Synthetic peptides derived from the Listeria monocytogenes p60 protein as antigens for the generation of polyclonal antibodies specific for secreted cell-free L. monocytogenes p60 proteins.

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.108.5.1577 ◽

1989 ◽

Vol 108 (5) ◽

pp. 1577-1588 ◽

Cited By ~ 89

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.

Download Full-text

The third member of the Tetrahymena CCT subunit gene family, TpCCTα, encodes a component of the hetero-oligomeric chaperonin complex

Biochemical Journal ◽

10.1042/bj3260021 ◽

1997 ◽

Vol 326 (1) ◽

pp. 21-29 ◽

Cited By ~ 7

Author(s):

Helena SOARES ◽

Luisa CYRNE ◽

Cristina CASALOU ◽

Bruno EHMANN ◽

Claudina RODRIGUES-POUSADA

Keyword(s):

Gene Expression ◽

Gene Family ◽

Polyclonal Antibody ◽

Similar Pattern ◽

Synthetic Peptides ◽

Polyclonal Antibodies ◽

Recombinant Yeast ◽

Subunit Gene ◽

The Third ◽

Tubulin Genes

The sequence of a third member of the Tetrahymenapyriformis chaperonin CCT (‘chaperonin containing TCP1’) subunit gene family is presented. This gene, designated TpCCTα, is the orthologue of the mouse chaperonin gene TCP1/CCTα. To characterize the CCT complex in this ciliate, we have produced polyclonal antibodies against synthetic peptides based on C-terminal sequences deduced from the primary sequences of the TpCCTα, TpCCTγ and TpCCTη subunits. We have also used polyclonal antibodies produced against recombinant yeast CCTα and CCTβ subunits. Using these antibodies, we show that Tetrahymena cells contain a hetero-oligomeric CCT chaperonin comprising at least seven distinct subunits. Three of these were assigned to specific TpCCT genes, whereas a fourth was recognized by the polyclonal antibody against yeast CCTβ, suggesting that this gene is also present in the ciliate. The CCT complex also contains other unidentified proteins that were recognized by the polyclonal antibody UM-1, raised against the putative ATP binding domain of the chaperonin proteins. TpCCTα gene expression was shown in exponentially growing cells and cells regenerating their cilia for different periods to have a similar pattern to the previously identified genes TpCCTγ and TpCCTη, and also to tubulin genes.

Download Full-text

Selection and application of peptide mimotopes of MPT64 protein in Mycobacterium tuberculosis

Journal of Medical Microbiology ◽

10.1099/jmm.0.025098-0 ◽

2011 ◽

Vol 60 (1) ◽

pp. 69-74 ◽

Cited By ~ 10

Author(s):

Hua Yang ◽

Zhong-Hua Liu ◽

Li-Ting Zhang ◽

Jie Wang ◽

Huan-Seng Yang ◽

...

Keyword(s):

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Sequence Alignment ◽

Synthetic Peptides ◽

Polyclonal Antibodies ◽

Diagnostic Testing ◽

Conformational Epitope ◽

Space Structure ◽

Serological Tests ◽

Large Variability

Antibody responses can be useful markers of tuberculosis (TB) infection, especially in the screening of extra-pulmonary TB. MPT64 is an important antigen in Mycobacterium tuberculosis (MTB) infection and is used in serological diagnosis. However, large variability in the diagnostic accuracy of MPT64 as a serological tool has limited its application. Phage-displayed random peptide libraries have emerged as a powerful technique to select peptides (epitopes) or mimotopes that may serve as surrogate diagnostic markers in serological tests. In the present study, this method was employed to identify mimotopes of the MPT64 protein of MTB by screening a linear heptapeptide library with rabbit antibodies raised against MPT64 protein. Two antigenic mimotopes (M2 and M6) resembling B-cell epitopes of MPT64 were identified that bound the affinity purified anti-MPT64 polyclonal antibodies and competed with MPT64 for antibody binding. From the results of sequence alignment and a structure modelling figure of MPT64, the sequence of the 2nd to 5th amino acids (DSML) of M2 was totally consistent with the sequence of the 224th to 227th amino acids of MPT64 and the peptide is located on the surface of the space structure of MPT64, suggesting that it might be a linear epitope of MPT64. The recognition of both phage-displayed and synthetic peptides of M2 by the anti-MPT64 polyclonal antibodies also supported this. Although no recurring sequence and no analogue to MPT64 of M6 were found for sequence alignment, the recognition of both phage-displayed and synthetic peptides of M6 by the anti-MPT64 polyclonal antibodies indicated that it might be a mimotope of a conformational epitope of MPT64. According to the results of the reactivity of human sera with synthetic M2 and M6 peptides and MPT64, M2 showed a significantly higher AUC and sensitivity than M6 and MPT64, especially for the sera from sputum-negative TB patients, suggesting that the M2 mimotope may be useful in serological diagnostic testing for TB.

