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. Bottger c, B. Micheel a,*

aInstitute of Biochemistry and Biology, Biotechnology, Potsdam University, Karl-Liebknecht-Str. 24-25, 14476 Golm, GermanybMax-Delbruck-Center for Molecular Medicine, Robert-Rossle-Str. 10, 13125 Berlin-Buch, Germany

cWilex Biotechnology GmbH, Grillparzer Str. 10 B, 81675 Munchen, 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 differ-

ent applications in research as well as medicine and

biotechnology (Van Regenmortel and Muller, 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-

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 se-

quence. On the other hand, peptide displaying phage

libraries (Scott and Smith, 1990) and synthetic pep-

tides (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 (Bottger 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 (Kieber-

Emmons et al., 2000).

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022-1759 (01 )00561 -0

Abbreviations: BSA, Bovine serum albumin; ELISA, Enzyme-

linked immunosorbent assay; FITC, Fluorescein isothiocyanate;

HRP, Horseradish peroxidase; PBS, Phosphate-buffered saline; M-

PBS, 1% milkpowder in PBS.* Corresponding author. Tel.: +49-331-977-5242; fax: +49-331-

977-5061.

E-mail address: bmicheel@rz.uni-potsdam.de (B. Micheel).

www.elsevier.com/locate/jim

Journal of Immunological Methods 261 (2002) 141–144

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 deoxyniva-

lenol. 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 an-

tibody.

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 fluores-

cein, 6-carboxyfluorescein and FITC-labelled proteins

and three orders of magnitude weaker with the struc-

turally related molecule phenol red.

2.2. Synthetic peptides

Themimotope peptideswhich bind to antibodyB13-

DE1 were originally identified by biopanning using

phage peptide library (Bottger et al., 1999). The pep-

tides 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 reac-

tivity with B13-DE1 in several different assays: ADG-

AGSWGEWGA-amid (the underlined insert sequence

was identified by phage display as mimotope).

2.3. Peptide peroxidase conjugates

The N-terminus of the peptides representing fluo-

rescein mimotopes were conjugated to horseradish

peroxidase (HRP; Roche Diagnostics, Mannheim) ac-

cording to slightly modified standard methods des-

cribed by Hermanson (1996). The amount of 0.5 mg

peptide was conjugated to 1.5 mg HRP.

2.4. Conjugation of FITC to BSA and HRP

The NH2 groups of the proteins BSA and HRP

was conjugated to fluorescein according to slightly

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 mlper 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 incu-

bated for 4 h at room temperature with 50 ml/well of amixture (incubated in advance for 30 min at room tem-

perature) of a HRP-peptide conjugate (1:500 inM-PBS

diluted) or FITC-HRP conjugate and different fluores-

cein or fluorescein derivative concentrations. All

stripes were then washed 10 times with 200 ml per wellof 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.

2.6. Assay validation

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 mimotope-

HRP conjugate was prepared which resulted in a slig-

htly higher assay sensitivity compared to the tests

using the first batch. For all these assays the materials

were identical, including the samples for the calibra-

tion curve. The fluorescein concentrations were deter-

mined 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 soft-

ware 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.

3. Results

A dilution of 1:500 was found to be the best work-

ing concentration for the conjugate S940-HRP in the

F. Sellrie et al. / Journal of Immunological Methods 261 (2002) 141–144142

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 competi-

tion 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-carboxy-

fluorescein showed a definitely lower inhibiting effi-

ciency 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 bind-

ing 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 (Bottger et al., 1999; Micheel et al.,

1988).

Higher sensitivities for the determination of fluo-

rescein could be obtained when altering some assay

conditions, especially the duration of incubation. The

calibration range in such optimized assays was bet-

ween 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, espe-

cially the duration of incubation.

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).

Fig. 1. Inhibition of S940-HRP conjugate binding to solid phase

B13-DE1 by fluorescein derivatives or related dyes.

Fig. 2. Inhibition of S940-HRP and FITC-HRP conjugate binding to

solid phase B13-DE1 by fluorescein.

