Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures.
In this study we describe a large-scale screening cell ELISA protocol which is suitable for the characterization of exogenic factor effects in mixed central nervous system (CNS) culture. The main novelty of the assay is that it permits the measurement of cellular responses in populations comprising as little as 2-4% of the total cell number. For standardization of the assay, we employed antibodies against opsin and microtubule-associated protein (MAP2) which label distinct retinal cell classes. Embryonic chick retinal neurons were grown in microtiter plates and directly processed for detection of antibody binding on the same plate. Binding of the antibodies was saturable and the ELISA signal was proportional to the number of immunoreactive cells comprising 2-4% and 16% of the total cell number with opsin and MAP2 antibodies, respectively. A minimum of 2000 opsin-positive cells could be reliably determined. Using our cell ELISA protocol, we demonstrate a developmental increase of both cell markers which reflected an increase in the number of opsin-positive cells but an enhanced expression per cell in the case of MAP2. We also show that growth-promoting activity-the presumed chick ciliary neurotrophic factor (CNTF)-stimulated the expression of opsin in retinal cultures (EC50; 2.3 pM) and that a corresponding activity is specifically expressed in the developing retina. Our results show that the cell ELISA protocol allows the rapid screening for distinct, low-percentage cell populations responding to exogenous factors in mixed CNS cultures.
Fuhrmann
S
S
Institute of Anatomy, University of Freiburg, Germany.
Kirsch
M
M
Wewetzer
K
K
Hofmann
H D
HD
D000818:Animals; D002452:Cell Count; D002478:Cells, Cultured; D002642:Chick Embryo; D020934:Ciliary Neurotrophic Factor; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007150:Immunohistochemistry; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins / Q000494:pharmacology*; D010786:Photoreceptor Cells / Q000187:drug effects / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects; D012160:Retina / Q000166:cytology* / Q000187:drug effects; D017948:Retinal Rod Photoreceptor Cells / Q000187:drug effects / Q000378:metabolism; D017299:Rod Opsins / Q000378:metabolism
D020934:Ciliary Neurotrophic Factor; D008869:Microtubule-Associated Proteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D017299:Rod Opsins