Binding Assays

A single binding assay is illustrated in Fig. 3, and the schematic (see Fig. 4) shows the changes occurring at the surface in the bulk phase.

3.5.1. Footprinting and Multimolecular Complexes

In all footprinting experiments, it is essential that the ligates are used at concentrations > Kd for the ligand, when the ligate will occupy >50% of the binding sites on the ligand. The main measurement made in footprinting experiments is the extent of ligate bound, which requires the binding reaction to be at or near equilibrium. Consequently, these experiments take a relatively long time. The extent of binding is calculated in two ways, the results of which should be identical. First, the extent of ligate binding can be determined directly from the binding curve, where it is equal to the response at equilibrium minus the response before the addition of ligate, minus the bulk shift. Second, the nonlinear curve-fitting software supplied by the manufacturer is used to calculate the extent of binding from the association curve.

The protocols described in Figs 5 and 6 can be adapted for the other instruments, though the high flow rates required for efficient mixing will require quite extensive use of ligates.

3.5.2. Footprinting with a Competitor of the Ligate

These experiments test the hypothesis that two ligates bind to sites on the ligand that are the same, overlap, or interfere with each other. In the example shown (see Fig. 5), the two ligates are bFGF and a synthetic peptide bFGF(127-140). The experiment illustrates how the dissociation rate constant of a ligate determines the experi-

Fig. 3. A binding assay. Binding of bFGF (final concentration 3 ^g/mL or 167 nM) to biotinylated heparan sulfate from Rama 27 cell culture medium, immobilized on a streptavidin derivatized carboxymethyl dextran cuvet. A response of 200 arc seconds is equal to 1 ng/mm2 of protein in the carboxymethyl dextran gel. The binding curve is described by a series of events (arabic numerals) and regions (letters). Event 1: Addition of ligate. Binding is initiated by the addition of 1 ^L of 90 ^g/mL bFGF to the 29 ^L of PBST in the cuvet. Event 2: PBSTwashes. At the end of the association reaction, the cuvet is washed 3 times with 50 ^L of PBST to initiate the dissociation reaction. Event 3: Regeneration. To regenerate the surface, the cuvet is washed 3 times with 2 M NaCl, 10 mM NaHPO4, pH 7.2, and left in this solution for 1 min. Event 4: Return to starting conditions. The cuvet is washed 3 times with 50 ^L of PBST and then once with 29 ^L of PBST. Region A is the baseline, 29 ^L of PBST in the cuvet. Region B is the 3-5 s region immediately after the addition of ligate (event 1), where mixing and bulk shifts occur—the latter result from the difference in refractive indices of PBST and PBST containing 3 ^g/mL bFGF. Region C is the association phase from which the kon and the extent of binding are calculated. Region D is the 3-5 s region immediately following the PBST washes (event 2), where mixing and bulk shifts occur. Region E is the dissociation phase from which kdiss is calculated. Region F is regeneration, which is initiated by the addition of 2 M NaCl (event 3). Region G is the new baseline with 29 ^L of PBST in the cuvet (event 4).

mental protocol. The bFGF dissociates slowly from heparin, whereas the peptide bFGF(127-140) dissociates more rapidly (4).

In the first footprinting experiment (Fig. 5, 7.9-104 min), once bFGF binding has reached a maximum, dissociation is initiated (Fig. 5, 29.7 min). At 43.8 minutes, the dissociation of bound bFGF from the immobilized heparin is negligible compared to the association of the peptide bFGF(127-140), so the peptide is added. In the second experiment (116.9-206.1 min) the order of addition of the ligates is reversed. This experiment has to contend with the relatively fast dissociation rate of the peptide from heparin. After the binding of the peptide has reached a maximum, 1 ^L of the bulk phase is replaced with 1 ^L of an identical solution (PBST containing 100 ^g/ mL bFGF (127-140) and 90 ^g/mL bFGF. In this way the equilibrium between the soluble and bound peptide is only perturbed by the bFGF binding to the immobilized heparin.

