Abstract
The 6B3‐Ag recognized by a monoclonal antibody 6B3 to human large cell lung carcinoma cell line (HLC‐2) is a high‐molecular‐weight glycoprotein of 1,000,000. Its serum level is increased in various adenocarcinoma patients. When a patient's serum with a high concentration of 6B3′Ag (54μg/ml) or concentrated 6B3‐Ag from normal human serum was analyzed by immunoelectrophoresis, 6B3‐Ag showed a long bimodal precipitin line extending from the per‐β to β globulin region. However, the precipitin line of 6B3‐Ag in the HLC‐2 culture medium was formed only in the pre‐β globulin region. The 6B3‐Ag was purified from pooled patients serum by salting out, precipitation by acidification at pH 4.5 and Sepharose 4B and immunoaffinity chromatographies. Western blotting indicated that the 6B3‐Ag from human serum contained IgG and/or IgM. The 6B3‐Ag from human serum showed a dose‐dependent reaction in a sandwich enzyme‐linked immunosorbent assay with anti‐6B3‐Ag antibody as a solid‐phase antibody and anti‐human IgG or anti‐human IgM antibody labeled with alkaline phosphatase. The 6B3‐Ag was concluded to be partly present as a complex with IgG and/or IgM in humanβ serum, and this complex showed a precipitin line in the /3 globulin region on immunoelectrophoresis.
Keywords: Glycoprotein, Lung cancer cell line, Human serum, Immunoglobulin‐binding
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REFERENCES
- 1. ) Nonaka , N. , Manaka , K. , Kobayashi , K. and Hirai , H.Detailed characterization of a high‐molecular‐weight glycoprotein secreted by lung cancer cells . Jpn. J. Cancer Res. , 84 , 982 – 988 ( 1993. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. ) Linsley , P. S. , Horn , D. , Marquardt , H. , Brown , J. P. , Hellström , I. , Hellström , K. E. , Ochs , V. and Tolentino , E.Identification of a novel serum protein secreted by lung carcinoma cells . Biochemistry , 25 , 2978 – 2986 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 3. ) Köhler , G. and Milstein , C.Continuous cultures of fused cells secreting antibody of predefined specificity . Nature , 256 , 495 – 497 ( 1975. ). [DOI] [PubMed] [Google Scholar]
- 4. ) Quaranta , V. , Walker , L. E. , Pellegrino , M. A. and Ferrone , S.Purification of immunologically functional subsets of human la‐like antigens on a monoclonal antibody (Q5/13) immunoadsorbent . J. Immunol. , 125 , 1421 – 1428 ( 1980. ). [PubMed] [Google Scholar]
- 5. ) Ouchterlony , O.Antigen‐antibody reaction in gels . Ark. Kemi. Mineral. Geol , 26B , 14 ( 1948. ). [Google Scholar]
- 6. ) Ruoslahti , E. , Hayman , E. G. , Pierchbacher , M. and Engvall , E.Fibronectin: purification, immunochemical properties, and biological activities . Methods Enzymol. , 82 , 803 – 831 ( 1982. ). [DOI] [PubMed] [Google Scholar]
- 7. ) Laemmli , U. K.Cleavage of structural proteins during the assembly of the head bacteriophage T4 . Nature , 227 , 680 – 685 ( 1970. ). [DOI] [PubMed] [Google Scholar]
- 8. ) Towbin , H. , Staehelin , T. and Gordon , T.Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets . Proc. Natl. Acad. Set. USA , 76 , 435 – 4354 ( 1979. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. ) Furukawa , K.Biological significance of IgG sugar chains . Igaku no Ayumi , 154 , 467 – 471 ( 1990. )( in Japanese ). [Google Scholar]
- 10. ) Ohori , K. , Fukamizu , H. , Matsushita , K. , Horiuchi , K. and Fujimoto , D.A cell‐binding, immunoglobulin‐like protein from human plasma. I. Isolation and subunit structure . J. Biochem. , 100 , 837 – 842 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 11. ) Fukamizu , H. , Ohori , K. , Naito , Y. , Horiuchi , K. and Fujimoto , D.A cell‐binding, immunoglobulin‐like protein from human plasma. II. Cell‐binding activity. . J.Biochem. , 100 , 843 – 848 ( 1986. ). [DOI] [PubMed] [Google Scholar]
- 12. ) Natali , P. G. , Wilson , B. S. , Imai , K. , Bigotti , A. and Ferrone , S.Tissue distribution, molecular profile, and shedding of a cytoplasmic antigen identified by the monoclonal antibody 465.12S to human melanoma cells . Cancer Res. , 42 , 583 – 589 ( 1982. ). [PubMed] [Google Scholar]