| Background:8 hydroxyl-2-deoxyguanosine(8-OHdG)is an oxidizing adduct produced by the active oxygen radicals,such as hydroxyl radical and inglet oxygen,which attack the eighth carbon atom of guanine base in DNA molecules.If it is not removed timely,the genetic mutation and cancerous can be caused by the G-C/A-T type base pairing errors in the process of DNA replication.Studies have shown that the oecurrence and development of diabetes,cancer and cardiovascular diseases is closely related to oxidative damage,and the increased 8-OHdG level can also be detected in the body.Therefore,the determination of the 8-OHdG level can not only assess the degree of oxidative damage of the body,but also help the diagnosis and reflection of the development of related diseases.Objective:The objective of this study was to obtain the specific paired antibody of 8-OHdG and to establish a biotin-avidin double anti sandwich ELISA for detecting 8-OHdG.On this basis,the content of 8-OHdG in gestational diabetes mellitus patients and normal healthy people was detected,meanwhile,the relationship between gestational diabetes and the level of 8-OHdG was compared.Methods:In this study,Balb/C mice were injected and immunized by a coupling 8-OHdG-mcKLH complex as immunogen,and monoclonal antibody was obtained by cell fusion.The positive hybridoma cell line was screened by ELISA method,then the cell strain was expanded,and the antibody of the ascites was purified and screened to get a highly specific 8-OHdG-paired antibody.The specificity and sensitivity were determined by detecting the purity,cross reactivity and titer of the monoclonal antibody.On the basis of this paired antibody,a biotin-avidin labeled double anti sandwich ELISA method for the quantitative detection of 8-OHdG was established by screening the concentration of coating buffer,blocking buffer,coating antibody and enzyme labeled antibody.The stability and accuracy of the method were evaluated by intra and inter batch experiments test and recovery percent.Moreover,the relationship between 8-OHdG and gestational diabetes mellitus was preliminarily explored.Results:1.In this study,a highly specific 8-OHdG-paired antibody was obtained.2.In this study,we established a biotin-avidin labeled double antibody sandwich ELISA method for the quantitative detection of 8-OHdG by using paired antibodies.3.In this study,the research methods was used to detect the samples of patients with gestational diabetes mellitus.Through the comparison of patients and healthy pregant women,it was found that the 8-OHdG level of the patients with gestational diabetes was higher than that of the control group,suggesting that the increase of 8-OHdG was related to the occurrence of gestational diabetes.Conclusions:1.Based on the obtain of a specific 8-OHdG-paired antibody,an avidin-biotin complex sandwich ELISA detection method was established and the detection conditions were optimized,thereby establishing a specific method for the detection of 8-OHdG.This method was used to detect some samples of gestational diabetes patients and healthy people,the level of 8-OHdG in gestational diabetes patients was higher than that in healthy pregant women,suggesting that the abnormal level of 8-OHdG in control group may be closely related to the occurrence and development of GDM.2.The specific paired antibody obtained in this study can be used not only for the development of the above ELISA kit,but also for the development of an immune-combined for the detection of oxidative damage.The avidin-biotin complex sandwich ELISA detection method established in this study made a complementation for the current blank of 8-OHdG detection and provided a more accurate and convenient detection method for the study of the relationship between oxidative damage and diseases. |
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