Bioconjugste Chem. 1902, 3, 225-229
225
Effect of Different Binding Proteins on the Detection Limits and Sensitivity of Assays Based on Biotinylated Adenosine Deaminase Minas S. Barbarakis, Sylvia Daunert, and Leonidas G. Bachas’ Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506-0055.Received December 9,1991
The properties of binding proteins that control the nature and magnitude of inhibition of the enzymeligand conjugates in homogeneous enzyme-linked competitive binding assays were investigated. An assay for biotin that employed adenosine deaminase as the enzyme-label was used as a model system because of the availability of several biotin-specific binders with different characteristics. It was found that the association constant between the ligand and the binding protein, as well as the depth of the binding pocket, affect the response characteristics of the assay. In addition, the reported data suggest that steric-hindrance effects also contribute toward the sensitivity and detection-limit capabilities of the assay.
INTRODUCTION
EXPERIMENTAL PROCEDURES
Homogeneous enzyme-linked competitive binding assays have found numerous applications in bioanalysis. Usually, in these assays, the activity of an enzyme-labeled ligand (conjugate) is inhibited upon binding to a ligandspecificbinder (e.g., antibody, binding protein, lectin, etc.). The inhibition of the enzymatic activity can be modulated by a competition between the unlabeled ligand (analyte) and the enzyme conjugate for the binding sites of the binder (1,2). The sensitivity and detection limits of these assays are controlled by (a) the ability to measure low levels of enzymatic activity, (b) the extent of the change in the activity of the enzyme-ligand conjugate upon binding to the ligand-specificbinder, (c)the binding constant between the ligand and its specific binder, and (d) the assay susceptibility to interference (3). Consequently, a variety of factors (e.g., assay conditions, enzyme-label employed ( 4 ) , affinity of the binding system involved (5), amplification of the detection scheme (61,use of better transducers capable of discriminating small signal changes from background noise) have been considered in order to improve the sensitivity and detection limits of these assays
Reagents. The reagents and procedure for the isoelectric focusing experiments have been described previously (12). Avidin (egg white, lyophilized) was obtained from Calbiochem (La Jolla, CA) and streptavidin from InFerGene (Benicia, CA). 4’-Hydroxyazobenzene-2-carboxylate (HABA) and 1,2,4,5-benzenetetracarboxylic dianhydride (PMDA), a reactive form of pyromellitic acid (PML),were obtained from Aldrich (Milwaukee,WI). Sulfosuccinimidyl 6-(biotinamid0)hexanoate (biotin-LCNHS) was from Pierce (Rockford, IL). Tris(hydroxymethy1)aminomethane (Tris) (ultra pure) was obtained from Research Organics (Cleveland, OH). N-Hydroxysuccinimide biotin (biotin-NHS),adenosine deaminase (ADA) from calf intestinal mucosa (type VI), bovine serum albumin (BSA), N-hydroxysuccinimide (NHS), l-ethyl3-[3’-(dimethylamino)propyllcarbodiimide hydrochloride (EDAC), and folic acid were purchased from Sigma (St. Louis, MO). The assay buffer was 0.0100 M Tris-sulfate M ethylenediaminetetraacetic acid containing 6.5 X (EDTA), pH 7.40. The biotin and avidin standards were prepared in assay buffer containing 0.10% (w/v) BSA. Deionized (Milli-Q Water Purification System; Millipore, Bedford, MA) distilled water was used for all solutions. Apparatus. A Perkin-Elmer (Lambda 6)UV/vis spectrophotometer (Norwalk, CT) was employed for all spectrophotometric experiments. An isoelectric focusing apparatus (Model 111Mini IEF Cell; Bio-Rad, Richmond, CAI, along with a standard power supply (Gelman Instrument, Ann Arbor, MI), was used to determine the isoelectric points of the avidin conjugates. The electrophoresis was performed in a nonsieving medium (polyacrylamide gel) in the presence of a carrier ampholyte (Bio-Lyte 3/10 or Bio-Lyte 4/6from Bio-Rad). Preparation of the ADA Conjugates. The ADAbiotin conjugate was prepared as described by Kjellstrom and Bachas ( 4 ) using an initial enzyme/biotin mole ratio of 1/500. The ADA-LC-biotin conjugate was prepared in a similar way. The solutions of the enzyme conjdgates were stored at 4 “C. The enzymatic activity of the conjugates was monitored potentiotnetrically ( 4 ) . Preparation of the Avidin-PML Conjugates. Avidin was dissolved in aO.lOO M NaHC03 (pH 8.50) solution. A 30-fold excess of HABA was added to protect the binding sites of avidin from modification. Aliquots of 2.00 mL of the above solution were used for conjugation. To each aliquot containing 1.0 mg/mL of avidin were added various amounts of a freshly prepared aqueous solution of PMDA.
