Control of antibody–antigen interaction using anion-induced conformational change in antigen peptide

Yoshio Katakura1, Takahiro Miyazaki, Hitomi Wada, Takeshi Omasa, Michimasa Kishimoto, Yuji Goto2 and Ken-ichi Suga

Department of Biotechnology, Graduate School of Engineering and 2 Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The binding of a monoclonal antibody to an epitope peptide was controlled by the conformational change of the epitope peptide induced by anions. We synthesized peptides in which the epitope sequence DTYRYI for the monoclonal antibody AU1 is located between amphiphilic peptides (KKLL)n and (LLKK)n. In the absence of an appropriate anion, the peptide was in a random coil state and the epitope was linear. In contrast, in the presence of an appropriate anion, the peptide exhibited an anti-parallel {alpha}-helical structure and the epitope was subsequently `bent'. In the presence of 41 µM triphosphate, the association constant between the antibody and the peptide was decreased by one order of magnitude in the case of n = 3 and at least three orders of magnitude in the case of n = 4 or 5. Oligo-DNAs, as anions, dissociated the antibody–peptide complex, whereas triphosphate did not. The DNA concentrations required for 50% dissociation of the antibody–peptide complex at pH 7.5 were 4x10–8, 1x10–7 and 6x10–6 M for decamer, octamer and hexamer DNA, respectively.

Keywords: affinity control/anion/antibody-antigen interaction/anti-parallel {alpha}-helical structure/fluorescence polarization


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The equilibrium of a reaction in which a complex (PL) is formed between a protein (P) and its ligand (L) is greatly shifted to the right under natural conditions:

Because of this one-sided balance, the specific reactions of a protein with its ligand, such as receptor–ligand and antibody–antigen reactions, assume important roles in vivo and have been applied in vitro for quantification, detection and separation. Control of these specific reactions is important for extending their applications. To shift the equilibrium to the left, it is necessary to reduce the concentration of the free protein and/or the free ligand or to reduce the affinity between the protein and the ligand. In the case of antibody–antigen interactions, the addition of competitive analogs of the antigen can shift the equilibrium to the left by decreasing the concentration of the free antibody. However, it is difficult to remove the analogs that bind strongly to the antibody. Although acidification or the addition of a strong chaotropic ion or a denaturant can dissociate the antibody–antigen complex, these harsh operations disrupt not only the intermolecular interaction between the antibody and the antigen but also the intramolecular interaction of the antibody and/or antigen. This disordered disruption of the intramolecular interaction sometimes causes an irreversible conformational change of the antibody and/or the antigen. Here, we report a method of controlling antibody–antigen interactions via an ordered reversible conformational change in the antigen peptide under natural conditions.

A peptide containing two tandem repeats of an LLKK sequence in a random coil state exists in the absence of anions owing to the positive charges on lysine residues repulsing each other. An appropriate anion that negates this repulsion induces the formation of an {alpha}-helix in the sequence. Subsequently, the hydrophobic interaction between leucine residues induces the formation of an anti-parallel {alpha}-helical structure in the peptide, at peptide concentrations in which association between peptide molecules is negligible (Goto and Aimoto, 1991Go; Goto et al., 1991Go; Hoshino and Goto, 1994Go; Hoshino et al., 1997Go). In the case of anti-peptide antibodies, an epitope peptide consisting of several amino acid residues is recognized by a groove of the antibody (MacCallum et al., 1996Go). Since a majority of the residues of the epitope are in contact with the antibody (Geysen et al., 1987Go; Lim et al., 1990Go), if the epitope bends and the conformation of the epitope is restricted, part of the epitope residues cannot contact the antibody and then the affinity between the antibody and the epitope decreases.

We synthesized a peptide in which the epitope sequence DTYRYI for the monoclonal antibody AU1 (Lim et al., 1990Go) is located between (KKLL)n and (LLKK)n (n = 3, 4, 5). In the absence of an appropriate anion, the antibody is expected to associate freely with the peptide because the peptide is in a random coil state and the epitope can be linear. In contrast, in the presence of an appropriate anion, the peptide exhibits an anti-parallel {alpha}-helical structure. Since the epitope is now `bent' and cannot fit into the groove of the antibody, the antibody dissociates from the peptide (Figure 1Go).



