| BackgroundParaquat is a herbicide that is widely used in worldwide currently, have strong toxicity to humans and animals. The mortality rate of Paraquat poisoning is very high in clinical and there is no effective treatment method. The death rate of paraquat poisoning is closely related to the amount of oral paraquat, and literature reported that > 20 mg/kg can lead to death generally. At present,our country usuallly detect concentration of PQ by uv spectrometry,high performance liquid chromatography(HPLC), high perfornance liquid tandem mass spectrometry(LC/MS) and capillary electrophoresis etc. But these methods require complicated sample pretreatment,and in the process will lose part of paraquat, res?lting in inaccurate test res?lts. And different samples have different detection methods, not only increase the diffic?lties of detection but also increased the cost of testing. Therefore there is a need to establish an efficient, reliable and simple detection method of paraquat residue, to guide the clinical treatment and prognosis.After paraquat enters the human body induced m?ltiple organ dysfunction syndrome thro?gh oxidative damage, inflammation etc.And breath failure caused by severe lung fibrosis is the main cause of death of paraquat poisoning.Therefore the key to treat paraquat poisoning is that slow down the process of p?lmonary fibrosis and reduce the mortality rate. At present, the first choice of treatment is blood purification in clinical, but the cost is expensive and found that altho?gh the blood purification can prolong the survival time, but not to reduce the rate of mortality. Therefore the treatment of paraquat poisoning has always been a clinical hard problem, and urgent need to establish a norms, standards and effective treatment to solve the problem. PurposeDevelop an indirect competitive ELISA kit which has simple pretreatment, high sensitivity, and can detect paraquat residues of food, blood, urine and gastric juice sim?ltaneously to meet the demands of rapid detection for agric?lture and clinical.Study the treatment effect of antibody against paraquat in paraquat induced acute lung injury and explore its feasibility in clinical application. Methods1.In this study,used indirect competitive ELISA method to detect the residue of paraquathe by the specific binding principle of antigen and antibody.Firstly synthetic PQ complete antigen(PQ-OVA) and then add target object which compete paraquat antibody with immobilized antigen, finally add enzyme labeled second antibody and TMB substrate, get the value of OD450 by ELIASA. And calc?late the content of small molec?les in the target object using the high specificity of paraquat antibody and paraquat small molec?le.In this study, the detection sensitivity is improved by optimizing the relative conditions of ELISA,it mainly includes:(1)the concentration of coating antigen and anti-paraquat antibody.(2) coating time and incubation temperature.(3) the kind,the concentration, the amount of blocking solution and the blocking time.(4) Optimization of antibody dilution.(5) competition time.(6) the concentration of HRP-antibody.(7)the HRP-antibody reaction time.(8) the ratio of substrate A to B.(9)the color reaction time.(10)optimization of antibody stabilizer.According to the optimized conditions to establish standard curve,select cabbage, corn, soybean, blood, urine and gastric juice as the sample,to determine the sample added recovery and coefficient of variation, and test its sensitivity, stability and specificity of the kit.At last, detect the validity of anti-PQ antibody and kit, to ensure that meet the application requirements.2.Exploring the paraquat dosage to establish rat model of paraquat poisoning, and observe the therapeutic effect of anti-paraquat sim?lated antibody in paraquat induced acute lung injury. Firstly purify anti-paraquat sim?lated antibody, and then establish rat model of paraquat poisoning.Selecting 65 SPF rats which are 6-8 weeks old, were randomly divided into 3 groups, the control group include 15 rats, the poisoning group and the treatment group each 25 rats. The control group was injected NS(1.8mL/kg) into abdomen;and the poisoning and treatment group was injected paraquat solution(18mg / kg) into abdomen( 20% paraquat diluted to 10 mg / ml); and 2 hours after injecting paraquat,treatment group was treated with PQ sim?lation antibody(1mg / kg) into tail vein, and continuous inject the antibody for 3 days. Observing the reaction of these rats in each group for 14 days, and respectively execute 3 rats at 1, 3, 7, 14 days,collect blood to test the expression level of TGF-? 1 and IL-6 and take left lobe of the lung to do H-E staining,observing the pathological character by microscope and contrasting the differences of three groups. Res?lts1. The optimal reaction conditions of the indirect competitive ELISA are as follows: the working concentrations of coating antigen and antibody respectively are 2.0?g / mL and 1: 28000; coating temperature and time is 4 14h; blocking buffer was 1% OVA;the amount of blocking buffer is 150??L; blocking time is 45min; competitive binding reaction time is 45 min, the secondary antibody reaction time is 30 min, the concentration of HRP-antibody is 1: 4000; the ratio of A to B = 1/19; the color reaction time is 10 min.2.The detection limit was 0.08ng/mL,and the IC50 value was 1.32ng/mL.The liner range of the inhibition curve was between 0.16ng/mL-10.76ng/mL. The cross reaction rate of PQ and DQ is only 0.01348%. The samples adding recovery rates are all higher than 85%,and the coefficient of variation is less than 14%.The coefficient of variation between the inner panel 0.86% ~ 9.57%, inter-plate variability coefficients between 2.18% ~ 11.09%..Res?lts show that the detection sensitivity is higher compared with the national standard method.3.The paraquat sim?lated antibody was expressed successf?lly, which was proved to have good competitive activity by the indirect competitive ELISA. The paraquat poisoning model was successf?lly established with the dose of 18mg/kg, while using anti-paraquat antibody to detoxify.In the poisoning group, the lung tissue injury was aggravated with the increase of the experimental days,and a large number of inflammatory cell infiltration, p?lmonary fibrosis gradually formed. In the treatment group, the inflammation of lung tissue was reduced, and there was no obvious fibrosis in the experimental process. The level of TGF- ?1 and IL-6 increased gradually with the increase of test time in poisoning group, while the treatment group showed a downward trend. Conclusion1.The indirect competitive ELISA kit can detect food, blood, urine and gastric juice at the same time,with high sensitivity, good specificity, simple sample pre-treatment, high add recovery rate and low variation rate.It can f?lly meet the demand for testing paraquat.2.The anti-paraquat sim?lated antibody can inhibit the secretion of TGF-?1 and IL-6,and reduce the lung injury caused by paraquat poisoning, and can prevent the development of p?lmonary fibrosis to some extent. |
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