scholarly journals Immunochemical Characterization of Temperature-Regulated Production of Enterocin L50 (EntL50A and EntL50B), Enterocin P, and Enterocin Q by Enterococcus faecium L50

2006 ◽  
Vol 72 (12) ◽  
pp. 7634-7643 ◽  
Author(s):  
Raquel Criado ◽  
Jorge Gutiérrez ◽  
María Martín ◽  
Carmen Herranz ◽  
Pablo E. Hernández ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
Polyclonal Antibodies ◽  
Keyhole Limpet Hemocyanin ◽  
Enzyme Linked Immunosorbent Assay ◽  
C Terminus ◽  
Bacteriocin Producer ◽  
Agric Food ◽  
Different Temperatures ◽  
Enterocin P

ABSTRACT Polyclonal antibodies with specificity for enterocin L50A (EntL50A), enterocin L50B (EntL50B), and enterocin Q (EntQ) produced by Enterococcus faecium L50 have been generated by immunization of rabbits with chemically synthesized peptides derived from the C terminus of EntL50A (LR1) and EntL50B (LR2) and from the complete enterocin Q (EntQ) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of these antibodies were evaluated by a noncompetitive indirect enzyme-linked immunosorbent assay (NCI-ELISA) and a competitive indirect ELISA (CI-ELISA). The NCI-ELISA was valuable for detecting anti-EntL50A-, anti-EntL50B-, and anti-EntQ-specific antibodies in the sera of the LR1-KLH-, LR2-KLH-, and EntQ-KLH-immunized animals, respectively. Moreover, these antibodies and those specific for enterocin P (EntP) obtained in a previous work (J. Gutiérrez, R. Criado, R. Citti, M. Martín, C. Herranz, M. F. Fernández, L. M. Cintas, and P. E. Hernández, J. Agric. Food Chem. 52:2247-2255, 2004) were used in an NCI-ELISA to detect and quantify the production of EntL50A, EntL50B, EntP, and EntQ by the multiple-bacteriocin producer E. faecium L50 grown at different temperatures (16 to 47°C). Our results show that temperature has a strong influence on bacteriocin production by this strain. EntL50A and EntL50B are synthesized at 16 to 32°C, but production becomes negligible when the growth temperature is above 37°C, whereas EntP and EntQ are synthesized at temperatures ranging from 16 to 47°C. Maximum EntL50A and EntL50B production was detected at 25°C, while EntP and EntQ are maximally produced at 37 and 47°C, respectively. The loss of plasmid pCIZ1 (50 kb) and/or pCIZ2 (7.4 kb), encoding EntL50A and EntL50B as well as EntQ, respectively, resulted in a significant increase in production and stability of the chromosomally encoded EntP.

Production and Characterization of Antibodies against Fumonisin B1

1996 ◽  
Vol 59 (9) ◽  
pp. 992-997 ◽  
Author(s):  
FENG-YIR YU ◽  
FUN S. CHU
Keyword(s):  
Detection Limit ◽  
Polyclonal Antibodies ◽  
Fumonisin B1 ◽  
Correlation Coefficients ◽  
Keyhole Limpet Hemocyanin ◽  
Hplc Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Indirect Competitive Elisa ◽  
Antibody Titers

Polyclonal antibodies against fumonisin B1 (FmB1) were produced in rabbits after immunizing the animals with either FmBl-keyhole limpet hemocyanin (KLH) or FmB1 bovine serum albumin (BSA). A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) and an indirect competitive ELISA (idc-ELISA) were used for the characterization of the antibodies and for analysis of the toxin in corn samples. The antibody titers in the serum of rabbits immunized with FmBl-KLH were considerably higher than in those immunized with FmBl-BSA. The antibodies from the rabbits immunized with FmBl-KLH were further characterized. The concentrations causing 50% inhibition of binding of FmB1-horseradish peroxidase (HRP) to the antibodies by FmB1, FmB2 and FmB3 in the ELISA were found to be 0.45, 0.72, and 25 ng/ml, respectively. The detection limit of FmBl, based on 95% confidence at 5% of inhibition of binding of FmBl-HRP conjugate, in buffer of the dc-ELISA was found to be 0.05 ng/ml. In the presence of a matrix such as corn, the detection limit was less than 50 ppb. The overall analytical recoveries of FmBl (50 to 1,000 ng/g) added to the ground corn and then extracted with CH3CN/H2O (1/1, vol/vol) with cleanup and without cleanup in the dc ELISA were found to be 70.5 and 85.9%, respectively. A good correlation was found between the FmBl levels in 2 starch and 10 naturally contaminated corn samples analyzed by the dc-ELISA and the high-pressure liquid chromatography (HPLC) method. The correlation coefficients between ELISA and HPLC were found to be 0.955 (y [ELISA] = 1.3 1x [HPLC] + 77 ppb; P < 0.001) and 0.811 (y = 1.13x + 34 ppb; P < 0.01) for the sample without and with cleanup treatment, respectively.