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Fig. 1. Control of antibody–antigen interaction. Interaction between the peptide and the antibody is controlled via the formation of an anti-parallel {alpha}-helix in the peptide induced by an anion. The bold line indicates the epitope sequence.

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Peptide synthesis and fluorescein labeling

The peptides, AG(KKLL)nDTYRYI(LLKK)nGAGC(Acm) (n = 0, 3, 4, 5), were prepared using a 9050 Plus PepSynthesizer (Millipore, Milford, MA) according to the manufacturer's standard Fmoc protocol using the PEG-PS support. When the peptides were labeled with fluorescein, the N-terminal amino group was labeled with fluorescein succinimidyl ester prior to the deprotection of the side chain, as follows. A 10 mg amount of the dried support, on which about 1 µmol of the peptide was synthesized, was resuspended in 100 µl of dimethylformamide and degassed. A 4 µmol amount of fluorescein succinimidyl ester dissolved in 25 µl of dimethyl sulfoxide was added to the suspension and reacted for 4 h in the dark at 37°C. The support was washed three times with dimethylformamide and once with dichloromethane by centrifugation (1000 g, 2 min) and then vacuum dried. The labeled and unlabeled peptides were deprotected and cleaved according to the manufacturer's instructions and then purified on a C18 reversed-phase column (µBondapack C18 Semiprep, 30 cmx7.8 mm i.d.) (Millipore). Synthesis and labeling were confirmed by mass spectrometry (Voyager RP, PerSeptive Biosystems, Foster City, CA).

Preparation of the antibody

Antibody AU1 prepared from ascites fluid was purchased from BAbCO (Richmond, CA). The antibody was loaded on to the antigen column in which 280 nmol/ml resin of the peptide (n = 0, unlabeled) was immobilized on cyanogen bromide-activated Sepharose 4B (Pharmacia, Uppsala, Sweden). After washing the column extensively with phosphate-buffered saline (PBS) containing 0.5% (w/v) Tween 20 and then with PBS, the bound antibody was eluted with 0.1 M glycine–HCl (pH 2.7). Following neutralization with Tris base, the eluted antibody was concentrated and washed with 10 mM Tris–Cl (pH 7.5) using an Ultrafree UFC3TTK (Millipore). The antibody concentration was estimated from the absorbance at 280 nm.

Circular dichroism (CD) measurements

CD measurements of the unlabeled peptides were carried out at 37°C in 10 mM Tris–Cl (pH 7.5) with a Jasco Model J-500A spectropolarimeter. The results were expressed as mean residue ellipticity [{theta}], which is defined as [{theta}] = 100{theta}obsd/lc, where {theta}obsd is the observed ellipticity in degrees, c is the concentration in residue mol/l and l is the length of the light path in cm. Peptide concentration was determined by amino acid analysis.

Binding assay

The binding of the peptide to the antibody was detected by a fluorescence polarization assay (Lundblad et al., 1996Go; Katakura et al., 1997Go) using a Beacon 2000 fluorescence polarization system (PanVera, Madison, WI). When fluorescent molecules are excited with plane-polarized light, they emit light in the same polarized plane, provided that the molecules remain stationary throughout the excitation. Since excited molecules actually rotate or tumble out of the plane of polarized light while they are in the excited state, the intensity of the emission in a plane different from that of the initial excitation increases depending on the rotational Brownian movement of the molecules. Since the movement of the molecules depends on their molecular size, the degree of the fluorescence polarization, defined by , reflects molecular size, where IH and IV are intensities of the emission observed through a horizontal and a vertical polarizer, respectively. That is, when the fluorescently labeled peptides dissociate from the antibody (or the peptides are monomers), P is low. In contrast, when the peptides associate with the antibody (or the peptides are polymerized by anions), P increases because the apparent molecular size of the labeled peptide increases.

