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Adsorption studies of tritium-labeled peptides on polystyrene surfaces
Journal of Immunological Methods 221 Ž1998. 131–139
Adsorption studies of tritium-labeled peptides on polystyrene surfaces Elma E.M.G. Loomans a , Tom C.J. Gribnau b, Henri P.J. Bloemers c , Wim J.G. Schielen b,) b
a ATO-DLO, Department of Industrial Proteins, Wageningen, Netherlands AKZO NOBEL, Pharma Group, Organon Teknika, Chemistry Research Unit, PO Box 84, 5280 AB, Boxtel, Netherlands c UniÕersity of Nijmegen, Department of Biochemistry, Nijmegen, Netherlands
Received 14 July 1998; revised 5 August 1998; accepted 26 August 1998
Abstract In this study, three presentation formats of an epitope peptide Žhepta-peptide., derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear ŽLys. 7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3 H y labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mgrm2 when 1.0 mmolrl was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mgrm2 Žat 1.5 mmolrl.. The lysyl ŽK 7 . peptide showed a maximum plateau value of 0.2 mgrm2 , and therefore the lysyl ŽK 7 . extension reduced the peptide surface coverage at relatively high coat concentrations Žabove 0.1 mmolrl. compared to the parent peptide. At lower input concentrations Žbelow 0.1 mmolrl., however, the packing density of the lysyl ŽK 7 . peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and Žfunctional. affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl ŽK 7 . extension. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Surface interaction; Polystyrene; Labeled peptide; Tritium label
1. Introduction The initial step in preparing a solid-phase immunoassay is the adherence of biomaterials to a solid
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Corresponding author. Tel.: q31-411-654435; Fax: q31411-654427; E-mail: [email protected]
phase, the process known as coating or adsorption. For example, when an enzyme-linked immunosorbent assay ŽELISA. is developed to detect or quantitate HIV antibodies in a patient’s serum, HIV antigens have to be coated to the polystyrene wells of the microELISA plates. The amount of coated antigen mass is an important factor determining final ELISA reactivity, along with such factors as the
0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 1 7 4 - 4
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antibody binding activity of the coated antigen Žorientation andror conformation. and affinity of the antigen for the antibody. Increasingly in the past decade, synthetic peptides have become the antigens used in ELISA. This reflects demands for specificity and ease of characterization, notwithstanding immobilization problems generally encountered with synthetic peptides ŽBriand et al., 1985; Dagenais et al., 1994.. We recently described a general method which substantially improved the ELISA reactivity Žreferred to in that article as coating efficiency. of small synthetic peptides by an N-terminal extension with an Ata- or oligo-lysyl ŽK 7 . group and adsorption to a plastic surface ŽLoomans et al., 1997a.. In another study, we found that an increase in binding capacity was the main cause of the improved ELISA reactivity ŽLoomans et al., 1998.. The goal of the present study is to assess the contribution of adsorption to the enhancement of the binding capacity. Suitable techniques to quantitate adsorbed peptide mass are limited especially in view of the fact that the experimental coating conditions should preferably correspond to standard ELISA coating conditions. The use of coating substrates other than polystyrene or using different times, temperature, or buffer conditions ŽpH and ionic strength. for coating can generate results which are not representative of the standard peptide ELISA ŽOldenzeel, 1993; Ruzgas et al., 1992; Malmsten et al., 1996; Stevens et al., 1995; Fukuzaki et al., 1996.. Reflectometry, a real-time measuring technique, can be used under conditions almost identical to the ELISA, but with this technique it is not possible to monitor peptide adsorption in the lower concentration range. Instrumental limitations and the use of low-molecular-weight compounds such as peptides Žin reflectometry the measured quantity is the bound mass. has restricted the use of the reflectometer to relatively high coat concentrations Ž250 mgrml or higher. ŽLoomans et al., 1997b.. Radioactive labeling of the peptides of interest is another widely used method to measure the adsorbed mass. Iodination of the peptides, however, would add a substantial molecular weight to small synthetic peptides ŽMW 739., possibly affecting the adsorptive characteristics of the peptide. Moreover, the peptides need to contain at least one tyrosine residue in order to be iodinated.
In comparison to iodination, the incorporation of tritium into the peptides by introducing a 3 H y labeled glycyl residue into three different formats of a hepta-peptide Ži.e., a parent peptide, an Ata-extended peptide, and a lysyl-extended ŽK 7 . peptide 3A. is attractive for several reasons: Ž1. any change in coating behavior due to labeling is unlikely since almost no molecular weight is added and there is no alteration of the chemical properties, Ž2. this technique does not depend on any affinity interaction or enzyme-generated absorbance signal afterwards, Ž3. tritium-labeled peptides can be used over a longer period Ž t 1r2 s 12.3 year., and, Ž4. although not as practical as iodination, tritium-labeled peptides can be prepared within 36 h. The absolute mass amounts of the three tritiated peptide versions adsorbed to the polystyrene wells could be readily determined and compared at various coat concentrations under standard ELISA coat conditions. Specific antibody binding to the peptides was also tested in an ELISA procedure.
2. Materials and methods 2.1. Biomaterials Peptide 3A ŽRLPGPSD., the epitope sequence recognized by anti-human chorionic gonadotropin monoclonal antibody OT-3A Žb-hCG 133-139. was synthesized using a Perkin Elmer Applied Biosystems 433A peptide synthesizer, with FastMoc 0.25 mmol procedures ŽFields and Nobel, 1990.. Half of this peptide 3A batch, attached to the resin, was further elongated N-terminally with a seven-residue lysyl ŽK 7 . extension in the peptide synthesizer. The purity of both peptides could not be checked because it was essential that they were not cleaved from the resin Žsee tritium-labeling of peptide 3A.. Bovine serum albumin ŽBSA, Boseral type R. was obtained from Organon Teknika ŽBoxtel, The Netherlands.. The mouse anti-hCG monoclonal antibody OT-3A was obtained as described in Loomans et al. Ž1997a.. Sheep anti-mouse lgG ŽSAM. was conjugated to horseradish peroxidase ŽHRP. according to the procedure of Wilson and Nakane Ž1978..
