Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.

2.50
Hdl Handle:
http://hdl.handle.net/2436/30219
Title:
Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.
Authors:
Waldron, E.E.; Murray, Paul G.; Kolar, Zdenek; Young, Lawrence S.; Brown, C.; Reynolds, Gary; Baumforth, Karl R. N.; Toomey, S.; Astley, S.J.; Perera, Shantha; Nelson, Paul N.
Abstract:
The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.
Citation:
Hybridoma and Hybridomics, 21(5): 393-398
Publisher:
New Rochelle (NY): Mary Ann Liebert, Inc.
Journal:
Hybridoma and Hybridomics
Issue Date:
2002
URI:
http://hdl.handle.net/2436/30219
DOI:
10.1089/153685902761022751
PubMed ID:
12470483
Additional Links:
http://www.liebertonline.com/doi/abs/10.1089%2F153685902761022751
Type:
Article
Language:
en
ISSN:
1536-8599
Appears in Collections:
Molecular Immunology Research Group

Full metadata record

DC FieldValue Language
dc.contributor.authorWaldron, E.E.-
dc.contributor.authorMurray, Paul G.-
dc.contributor.authorKolar, Zdenek-
dc.contributor.authorYoung, Lawrence S.-
dc.contributor.authorBrown, C.-
dc.contributor.authorReynolds, Gary-
dc.contributor.authorBaumforth, Karl R. N.-
dc.contributor.authorToomey, S.-
dc.contributor.authorAstley, S.J.-
dc.contributor.authorPerera, Shantha-
dc.contributor.authorNelson, Paul N.-
dc.date.accessioned2008-06-19T16:14:34Z-
dc.date.available2008-06-19T16:14:34Z-
dc.date.issued2002-
dc.identifier.citationHybridoma and Hybridomics, 21(5): 393-398en
dc.identifier.issn1536-8599-
dc.identifier.pmid12470483-
dc.identifier.doi10.1089/153685902761022751-
dc.identifier.urihttp://hdl.handle.net/2436/30219-
dc.description.abstractThe characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.en
dc.language.isoenen
dc.publisherNew Rochelle (NY): Mary Ann Liebert, Inc.en
dc.relation.urlhttp://www.liebertonline.com/doi/abs/10.1089%2F153685902761022751en
dc.subjectMonoclonal antibodiesen
dc.subjectPeptidesen
dc.subjectMultiple Antigenic Peptidesen
dc.subject.meshAnimalsen
dc.subject.meshAntibodies, Monoclonalen
dc.subject.meshBlotting, Westernen
dc.subject.meshEnzyme-Linked Immunosorbent Assayen
dc.subject.meshHybridomasen
dc.subject.meshImmunoglobulin Isotypesen
dc.subject.meshMiceen
dc.subject.meshMice, Inbred BALB Cen
dc.subject.meshPeptidesen
dc.subject.meshSpleenen
dc.subject.meshViral Matrix Proteinsen
dc.titleReactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.en
dc.typeArticleen
dc.identifier.journalHybridoma and Hybridomicsen
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