From a panel of 4 murine monoclonal antibodies directed against keratin 19 various antibody combinations were evaluated in solid-phase enzyme-linked sandwich immunoassays for detection of soluble keratin 19 fragments in patient sera. One of these antibody combinations, comprised of the monoclonal antibodies Ks 19.1 and BM 19.21, was selected for further development to a routine test (Enzymun-Test® CYFRA 21–1) because of its high diagnostic sensitivity and specificity for non-small cell lung carcinoma (NSCLC). Both antibodies are specific for keratin 19, no reactivity could be observed with cytokeratin 8 or 18. The epitopes of the two antibodies were determined to be within helix 2B of the rod romain. The epitope sequences lie within the sequence 311–335 for the catcher antibody Ks 19.1 and 346–367 for the detector antibody BM 19.21. These sequences are unique, as could be confirmed from sequence databases. The standard material for the assay was prepared from a cytoskeleton fraction of cultivated MCF-7 cells. Subsequent digestion of this fraction with chymotrypsin yielded a soluble and stable standard material. Both the standard material and the serum analyte appeared as oligomers when analysed on gel chromatography: the serum analyte appeared exclusively at a Mr of 100 ± 10 kD, whereas the standard material eluted in fractions corresponding to 100 ±10 kD and 450 kD. Due to the precise definition of the antigen and the localisation of the antibody binding sequences, Enzymun-Test® CYFRA 21–1 is one of the best characterised tumor markers so far.
The World Health Organisation: Histological typing of lung tumors.WHO, Geneva, Switzerland, 1982.
2.
CarneyN.D., De LeijL.Lung cancer biology.Sem Oncol1988; 15: 199–214.
3.
CarneyN.D., MarangosP.J., IhdeD.C.Serum neuron-specific enolase: a marker for disease extent and response to therapy of small cell lung cancer.Lancet1982; 1: 583–5.
4.
GouldV.E.Synaptophysin, a new and promising pan-endocrine marker.Arch Pathol Lab Med1987; 111: 791–3.
5.
O'ConnerD.T., DeftosL.J.Secretion of chromogranin A by peptide-producing endocrine neoplasms.-N Engl J Med1986; 314: 1145–51.
6.
BuchhagenD.L.Molecular mechanisms in lung cancer.Biochim Biophys Acta1991; 1072: 159–76.
7.
MollR., FrankeW.W., SchillerD.L., GeigerB., KreplerR.The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells.Cell1982; 31: 11–24.
8.
BroersJ.L.V., RamaekersF.C.S., RotM.K., OstendropT., HuysmansA., von MuijenG.N.P., WagenaarS.S., VoojesG.P.Cytokeratins in different types of lung cancer as monitored by chain-specific monoclonal antibodies.Cancer Res1988; 48: 3221–9.
9.
FuchsE., GraceM.P., KimK.H., MarchukD.Differential expression of two classes of keratins in normal and malignant epithelial cells and their evolutionary conservation. In: LevineA.J., Vande WoudeG.F., ToppW.C., WatsonJ.D., eds. Cancer cells, Vol. 1. The transformed phenotype.Cold Spring Harbour, N.Y: Cold Spring Harbour Laboratory, 1984; 161–8.
10.
HatzfeldM., MaierG., FrankeW.W.Cytokeratin domains involved in heterotypic complex formation determined by in-vitro binding assays.J Mol Biol1987; 197: 237–55.
11.
HatzfeldM., MaierG.W.W.Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides.J Cell Biol1985; 101: 1826–41.
12.
WeberK., GeislerN.Intermediate filaments - from wool α-keratin to neurofilaments: a structural overview. In: LevineA.J., Vande WoudeG.F., ToppW.D., WatsonJ.D., eds. Cancer cells, Vol. 1. The transformed phenotype.Cold Spring Harbour, NY: Cold Spring Harbour Laboratory, 1984; 153–9.
13.
SteinertP.M., StevenA.C., RoopD.R.The molecular biology of intermediate filaments.Cell1985; 42: 411–9.
14.
EbertW., LeichtweisB., SchapöhlerB., MuleyTh.The new tumor marker CYFRA is superior to SCC antigen and CEA in the primary diagnosis of lung cancer.Tumordiagn u Ther1993; 14: 91–9.
15.