( 7-9).
Studies performed to elucidate the parameters that control the activity of the enzyme-ligand conjugates demonstrated that the inhibition is a result of either steric exclusion of the substrate from the active site of the enzyme or a conformational change of the enzyme molecule (3,10, 11). Therefore, the response characteristics of these assays may be affected by the ability of a particular binder to induce such conformational changes. In that respect, it is important to know how different properties of the binder affect the inhibition of the enzyme-ligand conjugates. In this report, several properties of the binder that may control the nature and magnitude of the inhibition have been investigated. The studies were performed by using an assay for biotin based on the enzyme adenosine deaminase (ADA). This assay was chosen as a model system because of the commercial availability of several well characterized biotin-specific binders (e.g., avidin, streptavidin, anti-biotin antibody). These binders differ in size, isoelectric point, depth of the binding pocket, and association constant with biotin. This investigation revealed that both the depth of the binding pocket of the ligandspecific binder and the modification of the side chains of its amino acids affect the response characteristics of homogeneous enzyme-linked competitive binding assays. 1043-1802192l2903-0225$03.00l0
0 1992 American Chemical Society
226
Barbarakls et 81.
Bioconjugate Chem., Vol. 3, No. 3, 1992
The mixture was incubated with magnetic stirring at room temperature for 4 h. The pH was maintained between 8.0 and 8.5 throughout the reaction by adjusting with 1.0 M NaOH. The reaction mixture was dialyzed against four changes of 1.0 L of 0.0100 M Tris-sulfate (pH 7.40). The avidin-PML conjugateswere characterized by determining their isoelectricpoint (PO. The higher the amount of PML attached to avidin, the lower the PIof the conjugate. Preparation of the Avidin-Folate Conjugates. The avidin-folate conjugates were synthesized by the N-hydroxysuccinimideester method. The activated folate ester was prepared as described by Bachas et al. (13). To prepare the avidin-folate conjugates, the activated folate ester solution was added in aliquots at 5-min intervals into a solution of 3.0 mg mL-l of avidin in 0.100 M NaHC03 (pH 8.50). The conjugation reaction was run under magnetic stirring in an ice bath for 4 h as described for the avidinPML conjugates. Three avidin-folate conjugates were prepared using initial mole ratios of 1/20,1/30 and 1/40 of avidinlfolate. Inhibition Studies for the ADA-Biotin System. One hundred microliters of variable concentrations of a biotin-specific binder, 100 pL of 2.4 X M ADA-biotin conjugate (or ADA-LC-biotin), 100 pL of assay buffer, and 200 p L of a 0.10% (w/v) BSA solution in assay buffer (to prevent nonspecific binding) were incubated together for 15 min. Then, 1.20 mL of 2.7 X 10-4 M adenosine was added to the assay cup, followed by 15 min of incubation. The enzymatic reaction was stopped by adding 100 pL of 0.100 M AgN03, and then the amount of NH4+ produced was determined. An inhibition graph was generated by plotting the percent inhibition observed vs the moles of binding sites in the assay mixture. The moles of binding sites were determined by titrating each binder with biotin and using HABA as an indicator (14). Dose-Response Curves. The dose-response characteristics of the assay were studied by incubating biotin standards with the enzyme-biotin conjugate and a biotinspecific binder (4). One hundred microliters each of the enzyme conjugate, binder, and biotin standard were incubated for 20 min prior to the addition of 1.20 mL of 2.7 X M adenosine. Dose-response curves were prepared by plotting the percent inhibition observed vs the logarithm of the concentration of the biotin in the standards. RESULTS AND DISCUSSION
In conventional homogeneous enzyme-linked competitive binding assays the activity of an enzyme-ligand conjugate is inhibited by a ligand-specific binder. The ideal enzyme conjugate for these assays should have high enzymatic activityand should be inhibited at or near 100% by an excess of binder. In this study, native and modified biotin-specific binders were employed in a homogeneous enzyme-linked competitive binding assay for biotin in an attempt to understand how the properties of the binder affect the inhibition of enzyme-ligand conjugates. Adenosine deaminase was reacted with biotin-NHS to form the respective ADA-biotin conjugate. This enzyme conjugate could be fully inhibited by either avidin or streptavidin. However, for the same concentration of conjugate the inhibition curve obtained by using streptavidin as the binder was shifted toward lower concentrations compared to the one obtained using avidin (Figure 1).As shown in Figure 1, a lower concentration of streptavidin compared to avidin is needed to achieve the same level of inhibition. Consequently, the dose-response curve obtained for streptavidin allows for the determination of
loo
"
1
1 I
.