E.E.M.G. Loomans et al.r Journal of Immunological Methods 221 (1998) 131–139
2.2. Tritium-labeling of peptide 3A The Fmoc function of H y GlyŽ3 H. y OH ŽAmersham, Buckingshamshire. England. was introduced by way of the route described by ten Kortenaar et al. Ž1986.. The ethyl acetate layers were concentrated and diluted with 1-methyl 2-pyrrolidone ŽNMP. to the proper concentration. Fmocy GlyŽ3 H. y OH was introduced into the solid-phase bound peptide essentially as described in Fields and Nobel Ž1990.. The N-terminal Ata-group was introduced via an active ester coupling with Ata-N-hydroxysuccinimide. All reactions with the tritiumlabeled Gly Žmixed with non-tritium labeled Gly. were performed side by side with reactions performed only with non-tritium-labeled Ž‘cold’. Gly in order to check the purity Žwith HPLC and Maldi-Tof. of the peptides. Therefore, it was assumed that both types of peptides, the radio-active labeled peptides and the control peptides, had the same purity characteristics. The purity ŽHPLC. and molecular weight ŽMaldiTof. characteristics of the control peptides and the labeling efficiency of the radio-active peptides are listed in Table 1. 2.3. ELISA methods to compare immune reactiÕity and binding capacity To confirm that the radioactive labeling procedure did not damage the antigenicity of the peptides, the control peptides were tested in ELISA for immune reactivity as described in Loomans et al. Ž1997a.. The comparison of ELISA-reactivity between nonradioactive parent and N-linked peptide equivalents
was made in terms of the peptide coat concentration required to achieve 50% of the maximum ELISAsignal ŽEC 50 ., derived from a fitted curve according to a 3-parameter fit ŽRodbard and Feldman, 1978.. The binding capacity of the various control peptides was also determined by ELISA, as described in Loomans et al. Ž1998.. When binding capacity was measured, the affinity of the peptide-antibody interaction did not influence the ELISA result since the ELISA was performed with a ‘saturating’ OT-3A concentration Ž250 mgrml.. Notably, this high antibody concentration did not fully saturate the parent peptide surface Ževen 1 mgrml was still not saturating; see results in Loomans et al., 1998.. Comparison of binding capacity between the various control peptides was made by EC 50 values, derived from a fitted curve according to a 3-parameter fit ŽRodbard and Feldman, 1978.. 2.4. Determination of peptide mass on polystyrene surface The coating conditions used were identical to those described in Loomans et al. Ž1997a.: the tritium-labeled peptides, serially diluted Ž135 ml. in 0.05 molrl bicarbonate coating buffer ŽpH 9.6. in peptide concentrations of approximately 100 nM to 1.5 mM, were allowed to coat overnight at room temperature with constant shaking Ž600 rpm, TPM-2 shaker; Sarstedt, Numbrecht, Germany.. Earlier in¨ vestigations revealed that an overnight coating of parent and Ata-extended peptide 3A significantly enhanced the ELISA reactivity compared to shorter incubation times Ž10 min–4 h.ŽLoomans et al., 1997a.. From additional experiments it was con-
Table 1 Peptide characteristics of the Žcontrol. peptides Peptide sequence
Molecular weight
HPLC retention time Žin min.
Purity Žin %.
labeling efficiency 3 H) d Žin ‰.
GRLPGPSD Ata-GRLPGPSD GKKKKKKKRLPGPSD
796 912 1694
12.4 14.6 11.3
39.5a 34.5 b 72.3 c
0.34 0.58 0.40
a
The main peak Ž53.4%. consists of acetylated peptides Žwithout glycyl residue. instead of parent peptides. The main peak Ž51.4%. consists of acetylated peptides Žwithout glycyl residue. instead of Ata-extended peptides. c Other peaks were below 7%. d Determined in the presence of one solubilized well and 4 ml toluene. b
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Fig. 1. ELISA response of the control peptide analogs, all N-terminally extended by a glycyl residue. ELISA was performed using dilution series of parent peptide 3A Ž=., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Žv .. Binding was determined using a Mab OT-3A concentration of 1 mgrml Žnon-saturating. and a conjugate dilution of 1r5000 Ž2.5 mgrml.. Accuracy was within 8% throughout.
cluded that the EC 50 value increased by a factor of 2 after 3 days of coating compared to overnight coating Ždata not shown.. However, overnight coating Žbetween 16 and 19 h. was chosen for practical reasons. After coating, the polystyrene microELISA plates ŽU-shaped Breakfour; Greiner, Frickenhausen, Germany. were washed four times by manually aspirating Žvacuum pump; Thomas. and dispensing 150 ml PBS-Tween Ž6.7 mmolrl phosphate buffer pH 7.2; 0.13 molrl NaCl; 0.05% Žvrv. Tween-20.. Each
well was cut off the strip and separately dissolved by incubating it for 2.5 h in a plastic count-vial ŽMaxivial: Packard Instruments, Meriden, CT. with 4 ml toluene ŽJanssen, Geel, Belgium.. Prior to counting radioactivity in a liquid scintillation analyzer ŽPackard Instruments., 6 ml of scintillation liquid ŽUltra gold; Packard Instruments. were added and the mixture was vortexed. The amount of peptide coated to the polystyrene well was determined taking into account the counting efficiency Ž67%. and labeling efficiency ŽTable 1.. The possible contribution of
Table 2 ELISA-results of the control peptides Peptide sequence
EC 50 ŽmM. a
Improved ELISA reactivity factor b
EC50 ŽmM. c
Improved binding capacity factor b
GRLPGPSD Ata-GRLPGPSD GKKKKKKKRLPGPSD
1.1 = 10 2 6.6 9.8 = 10y2
1 17 1122
1.3 = 10 3d 2.2 = 10 2 7.9
1 6 165
a
EC 50 value of the ELISA reactivity ŽFig. 1. determined by a 3-parameter fit according to Rodbard and Feldman Ž1978.. Determined by comparing the EC 50 values of the various control peptides. c EC 50 value of the binding capacity ELISA ŽFig. 2. determined by a 3-parameter fit according to Rodbard and Feldman Ž1978.. d Since curve-fitting was not possible, the EC 50 value was estimated. This EC 50 value of the parent peptide could be somewhat overestimated since an OT-3A concentration of 250 mgrml did not fully saturate the peptide coating at a coat concentration of 350 mM or higher ŽLoomans et al., 1998.. b
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Fig. 2. Determination of the binding capacity of the control peptides, all N-terminally extended by a glycyl residue. ELISA was performed using a dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Ž=., and lysyl ŽK 7 . extended peptide 3A Že.. Binding was assessed with an Mab OT-3A concentration of 250 mgrml Ž‘saturating’. and a conjugate dilution of 1r448 000 Ž2.5 mgrml.. Accuracy was within 8% throughout.
the polystyrene well and 4 ml of toluene to the counting efficiency was controlled by adding both compounds to the blanks. A quenching experiment revealed, however, that the combination of one
polystyrene well and 4 ml toluene did not cause any positive or negative quenching. As controls, stock concentrations and several supernatants were also counted for the presence of b-particles. The adsorp-
Fig. 3. Adsorption isotherms for the tritium labeled peptide presentation formats of peptide 3A on polystyrene wells derived by plotting the peptide offered to each well against the peptide bound each well. Adsorption was performed overnight according to the ELISA coating experiment conducted using a dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Ž).. Accuracy was within 8% throughout.
