Development of Antibodies and Immunoassays for Monitoring of Nitrofuran Antibiotics in the Food Chain

Abstract

Nitrofurans are a broad group of drugs once widely used for treatment of microbial and protozoal infections in many livestock species. However, as concerns grew globally with regard to the potentially carcinogenic and mutagenic effects of their residues in foods, they have been banned from use in many parts of the world. In order to monitor compliance to these bans it is essential to have fitfor-purpose testing methods. Immunoassays are the screening tool of choice for many testing laboratories due to their relative low cost, ease of use and high sensitivity. As is the case with all immunoassays, the most important reagents required are high quality, high affinity antibodies that exhibit the required sensitivity and specificity. Generating such antibodies for the nitrofuran family of compounds has required a great deal of effort in the design of immunogens, as the compounds, due to size, are not capable of eliciting an immune response in hosts and are not easily conjugated to carrier proteins. This article reviews the range of strategies used to successfully generate suitable antibodies to a wide range of these drugs and their metabolites. In addition, the platform technologies for nitrofuran detection have moved from simple enzyme-linked immunosorbent assay (ELISA)-based procedures to more sophisticated multiplexing systems which can undertake faster and broader spectrum testing for the parent drugs and their metabolites. Reviews of the technologies used for immunochemical detection of the nitrofurans and of commercially available test kits are also presented.

Immunosensor of Nitrofuran Antibiotics and Their Metabolites in Animal-Derived Foods: A Review

“…The commonly used antibody format is IgG, which detects the intended targets qualitatively and quantitatively by binding to a specific epitop e . Usually, the higher the affinity of the antibody and the more stable it is, the higher the sensitivity of the method is ( Cooper et al, 2017c ). The key factor affecting the quality of the antibody is the structure of the antigen, involving the hydrophobicity ( Baldofski et al, 2018 ), size and structure of the incomplete antigen ( Cooper et al, 2017b ).…”

“…Usually, the higher the affinity of the antibody and the more stable it is, the higher the sensitivity of the method is ( Cooper et al, 2017c ). The key factor affecting the quality of the antibody is the structure of the antigen, involving the hydrophobicity ( Baldofski et al, 2018 ), size and structure of the incomplete antigen ( Cooper et al, 2017b ). Therefore, the basis and key to developing highly sensitive immunoassays is to obtain antigens and antibodies with high affinity and high specificity ( Dengl et al, 2016 ; Guillermo et al, 2019 ).…”

“…The key to develop antibodies with desired affinity and specificity is hapten design ( Shao et al, 2021 ). Since the molecular mass of nitrofuran metabolites is less than 1,000 Da, they are small molecule compounds that are not immunogenic and cannot easily bound to carrier protein ( Cooper et al, 2017b ). Therefore, when preparing antigens and assays, derivatization is usually performed first to increase the molecular mass and metabolite ionization efficiency before assaying and binding to carrier proteins.…”

“…Therefore, when preparing antigens and assays, derivatization is usually performed first to increase the molecular mass and metabolite ionization efficiency before assaying and binding to carrier proteins. Because the derived hapten was much larger than AOZ, it possessed enhanced immunogenicity, as well as a carboxyl group that allowed it to be conjugated to a carrier protein ( Cooper K. M. et al, 2017 ). The commonly used derivatization reagents are 4-carboxybenzaldehyde (4-CBA), 2-nitrobenzaldehyde (2-NBA), 4-nitrobenzaldehyde (4-NBA), etc.…”

Using the HPLC-MS/MS Method to Determine the Residual Amount of Nitrofuran Metabolites in Meat Products

“…Критерием оценки повторяемости служит коэффициент вариации. По результатам исследований определено, что коэффициент вариации воспроизводимости для растворов с концентрацией метаболита 5,10,20 мкг/кг составил 19,3, 21 Результаты исследований показали: -для метаболита 3-амино-2-оксазолидинон (НФ-А-ОЗ) уравнение линейности (0,745452*Х), коэффициент корреляции составил 0,9999876;…”