Antigen identification and characterization of lung cancer specific monoclonal antibodies produced by mAb proteomics.
A mass spectrometric (MS)-based strategy for antigen (Ag) identification and characterization of globally produced monoclonal antibodies (mAbs) is described. Mice were immunized with a mixture of native glycoproteins, isolated from the pooled plasma of patients with nonsmall cell lung cancer (NSCLC), to generate a library of IgG-secreting hybridomas. Prior to immunization, the pooled NSCLC plasma was subjected to 3-sequential steps of affinity fractionation, including high abundant plasma protein depletion, glycoprotein enrichment, and polyclonal antibody affinity chromatography normalization. In this paper, to demonstrate the high quality of the globally produced mAbs, we selected 3 mAbs of high differentiating power against a matched, pooled normal plasma sample. After production of large quantities of the mAbs from ascites fluids, Ag identification was achieved by immunoaffinity purification, SDS-PAGE, Western blotting, and MS analysis of in-gel digest products. One antigen was found to be complement factor H, and the other two were mapped to different subunits of haptoglobin (Hpt). The 2 Hpt mAbs were characterized in detail to assess the quality of the mAbs produced by the global strategy. The affinity of one of the mAbs to the Hpt native tetramer form was found to have a K(D) of roughly 10(-9) M and to be 2 orders of magnitude lower than the reduced form, demonstrating the power of the mAb proteomics technology in generating mAbs to the natural form of the proteins in blood. The binding of this mAb to the beta-chain of haptoglobin was also dependent on glycosylation on this chain. The characterization of mAbs in this work reveals that the global mAb proteomics process can generate high-quality lung cancer specific mAbs capable of recognizing proteins in their native state.
Barnett Institute, Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusettes, USA.
Dongdong
D
Wang
Marina
M
Hincapie
Mariana
M
Guergova-Kuras
Janos
J
Kadas
Laszlo
L
Takacs
Barry L
BL
Karger
D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D000941:Antigens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000097:blood / Q000276:immunology; D006031:Glycosylation; D006242:Haptoglobins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000276:immunology*; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008875:Middle Aged; D040901:Proteomics / Q000379:methods*
D000911:Antibodies, Monoclonal; D000941:Antigens; D014408:Biomarkers, Tumor; D006023:Glycoproteins; D006242:Haptoglobins