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tion experiments were performed twice with almost identical results; the results of one experiment are shown.
3. Results To compare the amount of adsorbed Ata- and lysyl-extended ŽK 7 . peptide 3A to that of the parent peptide 3A, the different peptides were radio-labeled by coupling a tritiated glycyl residue to each before coating on polystyrene wells. In addition, the control peptides which were subject to the same labeling procedure as the radioactive labeled peptides were tested for antigenicity when coated. All control peptides were immunoreactive with the specific antibody in the peptide-ELISA, as shown in Fig. 1. The ELISA reactivity of the Ata and the lysyl-extended ŽK 7 . peptide 3A variants Žexpressed in EC 50 -values. was increased by roughly one to three orders of magnitude, respectively, in comparison to the parent peptide 3A ŽTable 2.. The binding capacity of each of the three adsorbed control peptides was also determined in an
ELISA procedure using a ‘saturating’ antibody concentration ŽFig. 2.. According to the EC 50 values, the Ata-peptide layer and the K 7-peptide layer were 6 and 165 times, respectively, more active in specific antibody binding then the parent peptide coating layer ŽTable 2.. Thus, for the peptide concentrations used as input in the coat solution the K 7-peptide layer has the largest fraction of active molecules. A kinetic measurement of the chromogenic substrate reaction Ždata not shown. confirmed that the ELISA data for the K 7-peptide did not suffer from optical limitations, although the absorbance value was 2.5. Therefore the K 7-peptide layer reached its maximum binding capacity at 7.7 = 10 15 peptide molecules offered in the coating solution per well Žinput 94.5 mmolrl. ŽFig. 2.. Fig. 3 shows the adsorption isotherms of the three presentation formats of peptide 3A, which were readily reproducible. The adsorbed amounts of both the parent and the Ata-extended peptide continued to rise at increasing input concentrations. However, N-terminal linking of an Ata-group to the peptide consistently increased the amount of bound peptide molecules two to three times. The highest observed
Fig. 4. Efficiency of coating by plotting the input concentration for each well against the percentage of bound peptide for each well. Adsorption was performed overnight according to the ELISA coating experiment conducted using dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Ž).. Identical data series as in Fig. 3. Accuracy was within 8% throughout.
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surface coverages for the parent and the Ata-extended peptides amounted to 0.4 and 0.6 mgrm2 , respectively, at a maximum input coat concentration of 1.5 and 1.0 mmolrl, respectively. The adsorption isotherm of the K 7-peptide showed a very different picture: it reached a maximum plateau value of approximately 0.16 mgrm2 Žequal to 6 = 10 12 molecules per well. at a minimum input coat concentration of 94.5 mmolrl Žequal to 7.7 = 10 15 molecules per well.. The N-terminal extension of a peptide by a lysyl ŽK 7 . extension thus impaired the coating properties at relatively high coat concentrations Žabove an input of 94.5 mmolrl. as compared to the other two peptide presentation formats ŽFig. 3.. However, at lower peptide concentration Žbelow an input of 94.5 nmolrl. the packing density of the K 7-peptide was up to 25 times higher than that of the other two peptide analogs. Fig. 4 shows this effect more clearly by plotting the input concentrations against the percentage adsorbed both on a log scale. At very low K 7-peptide concentrations the process of adsorption almost reached 80% efficiency.
4. Discussion The adsorption of proteins on solid surfaces has received considerable attention due to its importance in the food and drug industry and the field of biocompatibility. However, limited studies are available on peptide adsorption ŽRuzgas et al., 1992; Tsai et al., 1996. although the peptide-polymer interaction is important to the diagnostic and pharmaceutical industries peptide coating is one of the critical factors in the development of diagnostic assays and the incorporation of peptides into polymer delivery systems is important for the controlled release of pharmaceuticals. The control peptides used in the study, synthesized to determine peptide characteristics and antigenicity, were assumed to be representative of the radioactive labeled peptides because they were subject to the same labeling procedure. The labeling procedure itself did not impair the antigenicity of the peptides since antibody binding in ELISA was evident ŽFigs. 1 and 2.. Moreover, the ELISA reactivity of the control parent peptide 3A was even slightly enhanced in comparison to previous results with a
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non-glycyl linked parent peptide 3A ŽLoomans et al., 1997a.. This difference could be caused by the addition of an extra glycyl residue since the addition of four extra glycyl residues increased the ELISA results by a factor of 10 ŽLoomans et al., 1997a.. The enhancement factors of the N-terminally extended control peptides, however, were one to three orders of magnitude lower when compared to the results of the equivalent peptides without an additional glycyl residue Žreported in Table 3 in Loomans et al., 1997a as factors of improved ‘binding capacity’.. Two reasons can be advanced. First, the significantly lower purity of all the glycyl-linked peptide analogs ŽTable 1. which results in large additional fractions of acetylated peptides. From previous results ŽLoomans et al., 1997a., it is known that EC 50 -values of a parent peptide and an acetylated peptide are comparable. In conclusion, only the impure batches of the N-terminally linked variants Žespecially the Atapeptide. manifest the impurity with acetylated peptides which drastically reduces their EC 50 value in comparison to the parent peptide ŽLoomans et al., 1997a.. Secondly, the addition of an extra glycine probably has relatively more effect on the ELISA reactivity of the parent peptide than on the larger N-terminally linked analogs. Since the influence of affinity was excluded in the binding capacity experiment in contrast to the ELISA reactivity experiment, it can be concluded from a comparison of the EC 50 values of equivalent peptides that by N-terminal linking of the glycyl-peptide functional affinity increases ŽTable 2.. This conclusion is confirmed by the functional affinity results from the direct ELISA, reported in Loomans et al. Ž1998.. When the radioactive coating results ŽFig. 3. and the binding capacity results ŽTable 2. are combined, it can be concluded that the increase in binding capacity of the Ata-extended peptide in comparison to the parent peptide can be almost completely explained by the increase in bound Ata-peptide molecules. The binding capacity of the K 7-peptide is also correlated to the amount of bound K 7-peptide. Notably, the maximum plateau value of 0.16 mgrm2 of the K 7-peptide layer reached at a minimum input concentration of 94.5 mmolrl agreed well with the amount of K 7-peptide molecules in the coating solution necessary to attain maximum binding capacity. However, the increase in the binding capacity of the
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K 7-peptide Ž165-fold. has to be explained by the sum of two contributing factors since ‘only’ a 25-fold increase in the amount of bound K 7-peptide molecules could be observed in the lower concentration range compared to the amount of bound parent peptide molecules. In addition to the packing density, the percentage of active Ži.e., able to bind the specific monoclonal antibody. peptide bound to the polystyrene surface had to be increased as well. This explanation is supported by the observation that the binding capacity of the K 7-peptide could already be observed in ELISA when about 3 = 10 12 molecules were coated on the polystyrene well while for the parent and the Ata-extended peptide, the amount of bound peptide had to be six times higher before any antibody binding could be observed in ELISA. It is remarkable that at the higher coat concentrations, the surface coverage of the parent and the Ata-peptides exceeded the surface coverage of the K 7-peptide layer while at the same time the binding capacity of the parent and the Ata-peptide layers was significantly lower than that of the K 7-peptide layer. Thus, an increase in activity has to fully cover the improved binding capacity of the K 7-peptide. It is known that activity is inversely related to the molar density probably because of orientational andror conformational improvements with respect to antibody binding ŽHeuvel van den et al., 1993.. However, the higher surface coverage of the K 7-peptide Žand the higher binding capacity. at dilute concentrations must be caused by the lysyl ŽK 7 . extension itself. That the addition of lysyl residues to the N-terminus of a peptide can enhance the antigenicity has been previously recognized ŽLeach, 1983.. It is interesting to speculate why a maximum plateau value was observed for the K 7-peptide and not for the two other peptide presentation formats. Possibly, the additional positive charge of seven lysyl residues per peptide electrostatically hampers a closer packing on the surface ŽRuzgas et al., 1992., although in the initial stage of the adsorption process the addition of lysyl residues and the Ata-group could electrostatically favor adsorption and therefore improve the peptide–polymer interaction ŽLoomans et al., 1997a.. The lysyl ŽK 7 . extension could also favor conformational extension of the peptide on the surface, thereby inhibiting a higher surface coverage ŽTsai et al., 1996..
