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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
SOLUBLE scFc ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA) IN MICROTITIRE PLATES
Soluble scFv Enzyme Linked Immunosorbant Assay (ELISA) in Microtitre Plates
Contributor: The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco Overview
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
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).
PBS 137 mM Sodium Chloride (NaCl)
2.7 mM Potassium Chloride (KCl)
Also see Protocol ID#2152
4.3 mM Sodium Phosphate, Dibasic (Na2HPO4
1.4 mM Potassium Phosphate, Monobasic (KH2PO4)
Anti-Mouse Solution 1:2000 Anti-Mouse Horseradish Peroxidase:2% MPBS 9E10 mAb Solution 1:1000 9E10 Monoclonal Antibody (Santa Cruz Biotech):2% MPBS Antigen Solution Also see Hint #1
10 μg/ml to 1 mg/ml Antigen in PBS
2% MPBS Prepared in PBS
2% (w/v) Non-Fat Milk Powder (Carnation™)
4% MPBS 4% (w/v) Non-Fat Milk Powder (Carnation™)
Prepared in PBS
PBS/Tween 0.1% (v/v) Tween 20
Prepared in PBS
BioReagents and Chemicals
Sodium Phosphate, Dibasic
Potassium Phosphate, Monobasic
Antibody, HRP Labeled, Anti-Mouse
Non-Fat Milk Powder
Monoclonal Antibody, 9E10
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).
Citation and/or Web Resources
1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press.