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Phage Capture Assay: Quantifying Binding of Receptor to Phage Clones
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
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 (see Protocol ID#2167) 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 biotinylated (see Protocol ID#2188) so 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 (see Hint #1).
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 μl portions of these standards to coat a row of ten wells in each ELISA plate (see Hints #3 and #4).
4. Place ELISA plate aside until needed.
B. Virion Preparation
Virions of varying degrees of purity are used as immobilized antigens. The virions prepared in the following protocols may be used (see Protocol ID#2169, Protocol ID#2177 and Protocol ID#2178 [see Hint #5]).
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 (see Hint #6). 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 (see Hint #7).
4. Load each well with a 50 μl 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 (see Hint #7).
6. Just before the ligate is added, wash the ELISA plate 5 times with TBS/Tween, preferably using an automated plate washer (see Hint #8).
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 (see Hint #9).
2. Add biotinylated ligate in 50 to 200 μl 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 (see Hint #10).
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 (see Hint #11).
2. A known ligand for the receptor-most often, 5 X 1010 virions of an affinity-selected clone in 50 μl 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 μl portions of biotinylated BSA standards (Section A).
3. Add 190 μl of TTDBA to the wells containing the biotinylated BSA standards. To the ligand-coated wells, add 190 μl of TTDBA containing the predetermined fixed concentration of biotinylated receptor and graded concentrations of inhibitor peptides-either free or in the form of virions (see Hint #12).
4. Incubate the plate overnight in a humidified plastic box at 4°C.
5. Wash and develop the ELISA plate as described in Section F.
F. Developing the ELISA (see Hint #13)
1. Pipette 5 ml of AP-SA diluent into a 15 ml tube.
2. Add 10 μl 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 μl.
5. Wash the ELISA plate (prepared earlier) 10 times with TBS/Tween.
6. Pipette 65 μl of the diluted AP-SA into the coated wells (see Hint #13 and 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 μl of MgCl2 to the Diethanolamine Buffer in the reservoir.
10. Add 100 μl 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 μl.
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 μl of diluted substrate solution into the wells (see Hint #14).
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 (see Hint #15).
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 (see Hint #16).
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 (see Hint #17).
Solutions
NPP Substrate
50 mg/ml p-nitrophenylphosphate
Dispense into 100 μl aliquots
Store at -20°C
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 (see Hint #18)
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 μl of 1 M MgCl2 and 2 μl 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 Hint #2
TBS/Tween
Prepare in TBS
0.5 % (v/v) Tween-20
Dialyzed BSA
Store at -20°C
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 μg/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 4°C
BioReagents and Chemicals
Bovine Serum Albumin
p-nitrophenylphosphate
Tris HCl
Sodium Nitrate
Diethanolamine
Alkaline phosphatase-conjugated Streptavidin
Tween-20
Hydrochloric Acid
Glycerol
Magnesium Chloride
Zinc Chloride
Sodium Chloride
Gelatin TBS/Tween
Protocol Hints
1. It is also greatly simplified with an automated plate washer that can rapidly wash all 96 wells of an ELISA plate.
2. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
3. The contributors of this protocol use Corning modified flat-bottomed ELISA plates, which provide an optically flat bottom surface, enhancing the accuracy of OD measurements by the plate reader.
4. As all the standard wells receive the same total concentration of BSA, the fraction of the input biotinylated BSA that becomes irreversibly immobilized to the plastic surface should be constant from well to well. Therefore, the amount of biotinylated BSA immobilized in each well is presumably directly proportional to the input concentration of biotinylated BSA. When these wells are developed (along with all the other wells), the ELISA signal is plotted as a function of the concentration of input Bio-BSA (proportional to the actual amount of immobilized biotin); the plot is typically roughly linear for the low inputs, tending to level off for the highest inputs. This provides a standard curve by means of which ELISA signals from the other wells (signals that are not necessary linearly related to the amount of immobilized biotin in those wells) can be converted into an equivalent number that is proportional to the actual amount of biotin immobilized in those wells.
5. Any of three methods for propagating virions in Protocol ID#2169 may be used for this protocol. The contributors suggest the use of either virions prepared by double PEG precipitation (Section B) or virions prepared by double PEG precipitation and acid precipitation (Section C). An alternative is to use virions propagated according to Protocol ID#2177, or Protocol ID#2178. Other antigens, such as purified or recombinant protein, can be used as the immobilized species when appropriate.
