Skip to main content

Validation Procedure for Multiplex Antibiotic Immunoassays Using Flow-Based Chemiluminescence Microarrays

Part of the Methods in Molecular Biology book series (MIMB,volume 1518)


Small molecules like antibiotics or other pharmaceuticals can be detected and quantified, among others, with indirect competitive immunoassays. With regard to multiplex quantification, these tests can be performed as chemiluminescence microarray immunoassays, in which, in principle, the analyte in the sample and the same substance immobilized on the chip surface compete for a limited number of specific antibody binding sites. The amount of the specific primary antibody that has been bound to the surface is visualized by means of a chemiluminescence reaction.

Validated quantitative confirmatory methods for the detection of contaminants, for example drug residues, in food samples usually comprise chromatographic analysis and spectrometric detection, e.g., HPLC-MS, GC-MS, or GC with electron capture detection. Here, we describe a validation procedure (according to the Commission Decision of the European Communities 2002/657/EC) for multiplex immunoassays performed as flow-through chemiluminescence microarrays, using the example of a small molecule microarray for the simultaneous detection of 13 antibiotics in milk. By this means, we suggest to accept multianalyte immunoassays as confirmatory methods as well, to benefit from the advantages of a fast automated method that does not need any pretreatment of the sample. The presented microarray chip is regenerable, so an internal calibration is implemented. Therefore, the analytical results are highly precise, combined with low costs (the aim for commercialization is less than 1 € per analyte per sample, this is significantly less than HPLC-MS).

Key words

  • Antibiotics
  • Microarrays
  • Competitive immunoassay
  • Chemiluminescence
  • Regeneration
  • Flow-based analysis platform
  • Screening methods
  • Residue analysis
  • Veterinary drugs

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-4939-6584-7_13
  • Chapter length: 18 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   89.00
Price excludes VAT (USA)
  • ISBN: 978-1-4939-6584-7
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   119.00
Price excludes VAT (USA)
Hardcover Book
USD   169.99
Price excludes VAT (USA)
Fig. 1
Fig. 2
Fig. 3
Fig. 4

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Kloth K, Niessner R, Seidel M (2009) Development of an open stand-alone platform for regenerable automated microarrays. Biosensor Bioelectron 24(7):2106–2112. doi:10.1016/j.bios.2008.11.005

    CAS  CrossRef  Google Scholar 

  2. Kloth K, Rye-Johnsen M, Didier A, Dietrich R, Märtlbauer E, Niessner R, Seidel M (2009) A regenerable immunochip for the rapid determination of 13 different antibiotics in raw milk. Analyst 134(7):1433–1439. doi:10.1039/b817836d

    CAS  CrossRef  PubMed  Google Scholar 

  3. Wutz K, Niessner R, Seidel M (2011) Simultaneous determination of four different antibiotic residues in honey by chemiluminescence multianalyte chip immunoassays. Microchim Acta 173(1-2):1–9. doi:10.1007/s00604-011-0548-9

    CAS  CrossRef  Google Scholar 

  4. Oswald S, Karsunke X, Dietrich R, Märtlbauer E, Niessner R, Knopp D (2013) Automated regenerable microarray-based immunoassay for rapid parallel quantification of mycotoxins in cereals. Anal Bioanal Chem 405(20):6405–6415. doi:10.1007/s00216-013-6920-3

    CAS  CrossRef  PubMed  Google Scholar 

  5. Szkola A, Campbell K, Elliott CT, Niessner R, Seidel M (2013) Automated, high performance, flow-through chemiluminescence microarray for the multiplexed detection of phycotoxins. Anal Chim Acta 787:211–218. doi:10.1016/j.aca.2013.05.028

    CAS  CrossRef  PubMed  Google Scholar 

  6. Szkola A, Linares EM, Worbs S, Dorner BG, Dietrich R, Märtlbauer E, Niessner R, Seidel M (2014) Rapid and simultaneous detection of ricin, staphylococcal enterotoxin B and saxitoxin by chemiluminescence-based microarray immunoassay. Analyst 139(22):5885–5892. doi:10.1039/c4an00345d

    CAS  CrossRef  PubMed  Google Scholar 

  7. Huebner M, Ben Haddada M, Methivier C, Niessner R, Knopp D, Boujday S (2015) Layer-by-layer generation of PEG-based regenerable immunosensing surfaces for small-sized analytes. Biosensor Bioelectron 67:334–341. doi:10.1016/j.bios.2014.08.047

    CAS  CrossRef  Google Scholar 

  8. Seidel M, Niessner R (2014) Chemiluminescence microarrays in analytical chemistry: a critical review. Anal Bioanal Chem 406(23):5589–5612. doi:10.1007/s00216-014-7968-4

    CAS  CrossRef  PubMed  Google Scholar 

  9. The Commission of the European Communities (2002) Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Available via EUR-Lex. Accessed 17 Oct 2015

Download references


The Milchprüfring Bayern that is already applying the MCR 3 to detect antibiotics in milk samples is gratefully acknowledged for taking part in this study.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Michael Seidel .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Meyer, V.K. et al. (2017). Validation Procedure for Multiplex Antibiotic Immunoassays Using Flow-Based Chemiluminescence Microarrays. In: Uttamchandani, M., Yao, S. (eds) Small Molecule Microarrays. Methods in Molecular Biology, vol 1518. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6582-3

  • Online ISBN: 978-1-4939-6584-7

  • eBook Packages: Springer Protocols