 0, 5, 10, 20, 30, 40, and 100 ppm before processing or preparation. Unprocessed and processed foods were tested with the handheld gluten sensor and by two gluten-specific enzyme-linked immunosorbent assays (ELISAs) on the basis of the R5 and G12 monoclonal antibodies, respectively. The portable gluten detection device detected gluten in all food types at the 30-ppm addition level, failing to detect gluten in only 5 (6.4%) of 78 subsamples. At the 20-ppm addition level, the portable gluten detection device failed to detect gluten in one type of pasta but detected gluten residues in 63 (87.5%) of 72 other subsamples. The device was able to detect gluten at the 10-ppm addition level in 9 of the 13 food matrices (41 of 54 subsamples, 75.9%) but not in the three types of pasta and the puffed corn snack. The gluten-sensing device did not perform reliably at the 5-ppm addition level in 11 of 13 food matrices (exceptions: ice cream and muffins). In contrast, the ELISA methods were highly reliable at gluten addition levels of ≥10 ppm in all food matrices. The portable gluten detection device yielded a low percentage of false-positive results (4 of 111, 3.6%) in these food matrices. Thus, this handheld portable gluten sensor performed reliably in the detection of gluten in foods having ≥20 ppm of added gluten with only 18 (5.9%) of 306 failures, if results of the one type of pasta are excluded. The device worked with greater reliability as the gluten levels in the foods increased.

scholarly journals RGD-independent cell adhesion to the carboxy-terminal heparin-binding fragment of fibronectin involves heparin-dependent and -independent activities.

1990 ◽  
Vol 110 (3) ◽  
pp. 777-787 ◽  
Author(s):  
J B McCarthy ◽  
A P Skubitz ◽  
Z Qi ◽  
X Y Yi ◽  
D J Mickelson ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Dependent Manner ◽  
Heparin Binding ◽  
Independent Manner ◽  
Concentration Dependent Manner ◽  
Extracellular Matrix Components ◽  
Carboxy Terminal

Cell adhesion to extracellular matrix components such as fibronectin has a complex basis, involving multiple determinants on the molecule that react with discrete cell surface macromolecules. Our previous results have demonstrated that normal and transformed cells adhere and spread on a 33-kD heparin binding fragment that originates from the carboxy-terminal end of particular isoforms (A-chains) of human fibronectin. This fragment promotes melanoma adhesion and spreading in an arginyl-glycyl-aspartyl-serine (RGDS) independent manner, suggesting that cell adhesion to this region of fibronectin is independent of the typical RGD/integrin-mediated binding. Two synthetic peptides from this region of fibronectin were recently identified that bound [3H]heparin in a solid-phase assay and promoted the adhesion and spreading of melanoma cells (McCarthy, J. B., M. K. Chelberg, D. J. Mickelson, and L. T. Furcht. 1988. Biochemistry. 27:1380-1388). The current studies further define the cell adhesion and heparin binding properties of one of these synthetic peptides. This peptide, termed peptide I, has the sequence YEKPGSP-PREVVPRPRPGV and represents residues 1906-1924 of human plasma fibronectin. In addition to promoting RGD-independent melanoma adhesion and spreading in a concentration-dependent manner, this peptide significantly inhibited cell adhesion to the 33-kD fragment or intact fibronectin. Polyclonal antibodies generated against peptide I also significantly inhibited cell adhesion to the peptide, to the 33-kD fragment, but had minimal effect on melanoma adhesion to fibronectin. Anti-peptide I antibodies also partially inhibited [3H]heparin binding to fibronectin, suggesting that peptide I represents a major heparin binding domain on the intact molecule. The cell adhesion activity of another peptide from the 33-kD fragment, termed CS1 (Humphries, M. J., A. Komoriya, S. K. Akiyama, K. Olden, and K. M. Yamada. 1987. J. Biol. Chem., 262:6886-6892) was contrasted with peptide I. Whereas both peptides promoted RGD-independent cell adhesion, peptide CS1 failed to bind heparin, and exogenous peptide CS1 failed to inhibit peptide I-mediated cell adhesion. The results demonstrate a role for distinct heparin-dependent and -independent cell adhesion determinants on the 33-kD fragment, neither of which are related to the RGD-dependent integrin interaction with fibronectin.

scholarly journals Antibodies specific to acetylated histones document the existence of deposition- and transcription-related histone acetylation in Tetrahymena.

1989 ◽  
Vol 108 (5) ◽  
pp. 1577-1588 ◽  
Author(s):  
R Lin ◽  
J W Leone ◽  
R G Cook ◽  
C D Allis
Keyword(s):  
Life Cycle ◽  
Histone Acetylation ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Gene Activation ◽  
Strong Support ◽  
Chromatin Assembly ◽  
Rapid Turnover ◽  
Core Histones