Baseline (regions A, G) PBST

Association phase (regions B, C) PBST

Baseline (regions A, G) PBST

sensor surface wash event with PBST

sensor surface

Association phase (regions B, C) PBST

(bFGF)

add bFGF ^

YbFGF)

event (1)

XT

sensor surface

wash event with PBST

wash event with PBST

wash event with PBST

Regeneration phase (region F) Dissociation phase (regions D, E) 2 M NaCl

Regeneration phase (region F) Dissociation phase (regions D, E) 2 M NaCl

Fig. 4. Schematic of molecular changes occurring at the sensor surface in the course of the experiment in Fig. 3. SA, streptavidin.

The concentration of the ligates and their molecular weight have a considerable effect on this type of footprinting experiment. The concentrations of peptide and bFGF relative to their Kd for heparin (4) were chosen such that the peptide in the first experiment (43.8 min) could displace bFGF, but in the second experiment bFGF was unlikely to displace the peptide. The signal produced by proteins and nucleic acids in optical biosensors is directly proportional to molecular weight. In this example the molecular weights of bFGF and bFGF(127-140), 18 and 1.6 kDa, respectively, differ by over 10-fold. Thus displacement of bFGF in the first experiment (43.8 min) by the peptide would cause a large decrease in signal, whereas bFGF is unlikely to displace the peptide in the second experiment and cause an increase in signal. Other experiments, however, may be set up differently due to the type of ligates, and the results not be so clearcut. In these cases, if ligate dissociation is slow, antibodies can be used at the end of the experiment to quantify the amount of each ligate that is bound. Thus an antibody to bFGF could be added at 94.1 min, and once this has bound maximally, antibody to the peptide would be added.

time (min)

Fig. 5. Footprinting with a competitor of the ligate in an IAsys optical biosensor. In the first experiment, the extent of binding of bFGF to biotinylated porcine mucosal heparin, captured on streptavidin immobilized on a carboxymethyl dextran surface, is measured. Then the synthetic peptide bFGF(127-140) is added to determine the extent of binding of the peptide to porcine mucosal heparin in which over half the binding sites or bFGF are occupied. In the second experiment the order of addition of the ligates is reversed, so the synthetic peptide is added first, followed by bFGF. This experiment measures the extent of binding of the synthetic peptide to porcine mucosal heparin and the extent of binding of bFGF to porcine mucosal heparin in which over half the peptide binding sites are occupied. The table below is a detailed description of experiment.

Calculation of extent of binding: In the first experiment the bulk shifts do not have to be explicitly subtracted, since the two binding reactions start and finish in PBST. The extent of binding of bFGF and the bFGF(127-140) peptide is denoted by the vertical two headed arrows. In the second experiment, the situation is a little more complex, since the cuvette is not returned to PBST after the bFGF(127-140) peptide has bound, due to the relatively fast rate of dissociation of the peptide-heparin complex. In this case the bulk shift is taken as the first 5 s of the binding reaction and an offset baseline (horizontal dotted lines) is used to calculate the extent of binding (two headed arrow).

Time (min)

Event

0

Establish a baseline with 29 ^L of PBST.

7.9

Add 1 ^L of 90 ^g/mL bFGF to the cuvet.

29.7

Maximal binding of bFGF, wash 3 x with 50 ^L of PBST; leave the

cuvet in 29 ^L of PBST.

43.8

Add 1 ^L of 3 ^g/mL bFGF(127-140) peptide.

77.7

Maximal binding of bFGF(127-140).

77.7

Wash 3 x with 50 ^L of PBST; leave the cuvet in 29 ^L of PBST.

94.1

Regenerate with multiple washes of 2 M NaCl.

104

Establish a baseline in 29 ^L of PBST.

116.9

Add 1 ^L of 3 mg/mL bFGF(127-140) peptide.

156.8

Maximal binding of bFGF(127-140); remove 1 ^L from the cuvet.

156.8

Add 1 ^L of 90 ^g/mL bFGF in PBST containing 100-^g/mL

bFGF(127-140).

184.6

Wash 5 x times with PBST and allow dissociation to proceed.

206.1

Regenerate with multiple washes of 2 M NaCl to recover the baseline.