Peptide surface coverages between 0.3 and 0.6 mgrm2 have been reported, in the literature and although the authors worked under different experimental conditions ŽRuzgas et al., 1992; Tsai et al., 1996. our values were similar. In terms of experimental conditions, the peptide surface coverages determined by reflectometry with the same peptides ŽLoomans et al., 1997b. would seem more relevant to this work. In the reflectometer, the surface coverages of the three presentation formats of peptide 3A at a coat concentration of 250 mgrml were considerably higher: 0.62, 0.76, and 1.72 mgrm2 for the parent, Ata-, and K 7-extended peptide 3A, respectively. The longer adsorption time in the ELISA could affect the extent of desorption and the presence of the detergent Tween in the washing buffer could also account for lower surface coverages. Nevertheless, the calculated amounts of bound peptide compared to the amounts which determine the final ELISA-results could be somewhat overestimated since possible desorption and displacement in subsequent washes and incubations is ignored. In summary, we have demonstrated that the Atapeptide 3A and the lysyl ŽK 7 . peptide 3A adsorb better to polystyrene surfaces than does the free parent peptide 3A. Furthermore, we have shown that N-terminal linking of an Ata-group or lysyl ŽK 7 . extension increases the activity andror affinity of the peptide-antibody interaction. Although no simple generalizations can be made, we expect that the above conclusions will also be valid for other small synthetic peptides. This new immobilization strategy may therefore encourage the increased use of peptides in research, medicine and industry.
5. Unlinked References Bally and Gribnau, 1989
Acknowledgements The authors would like to thank Rinie van Beuningen for his graphical assistance. We are also grateful to Dr. E. Sprengers for his contributions to the manuscript.
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References Bally, R.W., Gribnau, T.C.J., 1989. Some aspects of the chroX X mogen 3,3 ,5,5 -tetramethylbenzidine as hydrogen donor in a horseradish peroxidase assay. J. Clin. Chem. Clin. Biochem. 27, 791. Briand, J.P., Muller, S., Van Regenmortel, M.H.V., 1985. Synthetic peptides as antigens: pitfalls of conjugation methods. J. Immunol. Methods 78, 59. Dagenais, P., Desprez, B., Albert, J., Escher, E., 1994. Direct covalent attachment of small peptide antigens to enzyme-linked immunosorbent assay plates using radiation and carbodiimide activation. J. Immunol. Methods 222, 149. Fields, G.B., Nobel, R.L., 1990. Solid phase peptide synthesis utilizing 9-fluorenyl-methoxycarbonyl amino acids. Int. J. Peptide Protein Res. 35, 161. Fukuzaki, S., Urano, H., Nagata, K., 1996. Adsorption of bovine serum albumin onto metal oxide surfaces. J. Ferment. Bioeng. 81, 163. Heuvel van den, D.J., Kooyman, R.P.H., Drijfhout, J.-W., Welling, G.W., 1993. Synthetic peptides as receptors in affinity sensors: a feasibility study. Anal. Biochem. 215, 223. ten Kortenaar, P.B.W., van Dijk, B.G., Peeters, J.M., Raaben, B.J., Adams, P.J.H.M., Tesser, G.I., 1986. A rapid and efficient method for the preparation of Fmoc-amino acids starting from 9-fluorenylmethanol. Int. J. Pept. Protein Res. 27, 398. Leach, S.J., 1983. How antigenic are antigenic peptides?. Biopolymers 22, 425. Loomans, E.E.M.G., Petersen-van Ettekoven, A., Bloemers, H.P.J., Schielen, W.J.G., 1997a. Direct coating of polyŽLys. or Atapeptides to polystyrene; the effects in an enzyme-linked immunosorbent assay. Anal. Biochem. 248, 117. Loomans, E.E.M.G., Beumer, T.A.M., Damen, K.C.S., Bakker,
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M.A., Schielen, W.J.G., 1997b. Real-time monitoring of peptide-surface and peptide-antibody interaction by means of reflectometry and surface plasmon resonance. J. Colloid Interface Sci. 192. Loomans, E.E.M.G., Gribnau, T.C.J., Bloemers, H.P.J., Schielen, W.J.G., 1998. Binding capacity and affinity determination by ELISA-methods with respect to N-terminal elongation of hCG peptides with an Ata-group or ŽLys.7 extension. J. Immunol. Methods Žin press.. Malmsten, M., Lassen, B., Van Alstine, J.M., Nilsson, U.R., 1996. Adsorption of complement proteins C3 and C1q. J. Colloid Interface Sci. 178, 123. Oldenzeel, M., 1993. PhD thesis, University of Twente, The Netherlands. Rodbard, D., Feldman, Y., 1978. Kinetics of two-site immunoradiometric Ž‘sandwich’. assay. I Mathematical models for simulation, optimization and curve fitting. Immunochemistry 15, 71. Ruzgas, T.A., Kazlaukas, A.V., Razumas, V.J., Kulys, J.J., 1992. Adsorption of heme-containing peptides on silicon surfaces. J. Colloid Interface Sci. 154, 97. Stevens, P.W., Hansberry, M.R., Kelso, D.M., 1995. Assessment of adsorption and adhesion of proteins to polystyrene microwells by sequential enzyme-linked immunosorbent assay analysis. Anal. Biochem. 225, 197. Tsai, T., Mehta, R.C., DeLuca, P.P., 1996. Adsorption of peptides to polyŽD,L-lactideco-glycolide.: 1. Effect of physical factors on adsorption. Int. J. Pharm. 127, 31. Wilson, M.B., Nakane, P.K., 1978. Recent developments in the periodate method of conjugating horseradish peroxidase ŽHRPO. to antibodies. In: Knapp, W., Holubar, K., Wick, G. ŽEds.., Immunofluorescence and Related Staining Techniques. Elsevier, Amsterdam, p. 215.
