The Study On Enzyme-linked Immunosorbent Assay Of Fluoroquinolone Antibiotics And Immunoaffinity Chromatography Of Phenylethanolamine A In Food Samples

“Hunger breeds discontentment, food safety first", food safety is bound up withpeople living standard closely. In recent years, repeated food safety incidents havecompelled food safety to be a hot issue of society. The establishment of detection methodwith rapid, simple, accurate and reliable is the key of food safety. In this study, wedeveloped a highly sensitive enzyme-linked immunosorbent assay (ELISA) for thedetection of fluoroquinolone antibiotics in food, and we also set up a sampleimmunoaffinity chromatography for the detection of a new β-agonist phenylethanolamineA in samples.Fluoroquinolone antibiotics are a synthetic class of antibacterial drugs. They arewidely used in clinical practice because of their strong antibacterial activities and less crossresistance with other antibacterial drugs. They can be widely used as food additives for theprevention and treatment of various infectious diseases of animal and human. But theexcessive or improper use can lead to excessive fluoroquinolone antibiotics residues inanimal food, not only harm to the human body by its own toxicity, but also easy to induceresistance transfer by the long-term consumption of animal food which havefluoroquinolones with lower concentrations, thus affecting the clinical efficacy of the drugs.Therefore, the food safety of fluoroquinolone antibioticcs has attracted more and moreattention. So it is important to establish a high sensitivity, fast, simple, reliable detectionmethod for the simultaneous detection of several kinds of fluoroquinolone antibiotics. Inthis study, in order to detect more kinds of fluoroquinolone antibiotics and enhance thedetection sensitivity, we synthesized two different immunogens and four different kinds ofcoating antigens and produced two kinds of polyclonal antibodies. Under optimalexperimental conditions, the sensitivity of I antiserum was1.82-2.67ng mL-1and detectionlimit was0.089-0.22ng mL-1of ELISA for ciprofloxacin; the sensitivity of IV antiserumwas0.61-0.93ng mL-1and detection limit was0.031-0.067ng mL-1of ELISA for ciprofloxacin. It was seen that the cross-reactivity(CR) values ofⅠantiserum with ninekinds of fluoroquinolone antibiotics were more than5%and the CR values of Ⅳantiserum with seven kinds of fluoroquinolone antibiotics were more than10%. Theestablished ELISA could detect a variety of fluoroquinolone antibiotics with the advantageof high sensitive at ng g-1level. The milk, pork samples were spiked with enorfloxacin andnorfloxacin respectively, and detected by ELISA. The samples spiked recoveries werewithin86.16-118.25%with the intra-assay coefficients of variation of3.92-16.07%(n=3)and inter-assay coefficients of variation of1.66-14.7%(n=3). The ELISA was comfirmedby HPLC experiments, and the correlation coefficients were greater than0.9. Based on theconsistency between ELISA and HPLC, the developed ELISA for detectionfluoroquinolone antibiotics in food was proven to be a reliable analysis method.Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist that has beenillegally used as an animal feed additive for growth promotion. Some studies report thatwhen people eat the pork which containing PA, it can cause dizziness, weakness and othersymptoms of poisoning and the greater harm to patients with hypertension, heart disease. Itis likely to lead to chromosomal aberrations, induce malignant tumor by long consumption.Therefore, it is an urgent demond to develop a rapid, accurate detection method for PA insamples. The immunoaffinity column (IAC) integrated with liquid chromatographytechnology not only can simplifies sample handling steps, shorten analysis time, but alsocan validate the results to be accurate and reliable by chromatography detection. In thisstudy, an immunoaffinity chromatography (IAC) column for selective extraction of PAfrom swine feed, meat and liver samples was developed. The IAC column was constructedby coupling specificity antibody aginst PA to CNBr activated Sepharose4B. The extractionconditions including loading, washing and eluting solutions were carefully optimized.Under optimal conditions, the IAC column was characterized in terms of maximumcapacity, selectivity, extraction recovery and stability. The maximum capacity of the ICAfor PA extraction was found to be239.4ng. For selectivity testing,100ng of other threeβ-adrenergic agonists (clenbuterol, ractopamine and salbutamol) were separately loadedonto the column, and it was observed that the tested compounds could not be captured onthe column, e.g. the column could only selectively recognize PA. The recovery of the IACfor PA extraction was found within96.47%-101.98%when10,50and100ng PA wereseparately loaded onto IAC column. The IAC column was also applied to real sample extraction. Swine feed, meat and liver samples were collected and spiked with PA in rangeof1.0-20ng g-1. The extraction recoveries of the IAC for PA from the spiked samples werewithin89.48-104.89%measured by high performance liquid chromatography (HPLC), therelative standard deviation were within3.68%-13.16%. The stability of the column wasalso tested. It was showed that after35times repeated usage,60%of the maximumcapacity was still remained. The proposed IAC was proven to be a feasible extractionmethod for PA from different matrices with the properties of high maximum capacity,selectivity, extraction efficiency and stability.