Development of indirect competitive ELISA using egg yolk-derived immunoglobulin (IgY) for the detection of Gentamicin residues

Abstract

Gentamicin (Gent) is an aminoglycoside antibiotic being used in livestock sector. Gent residues could cause some genetic disorders by nonsense mutations. This study aimed to develop IgY-based ELISA for the detection of Gent in animal products. Gent was conjugated with Bovine serum albumin (BSA) by carbodiimide method for further immunization in the laying chickens. PEG-6000 extraction method was employed to extract IgY from the egg yolk. The titer of anti-Gent-IgY attained the peak of 1:256,000 after the 5(th) booster immunization. Checkerboard titration confirmed that, anti-Gent IgY in 1:2,000 dilution could give an Optical Density (OD) 1.0 at 2 µg mL(-1) of Gent-OVA coating concentration. IgY-based indirect competitive ELISA (Ic-ELISA) showed that, the IC50 value of anti-Gent IgY was 2.69 ng mL(-1) and regression curve equation was y = -16.27x + 56.97 (R(2) = 0.95, n = 3), confirming that, the detection limit (LOD, IC10 value) was 0.01 ng mL(-1). Recoveries from fresh milk, pork and chicken samples were ranged from 69.82% to 94.32%, with relative standard deviation lower than 10.88%. Our results suggested that generated anti-Gent IgY antibodies can be used in routine screening analysis of Gent residues in food samples.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment

“…Among the prevailing isolation methods, ultracentrifugation (including sucrose gradient fractionation method), polyethylene glycol (PEG) and SEC are three distinct methods frequently employed to isolate exosomes, each with its own advantages and limitations. In this investigation, polyethylene glycol (PEG) to precipitate lipid extracellular vesicles was avoided as PEG has traditionally been used to extract glucose metabolic enzymes and antibodies 47 – 49 . The use of SEC has been extensively examined for its superior capacity to eliminate common protein contaminants such as albumin 50 – 53 .…”

Egg yolk antibodies (IgY) and their applications in human and veterinary health: A review

“…IgY can also be used in assays to identify antibiotic residues in food products of animal origin, as demonstrated in a study by He et al, in which produced anti-gentamycin IgY specifically detected the target antibiotic present in animal origin products [123]. Following this rationale, Li et al used specific IgY to detect kanamycin and gentamycin residues in milk and meat samples by means of competition ELISA and FPIA (fluorescence polarization immunoassay) [124].…”

Evaluation of Human Anti Igg Polyclonal Antibody Production Conjugated with Peroxidase in Egg Yolk

“…Production of anti-IgG IgY is essential to detect and assess the activity of IgY and to design immunochemistry kits. A refined IgY extraction method could improve the quality of IgY product and make a breakthrough in IgY technology (31). In this study, injection of IgG along with Freund adjuvants to hens induced the production of anti-IgG antibodies, isolated from the lipids of the yolk via sedimentation with PEG 6000 that solubilized the proteins of the yolk; with centrifugation, the yolk proteins were purified.…”