HARP

+ 5 ng/ml heparin

,+ 500 ng/ml heparin

,+ 500 ng/ml heparin

HARP

+ 5 ng/ml heparin

time (min)

Fig. 6. Footprinting with a competitor of the ligand. In the control binding reaction, 3 ^L HARP (20 ^g/mL in PBST) is added to a cuvet containing 27 ^L of PBST. In the subsequent competitions, 3 ^L of PBST containing 20 ^g/mL HARP and 50-ng/mL, 5 ^g/mL, or 50 ^g/mL heparin is added. The signal from the last experiment is indistinguishable from the background. The fourth binding curve, which is again indistinguishable from background, controls for any signal that might be generated by the competitor alone; in this case 3 ^L of PBST containing no HARP and 50 ^g/mL of heparin is added.

3.5.3. Footprinting with a Competitor of the Ligand

These experiments test the hypothesis that the region of the ligate that recognizes the ligand can also recognize the soluble competitor. In the example shown (see Fig. 6), the effect of heparin on the binding of heparin affin regulatory peptide (HARP) (5,6) to immobilized heparin is determined.

3.5.4. Multimolecular Complexes

The analysis of multimolecular complexes, for example where ligate (1) binds to heparan sulfate and ligate (2) binds to ligate (1), is a simple extension of the above footprinting experiments. The only constraint is that the dissociation rate of ligate (1) from the ligand is sufficiently slow (as in the case of bFGF in Fig. 5) to allow the cuvette to be washed and returned to binding buffer prior to the addition of ligate (2).

3.5.5. Kinetics

One of the key uses of optical biosensors is the rapid determination of the kinetics of a molecular interaction. The instrument manufacturers provide ample information on the theoretical considerations behind the measurements of binding kinetics. This section will therefore deal only with artefacts.

The major artefact associated with the determination of kinetics in optical biosensors is the generation of second phase binding kinetics by diffusion limitations (the so-called mass-transport artefact) and steric hindrance (1,7). Diffusion limitations arise when the rate of diffusion of the soluble ligate (dependent on its diffusion coefficient, D) from the bulk, stirred solution, through the boundary layer of immobile solution, which exists next to the surface of the sensor, is equivalent or slower than the apparent on rate, kon. In this case, after the initial rapid depletion of soluble ligate from the solution near the surface, the observed association kinetics reflect diffusion rates rather than association rates. The diffusion artefact also affects the measurement of the dissociation rate constant, kdiss. kdiss should be independent of ligate concentration. If kdiss is found to increase with increasing ligate concentration, it is likely that dissociated ligate is rebinding to unoccupied ligand faster than it is diffusing into the bulk phase. Multivalent ligates, which possess a high avidity, represent a special case of this effect. The steric hindrance of binding sites arises when the immobilized ligand is at a high density and/or randomly oriented, and is most prominent on 3-dimensional carboxy-methyldextran surfaces (7).

There are two ways to investigate whether diffusion is indeed rate-limiting:

1. Decreasing the rate of flow or of stirring to determine the rate at which diffusion becomes limiting. A drawback is that this is rather insensitive and in stirred systems the relationship between diffusion and the rate of stirring may not be linear.

2. Increasing the viscosity of the binding buffer (diffusion is inversely proportional to viscosity) by the addition of glycerol. This is the more sensitive method.

To avoid these possible artefacts, experimental design should always include the following:

1. The minimum amount of ligand required to give a useful signal should be immobilized. This is determined empirically.

2. Oriented immobilization of ligand rather than random reduces steric hindrance between the surface and the binding site on the immobilized ligand.

3. Since kon depends directly on concentration, keeping the concentrations of ligate as low as possible reduces the possibility of the rate of diffusion controlling the binding reaction.

4. To avoid rebinding during dissociation, kdiss should be determined at ligate concentrations where the majority of the binding sites are occupied. If rebinding is still a problem, soluble ligand can be added as a competitor to the dissociation buffer [e.g., (8)].

5. Diffusion into the bulk phase from the stationary phase is faster with planar than three-dimensional, e.g., carboxymethyl dextran, surfaces, so some experiments should always use a planar surface (7).

