New monoclonal antibodies to mesothelin useful for immunohistochemistry, fluorescence-activated cell sorting, Western blotting, and ELISA.
OBJECTIVE
Mesothelin is a cell surface protein that is highly expressed in some malignant tumors, and is a promising target for immunotherapy. Recent data suggests that mesothelin is an adhesive protein and may have a role in the metastases of ovarian cancer. Although a few monoclonal antibodies (MAb) to mesothelin have been produced, they have limitations for the study of expression of native mesothelin because of their low affinity or reactivity only with denatured mesothelin protein. We have produced novel MAbs to mesothelin to help study mesothelin function and to develop improved diagnosis and immunotherapy of mesothelin-expressing tumors.


METHODS
Mesothelin-deficient mice were immunized with plasmid cDNA encoding mesothelin, and boosted with a mesothelin-rabbit IgG Fc fusion protein prior to cell fusion. Hybridomas were screened by an ELISA using plates coated with mesothelin-Fc protein.


RESULTS
Seventeen hybridomas producing anti-mesothelin antibodies were established and shown to react with two epitopes on mesothelin. One group reacts with the same epitope as the low affinity antibody K1 that was originally used to identify mesothelin. The other is a new group that reacts with a new epitope. One antibody from each group was chosen for further study and shown to react strongly on ELISA, on immunohistochemistry, and by fluorescence-activated cell sorting on living cells.


CONCLUSIONS
Our two newly established MAbs, MN and MB, have different and useful properties compared with current antibodies used for the detection of mesothelin by immunohistochemistry, fluorescence-activated cell sorting, ELISA, and Western blotting.
Onda
Masanori
M
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892-4264, USA.
Willingham
Mark
M
Nagata
Satoshi
S
Bera
Tapan K
TK
Beers
Richard
R
Ho
Mitchell
M
Hassan
Raffit
R
Kreitman
Robert J
RJ
Pastan
Ira
I
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D058851:GPI-Linked Proteins; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology; D007150:Immunohistochemistry / Q000379:methods; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D000090204:Mesothelin; D051379:Mice; D018345:Mice, Knockout; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D046888:Tissue Array Analysis
D000911:Antibodies, Monoclonal; D058851:GPI-Linked Proteins; D007141:Immunoglobulin Fc Fragments; D008562:Membrane Glycoproteins; C000715528:Msln protein, mouse; D011993:Recombinant Fusion Proteins; D000090204:Mesothelin