Related Experiment Video
Updated: Jul 16, 2026
The Use of a β-lactamase-based Conductimetric Biosensor Assay to Detect Biomolecular Interactions
Published on: February 1, 2018
Development of a receptor-based microplate assay for the detection of beta-lactam antibiotics in different food
1Department of Food Chemistry, University of Wuppertal, Gauss-Strasse 20, 42119 Wuppertal, Germany.
Abstract:
The penicillin-binding protein PBP 2x* from Streptococcus pneumoniae has been utilised to develop a novel microplate assay for the detection and determination of penicillins and cephalosporins with intact beta-lactam structure in milk, bovine and porcine muscle juice, honey and egg. In the assay, the receptor protein is immobilised to a microplate in the first step. To each sample a bifunctional reagent is added, with ampicillin and digoxigenin as functional groups (DIG-AMPI). The amount of bifunctional reagent, which is bound via its ampicillin part to the receptor protein, decreases with increasing beta-lactam concentration in the sample. The detection step uses anti-digoxigenin F(ab) fragments marked with horseradish peroxidase. The more bifunctional reagent is bound to the receptor protein, the more antibody fragments are bound via the digoxigenin part of the reagent. A maximum colour development with tetramethylbenzidine as chromogen for the peroxidase reaction is achieved, when no beta-lactam residues are present. A fractional factorial design was applied to detect chemometrically effects and interactions of the assay parameters. For optimisation of the significant parameters a Box-Behnken design was used. The assay has been developed for various food matrices as screening test with the option for a quantitative assay, when the identity of the residual beta-lactam is known (e.g. elimination studies). Cefoperazon, cefquinome, cefazolin, cloxacillin, ampicillin and benzylpenicillin could be detected at levels corresponding to 1/2 EU maximum residue limit (MRL) in milk, meat juice from muscle tissue of different species, egg and honey (where applicable) without needing lengthy and elaborate sample pre-treatment. Matrix calibration curves are presented, which show that quantitative analyses are possible.
Insights
A new microplate assay detects penicillin and cephalosporin residues in food using penicillin-binding protein 2x*. This method allows for sensitive screening and quantification of beta-lactam antibiotics.
Area of Science:
- Food Science
- Analytical Chemistry
- Microbiology
Background:
- Beta-lactam antibiotics, including penicillins and cephalosporins, are widely used in animal husbandry.
- Residues of these antibiotics in food products pose a potential risk to human health and can lead to antibiotic resistance.
- Accurate and sensitive detection methods are crucial for monitoring antibiotic residues in various food matrices.
Purpose of the Study:
- To develop and optimize a novel microplate assay for the detection and determination of penicillins and cephalosporins.
- To validate the assay's performance in diverse food matrices such as milk, meat juice, honey, and egg.
- To establish a screening test with the potential for quantitative analysis of beta-lactam residues.
Main Methods:
- Utilized penicillin-binding protein 2x* (PBP 2x*) from Streptococcus pneumoniae as the receptor.
- Developed a competitive immunoassay format using a bifunctional reagent (DIG-AMPI) and anti-digoxigenin antibody fragments labeled with horseradish peroxidase.
- Employed chemometric designs (fractional factorial and Box-Behnken) for parameter optimization and assay validation.
Main Results:
- The assay successfully detected various beta-lactam antibiotics (e.g., Cefoperazon, Cefazolin, Ampicillin, Benzylpenicillin) at levels as low as 1/2 EU maximum residue limit (MRL).
- The method demonstrated effectiveness across multiple food matrices (milk, meat juice, egg, honey) with minimal sample pre-treatment.
- Matrix calibration curves were generated, confirming the assay's capability for quantitative analysis when the specific antibiotic is known.
Conclusions:
- The developed microplate assay is a sensitive and robust tool for screening and quantifying beta-lactam antibiotic residues in a range of food products.
- The assay offers a rapid and efficient alternative to traditional methods, facilitating compliance with food safety regulations.
- This assay supports food safety monitoring and can be valuable for elimination studies in animal production.
More Related Videos
Related Concept Videos
01:17Microbial Biosensors
01:24Automated Microbial Diagnostics
01:33Enzyme-Linked Immunosorbent Assay
There are many different types of ELISAs, but they all involve an antibody molecule whose constant region binds an enzyme, leaving the variable region free to bind its specific antigen. Enzyme-substrate reaction allows the antigen to be visualized or quantified.