Studies on the primary structure and antigenic determinants of pilin isolated from Pseudomonas aeruginosa K

1985 ◽  
Vol 63 (4) ◽  
pp. 284-291 ◽  
Author(s):  
Parimi A. Sastry ◽  
Joyce R. Pearlstone ◽  
Lawrence B. Smillie ◽  
William Paranchych
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Protein A ◽  
Disulfide Bridge ◽  
Polyclonal Antiserum ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Fragments ◽  
Single Polypeptide Chain ◽  
Rabbit Polyclonal Antiserum ◽  
Single Polypeptide

The complete amino acid sequence of Pseudomonas aeruginosa K (PAK) pilin was determined using a combination of automated and manual Edman degradation techniques. Suitable peptides were derived from cyanogen bromide, tryptic, chymotryptic, peptic, thermolytic, and citraconylated tryptic cleavages of unmodified or carboxymethylated pilin. The protein, a single polypeptide chain, has N-methylphenylalanine at the NH2-terminus, a total of 144 residues, a molecular weight of 15 013, and an equal number of acid and basic amino acids. The NH2-terminal region (residues 1–43) is very hydrophobic with only three charged residues, suggesting a possible role in subunit–subunit interaction. The two half-cystines, residues 129 and 142, are shown to be linked through a disulfide bridge in the native protein. To delineate the antigenic regions of pilin, the protein was cleaved at Arg-30, Arg-53, and Arg-120 to produce peptide fragments cTI (residues 1–30), cTII (residues 31–53), cTIII (residues 54–120), and cTIV (residues 121–144). cTIII and cTIV were further degraded into several subfragments. The purified peptides were subjected to immunological analysis using direct and competitive enzyme-linked immunosorbent assay procedures. A major antigenic determinant was delineated in a region of the protein encompassing residues 82–101. Three other epitopes were also identified, but reacted with only minor amounts of antibody in the rabbit polyclonal antiserum.

scholarly journals Heterogeneity of type-specific and cross-reactive antigenic determinants within a single M protein of group A streptococci.

1980 ◽  
Vol 151 (5) ◽  
pp. 1026-1038 ◽  
Author(s):  
J B Dale ◽  
I Ofek ◽  
E H Beachey
Keyword(s):  
Protein Molecule ◽  
M Protein ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Fragments ◽  
M Proteins ◽  
The Cross ◽  
High Concentrations ◽  
Identical Type ◽  
Inhibition Studies

The heterogeneity of a pepsin extract of type-24 M protein (pep M24) was demonstrated by absorption of type-specific and cross-reactive human antisera with M protein fragments and heterologous serotypes of M proteins, pepsin extract of type-5 M protein (pep M5) and pepsin extract of type-6 M protein (pep M6). 2 of 12 individuals immunized with pep M24 developed significant rises in antibody titers against pep M5 and pep M6, as measured by the enzyme-linked immunosorbent assay. The sam individuals also developed opsonic antibodies against type-6, but not type-5, streptococci, which suggested the development of cross-protective immunity. Inhibition studies of one of these sera with the heterologous pep M proteins showed that the cross-reactive antibodies against pep M6 could not be blocked by high concentrations of pep M24, the immunizing antigen; these antibodies could be blocked, however, by cyanogen bromide-derived peptide fragments of pep M24, which suggested that the cross-reactive antibody was raised against an inaccessible site(s) in the pep M24 molecule. Inhibition studies of type-specific immune sera with pep M24 and peptides derived therefrom indicated that the M protein molecule contained multiple distinct as well as identical type-specific antigenic determinants that are unequally distributed among the seven derived peptide fragments.

scholarly journals Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)

2014 ◽  
Vol 60 (1) ◽  
pp. 51-62 ◽  
Author(s):  
E.F. Kolesanova ◽  
T.E. Farafonova ◽  
E.Yu. Aleshina ◽  
N.V. Pyndyk ◽  
M.V. Veremieva ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Amino Acid Sequences ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Peptide Fragments ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Eukaryotic Translation ◽  
Denatured States ◽  
Monospecific Antibodies

Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.

