A New Ligand for Immunoglobulin G Subdomains by Screening of a Synthetic Peptide Library
Antonio VerdolivaDaniela MarascoAntonia De CapuaAngela SaporitoPiero BellofioreVincenzo ManfrediRoberto FattorussoCarlo PedoneMenotti Ruvo
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Abstract By screening a synthetic peptide library of general formula (NH 2 ‐Cys1‐X2‐X3‐X4) 2 ‐Lys‐Gly‐OH, a disulfide‐bridged cyclic peptide, where X2‐X3‐X4 is the tripeptide Phe‐His‐His, has been selected as a ligand for immunoglobulin G (IgG). The peptide, after a preliminary chromatographic characterization, has proved useful as a new affinity ligand for the purification of polyclonal as well as monoclonal antibodies from biological fluids, with recovery yields of up to 90 % (90 % purity). The ligand is able to bind antibody fragments containing both Fab and Fc from different antibody isotypes, a fact suggesting the presence of at least two different antibody‐binding sites. While the recognition site on Fab is unknown, comparative binding studies with Fc, in association with the striking similarities of the peptide (named Fc‐receptor mimetic, FcRM) with a region of the human FcγRIII receptor, strongly indicate that the peptide could recognize a short amino acid stretch of the lower hinge region, which has a key role in autoimmune disease triggering. The unique properties make the ligand attractive for both the purification of antibody fragments and as a lead for the generation of Fc‐receptor antagonists.Keywords:
Tripeptide
Polyclonal antibodies
Tripeptide
Oligopeptide
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Tripeptide
Oligopeptide
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Two glycoproteins (HN and F) of parainfluenza virus were immunogold-labeled with polyclonal and monoclonal antibodies, respectively, and their labeling patterns were compared. Both glycoproteins HN and F were efficiently and homogeneously labeled with polyclonal antibodies, whereas they were labeled much less and heterogeneously with monoclonal antibodies. When either protein was initially labeled with monoclonal antibody, and then the other one, with polyclonal antibody, immunolabels of two glycoproteins were almost completely segregated. Although this segregation deformed virion morphology, it supported the concept of monoclonal antibody-mediated movements of glycoproteins.
Polyclonal antibodies
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SUMMARY We have examined the potential of isolating ligands for polyclonal antibodies from a nanopeptide phage library. The library was screened with a rabbit polyclonal antiserum raised against a synthetic peptide (ALWFRNHFVFGGGTKVT). Following screening, the positive phages were tested in an ELISA for their reactivity with the antiserum. Phages that showed positive reactivity with the antiserum compared with a normal rabbit serum were selected and their displayed peptides were determined. Among the 36 random positive clones, 31 clones carried the sequence AVFGGGTKL, PFFGGGSRA or APTGGSKRT that have a significant homology to the immunizing peptide. Five positive phages displayed the ATNIFIEGT sequence, which has no obvious linear homology with either the other selected peptides or with the peptide used for immunization. In contrast to the control peptide, the immunizing peptide inhibited binding of the antiserum to the peptide-displaying phages in a dose-dependent manner, thus demonstrating the specificity of the interaction. Furthermore, the rabbit B cell response to the peptide was found to be limited and focused on its C-terminal. Taken together, our data demonstrate the potential of random peptide phage libraries for defining epitopes for polyclonal antisera as well as for investigation of the nature of B cell responses to any given antigen.
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In order to test the specificity of the natural Delta-Sleep-Inducing-Peptide (DSIP), a tripeptide with the same N-terminal amino acid was synthesized. The synthesis of the new tripeptide L-Trp-L-Ser-L-Glu was carried out by the method of the mixed anhydride. Protecting groups were all oxygen-bound benzyl groups. The physical-chemical data of the newly synthesized peptides are reported. The biological activity of the tripeptide was assayed by intraventricular infusion in the rabbit under the same conditions as for the DSIP. The effects of the tripeptide on the EEG could not duplicate those of DSIP which induced a marked increase of delta activity, typical for orthodox 'Slow Wave Sleep' (SWS).
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Thirty two synthetic tripeptides structurally related both to thyrotropin releasing hormone (TRH) and anorexogenic tripeptide (Glp-His-Gly-OH) were investigated for anorexogenic effect in rats. While the two endogenous peptides, TRH and Glp-His-Gly-OH, were ineffective in rats deprived of food for 96 hours when they were administered intracerebroventricularly, some of the synthetic analogues showed significant food intake reducing effect under the same conditions. This anorexogenic effect of the tripeptides is similar--though much weaker--to that of satietin, a highly potent anorexogenic glycopeptide in human and mammalian serum. These tripeptides were ineffective when they were administered intravenously.
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Arm: to search for a protocol for synthesis of tripeptide. Method: dicarbonyl dichloride method was used to synthesize tripep-tides. Results: three kinds of tripeptides were successfully synthesized and identified by mass spectrometry, infrared spectrum, nuclear magnetic resonance and element analysis respectively. Conclusion: the protocol developed by the author is useful and suitable for synthesis of tripeptides from acidic aminoacids.
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