A 1 ml volume of 10 mM Tris–Cl (pH 7.5 at 37°C) containing AU1 or the unlabeled peptides, at various concentrations (0.4 to 210 nM or 0 to 8 µM, respectively), was prepared in a borosilicate test-tube (PanVera). After measuring the background intensity, the fluorescently labeled peptide was added to a final concentration of 7 nM. After 10 min of equilibration at 37°C, fluorescence polarization was measured using the three-read cycle mode. Affinity constants were estimated using a non-linear least-squares method.

Oligo DNAs

An equimolar mixture of synthesized 5'-AmCm and 5'-GmTm(0.2 mM each in 0.1 M Tris–Cl, 0.3 M NaCl, 5 mM MgCl2, pH 7.6, m = 3, 4, 5) was annealed by boiling and gradual cooling.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Formation of {alpha}-helical structure

It has been reported that the anion concentration required to induce {alpha}-helix formation of peptides containing the sequence LLKK depends on the species of the anion and that a micromolar concentration of triphosphate is sufficient since multivalent anions have a stronger effect than monovalent anions (Goto and Aimoto, 1991Go). Figure 2Go shows the CD spectrum of the peptide with n = 4 in the presence of various concentrations of triphosphate. The ellipticity observed at 222 nm induced by 41 µM triphosphate was –21 000° cm2/dmol. Similar results were observed in the cases of n = 3 and n = 5 (data not shown). These results indicate that our peptides containing the sequence LLKK exhibit an {alpha}-helical structure in the presence of 41 µM triphosphate.



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Fig. 2. Circular dichroism spectrum of the peptide (n = 4) in 10 mM Tris–Cl (pH 7.5 at 37°C). The numbers represent triphosphate micromolar concentrations.

 
Effects of anions on the association of antibody with peptide

Figure 3Go shows the binding of the antibody to the peptides, expressed as an increase in polarization (Lundblad et al., 1996Go; Katakura et al., 1997Go), in the absence or presence of 41 µM triphosphate. The affinity constant between the peptide of n = 3 and the antibody was decreased by one order of magnitude by 41 µM triphosphate (Figure 3BGo). The effect of triphosphate was clearer for the peptide of n = 4, where no binding was detected with up to 210 nM antibody in the presence of 41 µM triphosphate (Figure 3CGo). In the case of n = 5, the results were similar to those for n = 4 (data not shown). Since the affinity constant between the antibody and the peptide without the LLKK sequence (n = 0) was only 20% smaller in the presence of 41 µM triphosphate than in its absence (Figure 3AGo), the anion did not directly decrease the affinity between the peptides and the antibody.



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Fig. 3. Binding of peptides (n = 0, 3, 4) to antibody. {blacktriangleup}, In the presence of 41 µM triphosphate; •, in the absence of triphosphate.

 
Although we assume that the peptide exhibits an anti-parallel {alpha}-helical structure in the presence of triphosphate, other structures in the peptide to which the antibody does not bind should likewise be considered. Since multivalent anions such as triphosphate are known to oligomerize basic materials, the binding of the antibody to the peptide might be prevented by the steric hindrance caused by the oligomerization of the peptide in the presence of triphosphate. Oligomerization of the peptide can be monitored from the increase in fluorescence polarization of the labeled peptide. The increase in polarization of the peptide was observed only at peptide concentrations >10–6 M in the presence of 41 µM triphosphate (Figure 4Go). Under conditions where the anion is present in excess compared with the peptide, oligomerization should not occur because the excess anion molecules surround each peptide molecule. Therefore, we concluded that the effect of oligomerization of the peptide in our study is negligible because the peptide concentration was only 7 nM in our experiments.



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Fig. 4. Polymerization of peptide (n = 4). In the presence ({blacktriangleup}) or absence (•) of 41 µM triphosphate, various concentrations of the unlabeled peptide (n = 4) were added to the labeled peptide (n = 4).

