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d7a3952c-0cb6-4cf9-a089-42d41452609f.doc 01/15/13 Page 1 PHAGE ELISA NOTE: We use two main binding assays to confirm specific binding of “ligate” (the molecule whose binding to phage-borne peptide is to be tested) to phage-borne ligands: the ELISA procedure described in this document and the phage capture assay (alias “micropanning”) described in micropan.doc. Phage ELISA measures the capture of solution-phase ligate by phage immobilized on the plastic surface of ELISA dish wells. It thus has the advantage that it confirms specific binding of ligate to phage-borne ligand by the opposite of the the affinity selection procedure (AffinitySelection.doc) used to obtain the phage in the first place. ELISA also lends itself to semi-quantitative analysis of binding constants via inihibition ELISA (see below). On the other hand, ELISA is not very sensitive: even phage that have been successfully and specifically affinity-selected sometimes give a feeble ELISA signal that is indistiguishable from background, which probably reflects predominantly reaction of ligate with non- phage impurities. In contrast, the phage capture assay is far more sensitive than ELISA and is not obscured by reactions with non-phage components of the system. NOTE: It is assumed that the ligate is biotinylated (see biotinylation.doc), so that 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 ~20 time points spaced ~3 min apart. It is also greatly simplified with an automated plate washer that can rapidly wash all 96 wells of an ELISA dish. We use modified flat-bottomed ELISA dishes (Falcon 353915), which provide an optically flat bottom surface to enhance the accuracy of OD measurements by the plate reader. Bio-BSA standards 1. Dissolve biotinylated BSA (Sigma A6043) at 2 mg/ml in water, filter-sterilize, and store at 4°. 2. Using TBS containing 100 µg/ml BSA (non-biotinylated) as diluent, make ten dilutions containing 0, 4.5, 9,13.5,…, 31.5, 36, and 40.5 ng/ml. Use 50-µl portions of these standards to coat a row of ten wells in each ELISA dish. Because 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. Virions Virions of various degrees of purity are used as the immobilized antigens: d7a3952c-0cb6-4cf9-a089-42d41452609f.doc 01/15/13 Page 2 Singly PEG-precipitated virions: Propagation_1ml.doc, steps 1–4, 7. Doubly PEG-precipitated virions: Propagation_1ml.doc, steps 1–8. Doubly PEG-precipitated, acid-precipitated virions: Propagation_1ml.doc, steps 1–6, 7´–8´, 9–10 Doubly PEG-precipitated virions: SmallScaleVirions.doc CsCl-purified virions: VirionPurification.doc Other antigens—e.g., proteins—can also be used as the immobilized species when appropriate. Coating wells of the ELISA dish with virions 3. Dilute each virion preparation to 1012 virions/ml in TBS. Other buffers can be used, but they must not contain carrier protein, Tween, or any other ingredient that might interfere with non- specific absorption of the virions to the polystyrene surface. 4. Load each well with a 50-µl portion of phage dilution. If desired, some of the wells can be coated with Bio-BSA standards as described in steps 1–2. 5. Incubate the dish in a humidified plastic box at room temperature for at least 2 hr (overnight in the cold is better). 6. Just before the ligate is added, wash the dish 5 times with TBS/Tween, preferably using an automated plate washer. NOTE: We have recently stopped blocking unused non-specific adsoprtion sites on the plastic with BSA, as in a few circumstances this has led to an increase, rather than a decrease, in background signal. In order to prevent non-specific adsporption 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. Direct ELISA and titering NOTE: In 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 see how ELISA signal varies with ligate concentration. An important use of such titering reactions is to find a concentration of ligate that generates a suitable signal for inihibition ELISA (next subsection). As a rough guide to a range of ligate concentrations to try: We very seldom use monoclonal IgG’s at a concentration above 10 µg/ml (67 nM) or non-affinity-purified polyclonal IgG’s 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 <10 pM. d7a3952c-0cb6-4cf9-a089-42d41452609f.doc 01/15/13 Page 3 7. To the coated, washed ELISA dish (step 6) add biotinylated ligate in 50–200 µl TTDBA (or other Tween-20-containing buffer). 