scholarly journals Generation and Utilization of Polyclonal Antibodies to a Synthetic C-Terminal Amino Acid Fragment of Divercin V41, a Class IIa Bacteriocin

2004 ◽  
Vol 70 (1) ◽  
pp. 248-254 ◽  
Author(s):  
Christelle Richard ◽  
Djamel Drider ◽  
Ismael Fliss ◽  
Sandra Denery ◽  
Herve Prevost
Keyword(s):  
Polyclonal Antibodies ◽  
Tween 80 ◽  
Keyhole Limpet Hemocyanin ◽  
Single Step ◽  
Enzyme Linked Immunosorbent Assay ◽  
Terminal Amino Acid ◽  
Chromatography Method ◽  
Acid Fragment ◽  
Terminal Amino ◽  
Enterocin P

ABSTRACT Polyclonal antibodies have been generated by immunization of rabbits with a chemically synthesized C-terminal part of divercin V41 (DvnCt) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and reactivity of the DvnCt-KLH-generated antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA) using supernatant from cultures of 13 representative lactic acid bacterium strains, and specificity was confirmed by Western blot analysis. Anti-DvnCt-KLH antibodies were able to recognize not only divercin V41 but also enterocin P and piscicocin V1b, two other members of the class IIa bacteriocins. Production and activity of DvnV41 were evaluated by ELISA and activity tests during the growth of Carnobacterium divergens V41 in MRS medium containing or not containing Tween 80. Divercin V41, enterocin P, and piscicocin V1b were therefore purified by a single-step immunoaffinity chromatography method using a Sepharose matrix CNBr-activated directed binding of anti-DvnCt-KLH polyclonal antibodies.

scholarly journals Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

1998 ◽  
Vol 64 (12) ◽  
pp. 5033-5038 ◽  
Author(s):  
N. de Vries ◽  
K. A. Zwaagstra ◽  
J. H. J. Huis in’t Veld ◽  
F. van Knapen ◽  
F. G. van Zijderveld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Minimal Area

ABSTRACT Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC(H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Cloning, Expression, and Immunological Characterization of the P30 Protein of Mycoplasma pneumoniae

2007 ◽  
Vol 15 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Avanish Kumar Varshney ◽  
Rama Chaudhry ◽  
Sushil Kumar Kabra ◽  
Pawan Malhotra
Keyword(s):  
Fusion Protein ◽  
Mycoplasma Pneumoniae ◽  
Transmembrane Protein ◽  
Amino Acid Sequences ◽  
Gliding Motility ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Asthmatic Patients ◽  
C Terminus

ABSTRACT Mycoplasma pneumoniae, a self-replicating cell wall-deficient prokaryote, has a differentiated terminal organelle that is essential for cytadherence and gliding motility. P30, an important protein associated with the terminal organelle, is required for the cytadherence and virulence of M. pneumoniae. P30 is a transmembrane protein with an intracytoplasmic N terminus and an exposed C terminus. In the present study, we amplified and sequenced the full-length p30 gene of Mycoplasma pneumoniae directly from 18 Indian asthmatic patients. Sequence diversity was observed in the p30 genes from 16 clinical samples when the sequences were compared with the sequence of strain M-129. We also successfully expressed a fragment of the p30 gene (P30B) that includes the complete C-terminal proline-rich amino acid sequences in different Escherichia coli expression systems. The maltose binding protein (MBP)-P30B fusion protein was recognized by M. pneumoniae-infected patient sera in immunoblots, and the protein was immunogenic in mice. We further analyzed the reactivity of the MBP-P30B fusion protein with patient sera in an enzyme-linked immunosorbent assay (ELISA) and compared it with the reactivity obtained with a commercial kit (the Serion ELISA Classic kit). The sensitivity and the specificity of the in-house ELISA were 78.57% and 89.47%, respectively. This study suggests that the P30 protein can be used as an antigen along with other adhesin proteins for the immunodiagnosis of M. pneumoniae infection.

