SYNTHETIC PEPTIDE PROBES OF FACTOR VIII IMMUNOLOGY AND FUNCTION

1987 ◽  
Author(s):  
C A Fulcher ◽  
R A Houghten ◽  
S de Graaf Mahoney ◽  
J R Roberts ◽  
T S Zimmerman
Keyword(s):  
Amino Acid ◽  
Synthetic Peptide ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Keyhole Limpet Hemocyanin ◽  
Significant Inhibition ◽  
Terminal Region ◽  
Amino Terminal ◽  
Microtiter Plates ◽  
Peptide Immunogen

In order to develop specific immunologic reagents for mapping functionally important sites on FVIII, we have prepared rabbit polyclonal antibodies against synthetic peptides of FVIII derived from regions along the entire FVIII amino acid sequence. To date, a total of 70 peptides have been synthesized and characterized by amino acid and HPLC analysis. The peptides were coupled to keyhole limpet hemocyanin with glutaraldehyde as a linkage reagent and used to immunize rabbits. Antisera were tested by ELISA assay on polystyrene microtiter plates coated with either the peptide immunogen, or purified FVIII. The antisera were also tested for their ability to inhibit FVIII clotting activity and to react with separated FVIII polypeptides on immunoblots.Of the 70 peptides, all reacted with the peptide immunogen, 45 reacted with purified FVIII and 33 reacted with FVIII on immunoblots. Because we had obtained evidence that cleavage of the amino terminal region of the 80 kDa polypeptide may play a role in FVIII activation by thrombin, a series of partially overlapping peptides, 15 residues in length, were synthesized in this area. After affinity purifying these antibodies on columns of FVIII immobilized on agarose, adjusting the antibodies to equal antigen binding titers by dot immunoblotting and testing for inhibition of FVIII activity, only one antibody could strongly inhibit FVIII clotting activity. This inhibition could be blocked by the peptide itself at nanomolar concentrations and no significant inhibition could be shown by antibodies to partially overlapping peptides individually, or in combination. These data suggest that a site important to FVIII function can be localized to a 15 amino acid residue region of the 80 kDa polypeptide of FVIII. In addition, a second inhibitoryantibody was identified which was produced against a peptide in the carboxy terminal region of the 54 kDa thrombin fragment of FVIII and this area is currently being studied in a similar manner. In addition, two monoclonal anti-FVIII synthetic peptide antibodies have been produced which react with purified FVIII on immunoblots. One of these antibodies also functions as an immunoadsorbent when linked to agarose and FVII can be purified in this manner, using the synthetic peptide as eluant. It is evident that antibodies to synthetic peptides of FVIII can be useful probes of FVIII structure, function and interactions as well as being of use in FVIII purification.

Expression of Na+-d-glucose cotransporter in brush-border membrane of the chicken intestine

1999 ◽  
Vol 276 (2) ◽  
pp. R627-R631 ◽  
Author(s):  
Carles Garriga ◽  
Nativitat Rovira ◽  
Miquel Moretó ◽  
Joana M. Planas
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Western Blot ◽  
Brush Border ◽  
Synthetic Peptide ◽  
Brush Border Membrane ◽  
Polyclonal Antibodies ◽  
Membrane Vesicles ◽  
Dependent Transport

We have studied the expression of Na+-d-glucose cotransporter in brush-border membrane vesicles (BBMVs) of chicken enterocytes to correlate the changes in the apical Na+-dependent transport with the changes in the amounts of transporter determined by Western blot analysis. Two different rabbit polyclonal antibodies were used simultaneously. The antibody raised against amino acids 564–575 of the deduced amino acid sequence of rabbit intestinal SGLT-1 ( antibody 1) specifically detects a single 75-kDa band in the three segments, and this band disappeared when the antibody was preabsorbed with the antigenic peptide. The antibody raised against the synthetic peptide corresponding to amino acids 402–420 of the same protein ( antibody 2) only reacts with jejunal and ileal samples, but no signal is found in BBMVs of rectum. Only when antibody 1 was used was there a linear correlation between the maximal transport rates of hexoses in BBMVs and the relative protein amounts determined by Western blot. These results indicate that the Na+-d-glucose cotransport in the jejunum, the ileum, and the rectum of chickens is due to an SGLT-1 type protein.