Adsorption studies of tritium-labeled peptides on polystyrene surfaces Elma E.M.G. Loomans a , Tom C.J. Gribnau b, Henri P.J. Bloemers c , Wim J.G. Schielen b,) b
a ATO-DLO, Department of Industrial Proteins, Wageningen, Netherlands AKZO NOBEL, Pharma Group, Organon Teknika, Chemistry Research Unit, PO Box 84, 5280 AB, Boxtel, Netherlands c UniÕersity of Nijmegen, Department of Biochemistry, Nijmegen, Netherlands
Received 14 July 1998; revised 5 August 1998; accepted 26 August 1998
Abstract In this study, three presentation formats of an epitope peptide Žhepta-peptide., derived from the human chorionic gonadotropin amino acid sequence, were compared for adsorption to the polystyrene wells of a microELISA plate. The peptides had either a free N-terminus, an Ata-group or a linear ŽLys. 7-extension at the N-terminal. In order to measure the adsorption properties, all peptides were tritiated by synthesizing an additional 3 H y labeled glycyl residue to the N-terminus of their peptide sequence. Over a broad range of peptide concentrations used as coat solution, extension of the peptide by an Ata-group consistently increased adsorption by a factor of 1.5 to 3 compared to the free parent peptide. Of the three peptides studied, the Ata-peptide showed the highest surface coverage of 0.6 mgrm2 when 1.0 mmolrl was offered as the concentration of peptide in the coating solution. The highest surface coverage observed for the parent peptide was 0.4 mgrm2 Žat 1.5 mmolrl.. The lysyl ŽK 7 . peptide showed a maximum plateau value of 0.2 mgrm2 , and therefore the lysyl ŽK 7 . extension reduced the peptide surface coverage at relatively high coat concentrations Žabove 0.1 mmolrl. compared to the parent peptide. At lower input concentrations Žbelow 0.1 mmolrl., however, the packing density of the lysyl ŽK 7 . peptide was up to 25 times higher when compared to the other two peptide analogs. We conclude that better adsorption as well as improved antibody binding activity and Žfunctional. affinity could explain the higher reactivity observed in ELISA procedures when peptides are N-terminally extended by an Ata-group or lysyl ŽK 7 . extension. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Surface interaction; Polystyrene; Labeled peptide; Tritium label
1. Introduction The initial step in preparing a solid-phase immunoassay is the adherence of biomaterials to a solid
)
Corresponding author. Tel.: q31-411-654435; Fax: q31411-654427; E-mail: [email protected]
phase, the process known as coating or adsorption. For example, when an enzyme-linked immunosorbent assay ŽELISA. is developed to detect or quantitate HIV antibodies in a patient’s serum, HIV antigens have to be coated to the polystyrene wells of the microELISA plates. The amount of coated antigen mass is an important factor determining final ELISA reactivity, along with such factors as the
0022-1759r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 8 . 0 0 1 7 4 - 4
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antibody binding activity of the coated antigen Žorientation andror conformation. and affinity of the antigen for the antibody. Increasingly in the past decade, synthetic peptides have become the antigens used in ELISA. This reflects demands for specificity and ease of characterization, notwithstanding immobilization problems generally encountered with synthetic peptides ŽBriand et al., 1985; Dagenais et al., 1994.. We recently described a general method which substantially improved the ELISA reactivity Žreferred to in that article as coating efficiency. of small synthetic peptides by an N-terminal extension with an Ata- or oligo-lysyl ŽK 7 . group and adsorption to a plastic surface ŽLoomans et al., 1997a.. In another study, we found that an increase in binding capacity was the main cause of the improved ELISA reactivity ŽLoomans et al., 1998.. The goal of the present study is to assess the contribution of adsorption to the enhancement of the binding capacity. Suitable techniques to quantitate adsorbed peptide mass are limited especially in view of the fact that the experimental coating conditions should preferably correspond to standard ELISA coating conditions. The use of coating substrates other than polystyrene or using different times, temperature, or buffer conditions ŽpH and ionic strength. for coating can generate results which are not representative of the standard peptide ELISA ŽOldenzeel, 1993; Ruzgas et al., 1992; Malmsten et al., 1996; Stevens et al., 1995; Fukuzaki et al., 1996.. Reflectometry, a real-time measuring technique, can be used under conditions almost identical to the ELISA, but with this technique it is not possible to monitor peptide adsorption in the lower concentration range. Instrumental limitations and the use of low-molecular-weight compounds such as peptides Žin reflectometry the measured quantity is the bound mass. has restricted the use of the reflectometer to relatively high coat concentrations Ž250 mgrml or higher. ŽLoomans et al., 1997b.. Radioactive labeling of the peptides of interest is another widely used method to measure the adsorbed mass. Iodination of the peptides, however, would add a substantial molecular weight to small synthetic peptides ŽMW 739., possibly affecting the adsorptive characteristics of the peptide. Moreover, the peptides need to contain at least one tyrosine residue in order to be iodinated.
In comparison to iodination, the incorporation of tritium into the peptides by introducing a 3 H y labeled glycyl residue into three different formats of a hepta-peptide Ži.e., a parent peptide, an Ata-extended peptide, and a lysyl-extended ŽK 7 . peptide 3A. is attractive for several reasons: Ž1. any change in coating behavior due to labeling is unlikely since almost no molecular weight is added and there is no alteration of the chemical properties, Ž2. this technique does not depend on any affinity interaction or enzyme-generated absorbance signal afterwards, Ž3. tritium-labeled peptides can be used over a longer period Ž t 1r2 s 12.3 year., and, Ž4. although not as practical as iodination, tritium-labeled peptides can be prepared within 36 h. The absolute mass amounts of the three tritiated peptide versions adsorbed to the polystyrene wells could be readily determined and compared at various coat concentrations under standard ELISA coat conditions. Specific antibody binding to the peptides was also tested in an ELISA procedure.