.
.
.
. . . .,
I x 10-8
'
'
" . ' . . I
Ix
I x io-'
[Binding Sites]
Figure 1. Inhibition curves of the ADA-biotin conjugate obtained by using streptavidin ( 0 )and avidin (0). Error bars indicate f one standard deviation (n = 3).
0 I x 10-9
I x 10-8
I x IO-'
I x 10-6
[Biotin]
Figure 2. Dose-response curves for biotin obtained by using the ADA-biotin conjugate and streptavidin ( 0 )or avidin (0). Error bars indicate f one standard deviation (n = 3).
lower concentrations of biotin than the one for avidin (Figure 2). Specifically, the detection limit was found to be 1 X lo-' M and 2 X lo-* M biotin when using avidin and streptavidin, respectively. This observation provided the motivation for the present study, since a better understanding of the factors responsible for the lower detection limit obtained by using streptavidin may reveal ways to improve the detection limits of other homogeneous enzyme-linked competitive binding assays. Avidin and streptavidin are naturally occurring biotinspecific binders that, although they have very different origins, share the same high affinity for biotin. The dissociation constants of both proteins with biotin are on the order of 1 X lO-'5 M (15). With respect to physical properties, both molecules are approximately of the same size and both are tetramers comprised of identical subunits (15,161. The primary structures of the two proteins are almost homologous with respect to the amino acid residues involved in the construction of the biotin binding site ( 1 719). However, streptavidin is not a glycoprotein whereas avidin is. In addition, streptavidin has a p l of ca. 5 and therefore is negatively charged at pH 7.40; in contrast the p I of avidin is ca. 10, which causes avidin to be positively charged at pH 7.40 (20). Because of the large difference in the surface charge between streptavidin and avidin, it was thought that charge interactions may influence the ability of the binder to inhibit ADA-biotin. This led to the hypothesis that a modified avidin with reduced surface charge may improve the detection limits of the assay. In order to test this hypothesis, avidin was modified with PML by formation of an amide bond between the t-amino group of the lysine
Bloconjugate Chem., Vol. 3, No. 3, 1992 227
Inhlbltion of Blotinylated Enzymes
Table I. Isoelectric Points ( p f ) of Conjugates of Avidin with Pyromellitic Acid and Folate initial molar ratio conjugate PMDAIavidin folateiavidin pI avidin-PML-1 2i 1 9.3 avidin-PML-2 2011 8.3 avidin-PML-3 60i 1 5.7 2011 8.8 avidin-folate- 1 avidin-folate-2 3011 8.0 4011 6.0 avidin-folate-3
'" 1 80
0 E .c .-
e c
-
[Binding Sites]
Figure 4. Effect of the modification of the binder on the inhibition of ADA-biotin: avidin ( O ) ,avidin-folate-1 (e),avidinfolate-2 (a), and avidin-folate-3 ( 0 ) . Error bars indicate f one standard deviation (n = 3).