The Expression and Characterization of Gp85 Gene of Subgroup J Avian Leukosis Virus Associated with Hemangioma

2011 ◽  
Vol 343-344 ◽  
pp. 545-550
Author(s):  
Min Shi ◽  
Yan Lin ◽  
Yang Zhao ◽  
Ming Ping Guo ◽  
Luo Mei Sun ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Polyclonal Antibodies ◽  
Serum Antibody ◽  
Avian Leukosis Virus ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Western Blot Assay ◽  
Gp85 Gene ◽  
High Level

The subtype of avian leukosis virus (ALV) was mainly determined by the gp85 glycoprotein. A subtype J ALV strain SCDY1 associated with hemangioma was isolated from grandparent breeding chicken and the highly antigenic region of its gp85 gene was amplified and expressed in Rosetta Escherichia coli using the pET-32a(+)vector. The fusion protein, which was expressed at a high level, was similar antigenically to the native gp85 protein as determined by Western blot assay using polyclonal antibodies against ALV-J strain. The fusion protein was also purified. This research lays a foundation for using this recombinant protein for development of indirect enzyme-linked immunosorbent assay (ELISA) for serum antibody detection or for production of monoclonal antibodies against prevalent ALV-J.

scholarly journals Purification and characterization of glucosidase II involved in N-linked glycoprotein processing in bovine mammary gland

1987 ◽  
Vol 247 (3) ◽  
pp. 563-570 ◽  
Author(s):  
S Saxena ◽  
K Shailubhai ◽  
B Dong-Yu ◽  
I K Vijay
Keyword(s):  
Mammary Gland ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bovine Mammary Gland ◽  
Ph Optimum ◽  
Glucosidase Ii ◽  
Reducing Conditions ◽  
Glycoprotein Processing

Glucosidase II is an endoplasmic-reticulum-localized enzyme that cleaves the two internally alpha-1,3-linked glucosyl residues of the oligosaccharide Glc alpha 1----2Glc alpha 1----3Glc alpha 1----3Man5-9GlcNAc2 during the biosynthesis of asparagine-linked glycoproteins. We have purified this enzyme to homogeneity from the lactating bovine mammary gland. The enzyme is a high-mannose-type asparagine-linked glycoprotein with a molecular mass of approx. 290 kDa. Upon SDS/polyacrylamide-gel electrophoresis under reducing conditions, the purified enzyme shows two subunits of 62 and 64 kDa, both of which are glycosylated. The pH optimum is between 6.6 and 7.0. Specific polyclonal antibodies raised against the bovine mammary enzyme also recognize a similar antigen in heart, liver and the mammary gland of bovine, guinea pig, rat and mouse. These antibodies were used to develop a sensitive enzyme-linked immunosorbent assay for glucosidase II.

scholarly journals Characterization of antibodies directed against the Ankrd2 human muscle protein

2009 ◽  
Vol 61 (4) ◽  
pp. 683-691 ◽  
Author(s):  
Snezana Kojic ◽  
Elisa Medeot ◽  
Georgine Faulkner
Keyword(s):  
Monoclonal Antibody ◽  
Epitope Mapping ◽  
Muscle Protein ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Human Muscle ◽  
Deletion Mutants ◽  
C Terminus ◽  
Commercial Antibodies

In order to study the function of the Ankrd2 protein, for which commercial antibodies are not available, we report the production and analysis of polyclonal antibodies to full-length Ankrd2 and its C-terminal and N-terminal regions, as well as a monoclonal antibody to the C-terminus of the protein. Epitope mapping making use of recombinant deletion mutants showed that an epitope located in region 323-333 aa of Ankrd2 is detected by the monoclonal antibody. The high specificity of all four anti-Ankrd2 antibodies for recombinant and endogenous Ankrd2 protein is also demon?strated.

scholarly journals Induction and Characterization of Multianalyte Antibodies Against Penicillins in Egg Yolk

1997 ◽  
Vol 80 (6) ◽  
pp. 1220-1228 ◽  
Author(s):  
Peter de Leuw ◽  
Georg Kapa ◽  
Michael Petz
Keyword(s):  
Time Course ◽  
Immune Reaction ◽  
Keyhole Limpet Hemocyanin ◽  
Egg Yolk ◽  
Competitive Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microtiter Plates ◽  
Polyethylene Glycol Precipitation ◽  
Different Characteristics

Abstract Antibodies against penicillins were induced in eggs of laying hens after immunization with 6-aminopeniciIlanic acid (6-APA) coupled to keyhole limpet hemocyanin (KLH). Development of the antibody titer was monitored by an indirect enzyme-linked immunosorbent assay (ELISA), with 6-APA coupled to ovalbumin as antigen for coating microtiter plates. Different characteristics (time course, affinity) of the immune reaction were observed by testing eggs of individual hens. Titer values varied between 150 and 2000. Antibodies were isolated by polyethylene glycol precipitation and affinity chromatography, using a hapten sorbent with 6-APA as ligand. Glycine buffer, pH 3.0, was used to elute the immunoglobulins. Antibody specificity was determined in a competitive ELISA with 7 penicillins and the cephalosporin cephalexin as competitors. Cross reactivities for the penicillins were between 100 and 290% (6-APA = 100%). Cephalexin cross reacted only marginally (3%).

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