| Malachite green(MG)is has a high solubility in organic solutions such as ethanol,but is less soluble in water.It is often used in aquaculture to prevent and control many diseases,such as fungal diseases,parasitic diseases,bacterial diseases,etc.,or for long-distance transportation or preservation of aquatic products to ensure the survival rate of aquatic products.Malachite green and its metabolites(LMG)can remain in kidney,subcutaneous fat,muscle,liver and blood of animal tissues for a long time.After human consumption,it will cause toxic and side effects such as carcinogenesis and endanger human life and health.However,due to its low lost and high efficacy and the lack of alternative drugs at the same price,malachite green is still widely abused in aquaculture,transportation and storage of aquatic products.Malachite green has been listed as a banned drug in many countries including China,but it is still used illegally in the market because of its low cost and high efficacy.At present,the primary analytical method for MG and LMG is liquid chromatography tandem mass spectrometry(LC-MS)method,which required high price of detection instruments,long detection time,special training for operators and complicated operation.Therefore,it is extremely urgent to establish an accurate and highly sensitive method for rapid detection of malachite green.Immunological method is a commonly used rapid detection method with low price and high sensitivity.Antibody is the core of immunological methods,but malachite green itself has small molecular weight,uncomplicated spatial structure and no immunogenicity,so it is necessary to prepare complete antigen with immunogenicity firstly,then prepare antibody by immunizing animals,and adopt the principle of competition inhibition to establish immunological detection method for small molecule substances.1.Preparation of malachite green nanobody using MG coupled-antigenIn this study,carboxylated leuco-malachite green(CMG)was designed and synthesized by chemical method,and the production was coupled with carrier protein BSA or OVA to prepare complete antigen.After nuclear magnetic resonance(NMR),UV analysis and SDS-PAGE electrophoresis,it was found that the UV absorption peak of malachite green coupling protein CMG-BSA,was shifted.Result of SDS-PAGE showed that the coupling product protein was slightly larger than that of carrier BSA,and the immunogenicity of malachite green antigen was obtained.After immunizing mice,it was found that the antigen has a good immune effect,the titer of antiserum is more than 1:64000 and the inhibition concentration of antiserum is less than 10 ng/mL,which provides the base for the preparation of nanobody in the next step.We selected an immune library to screen malachite green nanobodies.Healthy alpaca was taken as immunized animal.After immunization,peripheral blood of 20 mL was collected from the alpaca.Total mRNA was extracted from the lymphocytes isolated by lymphocyte extract,and its purity was determined.mRNA was transcribed into cDNA using oligoDT12 as primer to be used as template to amplify the gene of alpaca antibody VHH with nested PCR.The gene fragment obtained was recombined with the coat protein gene of phage vector in vitro and transformed into expression system.With the assistance of helper phage,the immune library was constructed,and the target protein was displayed on the surface of the phage with the fusion expression of the target gene and the coat protein gene.Later on,the VHH library was produced by infecting the phage expression system.MG-specific phage selection needs to be performed.Finally,After the selection of the most suitable clone,the corresponding soluble VHH with HIS flag was produced.It is critical to select the most suitable phage displaying Nbs by SDS-PAGE electrophoresis.The size of MG nanobody was about 15 kd.2.Preparation of colloidal gold-based immunechromatography strip for the detection of malachite GreenIn this study,the rapid detection method for mallachite green was to prepared using colloidal gold-based immunechromatography.The colloidal gold solution of about 40 nm in diameter was obtained by producing the gold chloride solution using tri-sodium citrate reduction method.Based on the principle of competitive inhibition,nanobody was labeled with colloidal gold,sheep anti-mouse second antibody was labeled at the quality control line(C line)in NC membrane,and malachite green coupled antigen(MG-BSA)was coated on the detection line(T line)in NC membrane.When there was no malachite green in the sample,the colloidal gold labeled antibody would react with the coupled antigen(MG-BSA)at the T line to form an antigen-antibody complex with the color.If the sample contains malachite green,the gold labeled antibody competes with the coupling antigen.When the content of MG is high enough,the binding of the coupling antigen to the labeled antibody is completely inhibited,and there is no marker at the T line.The color depth of T-line is inversely proportional to the content of malachite green.The optimum ratio of antigen to antibody was determined in the orthogonal experiment.The labeled amount of antibody was 10 ?g/mL,the amount of colloidal gold labeled antibody was 12 ?L per well,the amount of antigen in NC membrane was 0.5mg/mL,and the concentration of second antibody was 0.4mg/mL.The colloidal gold test strip was prepared by this ratio,and the sample was pretreated and reacted for 15 minutes.The result was judged according to the chromaticity comparison of C and T lines.The detection strip is tested for 15 minutes,and limitation of detection is 0.5 ng/mL,which is quick and sensitive,and meets the national detection standard.It meets the testing requirements of the Ministry of Agriculture and the Food and Drug Administration,and provides a technical guarantee for cracking down on the illegal use of malachite green.3.Evaluation and application of colloidal gold-based immune-chromatography strip for the detection of malachite GreenIn order ot meet the requirements of the national standard,three batches of malachite green rapid detection strips of the same variety and specification were produced.140 strips in each batch of products wer provided,and 70 strips were randomLy selected for product testing.In blind sample preparation,the concentration of malachite green is cut-off 0%,50%and 100%,respectively,in which 0%and 50%concentration levels reflect the false positive rate of the product,and 100%concentration level examines the false negative rate of the product.Among them,there were 15 samples for 0%and 50%,and 30 samples with a qualitative value of 100%concentration level.The concentration residue content of the actual sample is equal to the requirement of the qualitative cut-off value,and did not exceed 10%of the qualitative critical value.After the setting of the sample,in the prescribed testing time,the detection strip meeted the testing requirements of the national testing department,which would determine whether the sensitivity and stability of the product meet the needs of field testing.When meeting the national requirements,this detection strip was used for the third-party detection in Jiangsu,Zhejiang,Jiangxi and other provinces.A total of 12179 samples were tested,including fish,shrimp,rice field eel and other aquatic products,and 38 samples were tested for positive with MG.The test results were the same with the national standard method,and the coincidence rate was 98.1%.The development of this product saved a lot of manpower and force,and detected quickly,which provides an effective technical support for ensuring the safety of agricultural products. |
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