3.6.6. Microaffinity Chromatography

Optical biosensors provide the opportunity to carry out microaffinity chromatogra-phy. In these experiments, the immobilized ligand is used to fish for a specific target in a mixture. Once the target is bound, the regeneration step is used to elute the target into the bulk phase. Recovery of the target is simplest in cuvet-based instruments. IAsys cuvets come in analytical and preparative formats. Analytical surfaces have a mask that covers all but 4 mm2 of the surface, so the total amount of ligand is low, thus preventing depletion of ligate from the bulk phase during the binding reaction. Preparative surfaces ("Select") do not have the mask and the area of the surface is 16 mm2, which allows the immobilization of fourfold more ligand and the capture of a correspondingly larger amount of ligate. Microaffinity chromatography can be used either as a means to identify the steps required to prepare an efficient conventional

Fig. 7. Selection of phage that bind heparin. Biotinylated porcine mucosal heparin was immobilized on a biotin select surface. Wild-type phage do not bind to this surface at NaCl concentrations as low as 10 mM (Rahmoune, H., and Fernig, D. G. unpublished). The cuvet is cleaned with phenol prior to the addition of phage, to eliminate contaminating wild-type phage from the environment and all solutions are autoclaved prior to use. Phage from the initial library (70 ^L at 1011 pfu/mL) are added to the cuvette (0 min, thin line). After a large bulk shift due to the high concentration of phage, a low but significant amount of binding is observed (about 20 arc s). After washing the surface with PBST, bound phage (A, the first generation) are eluted (equivalent to surface regeneration) with either 2 M NaCl or 1 mg/mL heparin in PBST, both of which were equally effective. These first-generation phage are grown up and added (70 ^L at 1011 pfu/mL) to the same surface (-2 min, thick line). Clearly the first generation is enriched in phage that are able to bind to the heparin compared to the initial library, since the extent of binding is double and the kon is considerably faster. Bound phage (B, the second generation) were collected as before and grown up.

Fig. 7. Selection of phage that bind heparin. Biotinylated porcine mucosal heparin was immobilized on a biotin select surface. Wild-type phage do not bind to this surface at NaCl concentrations as low as 10 mM (Rahmoune, H., and Fernig, D. G. unpublished). The cuvet is cleaned with phenol prior to the addition of phage, to eliminate contaminating wild-type phage from the environment and all solutions are autoclaved prior to use. Phage from the initial library (70 ^L at 1011 pfu/mL) are added to the cuvette (0 min, thin line). After a large bulk shift due to the high concentration of phage, a low but significant amount of binding is observed (about 20 arc s). After washing the surface with PBST, bound phage (A, the first generation) are eluted (equivalent to surface regeneration) with either 2 M NaCl or 1 mg/mL heparin in PBST, both of which were equally effective. These first-generation phage are grown up and added (70 ^L at 1011 pfu/mL) to the same surface (-2 min, thick line). Clearly the first generation is enriched in phage that are able to bind to the heparin compared to the initial library, since the extent of binding is double and the kon is considerably faster. Bound phage (B, the second generation) were collected as before and grown up.

chromatography column or as the preparative chromatography step itself. The only constraint on the use of optical biosensors as microchromatography systems is that nonspecific binding is undetectable.

The example shown is an experiment that used the optical biosensor to isolate hep-arin-binding phage from a library that displayed a constrained 7-amino acid peptide (see Fig. 7 and Chapter 50). Twenty clones were isolated from the second-generation phage and, compared to the initial starting library, the DNA sequences encoding the peptide library in second-generation phage always contained codons for basic amino acids, illustrating the success of the method. The clear advantage of using the optical biosensor as a microaffinity chromatography system is that the instrument provides a readout in real time of the binding events. It is therefore possible to troubleshoot protocols in real time and thus save considerable amounts of time and precious materials. In addition, since nonspecific binding is negligible, the fold-purification achieved is dramatic. The major inconvenience is that only small amounts of material are recovered, which require either a simple amplification step (as in the case of the phage) or suitable downstream microanalytical facilities.

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