Analysis of the performance of antibody capture methods using fluorescent peptides with capillary zone electrophoresis with laser-induced fluorescence.
A method to analyze the performance of an antibody capture method using fluorescent peptides by capillary zone electrophoresis using laser-induced fluorescence (CZE-LIF) for detection has been developed. Fluorescent peptides from the prion protein were synthesized and the corresponding antibodies were produced in rabbits against these peptides. The antibodies were used to capture the fluorescent peptides. The antibodies were then bound to protein A Sepharose. After elution, the amount of fluorescent peptide that was captured vs. the total amount placed in the assay was evaluated by CZE-LIF. Of the three peptides used in this evaluation, it was found that the recovery was approximately 25-35%. When the abnormal prion protein was prepared from scrapie-infected brain samples from hamsters and a sheep using the previously described extraction method and this method, the amount of abnormal prion protein that was measured in the fluorescence immunoassay correlated with amounts estimated from Western blot. We conclude that this method can be used to detect abnormal prion protein in a tissue sample.
Jackman
Roy
R
Veterinary Laboratory Agency, New Haw, Addlestone, Surrey, UK.
Schmerr
Mary Jo
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D001921:Brain / Q000378:metabolism; D006224:Cricetinae; D019075:Electrophoresis, Capillary / Q000379:methods*; D005455:Fluorescent Antibody Technique; D007834:Lasers; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D011328:Prions / Q000032:analysis* / Q000378:metabolism; D012608:Scrapie / Q000378:metabolism; D012756:Sheep
D000906:Antibodies; D010455:Peptides; D011328:Prions