Journal of Immunological Methods 261 (2002) 141 – 144 www.elsevier.com/locate/jim
A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer F. Sellrie a, J.A. Schenk a,b, O. Behrsing a, V. Bo¨ttger c, B. Micheel a,* a
Institute of Biochemistry and Biology, Biotechnology, Potsdam University, Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany b Max-Delbru¨ck-Center for Molecular Medicine, Robert-Ro¨ssle-Str. 10, 13125 Berlin-Buch, Germany c Wilex Biotechnology GmbH, Grillparzer Str. 10 B, 81675 Mu¨nchen, Germany Received 20 February 2001; received in revised form 4 October 2001; accepted 21 November 2001
Abstract Mimotope peptides — peptides which mimic the binding of a hapten to its corresponding monoclonal antibody — were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Mimotope; Enzyme conjugate; Competitive immunoassay; Fluorescein
1. Introduction Synthetic peptides have been used for many different applications in research as well as medicine and biotechnology (Van Regenmo¨rtel and Mu¨ller, 1999). Instead of using the whole protein such peptides have, e.g., been used as vaccines and in immunoassays with remarkable success. The synthesis of peptides can be performed fairly inexpensively and has become a routine methodology. Peptides have also become es-
Abbreviations: BSA, Bovine serum albumin; ELISA, Enzymelinked immunosorbent assay; FITC, Fluorescein isothiocyanate; HRP, Horseradish peroxidase; PBS, Phosphate-buffered saline; MPBS, 1% milkpowder in PBS. * Corresponding author. Tel.: +49-331-977-5242; fax: +49-331977-5061. E-mail address:
[email protected] (B. Micheel).
pecially interesting for the investigation of unknown gene products since they make it possible to identify the corresponding protein by antibodies raised against short synthetic peptides known from the DNA sequence. On the other hand, peptide displaying phage libraries (Scott and Smith, 1990) and synthetic peptides (Kramer and Schneider-Mergener, 1998) are now widely used for the identification of epitopes detected by monoclonal antibodies. In several cases using these methods peptides were identified which were structurally not identical to the real epitope but mimic the epitope by binding to the antibody. Such mimotopes were also detected for non-proteineceous epitopes (Bo¨ttger et al., 1999; Kieber-Emmons, 1998; Skerra and Schmidt, 1999). These mimotopes are of special interest, e.g. for immunization, in cases where the ‘‘original’’ epitopes are difficult to isolate and therefore available only in minute quantities (KieberEmmons et al., 2000).
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 1 ) 0 0 5 6 1 - 0
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Mimotopes should also be of value as surrogate antigens in immunoassays. A first assay using such a principle was described by Yuan et al. (1999) in an immunoassay to determine the mycotoxin deoxynivalenol. Here we describe a competitive immunoassay for the determination of the hapten fluorescein by applying the monoclonal anti-fluorescein antibody B13-DE1 and a mimotope peptide binding to this antibody.
2. Materials and methods 2.1. Antibodies The monoclonal antibody B13-DE1 produced in our laboratory (Micheel et al., 1988) was used for the experiments. B13-DE1 reacts strongly with fluorescein, 6-carboxyfluorescein and FITC-labelled proteins and three orders of magnitude weaker with the structurally related molecule phenol red.
modified standard methods described by Hermanson (1996). The amount of 1 mg protein was conjugated to 0.2 mg FITC. 2.5. Competitive immune assays ELISA stripes (Nunc) were coated with purified antibody B13-DE1 (incubation overnight with 50 ml per well containing 5 mg/ml in PBS), washed with tap water and blocked with 100 ml M-PBS per well for 30 min at room temperature. The wells were then incubated for 4 h at room temperature with 50 ml/well of a mixture (incubated in advance for 30 min at room temperature) of a HRP-peptide conjugate (1:500 in M-PBS diluted) or FITC-HRP conjugate and different fluorescein or fluorescein derivative concentrations. All stripes were then washed 10 times with 200 ml per well of 0.1% Triton X-100, 0.5 M NaCl, 10 mM Tris pH 7.5. ABTS (Roche Diagnostics, Mannheim; 50 ml/well) was used as substrate to detect solid phase-bound peroxidase. The reaction was measured after 20 min at 405 nm in an ELISA reader.
2.2. Synthetic peptides 2.6. Assay validation The mimotope peptides which bind to antibody B13DE1 were originally identified by biopanning using phage peptide library (Bo¨ttger et al., 1999). The peptides used here were synthesised by Biosyntan (Berlin). Out of the original three mimotopes, the peptide S940 with the following amino acid sequence was used in the experiments described here because of its reactivity with B13-DE1 in several different assays: ADGAGSWGEWGA-amid (the underlined insert sequence was identified by phage display as mimotope). 2.3. Peptide peroxidase conjugates The N-terminus of the peptides representing fluorescein mimotopes were conjugated to horseradish peroxidase (HRP; Roche Diagnostics, Mannheim) according to slightly modified standard methods described by Hermanson (1996). The amount of 0.5 mg peptide was conjugated to 1.5 mg HRP.
The between-assay precision was evaluated by performing a series of five assays on five different days. For the assays, a larger new batch of mimotopeHRP conjugate was prepared which resulted in a slightly higher assay sensitivity compared to the tests using the first batch. For all these assays the materials were identical, including the samples for the calibration curve. The fluorescein concentrations were determined for four different samples, prepared in advance by another person. Two samples were chosen within the calibration range of the assay, and two samples were chosen outside the calibration range. The software GraphPad Prism was applied for calculating the concentrations from measured optical densities in the separate assays as well as for performing the statistical analysis for all five assays to obtain the coefficients of variation.