 
Goto and Aimoto (1991) reported that 10–3 M monophosphate is necessary to induce {alpha}-helix formation in their peptide. When triphosphate, at a sufficient concentration to induce the formation of an {alpha}-helix, is degraded to monophosphate by the enzyme phosphatase, the antibody should bind again to the peptide, even though the molar anion concentration is increased threefold as a result of the degradation. The polarization of the labeled peptide solution (n = 4, antibody concentration 210 nM) containing 41 µM triphosphate or 123 µM monophosphate was 0.14 or 0.25, respectively. These results indicate that the peptide of n = 4 associated with the antibody in the presence of 123 µM monophosphate but not in the presence of 41 µM triphosphate. When phosphatase was added to a solution containing 41 µM triphosphate, the polarization of the solution increased with time and reached 0.25 (Figure 5Go). This increase in polarization was due to the binding of the antibody and not to the binding of phosphatase to the peptide because the polarization did not increase when phosphatase was added to the solution containing 123 µM monophosphate.



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Fig. 5. Binding of the antibody to the peptide by the degradation of triphosphate. The polarization of the peptide (n = 4) solution containing 210 nM antibody in the presence of 41 µM triphosphate or 123 µM monophosphate is indicated by a {triangleup} or {circ}, respectively. Filled symbols represent the time course of the polarization of each solution following the addition of 0.2 units of alkaline phosphatase (Takara Shuzo, Kyoto, Japan).

 
Effects of anions on the dissociation of the antibody–antigen complex

The antibody (210 nM ) was initially incubated with the labeled peptide (n = 4) and then anion solutions were added. Contrary to our expectations, when triphosphate was added to give a final concentration of 41 µM, the polarization of the labeled peptide did not decrease. Even in the presence of 10 mM triphosphate, the antibody did not dissociate from the peptide (data not shown). This indicates that triphosphate prevents the association of the antibody to the antigen but does not dissociate the antibody–antigen complex. This hysteresis might be due to an induced fit of the antibody to the antigen. To dissociate the antibody–antigen complex, we increased the chain length of the anion. Since it is difficult to obtain polyphosphates whose chain length is greater than that of triphosphate, we used oligo-DNAs as anions. Figure 6Go shows the results in which double-stranded DNAs were used as anions. In the absence of the antibody, the polarization of the peptide (n = 4) increased from 0.12 to 0.2 at decamer DNA concentrations between 10–8 and 10–7 M ({triangledown}), indicating that the DNA binds to the peptide and subsequently the epitope is expected to be bent. In the presence of the antibody, the polarization of the labeled peptide was 0.31 at decamer DNA concentrations <10–8 M but decreased to 0.11 at concentrations >10–7 M ({triangleup}). {Delta}P ({blacktriangleup}), which indicates the change in the apparent molecular size of the peptide by the binding of the antibody, was calculated by subtracting the polarization without antibody from that with antibody. The decrease in {Delta}P from 0.2 to 0 at DNA concentrations between 10–8 and 10–7 M indicates that the antibody dissociates from the peptide by the binding of the decamer DNA to the peptide. In the cases of octamer and hexamer DNAs (• and {blacksquare}, respectively), almost the same results as in the case of decamer DNA were obtained, although the DNA concentration required for the dissociation of the complex increased with decrease in DNA chain length. The DNA concentrations required for 50% dissociation of the antibody–peptide complex were calculated to be 4x10–8, 1x10–7 and 6x10–6 M in the cases of the decamer, octamer and hexamer DNAs, respectively.



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Fig. 6. Dissociation of the antibody–peptide (n = 4) complex by an oligo-DNA. Various concentrations of a decamer DNA were added to the solution containing the labeled peptide ({triangledown}) or the solution containing the antibody (210 nM) and the peptide ({triangleup}). The {blacktriangleup}, • and {blacksquare} indicate the difference in the polarization of the peptide with and without antibody in the case that decamer, octamer and hexamer DNAs were used as anions, respectively.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the presence of 41 µM triphosphate, the peptide exhibited an {alpha}-helical structure and the association of the peptide and the antibody was prevented. In the case when n >= 4, the affinity constant of the peptide for the antibody was decreased by at least three orders of magnitude in the presence of 41 µM triphosphate, although the decrease when n = 3 was one order of magnitude. Together with the result that the antibody–peptide (n = 4) complex is dissociated by the presence of oligo-DNA (Figure 6Go), four repeats of LLKK corresponding to four rounds of {alpha}-helix are necessary and sufficient to control the antibody–peptide interaction. When triphosphate was degraded to monophosphate by phosphatase, the antibody associated again with the peptide (Figure 5Go). These results indicated that the interference of the association of the antibody with the peptide by anions is reversible.