I x 10-0
I x 10-7
I x io-*
I x io-5
[Binding Sites]
Figure 3. Effect of the modification of the binder on the inhibition of ADA-biotin: avidin ( O ) ,avidin-PML-1 (m), avidinPML-2 (a),and avidin-PML-3 (+). Error bars indicate i one standard deviation (n = 3). residues on the avidin and PML. Three avidin-PML conjugates were prepared, which presented reduced PI values compared to that of avidin (Table I). The inhibition curves obtained with these modified binders demonstrate that a reduction of the positive surface charge of avidin by increasing the amount of conjugated PML (a fact indicated by the decrease in the PIvalue of the modified avidin) worsens the detection limit attainable by the system (Figure 3). This is also true for the modified avidin that had a PIless than the assay buffer (i.e., overall negatively charged under the conditions of the assay). Therefore, the observed differences in the inhibition caused by avidin and streptavidin cannot be attributed to differences in charge. The above data may be explained by considering that the PML molecules bound on avidin sterically hinder the binding between the enzyme-labeled biotin and avidin leading to assays with worse detection limits. These observations were further confirmed by modifying avidin with folic acid. Three avidin-folate conjugates were prepared that had a lower surface charge than avidin (Table I). In accordance to the results obtained with the avidin-PML conjugates, it was found that by decreasing the charge of avidin, the detection limits obtained with this system worsen as well (Figure 4). In addition and because of the bulkiness of the structure of folic acid (in comparison to PML), the induced steric hindrance in the interaction between the ADA-biotin and the modified avidin molecules was more pronounced when folate was used as the modifier. Indeed, a comparison of Figures 3 and 4 reveals that avidin-folate conjugates shift the inhibition curve to worse detection limits than avidin-PML conjugates with similar PIvalues. It should be noted that the affinity of avidin for biotin may decrease upon modification with PML and folic acid. In such an instance, the inhibition curve should also shift to worse detection limits, since it is known that the association constant between the binder and its specific ligand affects the detectability in binding assays (5). However,no significant difference in the inhibition curves
of ADA-LC-biotin obtained with avidin and avidin-folate3 was observed. ADA-LC-biotin was prepared from a derivative of biotin that contains one aminopentanoic acid residue attached to the carboxylic group of biotin by an amide bond. This provides an additional spacer of seven atoms between the attached biotin and the enzyme. The fact that the long-chain conjugate (ADA-LC-biotin) was not as affected by the modifications of avidin as was the short-chain conjugate (ADA-biotin) further supports the steric hindrance argument. Finally, control experiments indicated that an excess of either PML or folic acid does not affect the inhibition of ADA-biotin conjugate by avidin in solution. On the basis of the above results, it may be concluded that the increased inhibition of biotinylated ADA by streptavidin in comparison to avidin does not arise from its lower surface charge. Further, it should be mentioned that nonglycosylated avidin has been reported to have the same biotin-binding properties as native (glycosylated) avidin (21). Therefore, the difference in the carbohydrate content between the two proteins may not account for the displacement of the inhibition curves depicted in Figure 1. Rather, a possible explanation could involve the consideration that streptavidin binds with biotin in a stronger manner than avidin. The dissociation constants between biotin and avidin or streptavidin are considered M (15). Because of the lack of accuracy to be ca. 1X in the value of the reported dissociation constants, there is no direct support for the hypothesis that streptavidin may bind with biotin more strongly than avidin. However, there is some evidence in the literature that suggests the existence of a higher association constant between biotin and streptavidin than between biotin and avidin. Specifically, streptavidin and avidin were found to have different dissociation rate constants with biotin (22).In addition, Finn et al. found that a greater inhibition of the hormonal activity of biotinylated insulin is observed in the presence of streptavidin than in the presence of avidin (23). These authors proposed that streptavidin is capable of forming a more stable complex with biotinylinsulin than avidin. Besides the higher association constant with biotin, streptavidin appears to have a deeper binding pocket than avidin as indicated by differences observed in photochemically induced dynamic nuclear polarization experiments (24) and in the shielding of the disulfide bond of Bio-12SS-dUTP (a biotinylated nucleotide that contains a S-S bond in the linker arm joining biotin to the pyrimidine base) (25).
228
Barbarakls et el.
Bloconlugate Chem., Vol. 3, No. 3, 1992 60 50
I
-I
1
modification of the various assay reagent components (291, the possible impact of these modifications on the sensitivity and detection limit of the assay should be taken into consideration. ACKNOWLEDGMENT
This research was supported by a grant from the National Institutes of Health (GM 40510).
