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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

ANTIBODIES: ELISA

EXPRESSING SOLUBLE scFv IN MICROTITER PLATES

Expressing Soluble scFv in Microtiter 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 an alternative to assaying the binding properties of phage-displayed antibodies by enzyme linked immunosorbant assays (ELISA; see Protocol ID#2209). This procedure relies on the presence of soluble antibody fragments released from cells into medium. The expression of antibody (or other polypeptide) fragments from phagemid display vectors is controlled by the lacZ promoter. Glucose-free conditions stop catabolic repression of the lacZ promoter (mediated by the inhibition of cAMP synthesis); whereas, IPTG induces transcription by inactivating the lacQ repressor on the bacterial genome.

The method described here requires that the amount of glucose (0.1%) present in the starting medium is low enough to be metabolised by the time the inducer (IPTG) is added. This avoids any centrifugation steps to remove glucose reducing the risk of contamination.
 
Procedure
1. Add 100 μl of TYE/AMP/GLU to each well of a 96-well microtiter plate.

2. Using a 96-well sterile transfer device or pipette, transfer 2 μl from the master plate (prepared in Protocol ID#2208) into each well containing 100 μl of TYE/AMP/GLU.

3. Incubate on a shaking platform at 37°C until the absorbance at 600 nm (A600) is 0.9 (approximately 2 to 3 hr).

4. Using a 96-well sterile transfer device or pipette, add 50 μl of 2X TYE/AMP/IPTG into each well.

5. Incubate on a shaking platform at 30°C for an additional 16 hr.

6. Centrifuge the plates at 2,700 rpm for 15 min using a Beckman™ GH 3.8 rotor with a microtiter plate adaptor (1,200 X g).

7. Collect the supernatant (see Hint #1).

8. Use 50μl of the collected supernatant for ELISA (see Protocol ID#2111).

Solutions
TYE/AMP/IPTG   5 g NaCl
Prepare in 1 liter of ddH2O
10 g Bacto Yeast Extract (Difco)
Add Ampicillin to 100 μg/ml
16 g Bacto Tryptone (Difco)
Add IPTG to3 mM
Cool to 55°C
Autoclave to sterilize
TYE/AMP/GLU   5 g NaCl
Add Glucose to0.1% (w/v)
Prepare in 1 liter of ddH2O
10 g Bacto Yeast Extract (Difco)
16 g Bacto Tryptone (Difco)
Add Ampicillin to 100 μg/ml
Cool to 55°C
Autoclave to sterilize
 
BioReagents and Chemicals
Bacto Yeast Extract
IPTG
Ampicillin
Glucose
Bacto Tryptone
Sodium Chloride
 
Protocol Hints
1. Under these conditions, the gene coding for the scFv is transcribed, and soluble fragments are produced. The antibody leaks into the supernatant (in part, due to their toxicity in E. Coli) in a passive process such that the individual soluble fragments can usually be used to study binding properties. In some instances, more antibody fragments will remain inside the periplasm than in the supernatant. This occurs when the antibody is not very toxic and does not leak out significantly. If necessary, periplasmic extracts can be prepared to retrieve all the antibodies produced.

 
Citation and/or Web Resources
1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press.