Table 1

Between-assay validation data when inhibiting the binding of S940-

HRP conjugate to solid phase B13-DE1 by fluorescein

Number of

assay

Sample A Sample B Sample C Sample D

1 0.9339 0.6765 0.5089 0.1791

2 0.9144 0.6815 0.4935 0.2307

3 0.8716 0.6617 0.5035 0.2596

4 0.8557 0.6599 0.4792 0.2738

5 0.6131 0.5418 0.4765 0.1978

Mean of concentration

(ng/ml)

0.8377 0.6443 0.4923 0.2282

Coefficient of variation 15.45% 9.01% 2.91% 17.53%

F. Sellrie et al. / Journal of Immunological Methods 261 (2002) 141–144 143

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-fluores-

cein 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 sensi-

tive 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 conven-

tional competitive immunoassays by Wild (1994). The

validation data in addition to the results obtained by

testing the cross-reactivity and by comparing the hap-

ten and mimotope conjugates demonstrate the poten-

tial value of mimotope peptides in assay development.

The use of mimotope peptide conjugates in com-

petitive 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 with-

out hapten (or antigen) conjugates is the use of anti-

idiotypic 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 anti-

body 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 conju-

gate, especially valuable, dangerous or toxic in prac-

tical applications.

Acknowledgements

This research was supported in part by a grant to

the ‘‘Innovationskolleg: Biomolekulare Erkennungs-

systeme fur die biochemische Analytik’’, 16B1-1,

from Deutsche Forschungsgemeinschaft (DFG).

References

Bottger, V., Peters, L., Micheel, B., 1999. Identification of peptide

mimotopes for the fluorescein hapten binding of monoclonal

antibody B13-DE1. J. Mol. Recognit. 12, 191–197.

Hermanson, G.T., 1996. Bioconjugate Techniques. Academic Press,

San Diego.

Kieber-Emmons, T., 1998. Peptide mimotopes of carbohydrate anti-

gens. Immunol. Res. 17, 95–108.

Kieber-Emmons, T., Monzavi-Karbassi, B., Wang, B., Luo, P.,

Weiner, D.B., 2000. Cutting edge: DNA immunization with

minigenes of carbohydrate mimotopes induce functional anti-

carbohydrate antibody response. J. Immunol. 165, 623–627.

Kramer, A., Schneider-Mergener, J., 1998. Synthesis and screening

of peptide libraries on continuous cellulose membrane supports.

Methods Mol. Biol. 87, 25–39.

Langone, J.J., Bjercke, R.J., 1989. Idiotype–anti-idiotype hapten

immunoassays: assay for cotinine. Anal. Biochem. 182, 187–

192.

Micheel, B., Jantscheff, P., Bottger, V., Scharte, G., Kaiser, G.,

Stolley, P., Karawajew, L., 1988. The production and radioim-

munoassay application of monoclonal antibodies to fluorescein

isothiocyanate (FITC). J. Immunol. Methods 111, 89–94.

Scott, J.K., Smith, G.P., 1990. Searching for peptide ligands with an

epitope library. Science 249, 386–390.

Skerra, A., Schmidt, T.G., 1999. Applications of a peptide ligand for

streptavidin: the Strep-tag. Biomol. Eng. 16, 79–86.

Van Regenmortel, M.H.V., Muller, P., 1999. Synthetic peptides as

antigens. Laboratory Techniques in Biochemistry and Molecular

Biology, vol. 28. Elsevier, Amsterdam.

Wild, D., 1994. The Immunoassay Handbook. Stockton Press, New

York.

Yuan, Q., Pestka, J.J., Hespenheide, B.M., Kuhn, L.A., Linz, J.E.,

Hart, L.P., 1999. Identification of mimotope peptides which

bind to the mycotoxin deoxynivalenol-specific monoclonal anti-

body. Appl. Environ. Microbiol. 65, 3279–3286.

F. Sellrie et al. / Journal of Immunological Methods 261 (2002) 141–144144