Expression of Enzymic Activity by Exotoxin A from Pseudomonas aeruginosa

1980 ◽  
Vol 28 (2) ◽  
pp. 494-501
Author(s):  
Stephen Lory ◽  
R. John Collier
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nicotinamide Adenine Dinucleotide ◽  
Enzymic Activity ◽  
Proteolytic Processing ◽  
Nicotinamide Adenine ◽  
Exotoxin A ◽  
Whole Cells ◽  
Single Polypeptide Chain ◽  
Single Polypeptide ◽  
Active Fragments

Exotoxin A from Pseudomonas aeruginosa is a single polypeptide chain ( M r , 66,000) containing little if any adenosine 5′-diphosphate ribosyltransferase or oxidized nicotinamide adenine dinucleotide glycohydrolase activity. These activities have been demonstrated in the reduced intact toxin and in a peptide ( M r , 26,000) isolated from culture fluids or toxin preparations after storage. In this report we describe methods for generating enzymically active fragments by cleaving the fully or partially reduced exotoxin by proteolytic or chemical methods. Incubation of reduced toxin with chymotrypsin in the presence of oxidized nicotinamide adenine dinucleotide yielded an enzymically active peptide ( M r , 26,000) similar to the fragment characterized previously. Chemical cleavage by treatment of the reduced molecule with CNBr or 2-nitro-5-thiocyanobenzoate yielded fragments ( M r , 50,000 and 30,000, respectively) with similar activities. Also both adenosine 5′-diphosphate ribosyltransferase and oxidized nicotinamide adenine dinucleotide glycohydrolase activities were maximally expressed by the intact exotoxin after reduction of only two of its four disulfide bridges. Kinetic constants for activated whole toxin were similar to those of fragment A of diphtheria toxin. It is evident that in the native toxin the catalytic center is buried or distorted and that alterations in the covalent structure permit the center to become exposed or assume an active configuration. It is unknown whether reduction, proteolytic processing, or both occur during the course of toxin action on whole cells.

scholarly journals Immunochemical analysis of the released Leu-2 (T8) molecule.

1984 ◽  
Vol 160 (1) ◽  
pp. 116-124 ◽  
Author(s):  
J Fujimoto ◽  
S J Stewart ◽  
R Levy
Keyword(s):  
Cell Surface ◽  
Polypeptide Chain ◽  
Peptide Mapping ◽  
Rabbit Antiserum ◽  
Antigenic Determinants ◽  
Charge Heterogeneity ◽  
Single Polypeptide Chain ◽  
Reducing Conditions ◽  
Human T Cell ◽  
Single Polypeptide

We recently have found that the human T cell antigen Leu-2 was specifically released from Leu-2-bearing cells. The preliminary study showed that the released Leu-2 (RLeu-2) from HPB-ALL cells was composed of a single polypeptide chain of 27,000 molecular weight (mol wt), which was smaller than the subunit of the homodimeric molecule found on the cell surface. In the present study, RLeu-2 was further characterized and compared with cellular Leu-2 (CLeu-2). Metabolically radiolabeled Leu-2 was released from HPB-ALL cells and this released Leu-2 molecule had a mol wt of 27,000. Cell surface radioiodinated HPB-ALL cells were found to release radioactive Leu-2 molecules and this antigen also had the same mol wt of 27,000. In both experiments, the CLeu-2 was reconfirmed to be composed of a 33,000-mol wt subunit under reducing conditions. These experiments establish that the 27,000-mol wt single polypeptide chain of Leu-2 released from the cell is derived directly from the homodimeric Leu-2 molecule on the cell surface, presumably by a specific proteolytic cleavage. Two-dimensional gel analysis showed that CLeu-2 exhibited extensive charge heterogeneity with predominantly basic isoelectric points, whereas RLeu-2 was a group of more acidic proteins with less charge heterogeneity. Although CLeu-2 and RLeu-2 showed several different immunochemical characteristics, the homology between these two antigens was confirmed by the following results: CLeu-2 and RLeu-2 were found to share at least three different antigenic determinants, Leu-2a and Leu-2b, and those which were detected by a polyvalent rabbit antiserum. Significant similarities between CLeu-2 and RLeu-2 were demonstrated by peptide mapping analysis of these antigens. Therefore, RLeu-2 appears to be the specific, physiological product of the CLeu-2 protein.

