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Figure 1
1. In vitro phage antibody library screening with antigen (Ag)
2. Preparation of the scFv sub-library, enriched for antigen-specific scFv, from the first round phage infected TG1
Figure 2
3. Construction of yeast scFv-VP16 in vivo expression sub-library for antibody-antigen yeast interaction screening
4. E. coli transformation using electroporation
5. Yeast antibody-antigen interaction screening of the scFv-VP16 library with the antigen “bait”
6. Confirmation of antibody-antigen interaction in yeast clones by b-galactosidase filter assay
7. Plasmid DNA extraction from individual selected yeast colonies
8. Re-testing the scFv-VP16 plasmid using the yeast antibody-antigen interaction assay
9. Characterisation of the antigen-specific intracellular scFv
10. Binding of scFv to antigen by immunodetection assay (Western blot or ELISA).
Figure 3
11. Mammalian luciferase reporter assay for Ag-scFv intracellular interaction
References
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11. Mammalian luciferase reporter assay for Ag-scFv intracellular interaction
After the intracellular scFv have been rigorously characterised in the yeast (and perhaps also the in vitro assays), the ability of these intracellular scFv to bind to their target antigen in mammalian cells must be tested. This can conveniently be assessed by using mammalian reporter assays such as CAT assay or a more sensitive method is the luciferase reporter assay. It should be noted that these approaches will detect scFv which specifically bind to antigen but which do not induce rapid destruction of the Ag-Ab complex.
Preparation of mammalian vectors
For mammalian antigen-scFv interaction assays, antigen cDNA and scFv should be shuttled into suitable mammalian expression vectors. In this assay, pM1 vector may be used as a DBD-bait vector and pEF-VP16 as prey vector (maps shown in Figure 3). The pM1 and pBTM116 vectors have the same poly-linker to generate in-frame sequence. pEF-VP16 also has Sfi1-Not1 sites with the same protein translation reading frame as that of the yeast pVP16*, thus scFv can be directly shuttled into pEF-VP16 from pVP16*-scFv using Sfi1-Not1.
Luciferase reporter assay
a. Seed 2 x 105 cells per well in a six-well tissue culture plate (COS-7, CHO, NIH3T3, etc) in 2ml of the appropriate complete growth medium with 10% FCS.
b. Incubate the cells at 37°C in a CO2 incubator until the cells are about 70% confluent.
c. Prepare the following solutions in sterile 2ml Eppendorf tubes:
Solution A: For each transfection, dilute 8ul of LipofectAMINE™ Reagent into 100µl Opti-MEM® I Reduced Serum Medium.
Solution B: For each transfection, Mix 0.5µg of pM-antigen (GAL4-DBD-antigen), 0.5µg of pEF-scFv-VP16 (scFv-VP16), 0.5µg of pG5-Luciferase (5 x Gal4 binding sites-reporter) and 50 ng of pRL-CMV (control reporter vector, which serves as the baseline response) into 100µl Opti-MEM® Medium.
d. Combine the two solutions, mix gently, and incubate at room temperature for 30 min.
e. Wash the cells once with 2ml of Opti-MEM® Medium.
f. For each transfection, add 0.8ml of Opti-MEM® Medium to each tube. Mix gently and overlay this solution onto washed cells.
g. Incubate the cells for 8 hrs at 37°C in a CO2 incubator.
h. Add 1ml of the appropriate complete growth medium with 20% FCS without removing the transfection mixture.
i. At 24 hour after transfection, remove the transfection mixture and replace with new growth medium with 10% FCS.
j. At 48 hour from the start of transfection, remove the medium and wash twice with 2ml of PBS.
k. Luciferase assay is performed by using Dual-Luciferase Reporter Assay System according to Promega's instruction and detect with a luminometer.
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