Development of a Fluorescence Polarization Assay for Multi-Determination of 10 Aminoglycosides in Pork Muscle Sample Based on Ribosomal Protein S12 and Studying Its Recognition Mechanism

Abstract

The residues of aminoglycosides in foods of animal origin are a potential risk to consumers. There have been some immunoassays reported for the screening of aminoglycoside residues, but the method showing the broadest detection spectrum can only be used to detect two drugs. This is because a broad specific recognition reagent is not available. In the present study, the receptor of aminoglycosides (ribosomal protein S12 of Lysinibacillus sphaericus) was expressed, and its affinities and recognition mechanisms for 10 aminoglycosides were studied by using surface plasmon resonance and molecular docking, respectively. Then the receptor was used as a recognition reagent to develop a fluorescence polarization assay on a 96-well microplate for the detection of the 10 drugs in pork muscle samples. The limits of detection for the 10 drugs ranged from 5.25 to 30.25 ng/g. The sensitivities for the 10 drugs were generally consistent with their respective receptor affinities and binding energies. After comprehensive comparison, the method performances were better than all the previously reported immunoassays for aminoglycosides. This is the first study reporting the recognition mechanisms of ribosomal protein S12 of Lysinibacillus sphaericus for 10 aminoglycosides and the use of it as a recognition reagent to develop a pseudo-immunoassay for the multi-determination of aminoglycosides in food samples.

Production of AmpC β-Lactamase and Development of a Competitive Array for Discriminative Determination of Cephalosporins in Milk

“…coli AmpC β-lactamase was synthesized directly and inserted into pET32a to construct the express vector, and the above work was done at Sangon Biotech Co., Ltd. (Shanghai, China). The plasmids containing the express vector were transformed into BL21­(D3) to express the enzyme, and the detailed expression processes were done according to our recent report …”

“…The plasmids containing the express vector were transformed into BL21(D3) to express the enzyme, and the detailed expression processes were done according to our recent report. 17 Preparation of Enzyme-Labeled Conjugates. In this study, 5 randomly selected CPs were coupled with HRP to synthesize 5 conjugates.…”

“…During the past several years, receptors as novel recognition elements have attracted the interest of many scientists because a specific receptor can recognize all of its ligands, and the expression of a receptor is simple, cheap, and convenient. So, some research groups have used receptors to develop pseudoimmunoassays for the detection of veterinary drugs in foods of animal origin, including tetracyclines, sulfonamides, estradiol, beta-agonists, and aminoglycosides . It is well known that CPs exhibit their antibacterial effect by interacting with the penicillin binding proteins and impeding the synthesis of bacterial cell wall .…”

“…So, some research groups have used receptors to develop pseudoimmunoassays for the detection of veterinary drugs in foods of animal origin, 12 including tetracyclines, 13 sulfonamides, 14 estradiol, 15 beta-agonists, 16 and aminoglycosides. 17 It is well known that CPs exhibit their antibacterial effect by interacting with the penicillin binding proteins and impeding the synthesis of bacterial cell wall. 18 Therefore, penicillin binding proteins are the receptors of CPs that have been used as recognition elements to develop some methods for the determination of CPs, including pseudoimmunoassays, 19−21 test strips, 22 and biosensors.…”

Development of a Progesterone-Receptor-Based Pseudo-immunoassay for Multi-detection of Progestins in Milk and Studying Its Recognition Mechanism

“…Therefore, the receptor can be used to develop the broad specific analytical method. Up to now, several groups have used receptors as “special antibodies” to develop pseudo-immunoassays for detection of sulfonamides, tetracyclines, estrogen, β-lactams, β-agonists, and aminoglycosides . The results of these reports prove the following: the use of a laboratory animal is avoided; the expression of a receptor is simpler, more rapid, and less costly than the generation of an antibody; the detection spectra of receptor-based methods are usually broader than the antibody-based immunoassays.…”