Phage Capture Assay: Quantifying Binding of Receptor to Phag

Tuesday, October 21, 2003

Description
Two main binding assays can be used to confirm the specific binding of a target receptor (ligate) to phage-borne ligands: the phage capture assay and an ELISA-based procedure described in this protocol. Phage ELISA measures the capture of solution-phase ligate by phage immobilized on the plastic surface of ELISA plate wells.

It is assumed that the ligate is biotinylatedso that the bound ligate can be detected with streptavidin conjugated to alkaline phosphatase. As implemented here, the ELISA procedure requires a "kinetic" plate reader that can rapidly measure the OD in all 96 wells at each of approximately 20 time points spaced approximately 3 min apart

ELISA also lends itself to semi-quantitative analysis of binding constants via inhibition ELISA. Inhibition ELISA determines the affinity of a test peptide for a receptor by measuring the ability of various concentrations of the peptide in solution to competitively inhibit binding of a receptor to an immobilized ligand.

Procedure
A. Bio-BSA Standards

1. Thaw a vial of Biotinylated BSA Solution.

2. Dilute the Biotinylated BSA Solution in Non-Biotinylated BSA Solution to make ten dilutions containing 0, 4.5, 9, 13.5, 18, 22.5, 27, 31.5, 36, and 40.5 ng Biotinylated BSA/ml.

3. Use 50 l portions of these standards to coat a row of ten wells in each ELISA plate

4. Place ELISA plate aside until needed.

B. Virion Preparation

Virions of varying degrees of purity are used as immobilized antigens.

C. Coating the Wells of the ELISA plate with Virions

1. For this protocol, it is assumed that the outer 36 wells of the ELISA plate are not used . If all 96 wells are to be used, the volumes will need to be increased at Section F, Step #2.

2. Dilute each virion preparation to 1012 virions/ml in TBS

4. Load each well with a 50 l portion of the phage dilution. If desired, some of the wells can be coated with Bio-BSA standards as described in steps 1 to 2.

5. Incubate the plate in a humidified plastic box at room temperature for at least 2 hr

6. Just before the ligate is added, wash the ELISA plate 5 times with TBS/Tween, preferably using an automated plate washer

D. Direct ELISA and Titering ELISA

1. In a direct ELISA, biotinylated ligate is reacted directly with the phage-coated wells. In a titering ELISA, graded concentrations of biotinylated ligate are added to a series of wells with the same immobilized phage, in order to determine how ELISA signal varies with ligate concentration

2. Add biotinylated ligate in 50 to 200 l of TTDBA (or other Tween-20-containing buffer) to the coated, washed ELISA plate (Section C, Step #6).

3. Incubate the ELISA plate in a humidified plastic box for the desired time at the desired temperature

4. Wash and develop the ELISA plate as described below (Section F).

E. Inhibition ELISA

1. Inhibition ELISA determines the affinity of a test peptide for a receptor by measuring the ability of various concentrations of the peptide in solution to competitively inhibit binding of a receptor to an immobilized ligand .

2. A known ligand for the receptor-most often, 5 X 1010 virions of an affinity-selected clone in 50 l of TBS- is adsorbed to a series of wells of a 96-well microplate (Section C, Steps #3 to #6). At the same time, ten wells in the same microplate are coated with 50 l portions of biotinylated BSA standards (Section A).

3. Add 190 l of TTDBA to the wells containing the biotinylated BSA standards. To the ligand-coated wells, add 190 l of TTDBA containing the predetermined fixed concentration of biotinylated receptor and graded concentrations of inhibitor peptides-either free or in the form of virions

4. Incubate the plate overnight in a humidified plastic box at 4C.

5. Wash and develop the ELISA plate as described in Section F.

F. Developing the ELISA

1. Pipette 5 ml of AP-SA diluent into a 15 ml tube.

2. Add 10 l of 0.5 mg/ml AP-SA (multiply the volumes by 1.5 if all 96 wells of the plate are to be used).

3. Vortex gently but thoroughly to mix.

4. Pour into a dispensing reservoir for a multichannel pipetter. Place tips on the multichannel pipetter and set the dial to dispense 65 l.

