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A novel monoclonal antibody (MAb) against oxytetracycline (OTC) was generated and characterized. The MAb was used in the development of an enzyme-linked immunosorbant assay (ELISA)-based detection system. An OTC-bovine serum albumin (BSA) conjugate was prepared and used in the immunization of mice. A conventional somatic cell fusion technique was u...
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... (Fig.3-a). An antibiotic-carrier conjugate was obtained by T. Wongtangprasert et al. (2014) only when purified OTC was used ( Fig. 3-b). a b Figure 3 -Appearance of OTC-BSA conjugates: a -OTC HC for veterinary medicine; b -chemically purified OTC Spectrophotometric analysis was used to establish the crosslinking of the purified OTC into the carrier molecules (BSA, OVA). ...
Oxytetracycline is widely used in veterinary medicine for the treatment of animals, as well as growth promoters. They can have adverse effects on human health through animal products if the rules for the use of antibiotics are not followed. The World Health Organization has established maximum residue limits of antibiotics in milk and meat, which require accurate, rapid and inexpensive methods to determine. Immunochromatographic assay (ICA) is ideal for this purpose due to its speed of analysis, high sensitivity and ease of use. This paper describes the results of study on the obtaining an oxytetracycline (OTC) conjugate with bovine serum albumin (BSA) and/or ovalbumin (OVA). OTC has been chemically purified from the antibiotic hydrochloride salt (OTC HC), which is widely used in animal husbandry and is more affordable than its chemically pure analogue. Spectrophotometric analysis of the prepared conjugates and immunization of laboratory animals showed the suitability of OTC, chemically purified from OTC HC, for crosslinking into BSA and/or OVA molecules and obtaining hapten-specific antibodies. The results obtained could be used for the manufacture of ICA components - labeled OTC-specific antibodies and test line antigen.
... Essential characteristics of OTC, including its mode of action (i.e., inhibition of protein synthesis through reversible binding to the 30S subunit of the ribosome), bacteriostatic activity against a wide spectrum of pathogenic organisms, and its easy availability in both conventional and long-acting injectable formulations, collectively make this antibiotic particularly useful for the treatment of a great number of diseases in animals, including food-producing species [4]. Therefore, OTC is routinely used in veterinary practice in ruminants [5][6][7], swine [8,9], poultry [10][11][12], and aquaculture [13,14]. Additionally, this antibiotic is used for therapy in less obvious food-producing animal species, such as the honey bee [15,16] or horses [17,18]. ...
Even though modern analytical chemistry has developed a methodology enabling evaluation of the presence of OTC in milk, data regarding its concentration in the material collected from lactating sows are missing. Therefore, this paper was intended to provide new data on the transmission of OTC and its epimer, 4-epi-oxytetracycline (4-epi-OTC), in the milk of lactating sows after a singular intramuscular administration of a long-acting form of the antibiotic. The determination of OTC and 4-epi-OTC was carried out using ultrahigh-performance liquid chromatography with mass spectrometry (UHPLC–MS/MS). The highest average concentration of antibiotic (1132.2 µgL−1) was observed in samples collected 1 day after the administration of the drug. The average OTC level at day 3 was 358 µgL−1. The average concentration of the antibiotic found on the 21st day was 12.3 µgL−1. The highest average concentration of 4-epi-OTC—i.e., 54 µgL−1—was noted 1 day after the administration. Amongst samples collected at day 3, the average level of the substance in question was 26.4 µgL−1. The average value observed at day 21 was 1.5 µgL−1. Our results indicated considerable OTC and 4-epi-OTC transmission into the milk of lactating sows.
... Da), the conjugate molar ratio of CTC:BSA was calculated to be 4:1, indicating the occurrence of coupling molecules (Figure 1(a)). This conjugation occurred at a lower ratio than our report for oxytetracycline conjugated with BSA (7:1) (Wongtangprasert et al., 2014). The Mannich reaction is a simple and one-step reaction in which the hepten density of the conjugation is dependent on the reactivity of active hydrogen within the hepten molecule. ...
In this study, a monoclonal antibody (mAb) against chlortetracycline was prepared by immunisation using chlortetracycline conjugated to bovine serum albumin (CTC-BSA) and cell fusion. One monoclonal antibody (1-2B) against chlortetracycline was obtained with an IC50 of 0.33 ng/ml and a detection limit of 0.1 ng/ml. The cross reactivity of this antibody with structurally related and non-related compounds was less than 0.1%, demonstrating that the obtained mAb provided high specificity. Under optimal conditions for ic-ELISA, a linearity was obtained between 0.06 and 3.9 ng/ml of chlortetracycline. The quantification of CTC-spiked milk and honey samples was also performed, demonstrating recovery rates ranging from 95.2% to 105.6% and the coefficient of variation below 10% for both intra- and inter-variation assays. Based on the sensitivity and specificity measurement, mAb 1-2B could feasibility be used as a screening tool for CTC residue detection in milk and honey samples.
