Preparation Of Monoclonal Antibodies And Development Of An Enzyme-linked Immunosorbent Assay Kit For Tylosin And Tilmicosin

Tylosin and tilmicosin belong to macrolide antibiotics,which are used extensively to treat the infection of sensitive Gram positive bacteria and Mycoplasma in veterinary clinic.In Addition,a majority of tylosin are used as feed additive in stockbreeding. Recently,the tolerance of some Saphylococcus aureu,Streptococcus pneumonia and Haemophilus influenzae are increasing against?-lactam antibiotics.And the infection caused by Mycoplasma and Choamydiae which are sensitive to the macrolide antibiotics, are also increasing.These resulted in increasing usage for macrolide antibiotics in clinic. Macrolide antibiotics are easy to induce tolerance,and have cross tolerance with other antibacterial.The more extensive usage of macrolide antibiotics in veterinary medicine, the more serious people health is threaten.As concerned of human health,Tylosin was forbid as feed additive in European in 1999.Tylosin and tilmicosin were one of the most important antibiotics in macrolides,even in all antibiotics which were used in veterinary medicine and stockbreeding.Incorrect use of these antibiotics may leave residues in edible tissues,thus causing toxic effects for consumers,e.g.allergic reactions in hypersensitive individuals.In China,the maximal residue level?MRL? of tylosin and tilmicosin in animal tissues were established too.Therefore,it is necessary to study a rapid method to monitor their residues in animal tissues.Methods for the detection of tylosin and tilmicosin in animal tissues were mainly instrumental methods,which were based on high performance liquid chromatography ?HPLC? coupled with ultraviolet and mass spectrometry.Although sensitive and accurate, most of the chromatographic methods are expensive,time-consuming,and unsuitable for ananysis of many samples.Enzyme-linked immunosorbent assay?ELISA? is one of the most important methods for rapidly screening veterinary medicine residue in animal tissues and feed.Enzyme immunoassay is enough sensitive,specific,rapid and simple to be a good and economical alternative to instrumental methods for screening tylosin and tilmicosin residues.At present,only tylosin ELISA kit was available,and other ELISA methods for simultaneously detecting tylosin and tilmicosin were uncompleted.So, developing an ELISA kit for simultaneously detecting their residue could be significant.Desmycosin?DES? and 5-O-Mycaminosyltylonolide?OMT? were prepared by adjusting solution of tylosin tartrate to different acidity,and purified by column chromatography.The products were examined by ultraviolet spectrum,infrared spectrum and mass spectra.The results showed that these products were synthesized successfully. The relative content of DES and OMT,analysed by HPLC,was 95.0%and 97.6%. Haptens DES and OMT were conjugated to bovine serum albumin?BSA? and ovalbumin ?OVA? by three different linkers,O-carboxymethoxylamine?AOAA?,Adipic acid dihydrazide?ADH? and 4-Hydrazinobenzonic?HBA?.These conjugates were identified with ultraviolet spectrum.Their spectrum was far from different to carrier protein and haptens.The results showed that the conjugates were prepared successfully.The chemical stability of conjugates DES-AOAA-BSA and DES-AOAA-OVA was investigated.The results showed that they were stable in past 8 months.Conjugates OMT-ADH-BSA,OMT-HBA-BSA,OMT-AOAA-BSA,DES-ADH-BSA, and DES-AOAA-BSA were selected as immunogens.Balb/C mice were vaccinated with based and some intensified immunization.After successful immunization,the spleen cell coming from the mice with high titer was fused with SP2/0 myeloma.Following hybridoma screening and sub-cloning,a monoclonal hybridoma cell line,named 5.2.4/3C4,was established.The chromosome number was about 92.8.Monoclonal antibody?MAb? was prepared by ascites tumor,and was identified as lgG2a.The cross-reactivity of Mab with DES,Tylosin and tilmicosin was 100%,53.4%and 35.5% separately.The ascites containing Mab were mixed with equal glycerol and stored in -20?.The stability of Mab was investigated by antibody titers and IC₅₀ for DES.In past 6 months,antibody titers and IC₅₀ did not change obviously.Using the monoclonal antibodies prepared with DES-AOAA-BSA,a ciELISA method was developed to detect Tylosin and Tilmicosin.Standard curve ?y=-0.4592x+1.0671? was prepared for DES and good linearity?r=0.999? was achieved over the concentration?5?80?g/L?.The ciELISA method showed lowest detection limits of 1.2?g/L for DES.The procedures of extraction for tylosin and tilmicosin in muscle and liver were established.In brief,samples were extracted by 20%?v/v? acetonitrile in 0.3% metaphosphoric acid solution.The extract was adjusted to a pH of 8.5?9 with 5M NaOH, and extracted by ethyl acetate.The organic phase was evaporated to dyness.The residue was reconstituted in 10%?v/v? methonal in PBS,and analyzed by the ELISA.The lowest detection limits for tylosin in swine muscle and liver were 7.9?g/L and 8.4?g/L,and for tilmicosin were 11.9?g/kg and 12.7?g/kg respectibely.The lowest quantitation limits ?LOQ? of tylosin in swine muscle and liver were 20?g/L and 30?g/L respectively and the LOQ of tilmicosin in swine muscle were 30?g/L.Recoveries of tylosin and tilmicosin in fortified tissues at LOQ,0.5×MRL,MRL and 2×MRL concentration were in the range of 85%?115%,and inter-assay and intra-assay variability were all?20%.Eight healthy,crossbred pigs?about 15kg? were divided into 4 groups randomly, including blank and three treated groups.Pigs were administered 10 mg/kg tylosin tartaric intramuscularly on sides of the neck,every 24 h for 5 consecutive days.The animals were slaughtered in groups of 2 at 0,2 and 5 days post-administration of the drug.Muscle from injected site and loin,and liver were collected.After preparation,tissue samples were analyzed by established ELISA and high performance liquid chromatography?HPLC? method.The results showed that the ELISA method had a good correlation with HPLC for detecting muscle?r=0.997? and liver?r=0.996? samples.The values in loin muscle and liver measured by ELISA was accordant with Prats' result,but the concentration in injected site was far from Prats' result.It could be resulted from formulation of tylosin.In this study,the relations between the chemical structural difference of hapten and antibody characteristics were investigated,which could give others some useful advice. Several linkers with different chemical characters were used to prepare immunogens.The immune effect of antigen with different linker was first investigated in antibody preparation of Macrolides.The best immunization was achieved by the conjugate DES-AOAA-BSA,and monoclonal antibody was prepared.The Mab could combine with tylosin and tilmicosin.However,other available antibody which could combine with tylosin and tilmicosin was polyclonal antibody.Based on the MAb,ELISA method and kit were established.The kit could be used to simultaneously detect tylosin and tilmicosin residues,which was forecasted to have a good forground in future.In addition,the immunological characteristic of low molecular weight compound was discussed in this paper.The discussion could be useful for antigen design of other compounds.