Generation and Characterization of a Polyclonal Antipeptide Antibody to Human Glycodelin  Archna S Poddar, Jong G Kim, Kiran P Gill, Barry N Bates, Nalini Santanam, John A Rock, Ana A Murphy, Sampath Parthasarathy  Fertility and Sterility  Volume 69, Issue 3, Pages 543-548 (March 1998) DOI: 10.1016/S0015-0282(97)00558-X

Fig. 1 Western blot analysis of amniotic fluid for glycodelin. Different dilutions of amniotic fluid were run on 15% polyacrylamide gel electrophoresis. The gel was transferred to nitrocellulose membrane. Western blot analysis was performed (as described in Material and Methods) with the use of 1:1500 dilution of chicken antiglycodelin peptide antisera. The secondary antibody used was rabbit anti-chicken IgG conjugated with horseradish peroxidase (1:5,000). The proteins were detected with the ECL chemiluminescent detection system obtained from Amersham Life Sciences. Lanes 1, 2, and 3 refer to different dilutions of amniotic fluid (1:2, 1:4, and 1:8, respectively), and the arrow refers to the molecular weight of 45 kd. Top bands show scanning of polyacrylamide gel, in which the proteins were stained with Coumassie brilliant blue. Bottom bands show Western blot detection of glycodelin at 45 kd. Fertility and Sterility 1998 69, 543-548DOI: (10.1016/S0015-0282(97)00558-X)

Fig. 2 Immunocytochemistry on various cell lines for glycodelin. Glycodelin secreted by these cells is recognized by the antibody. Chamber slides were plated overnight with the various types of cells. After fixing and blocking, they were incubated with antiglycodelin antisera in 1:250 dilution. After wash, anti-chicken IgG conjugated with alkaline phosphatase was added in 1:500 dilution. After washing, fast red was added as chromogen in conjunction with alkaline phosphatase substrate Naphtol AS-TR phosphate. The immunostaining was observed under the microscope (original magnification, ×100). (A), Negative control of RL95-2 (human endometrial carcinoma cells), i.e., no primary antibody was added to the cells. (B), Experimental group of RL95-2, where the cells were incubated with chicken antiglycodelin antisera as the primary antibody. (C), Negative control of EM42-D (human endometrial epithelial cells). (D), Experimental group of EM42-D. (E), Negative control of HeLa (human cervical epitheloid carcinoma cells). (F), Experimental group of HeLa. (G), Negative control of OVCAR-3 (human ovarian adenocarcinoma cells). (H), Experimental group of OVCAR-3. Fertility and Sterility 1998 69, 543-548DOI: (10.1016/S0015-0282(97)00558-X)

Fig. 3 Recognition of glycodelin, secreted by the EM42-D cells in the medium. The medium alone, the culture medium with and without serum after a 48-hour culture (MS and M, respectively), and the culture medium with and without serum after an additional 4-hour culture (MS and M, respectively) were plated in a 96-well ELISA plate overnight. After that the plate was blocked, it was incubated with 1:500 dilution of antiglycodelin antisera. After washing, anti-chicken IgG conjugated with alkaline phosphatase in dilution of 1:35,000 was added to every well. After adding the substrate p-nitrophenyl phosphate, the optical density was measured at 405 nm with the use of a microplate reader. Values of culture medium minus medium were shown as the mean ± SD of three experiments. Fertility and Sterility 1998 69, 543-548DOI: (10.1016/S0015-0282(97)00558-X)