PujolJ-L, GrenierJ., DauresJ-P, DaverA., PujolH., MichelF.B.Serum fragment of cytokeratin subunit 19 measured by CYFRA 21–1 immunoradiometric assay as a marker of lung cancer.Cancer Res1993; 53: 61–6.
16.
StieberP., HasholznerU., BodenmüllerH., NagelD., Sunder-PlassmannL., DienemannH., MeierW., Fateh-MoghadamA.CYFRA 21–1 - a new marker in lung cancer.Cancer1993; 72: 707–13.
17.
KarstenU., PapsdorfG., RoloffG., StolleyP., AbelH., WaltherI., WeissH.Monoclonal anti-cytokeratin antibody from a hybridoma clone generated by electrofusion.Eur J Cancer Clin Oncol1985; 21: 733–40.
18.
AchstätterT.Koexpressionsmuster von Intermediärfilament-Proteinen. Doctoral Thesis, University of Heidelberg, 1988.
19.
Woodstock-MitchellJ., EichnerR., NelsonW.G., SunnT.Immunolocalization of keratin polypeptides in human epidermis using monoclonal antibodies.J Cell Biol1982; 95: 580–8.
20.
OsbornM., von LesenG., WeberK., KlöppelK., AltmannsbergerM.Differential diagnosis of gastrointestinal carcinomas by using monoclonal antibodies specific for individual keratin polypeptides.Lab Invest1986; 55: 497–504.
21.
WilsonM.B., NakaneP.K.Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies. In: KnappW., HolubarK., WickG., eds. Immunofluorescence and related staining techniques.Elsevier/North Holland Biomedical Press, 1978.
22.
EckertR.L.Sequence of the human 40 kD keratin reveals an unusual structure with very high sequence homology to the corresponding bovine keratin.Proc Natl Acad Sci1988; 85: 114–8.
23.
LuX., LaneE.B.Retrovirus-mediated transgenic keratin expression in cultural fibroblast: Specific domain functions in keratin stabilisation and filament formation.Cell1990; 62: 681–96.
24.
StasiakP.C., LaneE.B.Sequence of cDNA coding for human keratin 19.Nucl Acids Res1987; 15: 10058.
25.
EyP.L., ProwseS.J., JenkinC.R.Isolation of pure IgG1, IgG2a and IgG2b immunoglobulin from mouse serum using protein A-sepharose.Immunochemistry1978; 15: 429–36.
26.
PorterR.R.The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain.Biochem1959; 73: 119–26.
27.
BayerE.A., WichekM., SkutelskyE.Affinity cytochemistry: The localisation of lectin and antibody receptors via the avidin-biotin complex.FEBS Lett1976; 68: 240–4.
28.
BodenmüllerH., BanauchD., OfenlochB., JaworekD., DessauerA.Technical evaluation of a new automated tumor marker assay: the Enzymun-Test® CYFRA 21–1. In: KlapdorR., ed. Tumor associated antigens, oncogenes, receptors, cytokines in tumor diagnosis and therapy at the beginning of the 90s.Zuckerschwerdt Verlag, 1992; 137–8.
29.
SouleH.D., VazquezJ., LongA., AlbertS., BrennanM.A human cell line from a pleural effusion derived from a breast carcinoma.J Natl Cancer Inst1973; 51: 1409–16.
30.
AchstätterT., HatzfeldM., QuinlanR.A., ParmeleeD.C., FrankeW.W.Separation of cytokeratin polypeptides by gel electrophoretic and chromatographic techniques and their identification by immunoblotting.Meth Enzymol1986; 134: 355–71.
31.
MellerickD.M., OsbornM., WeberK.On the nature of serological TPA; monoclonal keratin 8, 18 and 19 antibodies react differently with TPA prepared from human cultured cells and TPA in serum.Oncogene1990; 5: 1007–17.
32.
ConradK., KarstenU., FrankK-H, PasternakG.Immunoblotting of monoclonal anticytoskeletal antibodies with cytoskeletal extracts of HeLa and MCF-7 cells.Biomed Biochim Acta1988; 47: 697–703.
33.
HuangC-C, BlitzerA., AbramsonM.Collagenase in human head and neck tumors and rat tumors and fibroblasts in monolayer cultures.Ann Otol Rhinol Laryngol1986; 95: 158–61.
34.
BloodH.C., ZetterB.Tumor interactions with the vasculature: angiogenesis and tumor metastasis.Biochim Biophys Acta1990; 1032: 89–118.