Download Full-text

Epitope map of two polyclonal antibodies that recognize amyloid lesions in patients with Alzheimer's disease

Biochemical Journal ◽

10.1042/bj2820517 ◽

1992 ◽

Vol 282 (2) ◽

pp. 517-522 ◽

Cited By ~ 4

Author(s):

J Ghiso ◽

T Wisniewski ◽

R Vidal ◽

A Rostagno ◽

B Frangione

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Amino Acids ◽

Synthetic Peptides ◽

Polyclonal Antibodies ◽

Sf9 Cells ◽

Beta Turn ◽

App Processing ◽

Conformational Epitopes ◽

N Terminus

Two synthetic peptides with sequences identical with those of fragments of the extracellular domain of the Alzheimer's-disease amyloid precursor protein (APP) were used to raise antibodies. SP28 comprises positions 597-624 of the APP695 isoform, whereas SP41 extends towards the N-terminus (amino acids 584-624) and contains the entire SP28 peptide. Using e.l.i.s.a. and inhibition experiments we identified the two beta-turn-containing segments 602-607 and 617-624 as the epitopes recognized by anti-SP41 and anti-SP28 respectively. Both antibodies immunolabelled amyloid lesions in brains from Alzheimer's-disease patients and patients with related disorders, whereas they were unreactive in control brains. However, when probed on immunoblots, anti-SP28 failed to detect full-length APP from baculovirus-infected Sf9 cells, and anti-SP41 reacted weakly compared with other anti-APP antisera. The data suggest that these antibodies are directed to conformational epitopes not existent in the native molecules but present after alternative APP processing.

Download Full-text

SYNTHETIC PEPTIDE PROBES OF FACTOR VIII IMMUNOLOGY AND FUNCTION

10.1055/s-0038-1644768 ◽

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1189 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1189-1196 ◽

Cited By ~ 146

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.

Download Full-text

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides

Blood ◽

10.1182/blood.v82.2.669.bloodjournal822669 ◽

1993 ◽

Vol 82 (2) ◽

pp. 669-676 ◽

Cited By ~ 37

Author(s):

P Hermand ◽

I Mouro ◽

M Huet ◽

C Bloy ◽

K Suyama ◽

...

Keyword(s):

Membrane Proteins ◽

Western Blot ◽

Red Blood Cells ◽

Blood Group ◽

Blood Cells ◽

Synthetic Peptides ◽

Rare Variants ◽

Polyclonal Antibodies ◽

Rh Proteins ◽

Rh Blood Group

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA- encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33–45 (MPC1), 224–233 (MPC4), 390–404 (MPC6), and 408–416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33– 45 and 408–416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.

Download Full-text

Kinesin-related proteins in eukaryotic flagella

Journal of Cell Science ◽

10.1242/jcs.107.6.1545 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1545-1550 ◽

Cited By ~ 6

Author(s):

L.A. Fox ◽

K.E. Sawin ◽

W.S. Sale

Keyword(s):

Synthetic Peptides ◽

Polyclonal Antibodies ◽

Immunoblot Analysis ◽

Motor Domain ◽

Flagellar Motility ◽

Central Pair ◽

Conserved Sequences ◽

Mutant Strains ◽

Related Proteins ◽

Antibodies To Synthetic Peptides

To identify kinesin-related proteins that are important for ciliary and eukaryotic flagellar functions, we used affinity-purified, polyclonal antibodies to synthetic peptides corresponding to conserved sequences in the motor domain of kinesin (Sawin et al. (1992) J. Cell Sci. 101, 303–313). Using immunoblot analysis, two antibodies to distinct sequences (LNLVDLAGSE, ‘LAGSE’ and, HIPYRESKLT, ‘HIPYR’) reveal a family of proteins in flagella and axonemes isolated from Chlamydomonas. Similar analysis of axonemes from mutant Chlamydomonas strains or fractionated axonemes indicates that none of the immunoreactive proteins are associated with dynein arm or spoke structures. In contrast, one protein, approximately 110 kDa, is reduced in axonemes from mutant strains defective in the central pair apparatus. Immunoreactive proteins with masses of 96 and 97 kDa (the ‘97 kDa’ proteins) are selectively solubilized from isolated axonemes in 10 mM ATP. The 97 kDa proteins co-sediment in sucrose gradients at about 9 S and bind to axonemes or purified microtubules in a nucleotide-dependent fashion characteristic of kinesin. These results reveal that flagella contain kinesin-related proteins, which may be involved in axonemal central pair function and flagellar motility, or directed transport involved in morphogenesis or mating responses in Chlamydomonas.

Download Full-text

Evaluation of Antibodies for Immunomagnetic Separation Combined with Flow Cytometry Detection of Listeria monocytogenes

Journal of Food Protection ◽

10.4315/0362-028x-66.7.1283 ◽

2003 ◽

Vol 66 (7) ◽

pp. 1283-1287 ◽

Cited By ~ 21

Author(s):

YONG SOO JUNG ◽

JOSEPH F. FRANK ◽

ROBERT E. BRACKETT

Keyword(s):

Flow Cytometry ◽

Listeria Monocytogenes ◽

Magnetic Beads ◽

Polyclonal Antibodies ◽

Immunomagnetic Separation ◽

Flow Cytometry Detection

Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry. The efficiency of immunocapturing using magnetic beads was also determined. None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L. monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples. Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23%. Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L. monocytogenes cells captured by the beads.

Download Full-text