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A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk

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Abstract

A simple, efficient and rapid method for the synthesis of cephalosporin–protein conjugates was established. These conjugates were used as immunogens to produce monoclonal antibodies (mAbs) and as solid phase antigens in competitive indirect enzyme immunoassays (EIAs). With this generic approach, a novel set of monoclonal antibodies for cephalosporins was prepared, including ceftiofur and cephalexin as well as, reported here for the first time, cefoperazone, cefquinome and cephapirin. All 5 EIAs were highly sensitive, with standard curve IC50 values of 0.7 (ceftiofur), 1.1 (cefquinome), 5.2 (cephalexin), 13.8 (cefoperazone) and 40.3 ng mL−1 (cephapirin). Detection limits (IC30) ranged from 0.3 (ceftiofur mAb 1D7) to 17.2 ng mL−1 (cephapirin mAb 2F10). Specificity studies revealed that cephalosporin–antibody binding was strongly determined by the side chain residues of the cephem nucleus. Therefore all mAbs, to some extent, recognized other beta-lactam antibiotics with similar side chain residues. Within the group of cephalosporins approved for use in veterinary medicine, however, the final EIAs were highly selective for their respective antigen, except for the ceftiofur EIA which showed cross-reactions with cefquinome. The applicability of the five assays for drug residue testing in milk was demonstrated. In each EIA the target drug could be determined in milk with high accuracy and precision at concentrations far below the European Union maximum residue limits.

Structures of cephalosporins for which an MRL has been set within the EU. Monoclonal antibodies were produced against those substances shown in green lettering.

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Abbreviations

7-ACA:

7-aminocephalosporanic acid

BSA:

bovine serum albumin

EDC:

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide)

EIA:

enzyme immunoassay

EPB:

(R)-(−)-α-[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]-4-hydroxybenzeneacetic acid

EU:

European Union

GlcOx:

glucose oxidase

HRP:

horseradish peroxidise

KLH:

keyhole limpet hemocyanin

LOD:

limit of detection

mAb:

monoclonal antibody

MES:

2-(N-morpholino)ethanesulfonic acid hydrate

MRL:

maximum residue limit

NHS:

N-hydroxysuccinimide

PBS:

phosphate buffered saline

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Acknowledgements

This research project was partly supported by the Bavarian State Ministry of Nutrition, Agriculture and Forestry. We thank Ms. Brunhilde Minich, Ms. Franziska Witzko and Mr. Mostefa Djeffal for their excellent technical assistance.

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Authors and Affiliations

  1. Department for Veterinary Sciences, Chair for Hygiene und Technology of Milk, Ludwig-Maximilians-University München, Schönleutnerstraße 8, 85764, Oberschleißheim, Germany

    Anna Bremus, Richard Dietrich, Lars Dettmar & Erwin Märtlbauer

  2. Veterinary Faculty, Chair of Dairy Science, Justus Liebig University, Ludwigstrasse 21, 35390, Giessen, Germany

    Ewald Usleber

Authors
  1. Anna Bremus
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  2. Richard Dietrich
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  3. Lars Dettmar
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  4. Ewald Usleber
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  5. Erwin Märtlbauer
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Corresponding author

Correspondence to Richard Dietrich.

Electronic supplementary material

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Table S1

Concentrations of immunoreagents used in the optimized competitive indirect EIAs for the determination of cephalosporins in buffer solution (DOCX 12 kb)

Table S2

Antiserum titres of mice immunized with cephalosporin-KLH conjugates (DOCX 11 kb)

Fig. S1

Standard curves for the detection of cefquinome using the four different mAbs in an indirect competitive EIA coated with cefquinome-NHS-BSA. The coefficients of variation for replicate standard concentrations were typically below 9.4% (DOCX 22 kb)

Fig. S2

Standard curves for the detection of cephalexin and cephapirin, using mAb 1F10 and 2F10, respectively, in an indirect competitive EIA coated with the accordant EDC conjugate. The coefficients of variation for replicate standard concentrations were typically below 6.0% for cephalexin coating and below 7.6% for cephapirin coating, respectively (DOCX 19 kb)

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Bremus, A., Dietrich, R., Dettmar, L. et al. A broadly applicable approach to prepare monoclonal anti-cephalosporin antibodies for immunochemical residue determination in milk. Anal Bioanal Chem 403, 503–515 (2012). https://doi.org/10.1007/s00216-012-5750-z

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  • DOI: https://doi.org/10.1007/s00216-012-5750-z

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