In this study, we have constructed synthetic peptides which are identical to hyperacetylated amino termini of two Tetrahymena core histones (tetra-acetylated H4 and penta-acetylated hv1) and used them to generate polyclonal antibodies specific for acetylated forms (mono-, di-, tri-, etc.) of these histones. Neither of these antisera recognizes histone that is unacetylated. Immunoblotting analyses demonstrate that both transcription-related and deposition-related acetate groups on H4 are recognized by both antisera. In addition, the antiserum raised against penta-acetylated hv1 also recognizes acetylated forms of this variant. Immunofluorescent analyses with both antisera demonstrate that, as expected, histone acetylation is specific to macronuclei (or new macronuclei) at all stages of the life cycle except when micronuclei undergo periods of rapid replication and chromatin assembly. During this time micronuclear staining is also detected. Our results also suggest that transcription-related acetylation begins selectively in new macronuclei immediately after the second postzygotic division. Acetylated histone is not observed in new micronuclei during stages corresponding to anlagen development and, therefore, histone acetylation can be distributed asymmetrically in development. Equally striking is the rapid turnover of acetylated histone in parental macronuclei during the time of their inactivation and elimination from the cell. Taken together, these data lend strong support to the idea that modulation of histone acetylation plays an important role in gene activation and in chromatin assembly.

scholarly journals Structure of the murine rab3A gene: correlation of genomic organization with antibody epitopes

1993 ◽  
Vol 293 (1) ◽  
pp. 157-163 ◽  
Author(s):  
M Baumert ◽  
G Fischer von Mollard ◽  
R Jahn ◽  
T C Südhof
Keyword(s):  
Monoclonal Antibodies ◽  
Molecular Mass ◽  
Polyclonal Antibodies ◽  
Protein Domains ◽  
Amino Acid Sequences ◽  
Sequence Motifs ◽  
Rab Protein ◽  
Gtp Binding ◽  
Gene Structures ◽  
Antibody Epitopes

Rab3A is a neuronal low-molecular-mass GTP-binding protein that is modified post-translationally by two geranylgeranyl groups and specifically targeted to synaptic vesicles. We have now cloned and characterized the murine gene coding for rab3A. With a size of less than 8 kb including the promoter, the rab3A gene is relatively small. It contains five exons, the first of which is non-coding. The organization of the rab3A coding sequence into exons in the gene is different from that of ras proteins, the only other low-molecular-mass GTP-binding proteins with currently characterized gene structures. Nevertheless, the intron placement in the primary structure of rab3A may be indicative of a domain division of the protein, since each coding exon contains one of the four major conserved rab protein sequence motifs. The epitopes of monoclonal and polyclonal antibodies to rab3A were mapped with the hypothesis that antibody epitopes might represent distinct exposed protein domains and correlate with exon structures. Two monoclonal antibodies, named 42.1 and 42.2, were found to recognize epitopes with a different degree of conservation between different rab3 isoforms. These epitopes were mapped to relatively short amino acid sequences corresponding to exons 4 and 5 respectively, whereas a polyclonal antibody recognized a complex epitope that required the presence of intact rab3A. Comparison of the sequence of rab3A with that of ras, whose crystal structure has been determined, revealed that the epitopes for the monoclonal antibodies correspond to regions in ras that are highly exposed. Taken together, these results suggest that exons 4 and 5 at least represent distinct exposed protein domains that also form major natural epitopes in rab3A.

scholarly journals Synthetic alpha-thrombin receptor peptides activate G protein-coupled signaling pathways but are unable to induce mitogenesis.

1992 ◽  
Vol 3 (1) ◽  
pp. 95-102 ◽  
Author(s):  
V Vouret-Craviari ◽  
E Van Obberghen-Schilling ◽  
U B Rasmussen ◽  
A Pavirani ◽  
J P Lecocq ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Phospholipase C ◽  
G Protein ◽  
Synthetic Peptides ◽  
Cleavage Site ◽  
Amino Acid Sequences ◽  
Thrombin Receptor ◽  
Thrombin Cleavage ◽  
Ccl39 Cells ◽  
G Protein Coupled

alpha-Thrombin (thrombin) stimulates phospholipase C and modulates the activity of adenylate cyclase in a number of cell types via G protein-coupled receptors. It is also a potent growth factor, notably for a line of hamster fibroblasts (CCL39 cells). Recently, predicted amino acid sequences for human and hamster thrombin receptors have been reported that display a putative thrombin cleavage site in the N-terminal extracellular domain. Synthetic peptides corresponding to 14 residues carboxyl to the presumed thrombin cleavage site of the human receptor have been shown to activate platelets as well as the thrombin receptor expressed in Xenopus oocytes. In the present study we have examined the effects of synthetic peptides corresponding to the same region of the hamster receptor (S-42-L-55) and shorter peptides (2-7 residues) on signal transducing systems in CCL39 cells. Our results indicate that hamster receptor peptides of greater than or equal to 5 residues effectively stimulate phospholipase C in CCL39 cells via the thrombin receptor and induce rapid desensitization of the response. The same peptides also inhibit adenylate cyclase in a pertussis toxin-sensitive manner. Although the peptides are potent agonists of serotonin release in platelets, unlike thrombin, by themselves they are not mitogenic. However, they potentiate DNA synthesis in cooperation with growth factors possessing tyrosine kinase receptors. Hence, we conclude that the potent mitogenic action of thrombin cannot be accounted for solely by the activation of the cloned receptor. We postulate the existence of an additional receptor activated by thrombin, which is required for its full mitogenic potential.

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