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals Determination of Citrinin in Barley by Indirect and Direct Enzyme Immunoassay

1996 ◽  
Vol 79 (6) ◽  
pp. 1325-1329 ◽  
Author(s):  
David Abramson ◽  
Ewald Usleber ◽  
Erwin Martlbauer
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Detection Limits ◽  
Keyhole Limpet Hemocyanin ◽  
Buffer Solutions ◽  
Microtiter Plates ◽  
Coefficients Of Variation ◽  
Optimum Extraction

Abstract Polyclonal antibodies against the mycotoxin citrinin were raised in rabbits after immunization with citrinin conjugated to keyhole limpet hemocyanin. The antibodies were used in a competitive indirect enzyme immunoassay (EIA) with citrinin coupled to glucose oxidase as the solid-phase antigen for coating microtiter plates. Detection limits of this indirect EIA for citrinin ranged from 0.4 to 0.8 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 100–2000 ng/g ranged from 105 to 112%, with coefficients of variation between 4.5 and 12%. A direct competitive EIA also was established, with citrinin coupled to horseradish peroxidase as the labeled antigen. Detection limits of this direct EIA for citrinin ranged from 2 to 4 ng/mL for buffer solutions. Recoveries of citrinin added to ground barley at 500–2000 ng/g ranged from 108 to 111%, with coefficients of variation between 8.4 and 26.9%. In naturally contaminated barley samples assayed with the indirect EIA, optimum extraction of citrinin was obtained in 30 min, and only one extraction was necessary to recover 72–76% of the analyte.

scholarly journals Identification and characterization of PF4varl, a human gene variant of platelet factor 4.

1989 ◽  
Vol 9 (4) ◽  
pp. 1445-1451 ◽  
Author(s):  
C J Green ◽  
R S Charles ◽  
B F Edwards ◽  
P H Johnson
Keyword(s):  
Amino Acid ◽  
Terminal Region ◽  
Leader Sequence ◽  
Platelet Factor 4 ◽  
Heparin Binding ◽  
Platelet Factor ◽  
Coding Sequences ◽  
Amino Terminal ◽  
Arginine Residues ◽  
Carboxy Terminal

A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.

Immunolocalization ofTaenia soliumgap junction innexins

Parasitology ◽  
2008 ◽  
Vol 135 (9) ◽  
pp. 1125-1131 ◽  
Author(s):  
R. ZURABIAN ◽  
A. LANDA ◽  
L. ROBERT ◽  
K. WILLMS
Keyword(s):  
Amino Acid ◽  
Western Blot ◽  
Small Molecules ◽  
Synthetic Peptide ◽  
Polyclonal Antibodies ◽  
Taenia Solium ◽  
Peptide Sequence ◽  
Rabbit Igg ◽  
Membrane Peptide ◽  
Cytoplasmic Glycogen

SUMMARYIn previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues ofTaenia soliumtapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adultT. soliumcontain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.

scholarly journals Identification of Epitope and Surface-Exposed Domains of Shigella flexneri Invasion Plasmid Antigen D (IpaD)

1998 ◽  
Vol 66 (5) ◽  
pp. 1999-2006 ◽  
Author(s):  
K. Ross Turbyfill ◽  
Jennifer A. Mertz ◽  
Corey P. Mallett ◽  
Edwin V. Oaks
Keyword(s):  
Amino Acid ◽  
Shigella Flexneri ◽  
Serum Antibody ◽  
Terminal Region ◽  
Virulence Plasmid ◽  
Amino Acid Residues ◽  
Unique Region ◽  
Amino Terminal ◽  
Convalescent Phase ◽  
Carboxyl Terminal

ABSTRACT Transport and surface expression of the invasion plasmid antigens (Ipa proteins) is an essential trait in the pathogenicity ofShigella spp. In addition to the type III protein secretion system encoded by the mxi/spa loci on the large virulence plasmid, transport of IpaB and IpaC into the surrounding medium is modulated by IpaD. To characterize the structural topography of IpaD, the Geysen epitope-mapping system was used to identify epitopes recognized by surface-reactive monoclonal and polyclonal antibodies produced against purified recombinant IpaD or synthetic IpaD peptides. Surface-exposed epitopes of IpaD were confined to the first 180 amino acid residues, whereas epitopes in the carboxyl-terminal half were not exposed on the Shigella surface. By using convalescent-phase sera from 10 Shigella flexneri-infected monkeys, numerous epitopes were mapped within a surface-exposed region of IpaD between amino acid residues 14 and 77. Epitopes were also identified in the carboxyl-terminal half of IpaD with a few convalescent-phase sera. Comparison of IpaD epitope sequences withSalmonella SipD sequences indicated that very similar epitopes may exist in the carboxyl-terminal region of each protein whereas the IpaD epitopes in the surface-exposed amino-terminal region were unique for the Shigella protein. Although the IpaD and SipD homologs may play similar roles in transport, the dominant serum antibody response to IpaD is against the unique region of this protein exposed on the surface of the pathogen.

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