2. Materials and methods 2.1. Biomaterials Peptide 3A ŽRLPGPSD., the epitope sequence recognized by anti-human chorionic gonadotropin monoclonal antibody OT-3A Žb-hCG 133-139. was synthesized using a Perkin Elmer Applied Biosystems 433A peptide synthesizer, with FastMoc 0.25 mmol procedures ŽFields and Nobel, 1990.. Half of this peptide 3A batch, attached to the resin, was further elongated N-terminally with a seven-residue lysyl ŽK 7 . extension in the peptide synthesizer. The purity of both peptides could not be checked because it was essential that they were not cleaved from the resin Žsee tritium-labeling of peptide 3A.. Bovine serum albumin ŽBSA, Boseral type R. was obtained from Organon Teknika ŽBoxtel, The Netherlands.. The mouse anti-hCG monoclonal antibody OT-3A was obtained as described in Loomans et al. Ž1997a.. Sheep anti-mouse lgG ŽSAM. was conjugated to horseradish peroxidase ŽHRP. according to the procedure of Wilson and Nakane Ž1978..
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2.2. Tritium-labeling of peptide 3A The Fmoc function of H y GlyŽ3 H. y OH ŽAmersham, Buckingshamshire. England. was introduced by way of the route described by ten Kortenaar et al. Ž1986.. The ethyl acetate layers were concentrated and diluted with 1-methyl 2-pyrrolidone ŽNMP. to the proper concentration. Fmocy GlyŽ3 H. y OH was introduced into the solid-phase bound peptide essentially as described in Fields and Nobel Ž1990.. The N-terminal Ata-group was introduced via an active ester coupling with Ata-N-hydroxysuccinimide. All reactions with the tritiumlabeled Gly Žmixed with non-tritium labeled Gly. were performed side by side with reactions performed only with non-tritium-labeled Ž‘cold’. Gly in order to check the purity Žwith HPLC and Maldi-Tof. of the peptides. Therefore, it was assumed that both types of peptides, the radio-active labeled peptides and the control peptides, had the same purity characteristics. The purity ŽHPLC. and molecular weight ŽMaldiTof. characteristics of the control peptides and the labeling efficiency of the radio-active peptides are listed in Table 1. 2.3. ELISA methods to compare immune reactiÕity and binding capacity To confirm that the radioactive labeling procedure did not damage the antigenicity of the peptides, the control peptides were tested in ELISA for immune reactivity as described in Loomans et al. Ž1997a.. The comparison of ELISA-reactivity between nonradioactive parent and N-linked peptide equivalents
was made in terms of the peptide coat concentration required to achieve 50% of the maximum ELISAsignal ŽEC 50 ., derived from a fitted curve according to a 3-parameter fit ŽRodbard and Feldman, 1978.. The binding capacity of the various control peptides was also determined by ELISA, as described in Loomans et al. Ž1998.. When binding capacity was measured, the affinity of the peptide-antibody interaction did not influence the ELISA result since the ELISA was performed with a ‘saturating’ OT-3A concentration Ž250 mgrml.. Notably, this high antibody concentration did not fully saturate the parent peptide surface Ževen 1 mgrml was still not saturating; see results in Loomans et al., 1998.. Comparison of binding capacity between the various control peptides was made by EC 50 values, derived from a fitted curve according to a 3-parameter fit ŽRodbard and Feldman, 1978.. 2.4. Determination of peptide mass on polystyrene surface The coating conditions used were identical to those described in Loomans et al. Ž1997a.: the tritium-labeled peptides, serially diluted Ž135 ml. in 0.05 molrl bicarbonate coating buffer ŽpH 9.6. in peptide concentrations of approximately 100 nM to 1.5 mM, were allowed to coat overnight at room temperature with constant shaking Ž600 rpm, TPM-2 shaker; Sarstedt, Numbrecht, Germany.. Earlier in¨ vestigations revealed that an overnight coating of parent and Ata-extended peptide 3A significantly enhanced the ELISA reactivity compared to shorter incubation times Ž10 min–4 h.ŽLoomans et al., 1997a.. From additional experiments it was con-
Table 1 Peptide characteristics of the Žcontrol. peptides Peptide sequence
Molecular weight
HPLC retention time Žin min.
Purity Žin %.
labeling efficiency 3 H) d Žin ‰.
GRLPGPSD Ata-GRLPGPSD GKKKKKKKRLPGPSD
796 912 1694
12.4 14.6 11.3
39.5a 34.5 b 72.3 c
0.34 0.58 0.40
a
The main peak Ž53.4%. consists of acetylated peptides Žwithout glycyl residue. instead of parent peptides. The main peak Ž51.4%. consists of acetylated peptides Žwithout glycyl residue. instead of Ata-extended peptides. c Other peaks were below 7%. d Determined in the presence of one solubilized well and 4 ml toluene. b
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Fig. 1. ELISA response of the control peptide analogs, all N-terminally extended by a glycyl residue. ELISA was performed using dilution series of parent peptide 3A Ž=., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Žv .. Binding was determined using a Mab OT-3A concentration of 1 mgrml Žnon-saturating. and a conjugate dilution of 1r5000 Ž2.5 mgrml.. Accuracy was within 8% throughout.
cluded that the EC 50 value increased by a factor of 2 after 3 days of coating compared to overnight coating Ždata not shown.. However, overnight coating Žbetween 16 and 19 h. was chosen for practical reasons. After coating, the polystyrene microELISA plates ŽU-shaped Breakfour; Greiner, Frickenhausen, Germany. were washed four times by manually aspirating Žvacuum pump; Thomas. and dispensing 150 ml PBS-Tween Ž6.7 mmolrl phosphate buffer pH 7.2; 0.13 molrl NaCl; 0.05% Žvrv. Tween-20.. Each
well was cut off the strip and separately dissolved by incubating it for 2.5 h in a plastic count-vial ŽMaxivial: Packard Instruments, Meriden, CT. with 4 ml toluene ŽJanssen, Geel, Belgium.. Prior to counting radioactivity in a liquid scintillation analyzer ŽPackard Instruments., 6 ml of scintillation liquid ŽUltra gold; Packard Instruments. were added and the mixture was vortexed. The amount of peptide coated to the polystyrene well was determined taking into account the counting efficiency Ž67%. and labeling efficiency ŽTable 1.. The possible contribution of
Table 2 ELISA-results of the control peptides Peptide sequence
EC 50 ŽmM. a
Improved ELISA reactivity factor b
EC50 ŽmM. c
Improved binding capacity factor b
GRLPGPSD Ata-GRLPGPSD GKKKKKKKRLPGPSD
1.1 = 10 2 6.6 9.8 = 10y2
1 17 1122
1.3 = 10 3d 2.2 = 10 2 7.9
1 6 165
a
EC 50 value of the ELISA reactivity ŽFig. 1. determined by a 3-parameter fit according to Rodbard and Feldman Ž1978.. Determined by comparing the EC 50 values of the various control peptides. c EC 50 value of the binding capacity ELISA ŽFig. 2. determined by a 3-parameter fit according to Rodbard and Feldman Ž1978.. d Since curve-fitting was not possible, the EC 50 value was estimated. This EC 50 value of the parent peptide could be somewhat overestimated since an OT-3A concentration of 250 mgrml did not fully saturate the peptide coating at a coat concentration of 350 mM or higher ŽLoomans et al., 1998.. b
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Fig. 2. Determination of the binding capacity of the control peptides, all N-terminally extended by a glycyl residue. ELISA was performed using a dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Ž=., and lysyl ŽK 7 . extended peptide 3A Že.. Binding was assessed with an Mab OT-3A concentration of 250 mgrml Ž‘saturating’. and a conjugate dilution of 1r448 000 Ž2.5 mgrml.. Accuracy was within 8% throughout.
