Development of a Lateral-Flow Immunochromatographic Strip for the Detection of Oxytetracycline Residues in Biological Fluids

Abstract

Oxytetracycline (OTC) is extensively used in veterinary medicine and for growth promotion around the globe. The indiscriminate use of OTC in food-producing animals leaves residues in animal products. The presence of these residues in animal products causes economic losses and harmful effects on consumers. Different regulatory bodies set maximum residue limits (MRLs) for different tetracyclines. To avoid harmful effects, there is a need for a simple, fast, and economical method for the screening of animal products. In this study, a fast, economical, and user-friendly lateral-flow immunochromatographic (LFIC) assay based on gold nanoparticles (AuNPs) was developed to detect the presence of OTC residues in biological fluids. AuNPs provided visual results as red lines in 6-15 min. Polyclonal rabbit IgG antibodies were produced using the immunogen of OTC. These antibodies were purified by the combined ammonium sulfate-octanoic acid precipitation method. Antibodies were conjugated to AuNPs as recognition biomolecules. A LFIC strip was optimized using borate buffer spiked with different concentrations of the OTC. The visual limit of detection (LOD) in different biological samples (milk, serum, and urine) was determined using samples spiked with OTC. The LOD was found to be 15 μg/L, which is very low from the MRL (100 μg/L) set by different regulatory authorities. This LFIC strip can be used to detect OTC residues in biological fluids for point-of-care testing (POCT). These strips are easy to use, cost-effective, and portable and provide quick results without the use of laboratory instruments.

Figure 1

Mannich reactions of OTC with BSA and OVA: (A) Mannich reaction of OTC and BSA and (B) Mannich reaction of OTC and OVA.

Figure 2

UV–visible spectra: (A) UV–visible spectra of OTC, BSA, and OTC-BSA, and (B) UV–visible spectra of OTC, OVA, and OTC-OVA.

Figure 3

Quantification of polyclonal rabbit IgG antibodies purified by a combined AS-OA precipitation method.

Figure 4

Separating gel containing different bands where “P”: prestained protein ladder, “C”: polyclonal anti-CFT IgG antibodies (part of other work), and “O”: polyclonal anti-OTC IgG antibodies.

Figure 5

Confirmation of gold-labeled pAb: (A) UV–visible spectra of AuNPs and gold-labeled pAb and (B) red line on the control line showing the formation of gold-labeled pAb.

Figure 6

(A) Schematic diagram of the LFIC strip. (B) Strips run with borate buffer spiked with OTC, (C) strips run with milk samples spiked with OTC, (D) strips run with serum samples spiked with OTC, and (E) strips run with urine samples spiked with OTC.