Development of immunochromatographic assay for simultaneous detection of tetracyclines and streptomycin in milk

Objectives. To optimize indirect antibody immobilization on gold nanoparticles (GNPs) using anti-species antibodies for enhanced conjugate stability and to develop an immunochromatographic assay (ICA) for antibiotic detection in milk.

Methods. The GNPs were synthesized by reduction of tetrachloroauric acid in the presence of seed particles. The size of GNPs was determined spectrophotometrically according to literature data using a Thermo Fisher Scientific Varioskan LUX instrument. Monoclonal mouse antibodies to tetracycline and streptomycin were immobilized on the surface of the GNPs via anti-mouse antibodies. Conjugates of bovine serum albumin with tetracycline and streptomycin were obtained through Mannich reaction and click-reaction, respectively. The coupling ratio was determined by MALDI-TOF mass spectrometry on a Bruker RapifleX instrument. Immunoreagents were dispensed onto a nitrocellulose membrane using a BioDot ZX1010 dispenser. The assembled multi-membrane composite was cut into test strips using a KinBio ZQ4500 guillotine cutter. The test results were interpreted visually and using an Allsheng TSR-100 test strip reader.

Results. Following conjugate formation via indirect immobilization using anti-species antibodies, it was necessary to block residual binding sites on the anti-species antibodies in order to enhance solution-phase conjugate stability. As a result of optimizing the concentrations of immunoreagents, an ICA was developed for the simultaneous detection of streptomycin and tetracyclines in milk. The detection limit of the optimized ICA for tetracyclines and streptomycin was 2–7.5 and 25 ng/mL, respectively, for visual result interpretation, and 0.29–2.15 and 1.34 ng/mL, respectively, when using a test strip reader.

Conclusions. It is shown that the stability of the resulting conjugates in solution can be enhanced by blocking the free binding sites of the anti-species antibodies to prevent cross-linking of the nanoparticles caused by anti-species antibody binding.

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