Two structures of the peptides to which the antibody does not bind are possible other than the anti-parallel {alpha}-helical structure. One is the oligomerization of the peptides by electrostatic or hydrophobic interaction. As shown in Figure 4Go, however, the peptides oligomerized at concentrations of almost the same order of magnitude as that of triphosphate but not at lower concentrations. No increase in polarization was observed at any concentrations of the peptides in the absence of triphosphate. These results show that the peptides do not oligomerize by either electrostatic or hydrophobic interaction at the peptide concentration used in this study (7 nM). The other is the conformational change of the epitope sequence induced by the formation of the {alpha}-helical structure in the LLKK sequence. Leder et al. (1995) reported a monoclonal antibody that forces an antigen peptide to exhibit an {alpha}-helical structure. The antibody was raised against a peptide that exhibits an {alpha}-helical structure under physiological conditions and the antibody cross-reacted with peptide containing two amino acid substitutions to proline which breaks {alpha}-helical structures. When their antibody bound to the substituted peptide existing in a random coil state under physiological conditions, binding of the antibody induced the formation of an {alpha}-helical structure in the substituted peptide. In contrast, if the AU1 antibody recognizes a non-helical and not a helical epitope, anions may force the epitope to exhibit the {alpha}-helical structure, resulting in a decrease in affinity between the antibody and the peptides. However, based on the prediction of the secondary structure of the epitope sequence by both the Chou–Fasman method (Chou and Fasman, 1978Go) and the Robuson method (Garnier et al., 1978Go), the potential for the formation of {alpha}-helical structures in the epitope is low (data not shown). Furthermore, if the peptide is linear, it is unfavorable from the viewpoint of free energy because all the leucine residues are exposed to the solvent. Accordingly, it is most likely that the decrease in affinity between the antibody and the peptide by the anions is due to the formation of the anti-parallel {alpha}-helical structure in the peptide. In conclusion, polyanions interfere with the association of the antibody with the peptide by inducing a conformational change in the peptide from the random coil state to the anti-parallel {alpha}-helical structure.

Triphosphate prevented the association of the antibody with the peptide. However, once the antibody had associated with the peptide, triphosphate did not dissociate the peptide from the antibody even at 10 mM, although oligo-DNA dissociated it at concentrations <1 µM. The difference in the effect between the oligo-DNAs and triphosphate can be explained as follows. The distance between the ith lysine and the i + 4th (or i + 3rd) lysine in the LLKK sequence is 6–7 Å (Figure 7Go). Since the distance between the phosphorus atoms on both ends of the triphosphate molecule is about 6 Å, at least n molecules of triphosphate are necessary to negate the positive charges in one (LLKK)n molecule. In contrast, since the distance between the adjacent phosphorus atoms in DNA is 6–7 Å, two (or one) double-stranded oligo-DNA molecules can negate the positive charges in one (LLKK)n molecule if the length of the oligo-DNA is more than n + 1 (both 3' and 5' ends of the synthesized oligo-DNAs used in this study were not phosphorylated). The anion–LLKK sequence complex should be upright in the case of oligo-DNA since each oligo-DNA molecule acts as a splinter for the LLKK sequence and can be flexible in the case of triphosphate since each triphosphate molecule binds to the LLKK sequence independently (Figure 8Go). As a result, in the case of triphosphate, the hydrophobic interaction between leucine residues existing near the epitope sequence cannot contribute to the bending of the epitope sequence. In the case of oligo-DNA, the hydrophobic interaction between leucine residues even at both ends of the peptide can contribute to the bending of the epitope sequence. Accordingly, the force required to change the conformation of the epitope sequence was sufficient to dissociate the complex in the case of oligo-DNA.



