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Open Access Highly Accessed Open Badges Methodology article

Rapid production of antigen-specific monoclonal antibodies from a variety of animals

Nobuyuki Kurosawa1, Megumi Yoshioka2, Rika Fujimoto3, Fuminori Yamagishi4 and Masaharu Isobe1*

Author Affiliations

1 Laboratory of Molecular and Cellular Biology, Faculty of Science and Engineering, Graduate School, University of Toyama, 3190 Gofuku, Toyama-shi, Toyama, 930-8555, Japan

2 Graduate School of Innovative Life Science, University of Toyama, Toyama-shi, Toyama, 930-8555, Japan

3 Department of Immunology, Kochi Medical School, Nangoku-shi, Kochi, 783-8505, Japan

4 Department of Surgery, Itoigawa General Hospital, Itoigawa-shi, Niigata, 941-0006, Japan

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BMC Biology 2012, 10:80  doi:10.1186/1741-7007-10-80

Published: 28 September 2012

Additional files

Additional file 1:

Klotz plots. Klotz plots of the binding of human insulin to guinea pig monoclonal antibodies (mAbs), as measured using ELISA. a0, the concentration of total antigen; A0, the chemiluminescence measured for the antibody in the absence of antigen; Ai, the chemiluminescence measured for bound antibody. The value in the parentheses represents the average of the different guinea pig mAb concentrations (0.5 nM and 1.0 nM).

Format: TIFF Size: 62KB Download file

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Additional file 2:

Phylogenetic analysis of VH and Vamino acid sequences of the highly binding guinea pig monoclonal antibodies (mAbs). (A) Guinea pig mAbs of the same lineage group are boxed and labeled as 1, 2 or 3. (B) Sequences of the corresponding VH and Vamino acid regions of the highly binding guinea pig mAbs.

Format: TIFF Size: 133KB Download file

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Additional file 3:

Epitope mapping of the highly binding guinea pig monoclonal antibodies (mAbs). (A) Western blots of epitope-glutathione S-transferase (GST) fusion proteins with guinea pig mAbs. About 0.1 μg of proteins were loaded on 15% SDS-PAGE. Lane 1: GST; lane 2: GST-insulin A; lane 3: GST-insulin B; lane 4: GST-insulin B1-20; lane 5: GST-insulin B1-13. (B) Epitope mapping by competitive enzyme-linked immunosorbent assay (ELISA). Excess amount of epitope-GST fusion proteins (10-fold molar excess relative to mAb) or overlapping peptides of human insulin (25-fold molar excess) were used as competitors. Binding of the antibodies to wild-type human insulin without competitors was set as 100%. Each experiment was repeated independently twice, and the mean values are shown.

Format: TIFF Size: 448KB Download file

Open Data