Study On Immunological Detection Of Clenbuterol Based On Novel Bioprobe

With the development of society and advances in science and technology,food safety problems have frequently broken out and threatened public health seriously.Sensitive and reliable analytical methods play an important role in ensuring food safety.Immunoassay is an analytical method based on the specific reaction of antigens and antibodies.It has good specificity and selectivity and has been widely used in food safety testing.In this dissertation,the veterinary drug Clenbuterol Hydrochloride?CL?,was used as the test object to prepare monoclonalantibody.Twokindsofanalyticalsensorswereconstructed:immunochromatographic test strip and enzyme-linked immunosorbent assay kit.The sensitive,reliable and stable detection of clenbuterol hydrochloride in feed,milk and swine urine was realized.The research results of this paper are as follows:1.Preparation of clenbuterol-resistant monoclonal antibody:Six-week-old female BALB/c mice were immunized with an artificial antigen,a commercial clenbuterol-bovine serum albumin conjugate?CL-BSA?,to induce mice to produce anti-clenbuterol antiserum.After four times of immunization,the titer of the anti-serum was the highest and stable.The mouse spleen cells were fused with mouse myeloma cells.The hybridoma cell strain 9A8capable of producing the desired antibody and having a good competitive effect was screened by indirect ELISA and indirect competition ELISA.The monoclonal cells strain was expanded and induced to produce ascites in BALB/c mice and purified using caprylic acid-ammonium sulfate to obtain antibodies.The antibody sensitivity?expressed as IC50?is1.25 ng/mL,which is not interfered by other structural analogs and has strong specificity for clenbuterol hydrochloride.2.Development of novel biological probe based and high sensitive immunochromatographic test strips and identification of their performance:In this study,a novel biological probe using bacteria as a signal carrier was developed.Bacteria can carry a large amount of gold nanoparticles?AuNPs?on their surface to amplify the signal and label extremely a small quantity of antibodies,triggering intense competition between free analytes and immobilized antigens to significantly increase assay sensitivity.By optimizing the analysis conditions,including bacterial species,coupling method and probe concentration ratio,the analytical performance was optimized to achieve a visual detection limit?VDL?of0.1 ng/mL for clenbuterol.Compared to traditional strips based on nanogold-labeled antibody probes,the sensitivity was increased for 20 times.The analysis platform had good specificity and stability,and it had been successfully used for the detection of clenbuterol in feed,milk and swine urine with VDLs of 0.2 ng/g,0.5 ng/mL and 0.1 ng/mL,respectively.This work has opened a new way for signal amplification and performance improvement of immunochromatographic strips.Moreover,inactivated bacteria can become outstanding signal carriers for more biosensors.3.Development of sensitive enzyme-linked immunosorbent assays?ELISAs?and identification of performance:Based on the previous section,bacteria were introduced as natural organic carriers to construct probes.Through the bacterial carrier,the ratio between the signaling molecule and the recognition molecule was flexibly adjusted to achieve the enrichment of horseradish peroxidase?HRP?and limitation of antibody.In this protocol,the enriched enzyme enhanced the signal by catalyzing the chromogenic reaction of the substrate,and a very small quantity of antibody triggered intense competition between the free small molecule target and the immobilized antigen to significantly improve the sensitivity.Based on this principle,an enzyme-linked immunoassay kit was constructed,and parameters were optimized,including the ratio of enzyme to antibody and reaction conditions.Under optimal conditions,the linear range for the analysis of clenbuterol was 0.02-1 ng/mL.The linear equation was:y=-0.542x+2.6826,R²=0.9957,the IC₅₀ was 0.5 ng/mL and the limit of detection?LOD?was 0.02 ng/mL.The kit was not interfered by other structural analogues and was used for the detection of of clenbuterol in feed,milk and swine urine,fully demonstrating that the sensor had good sensitivity,stability,specificity and practicality.