Study Of Tetracyclines Immunological Rapid Detection Method

Tetracyclines(TCSs)are a kind of broad-spectrum antibiotics,including tetracycline(TC),chlortetracycline(CTC),oxytetracycline(OTC),doxycycline(DC),demeclocycline(DMC),minocycline(MNC),metocycline(MTC),etc.In the clinical and veterinary fields,it is mainly used to treat various infectious diseases caused by bacteria,but the long-term abuse of illegal phenomenon leads to a large number of residues in animal derived food and environment,which seriously threatens human health and destroys the ecological environment.Therefore,it is of great significance to establish a high-throughput,fast and convenient detection method for TCs residue detection.Now,the detection methods of TCs drug residues mainly include high performance liquid chromatography(HPLC),high performance liquid chromatography tandem mass spectrometry(HPLC-MS),thin-layer chromatograph(TLC),capillary electrophoresis(CE),and so on.This kind of testing method is not suitable for on-site rapid testing because of its expensive instruments,complex sample pretreatment and tedious operation.The immunoassay based on the specific combination of antigen and antibody is not only simple,fast,sensitive and specific,but also has a great advantage in mass samples and on-site screening.In order to obtain broad-spectrum tcsmab,after successfully preparing TC artificial antigen and immunizing BALB/c mice,we selected the most sensitive mice and screened out a group of cell lines which can stably secrete TCsm Ab by cell fusion technology and monoclonal antibody technology.Then,a large number of TCsm Ab were prepared by ascites induction method,and an indirect competitive ELISA(ic-ELISA)was established to detect TCs residues in samples.1.Preparation of TCs artificial antigen and polyclonal antiserumIn order to prepare the broad-spectrum TCs monoclonal antibody,under the condition of retaining the TCs common structure to the greatest extent,TC were coupled with carrier protein by using Formaldehyde method and Diazotization method,UV scanning and SDS-PAGE were used to identify the coupling effect.The results showed that the peak position and peak type of the artificial antigen absorption were changed compared with TC and the carrier protein,and the migration rate of the conjugated protein was significantly lower than carrier protein.Therefore,it was preliminarily judged that the artificial antigen coupling was successful.Then the BALB/c mice were immunized with immunogen,and the effect of formaldehyde method was better.The titer of the polyclonal antibody was as high as 1:5.12×104,and the The IC₅₀ of TC,OTC,CTC and DC were 23.00?56.10?71.88?41.07 ng/m L respectively,There was no cross reaction with other antibiotics such as CAP,EM and GM.2.Preparation and characteristics identification of TCs monoclonal antibodyThe mice with the highest titer and sensitivity were selected as the fusion objects,and the spleen cells of the mice were fused with SP2/0 myeloma cells by cell fusion technology.The fusion cells were screened by ELISA,and the positive hybrid tumor cells were selected for cultured and expanded culture subcloning.Finally,three hybridoma cell lines which can stably secrete TCsm Ab were selected and named as2B6,3E5 and 7F11 respectively.Among them,the TCsm Ab produced by 3E5 cell line has the best inhibitory effect on TCs(TC,OTC,CTC and DC),which is the broad-spectrum monoclonal antibody needed for research.The cell supernatant titer was above 1:3200.Then the large-scale TCsm Ab was prepared by ascites induction method.The titer of ascites was as high as 1:256000,The subtype of the monoclonal antibody is Ig G1,and the affinity constant Ka is 3.94×10⁸L/mol.The IC₅₀ of TC,OTC,CTC and DC were 0.62?1.45?1.86?1.14 ng/m L respectively.There was a large cross reaction with OTC,CTC and DC,but was no cross reaction with other antibiotics such as CAP,EM and GM.3.The establishment of TCs ic-ELISABased on the obtained TCsmAb,ic-ELISA was established by optimizing the working concentration and working time.The optimal working concentration of ic-ELISA was 1:8000 times diluted of envelope antigen and 1:6000 times diluted of antibody 3E5,and the optimal working time was 25 minutes of primary antibody,45minutes of secondary antibody and 10 minutes of color development.Under these conditions,the standard inhibition curve of antibody 3E5 to TC was established.The linear regression equation was y=-0.3447x+0.3671,R²=0.9888,IC₅₀ to TC was 0.41ng/m L,and the linear range was 0.06?3.0 ng/m L.The IC₅₀ of OTC,CTC and DC were0.92?1.48?0.60 ng/ml respectively.There was no cross reaction with other antibiotics such as CAP,EM and GM.The recovery rates of TC,OTC,CTC and DC in milk were 86.3%?98.9%,85.7%?101.6%,85.3%?100.3%,82.8%?90.8%respectively,and the coefficient of variation was less than 15%.The method of ic-ELISA can be used to detect TCs residues in samples.