Study On Monoclonal Antibody Against Phenobarbital And Fluorescent Quantitative Test Strip

As a hypnotic,sedative,anticonvulsant and antiepilepitc drug,phenobarbital is often used in clinical treatment of mental anxiety,insomnia,epilepsy,hyperbilirubinemia and so on.Because of the great sedative and hypnotic effect,adding phenobarbital into the feed of edible animals can increase the livestock weight and economic benefits.Phenobarbital was strictly ruled not be detected in edible animals and animal food because it has teratogenic,carcinogenic and other adverse effets on human body.In the field of drug residue detection,immunological analysis has been widely used as its high efficiency,specificity,sensitivity,rapidity and convenience.Immunoassay products for rapid detection of phenobarbital residues have been developed in the United States,Germany and other countries.But the products have not yet appeared in China.In this study,molecular structure of phenobarbital was first analyzed,then prepared the corresponding antigen and monoclonal antibody.The fluorescent quantitative test strip of phenobarbital was developed.The main contents and research results of this stury were as follows:(1)Synthesis and identification of phenobarbital artificial antigen:Hapten-coated AA phenobarbital and immunohapten CA phenobarbital were prepared by substitution and hydrolysis of phenobarbital sodium.The synthesized haptens were all identified by mass spectrometry.The coated hapten CA phenobarbital and the immunological hapten AA phenobarbital were activated by active ester method and then conjugated with BSA or OVA to prepare immunogenic CA phenobarbital-BSA and coated original AA phenobarbital-OVA.With UV-scanning method,immunogenic CA phenobarbital-BSA and coated AA phenobarbital-OVA were peaked at 270nm and 276nm,respectively.It means the phenobarbital artificial antigen was successfully synthesized.(2)Preparation and identification of monoclonal antibodies against phenobarbitalFemale BALB/C mice aged 6-8 weeks were immunized with synthetic immunogen CA phenobarbital-BSA and coated AA phenobarbital-OVA,ci ELISA was used to select cell fusion spare mice.The 7H2,17A3,22H4,25C7,29B2 and 43E86 strain positive cell lines were screened by cell fusion technique,and the antibody secreted by the 29B2 cell line was the best.Female BALB/C mice aged 10-12 weeks were inoculated with 1*10⁶-1*10⁷CFU/m L hybridoma cells for preparing phenobarbital monoclonal antibody.The ascites was purified by affinity column method or octanoic acid-ammonium sulfate precipitation method.Optimal concentration was determined by square matrix tiration:the concentration of the enzyme-labeled secondary antibody was 2000 dilution,and the optiam L coating concentration of the original AA phenobarbital-OVA was 0.25?g/m L,the optimal concentration of the antibody was 0.125?g/m L,;at this antigen concentration,the antibody titer was 1:640000.Four-parameter logistic curve fitting was performed on the phenobarbital standard by indirect ELISA to prepare a standard curve.The IC₅₀value of phenobarbital was calculated to be24.90?g/L,which was linear at 5-405?g/L.The cross-reaction rate with the structural analogs such as barbital,pentobarbital and pentobarbital was less than 0.3%,indicating that the monoclonal antibody did not cross-react with other three barbiturates and had good specificity.(3)Development of fluorescent quantitative test stripsThe phenobarbital fluorescence quantitative test strip was developed by using monoclonal antibody to couple the Merck F1XC030 type carboxyl fluorescent nanospheres.The optimal amount of base for the coupling system was determined to be 80?L(0.1 M potassium carbonate solution);the optimal amount of monoclonal antibody-conjugated fluorescent nanospheres was 0.25 mg(0.5 m L fluorescent microsphere-labeled antibody);NC membrane The optimum drying time is 16 hours;the best system for the sample pad treatment solution is0.1M phenobarbital S(p H=7.4)buffer containing 2.0%sucrose,0.05%Tween 20,1%BSA;The optimal coating concentration of rabbit Ig G in the region C line was 0.25 mg/m L,and the optimal dilution factor after the goat anti-rabbit Ig G was coupled to the fluorescent nanospheres was 500 times;the coating area ofthe detection zone was coated with the antigen AA phenylbine ratio.The optimal coating concentration of barbital-OVA was 0.5 mg/m L,and the optimal dilution factor of fluorescent nanospheres after coupling anti-phenobarbital monoclonal antibody was 200 times.The sensitivity of phenobarbital fluorescence quantitative test strip to phenobarbital standard was 20?g/L.When the content of phenobarbital standard was 20?g/L,the T/C value of the strip was 77.32%of the standard T/C value(with no phenobarbital).It indicated the T/C value of the reagent card at this standard concentration was significantly different from that of the0.01M phenobarbital S test card.Samples of aquatic products(tilapia,fork tail,grass carp,bass,silver carp)and plant health foods(Houttuynia cordata,Panax notoginseng,Pueraria lobata,hawthorn,honeysuckle)were added and recovered.The recoveries of aquatic samples were relatively stable,ranging from82%to 130%,but the recoveries of fork tails fluctuated relatively large,ranging from 68%to127%;the recoveries of plant health foods fluctuated more than those of aquatic tissues,ranging from 60%to 137%.When the detection limit was 50 ug/L,the false negative rate and false positive rate were both 0%;the cross-reaction rate with barbiturate,pentobarbital and isopentobarbital was less than 0.3%;the variation coefficients of the test strips were less than8%;The developed phenobarbital fluorescence quantitative test strips were sealed and stored at 45?for 60 days.Fluorescent solutions conjugated with anti-phenobarbital monoclonal antibody and goat anti-rabbit fluorescent nanospheres were stored at 37?for 14 days in the dark.Compared with ELISA and colloidal gold method,the detection time of phenobarbital was shortened from 75 minutes to 10 minutes,and the quantitative detection of phenobarbital was achieved with the same high sensitivity.A quantitative quantitative test was achieved.The phenobarbital fluorescence quantitative test strip prepared in this study can meet the needs of the actual fast screening market,and also provides a reference for realizing the assembly of commercial fluorescent quantitative test strips.