Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Soluble scFv Enzyme Linked Immunosorbant Assay (ELISA) in Microtitre Plates |
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| Contributor: |
The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco | ||||||||||||||||||||||||||||||||||||||||||
| Overview |
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| This protocol describes the detection of bacteriophage-derived antibodies specific for an antigen of interest. The antigen is adsorbed on the plastic wells of a 96-well microtiter plate. An Enzyme Linked Immunosorbant Assay (ELISA) analysis to detect soluble scFv is similar in principle to the detection of phage antibodies (see Protocol ID#2209), with the exception that the soluble scFv is bound by an anti-epitope tag antibody and visualized with a secondary detection antibody. The phage display vector of this system encodes for the c-myc epitope. | |||||||||||||||||||||||||||||||||||||||||||
| Procedure |
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1. Coat microtiter plate wells overnight at 4°C with 100 μl of Antigen Solution per well (see Hint #1). 2. Discard the Antigen Solution and wash the microtiter plate wells two times with PBS. 3. Block the wells by adding 200 μl of 2% MPBS. 4. Incubate at 37°C for 2 hr. 5. Discard the MPBS Solution and wash the microtiter plate wells three times with PBS. 6. Add 50 μl of 4% MPBS to each of the microtiter plate wells. 7. Add 50 μl of culture supernatant containing soluble scFv (prepared in Protocol ID#2210). 8. Mix the solution well by pipetting up and down, then incubate for 1 hr at room temperature. 9. Discard the solution and wash the microtiter plate wells three times with PBS/Tween. 10. Wash the microtiter plate wells three times with PBS. 11. Add 100 μl of 9E10 mAb Solution to each microtiter plate well (see Hint #2). 12. Incubate at room temperature for 1 hr. 13. Discard the 9E10 mAb Solution and wash the microtiter plate wells three times with PBS/Tween. 14. Wash the microtiter plate wells three times with PBS. 15. Add 100 μl of Anti-Mouse Solution to each microtiter plate well. 16. Incubate at room temperature for 1 hr. 17. Discard the secondary antibody (Anti-Mouse Solution) and wash the microtiter plate wells three times with PBS/Tween. 18. Wash the microtiter plate wells three times with PBS. 19. Add 100 μl TMB System Solution. 20. Incubate at room temperature for 10 to 30 min in the dark. 21. A blue hue should develop within a couple of min. 22. Quench the reacton by adding 50 to 100 μl of Stop Solution. 23. Determine the adsorbance at 450 nm (A450). |
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| Solutions |
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| BioReagents and Chemicals |
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Sodium Phosphate, Dibasic Potassium Chloride Sodium Chloride Tween 20 Potassium Phosphate, Monobasic Antibody, HRP Labeled, Anti-Mouse Non-Fat Milk Powder Monoclonal Antibody, 9E10 |
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| Protocol Hints |
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1. 10 μg/ml Protein Antigen prepared in PBS is standard. Occasionally, a higher concentration or a different binding buffer is required (e.g. Carbonate buffer). 2. The anti-tag antibody described here is 9E10, which recognises the c-myc tag and is incorporated into the pHEN 1 vector. 9E10 is commercially available or the hybridoma cell line can be obtained from American Type Culture Collection (ATCC). |
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| Citation and/or Web Resources |
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1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press. |
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