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.
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.
  
  
  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 polyclonal antibodies and the use of monoclonal antibodies.
  
  
  RESULTS
  The factors of variance for the ELISA were 7% (intraassay 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 polyclonal antibodies successfully in Western blot analysis and immunoadsorption chromatography.
  
  
  CONCLUSIONS
  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.
authors: Department of Obstetrics and Gynecology, Ludwig-Maximilians-University of Munich, Maistrasse 11, 80337 Munich, Germany. udo.jeschke@med.uni-muenchen.de
authors: Udo
authors: U
authors: Jeschke
authors: Anja
authors: A
authors: Bischof
authors: Runa
authors: R
authors: Speer
authors: Volker
authors: V
authors: Briese
authors: Dagmar-Ulrike
authors: DU
authors: Richter
authors: Claudia
authors: C
authors: Bergemann
authors: Ioannis
authors: I
authors: Mylonas
authors: Naim
authors: N
authors: Shabani
authors: Klaus
authors: K
authors: Friese
authors: Uwe
authors: U
authors: Karsten
mesh_terms: D000906:Antibodies / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D000071816:Glycodelin; D006023:Glycoproteins / Q000032:analysis* / Q000097:blood / Q000276:immunology; D006801:Humans; D010051:Ovarian Neoplasms / Q000097:blood / Q000737:chemistry*; D011257:Pregnancy Proteins / Q000032:analysis* / Q000097:blood / Q000276:immunology
chemical_list: D000906:Antibodies; D000071816:Glycodelin; D006023:Glycoproteins; C045246:PAEP protein, human; D011257:Pregnancy Proteins