Production and Characterization of Polyclonal Antibodies in Rabbits to 4S-Limonene Synthase from Spearmint (Mentha spicata)

Background. Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. Materials and methods. Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. Results. On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. Conclusions. In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

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Characterization of the hydroxyproline-rich protein core of an arabinogalactan-protein secreted from suspension-cultured Lolium multiflorum (Italian ryegrass) endosperm cells

Biochemical Journal ◽

10.1042/bj2640857 ◽

1989 ◽

Vol 264 (3) ◽

pp. 857-862 ◽

Cited By ~ 30

Author(s):

P A Gleeson ◽

M McNamara ◽

R E H Wettenhall ◽

B A Stone ◽

G B Fincher

Keyword(s):

Polyclonal Antibodies ◽

Lolium Multiflorum ◽

Italian Ryegrass ◽

Arabinogalactan Protein ◽

Myeloma Protein ◽

Protein Core ◽

Liquid Suspension ◽

Peptide Component ◽

Immunodominant Epitopes

An arabinogalactan-protein (AGP) purified from the filtrate of liquid-suspension-cultured Italian-ryegrass (Lolium multiflorum) endosperm cells by affinity chromatography on myeloma protein J539-Sepharose was deglycosylated with trifluoromethanesulphonic acid to remove polysaccharide chains that are covalently associated with hydroxyproline residues in the peptide component of the proteoglycan. The protein core, which accounts for less than 10% (w/w) of the intact proteoglycan, was purified by h.p.l.c. It has an apparent Mr of 35,000, but reacts very poorly with both Coomassie Brilliant Blue R and silver stains. Amino-acid-sequence analysis of the N-terminus of the h.p.l.c.-purified protein core and of tryptic peptides generated from the unpurified protein reveals a high content of hydroxyproline and alanine. These are sometimes arranged in short (Ala-Hyp) repeat sequences of up to six residues. Polyclonal antibodies raised against the protein core do not cross-react with native AGP, the synthetic peptide (Ala-Hyp)4, poly-L-hydroxyproline or poly-L-proline. The results suggest that the polysaccharide chains in the native AGP render the protein core of the proteoglycan inaccessible to the antibodies and that the immunodominant epitopes include domains of the protein other than those rich in Ala-Hyp repeating units.

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Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽

10.1017/s0031182012000285 ◽

2012 ◽

Vol 139 (8) ◽

pp. 998-1004 ◽

Cited By ~ 17

Author(s):

X. CUI ◽

T. LEI ◽

D. Y. YANG ◽

P. HAO ◽

Q. LIU

Keyword(s):

Neospora Caninum ◽

Polyclonal Antibodies ◽

Functional Protein ◽

Reading Frame ◽

Protein Motifs ◽

Apicomplexan Parasites ◽

Potential Vaccine ◽

Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

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Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes

Biochemical Journal ◽

10.1042/bj2870443 ◽

1992 ◽

Vol 287 (2) ◽

pp. 443-446 ◽

Cited By ~ 13

Author(s):

O A Coso ◽

A Díaz Añel ◽

H Martinetto ◽

J P Muschietti ◽

M Kazanietz ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Pertussis Toxin ◽

Polyclonal Antibodies ◽

Gel Filtration ◽

Binding Activity ◽

Gi Protein ◽

Liver Membranes ◽

Blocking Capacity ◽

Polypeptide Band

A guanosine 5′-[gamma-[35S]thio]triphosphate-binding activity was detergent-extracted from Trypanosoma cruzi membranes. This binding activity was co-eluted from gel-filtration columns with a factor which, in a heterologous reconstitution system, blocks glucagon stimulation of adenylate cyclase activity in liver membranes. ADP-ribosylation of these membranes by pertussis toxin eliminated this blocking capacity. Incubation of T. cruzi membranes with activated pertussis toxin and [adenylate-32P]NAD+ led to the incorporation of radioactivity into a labelled product with an apparent M(r) of approx. 43,000. Crude membranes were electrophoresed on SDS/polyacrylamide gels and analysed, by Western blotting, with GA/1 anti-alpha common, AS/7 anti-alpha t, anti-alpha i1 and anti-alpha i2 polyclonal antibodies. These procedures led to the identification of a specific polypeptide band of about 43 kDa. Another polypeptide reacting with the SW/1 anti-beta antibody, of about 30 kDa, was also detected in the membrane fraction.

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Fractionation and Characterization of Polyclonal Antibodies Using Three Progressively More Chaotropic Solvents

Analytical Biochemistry ◽

10.1006/abio.1997.2376 ◽

1997 ◽

Vol 253 (2) ◽

pp. 246-252 ◽

Cited By ~ 23

Author(s):

Linda O. Narhi ◽

D.J. Caughey ◽

Thomas P. Horan ◽

Yoshiko Kita ◽

David Chang ◽

...

Keyword(s):

Polyclonal Antibodies

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Production and characterization of polyclonal antibodies recognizing the intracytoplasmic third loop of the 5-hydroxytryptamine1A receptor

Neuroscience ◽

10.1016/0306-4522(94)90472-3 ◽

1994 ◽

Vol 62 (3) ◽

pp. 721-739 ◽

Cited By ~ 30

Author(s):

C. Gérard ◽

X. Langlois ◽

J. Gingrich ◽

E. Doucet ◽

D. Vergé ◽

...

Keyword(s):

Polyclonal Antibodies

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Production and Characterization of Three Polyclonal Antibodies Raised Against Cyst Wall Proteins of a Hypotrichous Ciliate

The Journal of Protozoology ◽

10.1111/j.1550-7408.1992.tb04854.x ◽

1992 ◽

Vol 39 (5) ◽

pp. 584-588 ◽

Cited By ~ 1

Author(s):

ROSA M. RIOS ◽

JESUS MARTIN ◽

ANTONIO TORRES ◽

CONCEPCION FEDRIANI

Keyword(s):

Cyst Wall ◽

Polyclonal Antibodies

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Synthesis and Characterization of Mercapturic Acid (N-acetyl-L-cysteine)-Aflatoxin B1 Adduct and Its Quantitation in Rat Urine by an Enzyme Immunoassay

Journal of AOAC International ◽

10.1093/jaoac/92.2.487 ◽

2009 ◽

Vol 92 (2) ◽

pp. 487-495 ◽

Cited By ~ 1

Author(s):

Sujatha Nayak ◽

Penmatsa Tanuja ◽

Rao Beedu Sashidhar

Keyword(s):

Enzyme Immunoassay ◽

Aflatoxin B1 ◽

Polyclonal Antibodies ◽

Mercapturic Acid ◽

Rat Urine ◽

In Vitro Synthesis ◽

Layer Chromatography ◽

Serum Albumen

Abstract A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg100 ng of the analyte (y ab x). A 50 displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50)of 11.9 ng GSH-AFB1 (r2 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 0.98). Spiking 5 g/mL of reference standard to the control rat urine showed a recovery of 98 2. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.225.97 g/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

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