6. The outer 36 wells of the ELISA plate are not used in order to avoid "edge" effects. The contributors, however, have very little actual evidence of such edge effects, and see no strong reason not to use all 96 wells. In the development procedure (Section F) the contributors assume that only the central 60 wells have been used; if all 96 wells have been used, volumes will have to be increased at the beginning of Section F as noted.
7. Other buffers may be used, but they must not contain carrier protein, Tween, or any other agent that could interfere with non-specific absorption of the virions to the polystyrene surface.
8. The contributors have recently stopped blocking unused non-specific adsorption sites on the plastic with BSA, because in a few circumstances this has led to an increase, rather than a decrease, in background signal. In order to prevent non-specific adsorption of subsequent reactants to the plastic, the reactants are dissolved in buffers containing 0.5% Tween 20. The washing buffer, TBS/Tween, also contains 0.5% Tween 20.
9. An important use of such titering reactions is to determine a concentration of ligate that generates a signal suitable for inihibition ELISA (next subsection). The contributors seldom use monoclonal IgG at a concentration above 10 μg/ml (67 nM) or non-affinity-purified polyclonal IgG at a concentration above 100 μg/ml (667 nM). A hyperimmune non-affinity-purified polyclonal IgG can generate a strong ELISA signal against some phage clones at a concentration of the order of 1 nM; the concentration of the subspecificity that recognizes the particular epitope on the phage in these circumstances presumably represents only a tiny fraction of the total IgG population, probably 1% or less, corresponding to a subspecificity concentration of less than 10 pM.
10. Typically 1 to 2 hr is enough to reach equilibrium in most cases, but as a matter of convenience, the contributors tend to allow the reaction to continue overnight at 4°C.
11. If the affinity is sufficiently high (dissociation equilibrium constant KD less than 1 μM), peptides can be analyzed in the form of whole virions; otherwise (or in addition), peptides (or other non-phage-borne competitive ligands) can be synthesized chemically and analyzed free in solution. The strength of the reaction between the ligand and the biotinylated ligate should already have been titered as described in the previous subsection in order to find a suitable ligate concentration for the inhibition ELISA. This concentration should be as low as possible and yet still provide a sufficient ELISA signal (usually 10 to 20 mOD/min). The ligate concentration will remain fixed at this level in all the wells of the inhibition ELISA. The lower the ligate concentration, the more accurately inhibition reflects the actual solution-phase equilibrium between the ligate and the inhibitor. When several different competitive inhibitors are to be tested against a single ligate, it is convenient to use a single immobilized ligand. The immobilized ligand and the solution-phase competitive inhibitor need not be identical as long as they compete for the same binding site on the ligate.
12. The virions should have been purified at least to the extent necessary for accurate spectrophotometric quantitation, as described in the Protocol on Absorption Spectroscopy and Quantification of Filamentous Phage).
13. The following steps are facilitated by the use of a multichannel pipetter capable of delivering 65 and 90 μl volumes in at least 8 channels.
14. The contributors generally fill all 96 wells, even if only the central 60 have been coated with ligand as the outer wells can serve as negative controls.
15. The contributors find that the increase in OD with time for the alkaline phosphatase assay is invariably linear over the entire 57 min incubation period.
16. Some plate-reader software may be programmed to execute these calculations automatically, but a user can also perform them using a spreadsheet program like Excel. These Y values are not absolute measures of the amount of biotinylated ligate captured. Within one ELISA plate, however, they should be directly proportional to the amount of biotinylated ligate captured (unlike the slopes themselves, which show a non-linear dependence on the amount of immobilized Bio-BSA in the standard wells).
17. Under a broad range of conditions, however, the inhibition curve is insensitive to the nuisance parameters and sensitive to the KD of the inhibitor. A value of the latter that brings the curve into accord with the data should be close to reality (assuming the underlying kinetic model is realistic). The effects of varying the parameters within their plausible ranges can be checked by computer.
18. The alkaline phosphatase-conjugated streptavidin is supplied as 1 mg/ml conjugate solution in buffer from Jackson ImmunoResearch. The conjugate is supplied from Jackson ImmunoResearch as a lyophilized powder, dissolve the powder in 1 ml of water before adding the glycerol/salt solution in the preparation process. Keep the vial of alkaline-phosphatase-conjugated streptavidin ice-cold. Return the vial to ice after each manipulation to keep the solution cold.
