An affinity based bio-sensing technique was developed using an anti-transmissible spongiform encephalopathy monoclonal antibody as a bio-recognition molecule. Fluorescein iso-thio-cynate (FITC), labeled with a prion epitope (QYQRES), was used as a decoy for prions. Experiments done in 0.1M phosphate buffer revealed that the dye fluorescence increased with the pH of the buffer and was influenced by solvent polarity.
Binding studies conducted at pH 6, 7, and 8 showed that the optimum pH for the antibody-decoy binding was 7. Maximum differences between control and antibody samples were observed at pH 7. The optimum incubation time was found to be less than 4 hours for the control, antibody, and the prion samples at room temperature. Prion detection curves were established at 4 and 10 nM antibody decoy concentrations. The lowest detectable prion concentration in phosphate buffer was 8 nM.
Experimental conditions determined in the phosphate buffer were used to implement the technique in gelatin and baby formula. Prion detection curves were generated in 0.01, 0.4, 1.0 and 2.0 mg/ml of gelatin solution. The gelatin interfered with the binding and the displacement reaction of antibody, decoy and prion. Addition of an anionic surfactant, sodium dodecyl sulfate (SDS) at 0.3 mg/ml to gelatin samples facilitated prion detection in gelatin. The lowest detectable concentration of prion in gelatin was 0.5 nM at 0.4mg/ml gelatin. The baby formula samples produced light scattering and the intrinsic peak of baby formula at 526nm interfered with the dye peak at 514nm. Serial dilutions of baby formula were done to reduce the interference. Prion detection curves were then obtained at 1.31 and 5.34 mg/ml baby formula and 0.454 mg/ml of Triton-X-100 was added to the baby formula samples. The lowest detectable concentration of prion was 2 nM for baby formula.
This developed bio-sensing technique can be used to detect prion in gelatin and baby formula solutions. Addition of surfactants assisted prion detection in foods, while high concentrations of gelatin and baby formula had an adverse effect on the detection system.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/1503 |
| Date | 17 February 2005 |
| Creators | Anand, Ashish |
| Contributors | Castell-Perez, Elena, Moreira, Rosana |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Thesis, text |
| Format | 2083325 bytes, electronic, application/pdf, born digital |
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