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Fig.7. Distance of positive or negative charges in the peptide or the anions.

 


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Fig. 8. Effects of triphosphate and oligo-DNA on the antibody–peptide complex. The bold line in the peptide indicates the epitope sequence.

 
Since the oligo-DNA is a long molecule, it is possible for it to bind to the peptide parallel to the epitope sequence (Figure 8Go, right). The antibody may not bind to the peptide owing to the steric hindrance caused by the oligo-DNA even when the epitope sequence is linear and not `bent'. However, the dissociation of the peptide–antibody complex was observed even in the case of hexamer DNA containing five phosphate groups in each strand. Since the lysine residues adjacent to both ends of the epitope sequence are separated by 10 residues of non-positively charged sequence (consisting of the epitope sequence and two leucine residues at both ends), the number of salt bridges between the lysine side chains and the phosphate groups of the oligo-DNA in the case when the oligo-DNA binds parallel to the epitope is smaller than that in the case when the oligo-DNA(s) binds parallel to the LLKK sequence. Accordingly, the oligo-DNA molecule should bind parallel to the LLKK sequence because the interval of positive charges in the {alpha}-helix of the LLKK sequence is almost the same as that of negative charges in the oligo-DNA.

As the antibody, we used AU1 because its epitope sequence is the shortest among commercially available antibodies. However, our method can be applied not only to other antibodies but also to peptide receptors because the epitope sequences can be determined by using phage display peptide libraries (Pasqualini et al., 1995Go; Yu and Smith, 1996Go). By connecting the LLKK sequence to both ends of the identified epitope sequence against the target molecule, one can construct a functional peptide. Then the interaction between the functional peptide and its target can be controlled by adjusting the concentration of anions. When one isolates an antibody recognizing an oligopeptide consisting of D-amino acids from phage antibody libraries (Winter et al., 1994Go) and connects it with LLKK consisting of D-amino acids, the resultant functional peptide will be tolerant to proteolytic degradation (Schumacher et al., 1996Go).

We controlled the antibody–antigen interaction using the anion-induced conformational change in the antigen peptide. Although the details of the mechanism involved in the dissociation of the antibody–peptide complex should be studied further, the conditions for the dissociation are different from those of conventional methods. Our method dissociates antibody–antigen complexes by inducing an ordered conformational change in the antigen at neutral pH and low ionic strength, whereas conventional methods, such as the addition of high concentrations of chaotropic ions or denaturants and extreme change of pH, induce a disordered conformational change of the antibodies and/or the antigens. Competitors added for the dissociation of antibody–antigen complexes bind to the antibodies instead of the antigens; in contrast, the anions bind to the LLKK peptide but not to the antibody. The anions that bind to the LLKK peptide can be removed easily by the addition of salts (e.g. 2 M NaCl; data not shown), whereas the competitors that bind strongly to the antibodies are difficult to remove.

Since the results shown in Figure 6Go demonstrate directly that the antibody–antigen interaction can be turned on and off, our method will have the following applications. Cells displaying a peptide receptor (e.g. a B cell displaying an antigen receptor) can be captured specifically by the functional peptide consisting of the epitope and LLKK sequences immobilized on a solid phase. The captured cells can be recovered without damage by the addition of an oligo-DNA because the concentration required for the dissociation of the receptor from the functional peptide is only <10–6 M. Enzymes can be modified by the functional peptide via its C-terminal Cys using bifunctional cross-linking reagents [e.g. N-succinimidyl-6-maleimidohexanoate (Bos et al., 1994Go) connects amines of proteins and Cys of the peptide]. The modified enzymes will be attached to the receptor on a solid phase and be removed by the addition of anions.


    Notes
 
1 To whom correspondence should be addressed. E-mail: katakura{at}bio.eng.osaka-u.ac.jp Back


    Acknowledgments
 
We thank Dr E.Fukusaki and S.Kajiyama for their helpful advice.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received May 17, 2000; revised July 17, 2000; accepted August 24, 2000.





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