the polystyrene well and 4 ml of toluene to the counting efficiency was controlled by adding both compounds to the blanks. A quenching experiment revealed, however, that the combination of one
polystyrene well and 4 ml toluene did not cause any positive or negative quenching. As controls, stock concentrations and several supernatants were also counted for the presence of b-particles. The adsorp-
Fig. 3. Adsorption isotherms for the tritium labeled peptide presentation formats of peptide 3A on polystyrene wells derived by plotting the peptide offered to each well against the peptide bound each well. Adsorption was performed overnight according to the ELISA coating experiment conducted using a dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Ž).. Accuracy was within 8% throughout.
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tion experiments were performed twice with almost identical results; the results of one experiment are shown.
3. Results To compare the amount of adsorbed Ata- and lysyl-extended ŽK 7 . peptide 3A to that of the parent peptide 3A, the different peptides were radio-labeled by coupling a tritiated glycyl residue to each before coating on polystyrene wells. In addition, the control peptides which were subject to the same labeling procedure as the radioactive labeled peptides were tested for antigenicity when coated. All control peptides were immunoreactive with the specific antibody in the peptide-ELISA, as shown in Fig. 1. The ELISA reactivity of the Ata and the lysyl-extended ŽK 7 . peptide 3A variants Žexpressed in EC 50 -values. was increased by roughly one to three orders of magnitude, respectively, in comparison to the parent peptide 3A ŽTable 2.. The binding capacity of each of the three adsorbed control peptides was also determined in an
ELISA procedure using a ‘saturating’ antibody concentration ŽFig. 2.. According to the EC 50 values, the Ata-peptide layer and the K 7-peptide layer were 6 and 165 times, respectively, more active in specific antibody binding then the parent peptide coating layer ŽTable 2.. Thus, for the peptide concentrations used as input in the coat solution the K 7-peptide layer has the largest fraction of active molecules. A kinetic measurement of the chromogenic substrate reaction Ždata not shown. confirmed that the ELISA data for the K 7-peptide did not suffer from optical limitations, although the absorbance value was 2.5. Therefore the K 7-peptide layer reached its maximum binding capacity at 7.7 = 10 15 peptide molecules offered in the coating solution per well Žinput 94.5 mmolrl. ŽFig. 2.. Fig. 3 shows the adsorption isotherms of the three presentation formats of peptide 3A, which were readily reproducible. The adsorbed amounts of both the parent and the Ata-extended peptide continued to rise at increasing input concentrations. However, N-terminal linking of an Ata-group to the peptide consistently increased the amount of bound peptide molecules two to three times. The highest observed
Fig. 4. Efficiency of coating by plotting the input concentration for each well against the percentage of bound peptide for each well. Adsorption was performed overnight according to the ELISA coating experiment conducted using dilution series of parent peptide 3A ŽB., Ata-extended peptide 3A Žq., and lysyl ŽK 7 . extended peptide 3A Ž).. Identical data series as in Fig. 3. Accuracy was within 8% throughout.
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surface coverages for the parent and the Ata-extended peptides amounted to 0.4 and 0.6 mgrm2 , respectively, at a maximum input coat concentration of 1.5 and 1.0 mmolrl, respectively. The adsorption isotherm of the K 7-peptide showed a very different picture: it reached a maximum plateau value of approximately 0.16 mgrm2 Žequal to 6 = 10 12 molecules per well. at a minimum input coat concentration of 94.5 mmolrl Žequal to 7.7 = 10 15 molecules per well.. The N-terminal extension of a peptide by a lysyl ŽK 7 . extension thus impaired the coating properties at relatively high coat concentrations Žabove an input of 94.5 mmolrl. as compared to the other two peptide presentation formats ŽFig. 3.. However, at lower peptide concentration Žbelow an input of 94.5 nmolrl. the packing density of the K 7-peptide was up to 25 times higher than that of the other two peptide analogs. Fig. 4 shows this effect more clearly by plotting the input concentrations against the percentage adsorbed both on a log scale. At very low K 7-peptide concentrations the process of adsorption almost reached 80% efficiency.
