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Development of monoclonal and polyclonal antibodies and an ELISA for the determination of glycodelin in human serum, amniotic fluid and cystic fluid of benign and malignant ovarian tumors

Item Type:Article
Title:Development of monoclonal and polyclonal antibodies and an ELISA for the determination of glycodelin in human serum, amniotic fluid and cystic fluid of benign and malignant ovarian tumors
Creators Name:Jeschke, U. and Bischof, A. and Speer, R. and Briese, V. and Richter, D.U. and Bergemann, C. and Mylonas, I. and Shabani, N. and Friese, K. and Karsten, U.
Abstract:Objective: The role of glycodelin in human reproduction and gynecological malignancies has been investigated in a large number of studies in recent years. Three dominant functions of this glycoprotein were identified. Glycodelin is immunosuppressive, a morphological marker of differentiation and a contraceptive. Glycodelin is a glycoprotein with a molecular weight of 28 kDa and a carbohydrate content of 17.5%. Unusual LacdiNAc structures were identified on glycodelin A, isolated from amniotic fluid. Because no kit for glycodelin quantification is commercially available, we developed all reagents and a functional ELISA. Materials and Methods: Glycodelin A was purified from amniotic fluid by chromatographic methods. The purity of the isolated protein was checked with SDS-PAGE. Polyclonal antibodies against glycodelin were generated in rabbits. Monoclonal antibodies against glycodelin were generated from immunized BALB/c mice. Positive hybridomas were cloned and cultured. Monoclonal antibodies were isolated by immunoadsorption chromatography from culture supernatants. The glycodelin ELISA was developed in two formats, namely coating with polydonal antibodies and the use of monoclonal antibodies. Results: The factors of variance for the ELISA were 7% (intra-assay variance) and 15% (inter-assay variance). The glycodelin ELISA was used to determine the glycodelin A concentration in sera of fertile women during the proliferative and secretory phases of the endometrium. The glycodelin A concentration was insignificantly elevated in the secretory phase compared to the proliferative phase. Significantly higher levels of glycodelin A were found in women using oral contraceptives compared to women who were not (p<0.001). This is probably due to progesterone, which stimulates glycodelin production. We also found significantly increased glycodelin concentrations in the fluids of malignant ovarian cysts compared to benign ovarian tumors (p<0.001). Furthermore, we tested the monoclonal and polydonal antibodies successfully in Western blot analysis and immunoadsorption chromatography. Conclusion: We consider the described ELISA for the quantification of glycodelin as a useful tool for the determination of glycodelin in amniotic fluid, serum and cystic fluids. Its most promising application is expected in the diagnosis of ovarian cancer. The antibodies generated are applicable to multiple techniques.
Keywords:ELISA, Glycodelin, Monoclonal antibodies, Ovarian cancer
Source:Anticancer Research
ISSN:0250-7005
Publisher:Delinassios (Greece)
Volume:25
Number:3
Page Range:1581-1589
Date:1 January 2005
PubMed:View item in PubMed

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