... The current reliable analytical methods to detect OTC residues in fish edible tissues include high-performance liquid chromatography (HPLC) (Wagil et al., 2015), liquid chromatography mass-spectroscopy (LC-MS/MS) (Monteiro et al., 2015) and antibody-based enzyme-linked immunosorbent assay (ELISA) (Wongtangprasert et al., 2014;Le, Yu, Zhao, & Wei, 2012). These sophisticated tools have demerits of being laboratory-based, expensive, complicated sample preparation, tedious and need instrumental assistance, professional expertise to analyze and interpret chromatograms. ...
... Even the use of toxic solvents may pollute the environment. The ELISAs have often complained of specificity issues (Wongtangprasert et al., 2014;Le et al., 2012). Recently, quantum dot (QD) based, an electrochemical aptamer-based sensor has been introduced to determine tetracyclines (García-Fernández, Trapiella-Alfonso, Costa-Fernández, Pereiro, & Sanz-Medel, 2014;Wang et al., 2015). ...
... ten times) in the immunoassays than in the MS analysis. Finally, recently, Wongtanprasert et al. [103] developed a new monoclonal antibody against oxytetracycline and applied it for OTC quantification in shrimp using an indirect competitive ELISA with recoveries of 82 to 118%. The antibody showed high cross-reactivity to rolitetracycline but no cross-reactivity to other unrelated antibiotics. ...
Antibiotics are widely used as growth promoters in animal husbandry; among them, the tetracyclines are a chemical group of relevance, due to their wide use in agriculture, surpassing in quantities applied almost every other antibiotic family. Seeing the considerable amounts of tetracyclines used worldwide, monitoring of these antibiotics is paramount. Advances must be made in the analysis of antibiotics to assess correct usage and dosage of tetracyclines in food and feedstuffs and possible residues in pertinent environmental samples. The tetracyclines are still considered a clinically relevant group of antibiotics, though dissemination of tolerance and resistance determinants have limited their use. This review focuses on four different aspects: (i) tetracyclines, usage, dosages, and regulatory issues that govern their food-related application, with particular attention to the prohibitions and restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union, (ii) analytical methods for tetracyclines, determination, and residues thereof in feedstuffs and related matrices with an emphasis on the most relevant and novel techniques, including both screening and confirmatory methods, (iii) tetracycline resistance and tetracycline-resistant bacteria in feedstuff, and (iv) environmental and health risks accompanying the use of tetracyclines in animal nutrition. In the last two cases, we discuss the more relevant undesirable effects that tetracyclines exert over bacterial communities and nontarget species including unwanted effects in farmers.
... Additionally, compared with the chance to obtain high-affinity antibodies against large molecules (e.g. protein antigens), the chance to get the effective mAbs with free hapten-binding ability is relatively low. For instance, only three hybridomas of interest were found among a total of 8700 cultured wells from ten fusions in a previous report on the production of mAbs against oxytetracycline, an antibiotic with the molecular weight of 460.4 Da (Wongtangprasert et al., 2014). ...
A water-soluble adjuvant named QuickAntibody (QA) was introduced into the procedure of mouse immunization for the development of hapten-specific monoclonal antibodies (mAbs), using four kinds of pesticides as model compounds. Compared with conventional Freund;’s adjuvants, QA treatments offered relatively low but acceptable antiserum titers after three inoculations, gave little adverse effects to the experimental animals, and were preferable in harvesting splenocytes during the steps of cell fusion. Afterwards, hybridomas from the QA group were prepared and screened by both non-competitive and competitive indirect enzyme-linked immunosorbent assays (ELISAs). The efficiency of gaining immune-positive hybridomas was satisfactory, and the resultant mAbs showed sensitivities (half maximal inhibitory concentration (IC50)) of 0.91, 2.46, 3.72, and 6.22 ng/ml to triazophos, parathion, chlorpyrifos, and fenpropathrin, respectively. Additionally, the performance of QA adjuvant was further confirmed by acquiring a high-affinity mAb against okadaic acid (IC50 of 0.36 ng/ml) after three immunizations. These newly developed mAbs showed similar or even better sensitivities compared with previously reported mAbs specific to the corresponding analytes. This study suggested that the easy-to-use adjuvant could be applicable to the efficient generation of highly sensitive mAbs against small compounds. © 2016, Zhejiang University and Springer-Verlag Berlin Heidelberg.
... However, these instruments are expensive and the preparation and clean up procedures are time consuming. Enzyme-linked immunosorbent assay (ELISA) is a detection method that offers some advantages as it is simple, rapid, inexpensive, can detect samples in large quantities and has been applied to detect OTC residues in different food sample types such as shrimp (Wongtangprasert et al., 2014), milk (Gaurav et al., 2014) and honey (Mahmoudi et al., 2014). The purpose of this study was to conduct a method validation of an indirect competitive ELISA (icELISA) for OTC residue detection in broiler chicken meat using locally generated polyclonal OTC-BSA antibodies (Widiastuti et al., 2103) and to determine the OTC residue level in broiler chicken meat samples marketed in traditional markets and supermarkets from several cities in Java by using the validated method. ...