‘“1 u LITERATURE CITED
I!
IO
0
1x10-9
lxlo-E
Ixlo-’
1x10-‘
1x10-5
[Binding Sites] Figure 5. Inhibition curves of the ADA-LC-biotin conjugate obtained by using streptavidin ( 0 )and avidin (0). Error bars indicate one standard deviation ( n = 3).
*
It is well-established that in the avidin-biotin complex the carboxylic group of biotin is at least 9 A beneath the van der Waals surface of the binding protein (15, 26). However, the link provided by the lysine residue in the biotinylated ADA allows only for 7.1 A between the carboxylic group of biotin and the polypeptide chain of the enzyme. Therefore, formation of the avidin-biotin complex may bring the avidin right to the polypeptide backbone of the ADA-biotin conjugate and either cause a conformational change of the enzyme or physically block its active site (4, 27). In that respect, the depth of the binding pocket of the binder should affect the inhibition of biotinylated ADA. To investigate this, biotin was coupled to the enzyme through a spacer arm of seven atoms to yield the ADA-LC-biotin conjugate. It was found that this conjugate was inhibited to a significantly different extent by streptavidin than by avidin (Figure 5). Indeed, ADA-LC-biotin was inhibited by streptavidin up to 54%, whereas avidin inhibits this enzyme conjugate up to 43 5%. This observation is consistent with streptavidin having a deeper binding pocket than avidin. Thus, formation of the streptavidin-biotin complex may bring the streptavidin closer (compared to avidin) to the polypeptide backbone of the enzyme conjugate and either cause a conformational change of the enzyme or physically block its active site. Finally, incubation of the ADA-biotin conjugate with an excess of an anti-biotin antibody did not inhibit the enzymatic activity. Although the lower binding constant between the antibody and the conjugate (28) may play a role in the inhibition of the enzymatic activity, this observation may also be explained by the depth of the binding pocket of the binder, which is lower in the case of antibodies (26). In conclusion, it was found that the characteristics of the binder significantly affect the detection capabilities of a homogeneous enzyme-linked competitive binding assay for biotin. Such characteristics include the affinity of the binder for its specific ligand and the depth of its binding pocket. It was also found that chemical modification of the binder can lead to undesirable steric hindrance effects. Finally, the information gained through these studies may be useful in the design of optimization strategies for homogeneous enzyme-linked binding assays. In particular, when developing this type of assays one should not only be concerned about the type of ligand derivative chosen for conjugation to the enzyme-label but also about the binding properties of the binder, especially in cases where many ligand-specific binders are available. Moreover, when the assay protocol involves chemical
(1) Ngo, T. T., and Lenhoff, H. M. (1981) Recent advances in homogeneous and separation-free enzyme immunoassays. Appl. Biochem. Biotechnol. 6, 53-64. (2) Monroe, D. (1984) Enzyme immunoassay. Anal. Chem. 56, 920A-931A. (3) Ullman, E. F., and Maggio, E. T. (1980) Principles of homogeneous enzyme-immunoassay. Enzy me-Immunoassay (E. T. Maggio, Ed.) pp 105-134, CRC Press, Boca Raton, FL. (4) Kjellstrom, T. L., and Bachas, L. G. (1989) Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label. Anal. Chem.61,1728-1732. (5) Bachas, L. G., and Meyerhoff, M. E. (1986) Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme-immunoassay doseresponse curves. Anal. Biochem. 