Development of polyclonal antibodies and evaluation of a sensitive radioimmunoassay for detection and measurement of synaptophysin.
Polyclonal antibodies directed towards synaptophysin were raised against a synthesised peptide corresponding to amino acids 246 to 260 of the human synaptophysin sequence. The antibodies, when applied for immunocytochemical staining, showed a staining pattern identical to that of the commercially available monoclonal antibody SY-38. A radioimmunoassay for measurements of synaptophysin was developed using these antibodies and the peptide as standard and tracer. The radioimmunoassay was used for optimising the conditions for purification of synaptophysin from rat brain. No synaptophysin was detected in blood plasma in humans, not even during an embolisation treatment of tumour metastases in the liver, which induced tumour cell necrosis, in a patient with carcinoid tumours. By radioimmunoassay, synaptophysin was detected in cell homogenate from the PC-12 (160 ng/mg) and LCC-18 (40 ng/mg) cell lines and in the cell culture media. In the LCC-18 cell line the synaptophysin immunoreactivity was found in the plasma membrane, and the presence of synaptophysin was confirmed both by radioimmunoassay measurements and by the Northern blot technique. These data indicate that measurements of synaptophysin using this radioimmunoassay are reliable and that the assay can serve as a useful tool in further explorations of the biological effects of synaptophysin.
Stridsberg
M
M
Department of Clinical Chemistry, University Hospital, Uppsala, Sweden.
Lundqvist
G
G
Engström
U
U
Wilander
E
E
Su
H
H
Gobl
A
A
Oberg
K
K
D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000917:Antibody Formation; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D016716:PC12 Cells; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D016708:Synaptophysin / Q000032:analysis* / Q000276:immunology
D000906:Antibodies; D003470:Culture Media; D016708:Synaptophysin