scholarly journals An Immunoaffinity Column for the Determination of Peanut Protein in Chocolate

1999 ◽  
Vol 82 (3) ◽  
pp. 666-668 ◽  
Author(s):  
Harvey W Newsome ◽  
Michael Abbott
Keyword(s):  
Detection Limit ◽  
Immunoaffinity Chromatography ◽  
Polyclonal Antiserum ◽  
Peanut Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoaffinity Column ◽  
Rabbit Polyclonal Antiserum ◽  
The Matrix ◽  
Direct Elisa

Abstract An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2–3.2 μg/g of peanut protein averaged 77% (range, 72–84%), and the minimum detection limit was 0.1 μg/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.

scholarly journals Conversion of proinsulin to insulin occurs coordinately with acidification of maturing secretory vesicles.

1986 ◽  
Vol 103 (6) ◽  
pp. 2273-2281 ◽  
Author(s):  
L Orci ◽  
M Ravazzola ◽  
M Amherdt ◽  
O Madsen ◽  
A Perrelet ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Islet Cells ◽  
Protein A ◽  
Secretory Vesicle ◽  
Average Density ◽  
Secretory Vesicles ◽  
Gold Particles ◽  
Single Polypeptide Chain ◽  
Igg Binding ◽  
Single Polypeptide

Proinsulin is a single polypeptide chain composed of the B and A subunits of insulin joined by the C-peptide region. Proinsulin is converted to insulin during the maturation of secretory vesicles by the action of two proteases and conversion is inhibited by ionophores that disrupted intracellular H+ gradients. To determine if conversion of prohormone to hormone actually occurs in an acidic secretory vesicle, cultured rat islet cells were incubated in the presence of 3-(2,4-dinitroanilino)-3' amino-N-methyldipropylamine (DAMP), a basic congener of dinitrophenol that concentrates in acidic compartments and is retained there after aldehyde fixation. The cells were processed for indirect protein A-gold colocalization of DAMP, using a monoclonal antibody to dinitrophenol, and proinsulin, using a monoclonal antibody that exclusively reacts with the prohormone. The average density of DAMP-specific gold particles in immature secretory vesicles that contained proinsulin was 71/micron 2 (18 times cytoplasmic background), which indicated that this compartment was acidic. However, the density of DAMP-specific gold particles in the insulin-rich mature secretory vesicle averaged 433/micron 2. This suggests that although proinsulin conversion occurs in an acidic compartment, the secretory vesicles become more acidic as they mature. Since the concentration of anti-proinsulin IgG binding in secretory vesicles is inversely proportional to the conversion of proinsulin to insulin, we were able to determine that maturing secretory vesicles had to reach a critical pH before proinsulin conversion occurred.

scholarly journals Antigenicity of Leishmania braziliensis Histone H1 during Cutaneous Leishmaniasis: Localization of Antigenic Determinants

2002 ◽  
Vol 9 (4) ◽  
pp. 808-811 ◽  
Author(s):  
Emma Carmelo ◽  
Enrique Martínez ◽  
Ana Cristina González ◽  
José Enrique Piñero ◽  
Manuel E. Patarroyo ◽  
...  
Keyword(s):  
Cutaneous Leishmaniasis ◽  
Chagas Disease ◽  
Expression System ◽  
Protein A ◽  
Histone H1 ◽  
Antigenic Determinants ◽  
Enzyme Linked Immunosorbent Assay ◽  
Leishmania Braziliensis ◽  
Serum Samples ◽  
Prokaryotic Expression System

ABSTRACT The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.

