Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner's fiber.
The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner's fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.
Estivill-Torrús
G
G
Departamento de Biología Animal, Facultad de Ciencias, Universidad de Málaga, E-29071 Málaga, Spain.
Cifuentes
M
M
Grondona
J M
JM
Miranda
E
E
Bermúdez-Silva
F J
FJ
Fernández-Llebrez
P
P
Pérez
J
J
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D002417:Cattle; D015816:Cell Adhesion Molecules, Neuronal; D017077:Culture Media, Conditioned / Q000032:analysis; D046508:Culture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D009419:Nerve Tissue Proteins / Q000134:cerebrospinal fluid / Q000276:immunology / Q000378:metabolism*; D013351:Subcommissural Organ / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015816:Cell Adhesion Molecules, Neuronal; D017077:Culture Media, Conditioned; D009419:Nerve Tissue Proteins; C108402:SCO-spondin