2.4. Conjugation of FITC to BSA and HRP
3. Results
The NH2 groups of the proteins BSA and HRP was conjugated to fluorescein according to slightly
A dilution of 1:500 was found to be the best working concentration for the conjugate S940-HRP in the
F. Sellrie et al. / Journal of Immunological Methods 261 (2002) 141–144
assays. Since the binding of the S940-HRP conjugate to solid phase-immobilised antibody B13-DE1 could be inhibited by FITC-labelled BSA, the same competition was tested with unconjugated fluorescein and a variety of related compounds. Fluorescein turned out to be the most potent inhibitor of HRP-peptide conjugate binding to B13-DE1. The closely related 6-carboxyfluorescein showed a definitely lower inhibiting efficiency and phenol red inhibited even less efficiently. Cresol red almost failed to inhibit the binding of the HRP-peptide conjugate (Fig. 1). The strength of binding of the different compounds to B13-DE1 was in correspondence with previous publications with the exception of the binding of 6-carboxyfluorescein, which was obviously weaker in the experiments described here (Bo¨ttger et al., 1999; Micheel et al., 1988). Higher sensitivities for the determination of fluorescein could be obtained when altering some assay conditions, especially the duration of incubation. The calibration range in such optimized assays was between 5 and 30 ng/ml (data not shown). The assay was optimised to reach a calibration range for the determination of fluorescein between 5 and 30 ng/ml by altering the assay conditions, especially the duration of incubation.
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Fig. 2. Inhibition of S940-HRP and FITC-HRP conjugate binding to solid phase B13-DE1 by fluorescein.
Since the S940-HRP-conjugate could effectively be used for the determination of fluorescein we compared it with the FITC-HRP conjugate (dilution 1:100,000) as tracer substance. The sensitivity of both assays was comparable and the dilution curves for fluorescein using both conjugates as tracers showed almost no difference. The mimotope-peptide conjugate showed an even slightly better sensitivity (Fig. 2). The between-assay validation for the competitive assay resulted in coefficients of variation of 3% and 9% for fluorescein samples within the calibration range (i.e. within the linear range of the inhibition curves) and of 15% and 17% for the fluorescein samples below and above the working range (Table 1).
Table 1 Between-assay validation data when inhibiting the binding of S940HRP conjugate to solid phase B13-DE1 by fluorescein Number of assay
Fig. 1. Inhibition of S940-HRP conjugate binding to solid phase B13-DE1 by fluorescein derivatives or related dyes.
1 2 3 4 5 Mean of concentration (ng/ml) Coefficient of variation
Sample A Sample B Sample C Sample D 0.9339 0.9144 0.8716 0.8557 0.6131 0.8377
0.6765 0.6815 0.6617 0.6599 0.5418 0.6443
0.5089 0.4935 0.5035 0.4792 0.4765 0.4923
0.1791 0.2307 0.2596 0.2738 0.1978 0.2282
15.45%
9.01%
2.91%
17.53%
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4. Discussion This paper describes the use of mimotope peptides in competitive immunoassay for the determination of hapten concentrations. The assay was built up using as model system the hapten fluorescein, the anti-fluorescein antibody B13-DE1 and the peptide mimotope S940. The peptide mimotope was conjugated to HRP and used to compete with fluorescein or related substances for binding to monoclonal antibody B13-DE1, which was immobilised on plate surfaces. Only one of the three original mimotope-peptide conjugates (S940, mimotope sequence GSWGEW) could be used for this type of assay. Our results definitely show that mimotopes can be used to build up simple and sensitive immune assays to quantitatively determine low molecular weight substances. The assay showed the same sensitivity as a competitive immunoassay using a FITC-HRP conjugate as tracer molecule. It might even have a better specificity since 6-carboxyfluorescein did not show the same competition as fluorescein. But this has to be clarified in further experiments. The reliability and reproducibility of the assay was proved by our assay validation data. They were found to be in the range described in literature for conventional competitive immunoassays by Wild (1994). The validation data in addition to the results obtained by testing the cross-reactivity and by comparing the hapten and mimotope conjugates demonstrate the potential value of mimotope peptides in assay development. The use of mimotope peptide conjugates in competitive immune assays might, therefore, be of interest in those cases when conventional hapten assays fail or are difficult to build up. An alternative to building up immune assays without hapten (or antigen) conjugates is the use of antiidiotypic antibodies (Langone and Bjercke, 1989). Since, however, the affinity of the anti-idiotype to its antibody is in most cases much higher than the affinity of the hapten (or the antigen) to its antibody, such competition assays using anti-idiotypes are often not sensitive enough and therefore applied only in special cases. It has still to be clarified whether mimotopes can be selected for many different haptens or whether the mimotopes known so far are rather an exception than the rule. Since further antibodies can easily be selected against haptens of practical interest
by producing either hybridomas or using large antibody libraries, it can also be anticipated that at least one of them would also react with a peptide mimotope detectable in peptide libraries. The tedious search for the mimotopes can be promising in those cases where the hapten is especially unstable, difficult to conjugate, especially valuable, dangerous or toxic in practical applications.
Acknowledgements This research was supported in part by a grant to the ‘‘Innovationskolleg: Biomolekulare Erkennungssysteme fu¨r die biochemische Analytik’’, 16B1-1, from Deutsche Forschungsgemeinschaft (DFG).
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