| Nowadays, China is a great aquaculture power,fishery drugs is used essentially,particularly depending on the use of antibiotics, but it will cause many problems such as antibiotic residues,antibiotic residues not only harms to the aquaculture environment,but also poses a threat to human health.Doxycycline is a kind of the most common fishery drugs antibiotics,and doxycycline is a kind of tetracycline antibiotics,it can treat effectively for chlamydia and mycoplasma infection,also has effects on gram positive bacteria and gram negative bacteria.Doxycycline has the characteristics of wide antimicrobial spectrum and strong antibacterial effect etc,thus doxycycline is widely used in aquaculture industry,but tetracycline drugs harm seriously human body,tetracycline drugs can stimulate gastrointestinal mucosa.liver and kidney,people usually have adverse reaction of emesis.nausea.liver ascites and liver swelling.The Ministry of Agriculture stipulated that the maximum residue limit of tetracycline drugs in muscle.liver and kidney were respectively 100mg/kg.300mg/kg and600mg/kg.Rapid detection can achieve the purpose of pool monitoring,if finding fishery drugs exceed standard,people can prolong withdrawal period,nipping harm in the bud.The purpose of this article is that preparing doxycycline monoclonal antibody and developing rapid detection reagent strips.1.Synthesis and identification of Doxycycline artificial antigens: doxycycline is a small molecular hapten,it has no immunogenicity,doxycycline was coupled respectively with bovine serum albumin(BSA) and chicken ovalbumin(OVA) in the method of glutaraldehyde(GA) to obtain DC-BSA and DC-OVA.The test result of UV spectral scanning is:The characteristic absorption peaks of DC-BSA(260nm) and DC-OVA(265nm) skew obviously compared with standard protein(BSA 278 nm,OVA280nm) and doxycycline(378nm),we preliminary concluded that the synthesis of artificial antigen was successful,the synthesis of artificial antigen was successful via further test in SDS-PAGE electrophoresis,the electrophoretic bands of DC-BSA and DC-OVA lag obviously behind the bands of standard protein.2.Doxycycline artificial antigen immunogenicity evaluation:DC-BSA was as the immunogen and DC-OVA was as the coating antigen in the experiment of indirect ELISA,the antibody titer is the positive serum maximum valid dilute double that theratio of positive serum and negative serum(P/N)≥2.1.The result showed:P/N of mice No.1 is 2.3>2.1,P/N of mice No.2 is 2.5>2.1,the serum antibody titer is 1×104.The result of competitive ELISA is: the mice serum was diluted by 10000 times,adding respectively the different concentration of doxycycline(50μg/ml.100μg/ml.200μg/ml).The OD value is:the higher concentration of DC,the lower OD value of positive serum of mice No.1 and mice No.2,P<0.05,the OD value added DC has significant difference from the positive control hole.Therefore,doxycycline artificial antigen has good immunogenicity.3. Doxycycline monoclonal antibody preparation:(1)The cultivation of myeloma cells(sp2/0):after recovering the sp2/0, we cultured sp2/0 with 15% FBS 1640,sp2/0 was cultured to the logarithmic phase,it can be used for hybridoma preparation.(2)The acquisition of spleen cells:we immuned about 4 weeks BALB/c mice for 4 times with DC-BSA as immunogen,then taking mice spleen in the fifth day after the last immune,spleen cell can be used in hybridoma preparation.(3)preparation of hybridoma:we fused the sp2/0 and spleen cells with high antibody titer under 50%PEG(molecular weight 4000),the fusion cells were cultured in HAT-FBS-RPMI-1640,the fused cells could be observed to began spliting after3 days,counted after 7 days,the count of hybridoma was 166 strains,the fusion rate was0.017 ‰.(4)screening of monoclonal antibody cell lines:screening 4 holes of positive hybridoma by indirect ELISA,they were B7,C3,D10 and G8,B7 and D10 is positive hybridoma after rechecking, the positive of B7 was more than D10, we cloned B7 cells2 times in the method of limited dilution,we screened 3 positive cell lines of doxcycline monoclonal antibody,they were 1B3,1F9 and 1G2,1F9 had the most strong positive,culturing lagely 1F9 cells.(5)preparation of monoclonal antibody:we injected paraffin oil into 3 BALB/c mices that were about 6 weeks 5 days in advance,then diluting the monoclonal antibody cells to 1x106/ml,we took mice ascites after 12 days under germ-free condition,the mice ascites was about 15 ml.(6)the mice ascites was purified in octanoic acid-ammonium sulfate method,the concentration of purified antibody is 3.1 mg/ml.(7)the result of competitive ELISA detection:we confected respectively 0.1 ug/ml of doxycycline.oxytetracycline.sulfamethoxazole.enrofloxacin and florfenicol,the OD value of cross experiment withoxytetracycline,sulfamethoxazole, enrofloxacin and florfenicol was 0.154,0.153,0.149 and 0.152,they had no outstanding difference from positive value 0.156,this showed that there were no cross reaction between the 4 kinds of antibiotics with antibody,while the average OD value of reacting with doxcycline was 0.093,there were significant difference from positive value 0.156,P<0.05.4. Preparation of rapid detection reagent strips:(1)doxycycline monoclonal antibody marked by colloidal gold:we prepared the limpid colloidal gold solution,then finding the optimal marked amount in tube test method,doxycycline monoclonal antibody was added into colloidal gold to prepare gold-labelled antibody.(2)confirming the optimal working concentration:gold-labelled antibody and sample were diluted to different concentration,the optimal working concentration was confirmed in chessboard titration.(3)the structure of reagent strip: the bottom of strip was PVC carrier board,front-end to back-end of strip was respectively sample pad,colloidal gold pad,nitrocellulose membrane(NC) and absorbent pad,test line and quality control line were on the nitrocellulose membrane,gold-labelled antibody was coated on colloidal gold pad,DC-OVA was coated on test line,Goat anti Mouse IgG was coated on quality control line.5.Application of doxycycline rapid detection reagent strip:doxycycline was diluted to different concentrations,doxycycline of different concentration was dropwise added on sample pad.Eventually,detectable concentration of doxcycline was 0.2μg/ml,when the concentration of doxcycline in sample was higher than 0.2μg/ml,T line was no color,C line showed red color,when the concentration of doxcycline in sample was lower than0.2μg/ml,T line showed visible to the naked eyes red color,C line was no color.Conclusion:the doxycycline rapid derection reagent strip prepared in this experiment can detect doxcycline leaving in water or fish body,the operation is simple,aquaculture famers can make rapidly preliminary detection,but we also do further optimization experiment to color development definition of reagent strips,detection limit can meet the national maximum residue limit in liver.kidney and sebum. |
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