Abstract
Introduction: The quality of immunodiagnostic and ?therapeutic methods greatly depends on the specificity and sensitivity of the antibodies employed. The objective was to develop new monoclonal antibodies against proteins from colorectal carcinoma cell membranes. The respective antigens should be localized in the tissue, chromatographically accumulated and biochemically analysed.
Results: After immunization of mice with membrane-proteins from human colorectal carcinoma, antibody producing hybridoma cell clones were established. After affinity assays were performed with ELISA, two cell clones with the most specific detection of carcinoma tissue compared with normal tissue were selected and further characterized.
MAK 03.1.1 detected an antigen with a relative molecular weight of 143kD. The antigen was accumulated after multiple chromatographic steps. Tryptic antigenic peptides were fractioned via RP18-HPLC and analyzed with mass-spectrometry and amino-acid sequencing. The resulting sequence corresponded with the alpha-chain of collagen I and explained the staining of interstitial tissue by the antibody in histological tissue slices. Further investigation proved the detection of collagen I with slight cross-reactivity towards collagen III.
MAK 05.1.14 showed an inhomogenic pattern between 150 and 200kD in immunoblot detection. The antigen could be isolated by immuno-affinity chromatography. After sequence analysis, it could be identified as a member of the CEA-family. Correspondingly, immunhistochemistry showed intense staining of colorectal carcinoma cells. After deglycosylation the antigen was still detectable in the immunoblot, desialylation even led to stronger signals.
Discussion: With the strategy described above, tumorspecific antibodies can be established. Furthermore it is possible to investigate correlations between expression and localisation of the antigen as well as tumor stage and prognosis. Collagen I and III as parts of the extracellular matrix play an important role in tumorbiology. Biochemical alterations of chains and fibrils as well as pathologic distribution patterns allow conclusions about tumor organisation and malignancy.
The establishment of MAK 05.1.14 verifies the importance of CEA as an antigen expressed during neoplastic development. |