156, 223-238. (6) Gleria, K., Hill, H. A. O., and Chambers, J. A. (1989) Homogeneous amperometric ligand-binding assay amplified by a proteolytic enzyme cascade. J. Electroanal. Chem. 267, 83-9 1. ( 7 ) Mattiasson, B., and Borrebaeck, C. (1980) Novel approaches to enzyme-immunoassay. Enzyme-Zmmunoassay(E. T. Maggio, Ed.) pp 213-248, CRC Press, Boca Raton, FL. (8) Chan, W. D. (1987) General principle of immunoassay. Zmmunoassay (W. D. Chan, and T. M. Perlstein, Eds.) pp 1-23, Academic Press, San Diego, CA. (9) Feldkamp, S. C., and Stuart, W. S. (1987) Practical guide to immunoassay method evaluation. Immunoassay (W. D. Chan, and T. M. Perlstein, Eds.) pp 49-95, Academic Press, San Diego, CA. (10) Rubenstein, K. E.,Schneider,R. S.,andUllman,E. F. (1972) Homogeneous enzyme immunoassay. A new immunochemical technique. Biochem. Biophys. Res. Commun. 47, 846851. (11) Rowley, G. L., Rubenstein, K. E.; Huisjen, J., and Ullman, E. F. (1975) Mechanism by which antibodies inhibit haptenmalate dehydrogenase conjugates. An enzyme immunoassay for morphine. J. Biol. Chem. 250, 3759-3766. (12) Barbarakis, M. S., and Bachas, L. G. (1991) Isoelectric focusing electrophoresis of protein-ligand conjugates; Effect of the degree of substitution. Clin. Chem. 37, 87-90. (13) Bachas, L. G., Lewis, P. F., and Meyerhoff, M. E. (1984) Cooperative interaction of immobilized folate binding protein with enzyme-folate conjugate: An enzyme-linked assay for folate. Anal. Chem. 56, 1723-1726. (14) Green, N. M. (1965) A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Biochem. J. 94, 2 3 ~ - 2 4 ~ . (15) Green, N. M. (1975) Avidin. Adu. Protein Chem. 29, 85133. (16) Chaiet, L., and Wolf, F. J. (1964) The properties of streptavidin, a biotin-binding protein produced by streptomycetes. Arch. Biochem. Biophys. 106, 1-5. (17) Argaraiia, C. E., Kuntz, I. D., Birken, S.,Axel, R., and Cantor, C. R. (1986) Molecular cloning and nucleotide sequence of the streptavidin gene. Nucleic Acids Res. 14, 1871-1882. (18) DeLange, R. J., and Huang, T. S. (1971) Egg white avidin. 111. Sequence of the 78-residue middle cyanogen bromide peptide. Complete amino acid sequence of the protein subunit. J. Biol. Chem. 246, 698-709. (19) Bayer, E. A., and Wilchek, M. (1990) Application of avidinbiotin technology to affinity-based separations. J.Chromatogr. 510, 3-11. (20) Buckland, R. M. (1986) Strong signals from streptavidinbiotin. Nature 320, 557-558.
Inhibition of Biotlnylated Enzymes
(21) Hiller, Y.,Bayer, E. A., and Wilchek, M. (1990)Nonglycosylated avidin. Methods Enzymol. 184,68-70. (22)Piran, U.,and Riordan, W. J. (1990) Dissociation rate constant of the biotin-streptavidin complex. J. Immunol. Methods 133,141-143. (23) Finn, F. S.,Gail, T., and Hofmann, K. (1984)Ligands for insulin receptor isolation. Biochemistry 23, 2554-2558. ( 2 4 ) Gitlin, G., Khait, I., Bayer, E. A., Wilchek, M., and Muszkat, K. A. (1989)Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues. Biochem. J . 259,493-498. (25)Herman, T. M., Lefever, E., and Shimkus, M. (1986)Affinity chromatography of DNA labeled with chemically cleavable biotinylated nucleotide analogs. Anal. Biochem. 156,48-55.
Bioconlugate Chem., Vol. 3, No. 3, 1992
229
(26) Green, N. M., Konieczny, L., Toms, E. J., and Valentine, R. C. (1971)The use of bifunctional biotinyl compounds to determine the arrangement of subunits in avidin. Biochem. J. 125, 781-791. (27) Daunert, S.,Bachas, L. G., and Meyerhoff, M. E. (1988) Homogeneous enzyme-linked competitive binding assay for biotin based on the avidin-biotin interaction. Anal. Chim. Acta 208,43-52. (28) Bayer, E.A.,and Wilchek, M. (1990)Application of avidinbiotin technology to affinity-based separations. J.Chromutogr. 510,3-11. (29) Bacquet, C.A,, and Twumasi, D. Y. (1984)A homogeneous enzyme immunoassay with avidin-ligand conjugate as the enzyme-modulator. Anal. Biochem. 136,487-490. Registry No. PMDA, 89-32-7; streptavidin, 9013-20-1.