4. Discussion The adsorption of proteins on solid surfaces has received considerable attention due to its importance in the food and drug industry and the field of biocompatibility. However, limited studies are available on peptide adsorption ŽRuzgas et al., 1992; Tsai et al., 1996. although the peptide-polymer interaction is important to the diagnostic and pharmaceutical industries peptide coating is one of the critical factors in the development of diagnostic assays and the incorporation of peptides into polymer delivery systems is important for the controlled release of pharmaceuticals. The control peptides used in the study, synthesized to determine peptide characteristics and antigenicity, were assumed to be representative of the radioactive labeled peptides because they were subject to the same labeling procedure. The labeling procedure itself did not impair the antigenicity of the peptides since antibody binding in ELISA was evident ŽFigs. 1 and 2.. Moreover, the ELISA reactivity of the control parent peptide 3A was even slightly enhanced in comparison to previous results with a
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non-glycyl linked parent peptide 3A ŽLoomans et al., 1997a.. This difference could be caused by the addition of an extra glycyl residue since the addition of four extra glycyl residues increased the ELISA results by a factor of 10 ŽLoomans et al., 1997a.. The enhancement factors of the N-terminally extended control peptides, however, were one to three orders of magnitude lower when compared to the results of the equivalent peptides without an additional glycyl residue Žreported in Table 3 in Loomans et al., 1997a as factors of improved ‘binding capacity’.. Two reasons can be advanced. First, the significantly lower purity of all the glycyl-linked peptide analogs ŽTable 1. which results in large additional fractions of acetylated peptides. From previous results ŽLoomans et al., 1997a., it is known that EC 50 -values of a parent peptide and an acetylated peptide are comparable. In conclusion, only the impure batches of the N-terminally linked variants Žespecially the Atapeptide. manifest the impurity with acetylated peptides which drastically reduces their EC 50 value in comparison to the parent peptide ŽLoomans et al., 1997a.. Secondly, the addition of an extra glycine probably has relatively more effect on the ELISA reactivity of the parent peptide than on the larger N-terminally linked analogs. Since the influence of affinity was excluded in the binding capacity experiment in contrast to the ELISA reactivity experiment, it can be concluded from a comparison of the EC 50 values of equivalent peptides that by N-terminal linking of the glycyl-peptide functional affinity increases ŽTable 2.. This conclusion is confirmed by the functional affinity results from the direct ELISA, reported in Loomans et al. Ž1998.. When the radioactive coating results ŽFig. 3. and the binding capacity results ŽTable 2. are combined, it can be concluded that the increase in binding capacity of the Ata-extended peptide in comparison to the parent peptide can be almost completely explained by the increase in bound Ata-peptide molecules. The binding capacity of the K 7-peptide is also correlated to the amount of bound K 7-peptide. Notably, the maximum plateau value of 0.16 mgrm2 of the K 7-peptide layer reached at a minimum input concentration of 94.5 mmolrl agreed well with the amount of K 7-peptide molecules in the coating solution necessary to attain maximum binding capacity. However, the increase in the binding capacity of the
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K 7-peptide Ž165-fold. has to be explained by the sum of two contributing factors since ‘only’ a 25-fold increase in the amount of bound K 7-peptide molecules could be observed in the lower concentration range compared to the amount of bound parent peptide molecules. In addition to the packing density, the percentage of active Ži.e., able to bind the specific monoclonal antibody. peptide bound to the polystyrene surface had to be increased as well. This explanation is supported by the observation that the binding capacity of the K 7-peptide could already be observed in ELISA when about 3 = 10 12 molecules were coated on the polystyrene well while for the parent and the Ata-extended peptide, the amount of bound peptide had to be six times higher before any antibody binding could be observed in ELISA. It is remarkable that at the higher coat concentrations, the surface coverage of the parent and the Ata-peptides exceeded the surface coverage of the K 7-peptide layer while at the same time the binding capacity of the parent and the Ata-peptide layers was significantly lower than that of the K 7-peptide layer. Thus, an increase in activity has to fully cover the improved binding capacity of the K 7-peptide. It is known that activity is inversely related to the molar density probably because of orientational andror conformational improvements with respect to antibody binding ŽHeuvel van den et al., 1993.. However, the higher surface coverage of the K 7-peptide Žand the higher binding capacity. at dilute concentrations must be caused by the lysyl ŽK 7 . extension itself. That the addition of lysyl residues to the N-terminus of a peptide can enhance the antigenicity has been previously recognized ŽLeach, 1983.. It is interesting to speculate why a maximum plateau value was observed for the K 7-peptide and not for the two other peptide presentation formats. Possibly, the additional positive charge of seven lysyl residues per peptide electrostatically hampers a closer packing on the surface ŽRuzgas et al., 1992., although in the initial stage of the adsorption process the addition of lysyl residues and the Ata-group could electrostatically favor adsorption and therefore improve the peptide–polymer interaction ŽLoomans et al., 1997a.. The lysyl ŽK 7 . extension could also favor conformational extension of the peptide on the surface, thereby inhibiting a higher surface coverage ŽTsai et al., 1996..
Peptide surface coverages between 0.3 and 0.6 mgrm2 have been reported, in the literature and although the authors worked under different experimental conditions ŽRuzgas et al., 1992; Tsai et al., 1996. our values were similar. In terms of experimental conditions, the peptide surface coverages determined by reflectometry with the same peptides ŽLoomans et al., 1997b. would seem more relevant to this work. In the reflectometer, the surface coverages of the three presentation formats of peptide 3A at a coat concentration of 250 mgrml were considerably higher: 0.62, 0.76, and 1.72 mgrm2 for the parent, Ata-, and K 7-extended peptide 3A, respectively. The longer adsorption time in the ELISA could affect the extent of desorption and the presence of the detergent Tween in the washing buffer could also account for lower surface coverages. Nevertheless, the calculated amounts of bound peptide compared to the amounts which determine the final ELISA-results could be somewhat overestimated since possible desorption and displacement in subsequent washes and incubations is ignored. In summary, we have demonstrated that the Atapeptide 3A and the lysyl ŽK 7 . peptide 3A adsorb better to polystyrene surfaces than does the free parent peptide 3A. Furthermore, we have shown that N-terminal linking of an Ata-group or lysyl ŽK 7 . extension increases the activity andror affinity of the peptide-antibody interaction. Although no simple generalizations can be made, we expect that the above conclusions will also be valid for other small synthetic peptides. This new immobilization strategy may therefore encourage the increased use of peptides in research, medicine and industry.
5. Unlinked References Bally and Gribnau, 1989
Acknowledgements The authors would like to thank Rinie van Beuningen for his graphical assistance. We are also grateful to Dr. E. Sprengers for his contributions to the manuscript.
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M.A., Schielen, W.J.G., 1997b. Real-time monitoring of peptide-surface and peptide-antibody interaction by means of reflectometry and surface plasmon resonance. J. Colloid Interface Sci. 192. Loomans, E.E.M.G., Gribnau, T.C.J., Bloemers, H.P.J., Schielen, W.J.G., 1998. Binding capacity and affinity determination by ELISA-methods with respect to N-terminal elongation of hCG peptides with an Ata-group or ŽLys.7 extension. J. Immunol. Methods Žin press.. Malmsten, M., Lassen, B., Van Alstine, J.M., Nilsson, U.R., 1996. Adsorption of complement proteins C3 and C1q. J. Colloid Interface Sci. 178, 123. Oldenzeel, M., 1993. PhD thesis, University of Twente, The Netherlands. Rodbard, D., Feldman, Y., 1978. Kinetics of two-site immunoradiometric Ž‘sandwich’. assay. I Mathematical models for simulation, optimization and curve fitting. Immunochemistry 15, 71. Ruzgas, T.A., Kazlaukas, A.V., Razumas, V.J., Kulys, J.J., 1992. Adsorption of heme-containing peptides on silicon surfaces. J. Colloid Interface Sci. 154, 97. Stevens, P.W., Hansberry, M.R., Kelso, D.M., 1995. Assessment of adsorption and adhesion of proteins to polystyrene microwells by sequential enzyme-linked immunosorbent assay analysis. Anal. Biochem. 225, 197. Tsai, T., Mehta, R.C., DeLuca, P.P., 1996. Adsorption of peptides to polyŽD,L-lactideco-glycolide.: 1. Effect of physical factors on adsorption. Int. J. Pharm. 127, 31. Wilson, M.B., Nakane, P.K., 1978. Recent developments in the periodate method of conjugating horseradish peroxidase ŽHRPO. to antibodies. In: Knapp, W., Holubar, K., Wick, G. ŽEds.., Immunofluorescence and Related Staining Techniques. Elsevier, Amsterdam, p. 215.