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Development of Colloid Gold-Based Lateral Flow Immunochromatographic Kits for Screening and Rapid Detection of Beta-Lactams Antibiotic Residues in Dairy Milk

Development of Colloid Gold-Based Lateral Flow Immunochromatographic Kits for Screening and Rapid Detection of Beta-Lactams Antibiotic Residues in Dairy Milk

Sara Mohamed Hemeda1, R.H. Sayed2, Hani Hassan1, Sheima A.E.2, Hassan Aboul-Ella3, R. Soliman3*

1Institute for Animal Reproduction, Ministry of Agriculture, Egypt; 2Institute of animal vaccines and serum production, Ministry of agriculture, Abbasia, Egypt; 3Department of Microbiology, Faculty of Veterinary medicine, Cairo University, Egypt.

 
*Correspondence | R. Soliman, Department of Microbiology, Faculty of Veterinary medicine, Cairo University, Egypt; Email: rafiksoliman108@hotmail.com

ABSTRACT

An affordable, sensitive, specific, fast, and easy to be delivered to end-users immunochromatographic lateral flow kit (LFK) has been developed for the detection of β-lactams antibiotic residues in dairy milk. The developed LFK is based on a competitive immunochromatographic format using anti-antibiotic-specific polyclonal antibodies. Specific artificially induced active acquired polyclonal antibodies production were performed in female New Zealand rabbits against the following β-lactam antibiotics; Amoxicillin (AMX)- Keyhole Limpet Hemocyanin (KLH) and Penicillin-G (Pen G)-KLH conjugates. The prepared anti-β-lactam antibodies were conjugated with gold chloride nanoparticles and used in the development of competitive lateral flow kits for the detection of β-lactams antibiotic residues in dairy milk. The concentration of the gold chloride-antibody conjugate in the conjugation pad was adjusted to detect β-lactam antibiotics in milk samples that exceed the permissible limit. In the newly developed kits, each involved β-lactam antibiotic conjugated with KLH was placed in the test line to compete for the gold chloride anti-β-lactams antibodies conjugate with the free beta-lactam antibiotics supposedly exceeding the permissible limit and existing in the tested samples or vice versa. The control line of the developed lateral flow kits was coated with polyclonal unlabeled goat anti-rabbit antibodies. If the tested milk sample contains a high antibiotic level that exceeds the permissible limits it will be consumed by reaction with the gold anti-β-lactam antibody conjugate in the conjugation pad and the test line read negative with no color development, while if the tested samples contain antibiotics lower than the permissible limits it will not consume the whole conjugate that will, in turn, reacts with the β-lactam antibiotic in the test line producing a deep red color in the test line, so the presence of two deep ruby red lines indicates acceptable Beta-lactam antibiotic residues beneath the permissible limit and vice versa in case of one deep ruby red line. The results could be accomplished within 8 minutes without the need for any other equipment. The recorded lower detection limit of β-lactams by the developed kits was 10ppb in skimmed milk and 5ppb in phosphate buffer saline. 75 cattle milk samples were examined by the developed kits and by the commercially available new existing and commercially available SNAP β-Lactam test kit. The results were compared, and the determined specificity, sensitivity, and accuracy of the locally prepared kits were 93.7%, 95.3%, and 94.6%, respectively.
 
Keywords | Polyclonal antibodies (PAbs) production, Nano-gold applications, Lateral flow immunochromatographic assay (LFA), Antibiotic residues, Rapid detectors

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Advances in Animal and Veterinary Sciences

June

Vol. 12, Iss. 6, pp. 994-1205

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