| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2006: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2005: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Isoelectric immunoassay is the quantification method for protein antigens as fluorescent complexes with fluorescence-labele antibody fragments using micro isoelectric focusing. Since antigens are identified by the isoelectric points of the complexes, the method can be highly accurate without being affected by nonspecific adsorption or crossreactivity of antibodies. In addition to this advantage, the analysis can be completed in a short time and with a small amount of a sample owing to the use of micro electrophoresis. Although this method is applicable to soluble protein antigens, some antigens are hard to be solubilized or tend to form aggregates forming a precipitate. Fragmentation of such proteins into peptides may improve the solubility and convert them into more suitable analytes for the application of isoelectric immunoassay. The conversion into the antigen peptides may also improve the stability of the samples and may lead to higher reproducibility of the quantification.
The prio
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n protein is a membrane protein and its conformational abnormality is believed to be related to the prion diseases, such as bovine spongiforrn encephalopathy. When isoelectric immunoassay was applied to quantification of a recombinant normal mouse prion protein (rMoPrP), we encountered a difficulty in the detection of the complex with a labeled anti MoPrP antibody fragment. The difficulty appeared to be related to the low solubility of the protein. We, then, applied isoelectric immunoassay after fragmentation of rMOPrP to the peptides. The use of protease, such as trypsin, chymotrypsm, pepsin, and lysylendopeptidase, was found to be unsuccessful resulting in the destruction of the epitope to the labeled antibody fragment used. On the other hand, the use of CNBr degradation resulted in the successful formation of the soluble peptide that can be detected by the isoelectric immunoassay. The investigation on the condition of CNBr degradation revealed that the reaction for 1 h at 50℃ with 1%CNBr in 70% formic acid is sufficient to complete the degradation. It was also found that the degradation proceeds identically even in the presence of bovine serum albumin at 10,000 times excess over the antigen and the peptide was quantified by the isoelectric immunoassay. The degradation was also effective for 60 ng of rMOPrP in the presence of 20 μ 1 of human serum. These results indicated that the fragmentation of proteins and subsequent analysis by isoelectric immunoassay is very promising for the quantification method of insoluble proteins. The applicability of ELISA for such peptide fragments should be more limited, since these peptides would be too small to fit in the sandwich assay system. Less
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