Antigen-Specific Cell Adherence Assay: A New Method for Separation of Antigen-Specific Hybridoma Cells

Najbauer, Joseph; Tigyi, Gábor; Nemeth, Peter

doi:10.1089/hyb.1986.5.361

Cited by 12 publications

(4 citation statements)

References 8 publications

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“…Recombinant human trypsin 4, and the synthetic 28‐amino acid leader peptide, were used to immunize female BALB/c mice (Charles River Laboratories, Raleigh, NC, USA). Antigen‐specific B lymphocytes, prepared from the spleens of high‐responder animals, were preselected using a method developed in our laboratory and published previously [29]. The fusion partner was the Sp‐2/0 Ag14 (ATCC, Manassas, VA, USA) mouse myeloma cell line, and the hybridoma cells were prepared and cloned as described previously [30].…”

Section: Methodsmentioning

confidence: 99%

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N‐terminal leucine

et al. 2007

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Recent genome programmes have explored an increasing number of new genes with unknown function. The estimated 35 000 human genes encode more than 10 5 expressed proteins as the result of various mechanisms, such as alternative promotion of transcription, alternative splicing of the transcripts and alternative translational initiation. Chromosome rearrangement can also serve as a source for evolutionary heterogeneity. SummaryChromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.Abbreviations GFP, green fluorescent protein; PRSS3, protease serine 3.

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Section: Methodsmentioning

confidence: 99%

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N‐terminal leucine

et al. 2007

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“…23 The fusion partner was the Sp-2/0 Ag14 (ATCC, USA) mouse myeloma cell line and the hybridoma cells were prepared and cloned as usual. 23…”

Section: Immunisation and Hybridoma Developmentmentioning

confidence: 99%

Development and characterisation of a monoclonal antibody family against aquaporin 1 (AQP1) and aquaporin 4 (AQP4)

Nagy

Szekeres²,

Kvell

et al. 2002

Pathol. Oncol. Res.

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Recent studies have discovered the existence of water-channel molecules, the so called aquaporins (AQP) presumably involved in active, ATP dependent water transport between the intracellular and extracellular compartments. Both genetic and protein sequences and structures of the AQPs are known and crystallographic analyses of some members of the AQP family have been performed. Specific antibodies are required to examine their histological locations and analyse their roles in physiological and pathological pathways of water transportation and osmotic regulation. Until recently some polyclonal antibodies have been developed against certain members of the AQP family. However, to date highly specific monoclonal antibodies against aquaporins do not exist. We have developed a monoclonal antibody family against the aquaporin 1 (AQP1) and aquaporin 4 (AQP4) molecules. Well-conserved epitop sequences of AQP1 and AQP4 proteins were selected by computer analysis and their synthetic peptide fragments were used as the antigens of immunisation and the following screening. Antibodies were characterised by immunoserological methods (ELISA, dot-blot and immunoblot), flow cytometry and immunohistochemistry of formaldehyde-fixed and paraffin-embedded tissue samples. RT-PCR tests controlled the specificity of the immune reactions. Our monoclonal antibodies recognised AQP1 and AQP4 in all the techniques mentioned above and apparently they are useful both in various quantitative and qualitative measurements of the expressions of AQP1 and AQP4 in several species (human, rat, mouse, invertebrates, even plants). According to preliminary immunohistochemical studies our monoclonal anti-AQP1 and anti-AQP4 antibodies are appropriate tools of patho-morphological examinations on routine formol-paraffin tissue samples.

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“…Mouse hybridoma cell lines producing ovalbuminspecific IgE (a-Ova) [9] and wheat-germ-agglutinin-antigen-specific IgG1 (a-WGA) [26,27] were used for in vitro studies. The a-PNAr-I (IgG1) monoclonal antibody was produced and selected in the usual way as described [21,30] against peanut-agglutinin-antigen-binding receptor (containing galactose-~3-galactosamine disaccharide) isolated from human gastric mucosal cells by lectin-affiulty chromatography [28].…”

Section: Hybridoma Cell Linesmentioning

confidence: 99%

Photo-immunotargeting with haematoporphyrin conjugates activated by a low-power He-Ne laser

Berki

Németh

1992

Cancer Immunol Immunother

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A combined method has been developed for selective cytolysis in vitro as well as in vivo using a photosensitizer haematoporphyrin-protein conjugate as the targeting molecule and low-power He-Ne laser (632.8 nm) irradiation in order to activate the sensitizer to its excited, toxic triplet energy state. The specificity of the procedure was demonstrated in vitro by purging a mixed cell population from one component, and in vivo in an animal (nude mice) xenograft tumour model, where human cancer cells were destroyed by the immunotargeting method using monoclonal-antibody-haematoporphyrin (mAb-HP) conjugate (a-PNAr-I mAbs, which bind to the cell surface antigens of gastric cancer cells) and soft laser irradiation. The cell destruction was dependent on the doses of both mAb-HP and He-Ne laser light energy, and occurred only in target cell populations.

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Antigen-Specific Cell Adherence Assay: A New Method for Separation of Antigen-Specific Hybridoma Cells

Cited by 12 publications

References 8 publications

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N‐terminal leucine

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N‐terminal leucine

Development and characterisation of a monoclonal antibody family against aquaporin 1 (AQP1) and aquaporin 4 (AQP4)

Photo-immunotargeting with haematoporphyrin conjugates activated by a low-power He-Ne laser

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