Enzyme-linked Immunosorbent Assay of Versicolorin A and Related Aflatoxin Biosynthetic Precursors

1991 ◽  
Vol 54 (2) ◽  
pp. 105-108 ◽  
Author(s):  
GWEN REYNOLDS ◽  
JAMES J. PESTKA
Keyword(s):  
High Performance ◽  
Cross Reactivity ◽  
Competitive Elisa ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Enhanced Production ◽  
Rabbit Polyclonal Antiserum ◽  
Norsolorinic Acid ◽  
Versicolorin A ◽  
Culture Procedure

An immunochemical approach is described for the detection of versicolorin (VA) and other aflatoxin precursors in Aspergillus parasiticus cultures. VA was purified from A. parasiticus ATCC 36537 cultures by semipreparative high performance liquid chromatography and confirmed by mass spectrometry and ultraviolet (UV) absorption. To be rendered immunogenic, VA was converted to a hemiacetal and conjugated to bovine serum albumin (BSA) by reductive alkylation. Rabbit polyclonal antiserum prepared against the VA hemiacetal-BSA conjugate was employed in a competitive ELISA using VA hemiacetal-horseradish peroxidase conjugate as the marker ligand. Based on the amount of VA analogue required to inhibit binding by 50% in competitive ELISA, cross-reactivity relative to VA for VA hemiacetal, averufanin, averantin, norsolorinic acid, averufin, sterigmatocystin, and aflatoxin B1 were 106, 85, 7, 6, 2, <1, and <1%, respectively. The ELISA was used to monitor enhanced production of VA equivalents by A. parasiticus ATCC 36537 in a modified culture procedure. The VA antibody should be extremely useful in the biochemical and genetic investigation of aflatoxin biosynthesis.

scholarly journals First Report of Banana Streak Badnavirus in Plantain Landraces in Southern Cameroon, Central Africa

Plant Disease ◽  
1997 ◽  
Vol 81 (11) ◽  
pp. 1335-1335 ◽  
Author(s):  
F. Gauhl ◽  
C. Pasberg-Gauhl ◽  
J. d'A. Hughes
Keyword(s):  
Central Africa ◽  
Polyclonal Antiserum ◽  
Enzyme Linked Immunosorbent Assay ◽  
University Of Minnesota ◽  
African Countries ◽  
Musa Spp ◽  
Rabbit Polyclonal Antiserum ◽  
Southern Cameroon ◽  
Healthy Control ◽  
Banana Streak Badnavirus

Banana streak badnavirus (BSV) has been reported from Musa spp. in many parts of West Africa, including Benin, Côte d'Ivoire, Ghana, and Nigeria (1). Symptoms of BSV infection in Musa spp. are sometimes similar to and confused with those caused by cucumber mosaic virus (CMV). BSV is prevalent in areas of southern Nigeria bordering Cameroon, and the disease may also be present in other Central African countries. In June 1996, six leaf samples with viruslike yellow/chlorotic streak symptoms were collected from plantain in the four villages, Awae, M'Balmayo, Nkolfep, and Nkolfoulou, within a 60-km radius of Yaoundé, Cameroon's capital. The samples were indexed for BSV and CMV by both triple antibody sandwich indirect enzyme-linked immunosorbent assay (TAS-ELISA) and immunosorbent electron microscopy (ISEM) to ascertain the presence of these two viruses. The TAS-ELISA was performed with rabbit polyclonal antiserum (obtained from B. E. L. Lockhart, University of Minnesota) for trapping and mouse polyclonal antiserum (obtained from G. Thottappilly, IITA) for detection. Out of the six samples, one tested strongly positive (>×2 A405 of the healthy control) and four were weakly positive (<×2 but >×1.5 A405 of the healthy control) for BSV by TAS-ELISA. However, all six samples contained BSV particles when examined by ISEM with rabbit polyclonal antiserum (from B. E. L. Lockhart) for trapping. None of the samples tested positive for CMV. These results confirm that BSV is present in Cameroon and that BSV is likely to be the causal agent of the symptoms. Reference: (1) C. Pasberg-Gauhl et al. Plant Dis. 80:224, 1996.

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