5. Wash the ELISA plate (prepared earlier) 10 times with TBS/Tween.

6. Pipette 65 l of the diluted AP-SA into the coated wells ( Section F, Step #4).

7. Allow the plate to react at room temperature for 30 min.

8. Add 10 ml of Diethanolamine Buffer to a reservoir.

9. Add 10 l of MgCl2 to the Diethanolamine Buffer in the reservoir.

10. Add 100 l of NPP Substrate to the reservoir.

11. Mix thoroughly by tipping the reservoir back and forth repeatedly. This constitutes the diluted substrate solution. Also prepare a multichannel pipetter dialed to 90 l.

12. When the 30 min incubation is finished, wash the ELISA plate 10 to 15 times with TBS/Tween.

13. Using the multichannel pipetter and reservoir prepared in Step #11, pipette 90 l of diluted substrate solution into the wells

14. Read the plate on a kinetic plate reader programmed to read the wells every 3 minutes for 20 reads altogether. Program the reader to record the Optical Density (OD) at wavelengths of 405 and 490, reporting the difference as the net OD reading. After all 20 reads, the machine calculates and reports the slope for each well (mOD/min). This is the ELISA signal

G. Calculations

1. Converting slopes to equivalent relative amounts of captured biotin

The dependence of slope on input concentration for the biotinylated BSA standards is modeled by a cubic equation, which is used in turn to transform the slopes for the other wells to equivalent relative amounts of biotinylated protein captured, which we call Y

2. Calculating percent inhibition in inhibition ELISA

The Y values calculated in the previous subsection (Section G, Step #1) are averaged for wells containing no inhibitor to give the maximum value, Ymax. Percent inhibition for the other wells is calculated as 100 x (Ymax- Y)/ Ymax.

3. Theoretical curves in inhibition ELISA

Theoretical inhibition curves can be calculated assuming unbound ligate is simultaneously in equilibrium both with ligate bound to the solution-phase inhibitor and with ligate bound to the immobilized ligand. The curves are governed not only by the parameter of interest-the inhibitor's KD for the receptor-but also by two nuisance parameters concerning the immobilized ligand that are not generally known: its effective concentration (the amount actually available for reaction with ligate divided by the reaction volume) and its KD for the ligate

Recipes
NPP Substrate 50 mg/ml p-nitrophenylphosphate
Dispense into 100 l aliquots
Store at -20C
Prepare in ddH2O

TTDBA Prepare in TBS/Tween
1 mg/ml BSA
0.02% NaN3

AP-SA Store in the refrigerator.
Tare the vial, then add 1.26 g (1 ml) of the above ice-cold mixture.
Close the cap and mix thoroughly by vigorous vortexing and repeated inversion. Place tube on ice.
Centrifuge the vial briefly. Open the septum and divide the solution equally between two 1.5 ml microcentrifuge tubes.
0.5 mg/ml stock of Alkaline Phosphatase-Conjugated Streptavidin
Centrifuge the commercial vial of streptavidin briefly.
Close the rubber septum, wrap the top of the vial with parafilm.
In a 1.5 ml microcentrifuge tube, measure 30 l of 1 M MgCl2 and 2 l of ZnCl2. Then weigh in 1.89 g of ultrapure Glycerol.
Tape the vial to a rotater and rotate approximately 30 min in the cold to thoroughly mix the contents.
Open the vial, keeping the rubber septum clean.

1 M MgCl2

Diethanolamine pH 9.8 Adjust pH with HCl
Need not be autoclaved
1 M Diethanolamine stock for ELISA
Also see

TBS/Tween Prepare in TBS
0.5 % (v/v) Tween-20

Dialyzed BSA Store at -20C
Filter sterilize
Prepare in ddH2O
50 mg/ml Biotin-free Bovine Serum Albumin (BSA) (Sigma)

TBS/Gelatin Store at room temperature
Autoclave 0.1 g Gelatin in 100 ml TBS
After autoclaving, swirl to mix in the melted gelatin

AP-SA Diluent 1 mg/ml dialyzed BSA
0.1% Tween 20
50 mM Tris-HCl pH 7.5
150 mM NaCl

TBS (1X) 50 mM Tris HCl, pH 7.5
Store at room temperature
Autoclave if desired
150 mM NaCl

Non Biotinylated BSA Solution 100 g/ml BSA (non-biotinylated)
Prepare in TBS

Biotinylated BSA Solution 2 mg/ml Biotinylated BSA (Sigma A6043)
Prepare in ddH2O
Filter sterilize and store at 4C

Supplies

Tips

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Phage Capture Assay: Quantifying Binding of Receptor to Phag