8. Incubate the ELISA dish in a humidified plastic box for the desired time at the desired temperature. Probably 1–2 hr is enough to reach equilibrium in most cases, but as a matter of convenience we usually react overnight in the cold. 9. Wash and develop the ELISA dish as described below (steps 13–19). Inhibition ELISA NOTE: Inhibition ELISA determines the affinity of a test peptide for ligate by measuring the ability of various concentrations of the peptide in solution to competitively inhibit binding of ligate to an immobilized ligand. If affinity is high enough (dissociation equilibrium constant KD<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 give a usable ELISA signal—usually 10–20 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. If several different competitive inhibitors are to be tested against a single ligate, it is convenient to use a single immobilized ligand; there is no necessity that the immobilized ligand and the solution-phase competitive inhibitor be the same as long as they compete for the same binding site on the ligate. 10. A known ligand for the ligate—most often, 5 × 1010 virions of an affinity-selected clone in 50 µl TBS—is adsorbed to a series of wells of a 96-well microplate (step 3–6). At the same time, ten wells in the same microplate are coated with 50-l portions of biotinylated BSA standards (steps 1–2). 11. To the wells with the biotinylated BSA standards add 190 l TTDBA; to the ligand-coated wells add 190 l TTDBA containing the predetermined fixed concentration of biotinylated ligate and graded concentrations of inhibitor peptides—either free or in the form of virions (the virions should have been purified at least to the extent necessary for accurate spectrophotometric quantitation, as described in AbsorptionSpectrum.doc). Incubate the dish overnight in a humidified plastic box—usually at 4º, but other temperatures may be appropriate in some circumstances. 12. Wash and develop the ELISA dish as in the next subsection (steps 13–19). d7a3952c-0cb6-4cf9-a089-42d41452609f.doc 01/15/13 Page 4 DEVELOPING THE ELISA DISH NOTE: We assume here that the investigator has a multichannel pipetter capable of delivering 65- and 90-µl volumes in at least 8 channels; we assume, too, that plastic reservoirs specially designed for these pipetters are available. These accessories are not strictly necessary, however. 13. Into a 15-ml tube pipette 10 ml TBS/Tween and 20 µl 0.5 mg/ml AP-SA; vortex gently but thoroughly to mix; pour into a dispensing reservoir for a multichannel pipetter. Put tips on the multichannel pipetter and dial to 65 µl. 14. Wash ELISA dish (step 9 or 12) 10 times with TBS/Tween, preferably using an automated plate washer. 15. Using the multichannel pipetter and the reservoir prepared in step 13, pipette 65 µl of the diluted AP-SA into the wells. Allow the dish to react at room temperature 30 min. 16. Meanwhile, prepare diluted substrate solution in a reservoir: 10 ml 1 M diethanolamine buffer pH 9.8 (pH adjusted with HCl) 10 µl 1 M MgCl2 100 µl (all) of a tube of NPP substrate (50 mg/ml p-nitrophenylphosphate) Mix thoroughly by tipping the reservoir back and forth repeatedly. Also prepare a multichannel pipetter dialed to 90 µl and with tips firmly attached. 17. When the 30-min incubation is finished, wash the ELISA dish using wash the dish 10–15 times with TBS/Tween. 18. Using the multichannel pipetter and reservoir prepared in step 16, pipette 90 µl diluted substrate solution into the wells (we generally fill all 96 wells, even if only the central 60 have been coated with ligand; the outer wells serve as negative controls). 19. Read the dish on a kinetic plate reader programmed to read the wells every 3 minutes for 20 reads altogether. We program our reader to record the OD at both 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), which is what we regard as the ELISA signal; we find that the rise in OD with time for alkaline phosphatase is invariably linear over the entire 57-min incubation period. CALCULATIONS Converting slopes to equivalent relaive 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. Our plate reader d7a3952c-0cb6-4cf9-a089-42d41452609f.doc 01/15/13 Page 5 software does these calculations automatically, but a user can do them for him- or herself using a spreadsheet program like Excel. These Y values are not absolute measures of the amount of biotinylated ligate captured; within one ELISA dish, 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). Calculating percent inhibition in inhibition ELISA The Y values calculated in the previous sub-subsection are averaged for wells containing no inhibitor to give the maximum value, Ym ax ; and percent inhibition for the other wells is calculated as 100 (Ymax Y ) / Ymax. 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 ligate—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. Under a broad range of conditions, however, the inhibition curve is insensitive to the nuisance parameters and sensitive to the inhibitor KD, and 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 effect of varying the parameters within their plausible ranges can be checked by computer.
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