Production of monoclonal antibody against doxycycline for immunoassay of seven tetracyclines in bovine muscle and milk

Abstract

The objective of this study was to produce a generic monoclonal antibody for multi-determination of the residues of tetracycline drugs in bovine muscle and milk. Two new immunogens of doxycycline were prepared that were used to produce the monoclonal antibodies. Results showed the obtained antibodies simultaneously recognized seven tetracycline drugs (doxycycline, tetracycline, chlortetracycline, oxytetracycline, minocycline, methacycline, demeclocycline). The obtained antibodies and three coating antigens were arranged into six combinations to optimize the reagents combination. After comparison of the performances of these combinations, a heterologous indirect competitive ELISA was then used to determine the seven tetracyclines in bovine muscle and milk. The crossreactivities to the seven analytes were in the range of 47%-102% and the limits of detection were in the range of 1.5-6.9 ng/mL depending on the compound. The recoveries of the seven drugs from fortified blank samples were in the range of 75.3%-106.8% with coefficients of variation lower than 10.9%. Therefore, this method could be used as a multi-analytes screen tool for routine monitoring of the residues of these tetracycline drugs in bovine muscle and milk.

Development of an ELISA for nitrazepam based on a monoclonal antibody

“…The hapten 7-NZP was conjugated to carrier protein using the diazotization method (Ahn et al, 2012;Gao, Zhao, Zhang, Wang, & Wang, 2013). A 300 µL aliquot of 1 N HCl was added to a solution of 5 mg of 7-NZP in 300 µL dry dimethylformamide, and stirred for 15 minutes at 4°C.…”

Production of a monoclonal antibody against oxytetracycline and its application for oxytetracycline residue detection in shrimp

“…Therefore, sample preparation must be modified for other types of matrix. Despite the specific sample separation for each matrix, ELISA is still considered as a suitable method for routine screening detection due to its simplicity compared to the chemical methods which are commonly used as the confirmation method (Gao et al, 2013).…”

Methods for the separation, preconcentration, and determination of tetracycline antibiotics

“…HPLC makes it possible to determine tetracycline at a level of hundredths of μg/mL. Food products and feed IC [46] Meat HPLC [15], [19], [21][22][23], [27], [51] SP [85] IC [16][17][18], [20], [27] VA [24], [31] SP [85] Environmental samples VA [24] Water HPLC [72], [86], [89], [93], [94], [100], [ Eggs HPLC [27], [32], [53], [68], [134] FL [117], [119], [156] IC [18], [27] …”

“…Liquid extraction from solid matrices seems the most common method for the separation of tetracy clines from the solid samples of food products. This method is used for the extraction of tetracycline anti biotics from meat [15][16][17][18][19][20][21][22][23][24][25][26][27], fish [27][28][29][30][31], eggs [18,27,32], and honey [33][34][35][36][37][38][39], and it is performed as follows: A weighed portion of a thoroughly ground solid sam ple is placed in a vessel for agitation, and a selected sol vent is added; the contents are stirred for a specified time (from several minutes to several hours). The phases are separated by filtration.…”

“…The phases are separated by filtration. Phosphate [16-18, 33, 38], citrate [19,29,33], hexamethylene tetramine [39], oxalate [20], and succinate [21,32] buffer solu tions and also the McIlvaine buffer solution [15,[34][35][36][37] are most frequently used as solvents. Furthermore, 5% trichloroacetic acid [23], a mixture of acetonitrile with a citrate buffer [28] and the McIlvaine buffer solution (60 : 40) [24,31], and the mixtures of metha nol with water (70 : 30) [30] and with 0.1 M succinic acid (50 : 50) [22,27] or acetonitrile are used [25,26].…”