Oxytetracycline (OTC) is one of the tetracycline (TCs) broad-spectrum antibiotics widely used inthe chicken industry. However, improper use of OTC with excessive doses potentially leads to residueformation in animal products that can be harmful to consumers in the form of allergic reaction orresistance. This study aimed to detect OTC residues in broiler chicken meat, marketed in traditionalmarkets and supermarkets in Depok, Bekasi, Bandung, Cilegon, Surakarta and Yogyakarta using indirectcompetitive enzyme-linked immunosorbent assay (icELISA) method. The analyses of 67 broiler meatsamples showed only 1 (1.5%) sample was positive for the OTC residue at 86.1 ng/g which meant belowthe maximum residue limits of permissible OTC (100 ng/g). Nevertheless, a stricter regulation for theuse of OTC in the poultry industry and the monitoring of its residue in chicken products prior tomarketing is still necessary to avoid the adverse effects of the residue present in animal products.
The stability of the fire blight control material, oxytetracycline, in water is strongly affected by pH, increasing with increasing acidity. From 2017-21, pear and apple orchard trials were conducted to evaluate if acidic amendments to oxytetracycline sprays improve fire blight control. Compared to the water-treated control, infection suppression after two bloom applications of an acidified commercial oxytetracycline formulation averaged 85.9% + 0.4 compared to 72.2% + 1.7 without an acidifier, but individual trials frequently had insufficient statistical power to separate among acidified and non-acidified antibiotic treatments. Across trials, a significant linear relationship was observed for regression of relative infection suppression from oxytetracycline (hydrochloride formulation) on spray tank pH. Similar relationships were observed for oxytetracycline (calcium complex formulation) and kasugamycin (P-values were 0.055 and 0.069, respectively). Also based on regression, acidified oxytetracycline and kasugamycin suppressed epiphytic populations of E. amylovora on flowers to a greater degree than the antibiotic only. As spray suspensions, commercial oxytetracycline formulations at label rate and amended with citric acid (1.2 g/liter) in well water had pH-values near 3.4, but after spraying, the pH of flowers washed in deionized water (1 ml/flower) measured in a range of 5.2 to 5.5 compared to a pH range of 5.8 to 6.0 after a treatment of oxytetracycline only. In pear fruit finish trials, sprays acidified with citric acid-based materials had negligible effects on fruit russeting. Based on a serological assay, the detectable residual of oxytetracycline on apple foliage was increased by co-application with citric acid compared to a non-acidified control.
In this work, we describe a new “signal-on” electrochemical biosensor based on a competitive immunoassay with a label-free format for the facile, sensitive and cost-effective determination of oxytetracycline (OTC). The device pattern was designed and printed on low-cost filter paper to construct a disposable paper-based analytical device (PAD) using a wax printing technique. The sensor consisted of a capture anti-OTC antibody (anti-OTC) and OTC-conjugated bovine serum albumin (OTC-BSA). Anti-OTC was first anchored onto the functionalized PAD, followed by the competitive binding of the OTC target and OTC-BSA. The amount of the captured OTC was examined by observing the signal response of the redox [Fe(CN)6]3-/4-. In the presence of OTC, the current response was significantly increased with increasing OTC concentration, whereas the signal response was negligible in the absence thereof. The detected OTC concentration was linear over a range of 1-200 ng·mL⁻¹, and the corresponding values of 0.33 and 1.1 ng·mL⁻¹ were obtained for the limit of detection (LOD) and limit of quantitation (LOQ), respectively. This introduced sensor displayed high sensitivity and specificity, and the amounts of OTC were successfully quantified in agricultural products. This developed signal-on sensor may be an alternative tool for determining OTC residues and broader antibiotic targets.
Antibiotics are a class of secondary metabolites produced by microorganisms (including bacteria, fungi and actinomyces) or advanced animals and plants in the process of life, and can interfere with the development of other living cells. The immunological methods and development status of antibiotics have been introduced in recent years in this chapter. We briefly reviewed the types and physicochemical properties, the toxicological properties, and harmness of representative antibiotics, as well as the maximum residue limits established by different countries. Then, the design and synthesis of haptens to the AGs, the establishment and evaluation of ELISA, and the pretreatment of foodstuffs were introduced in detail. Finally, the construction of flow immunochromatographic test strips for antibiotic detection, the pretreatment of the sample matrix, cutoff values, detection time, and other performance parameters are fully demonstrated. Examples from recent studies are drawn in the process of elaboration, and the probable perspectives of immunoassay for antibiotics are prospected in this chapter.
