Development Of The Test Kit For Dietary Structure-related Atherosclerosis

Many reports have confirmed that atherosclerotic disease is related to the dietary structure.However,dietary structure has different affect to individuals because of gene heterogenicity and various living environment.Lipoprotein-associated phospholipase A2(Lp-PLA2)is the best independent predictor of atherosclerosis currently,which directly reflects the vascular endothelial injury and predicts the risk of cardiovascular diseases.Therefore,risk of atherosclerotic disease can be evaluated by measuring Lp-PLA2 concentrations in people with different dietary structures,and the incidence of cardiovascular diseases can be lowered by providing personalized dietary instructions.Precise measurement of Lp-PLA2 are of great valuable for preventing atherosclerosis.Accordingly,the expression strategy of Lp-PLA2 recombinant protein was screened using prokaryotic and eukaryotic expression system,and Lp-PLA2 recombinant protein was successfully expressed with eukaryotic expression system.A novel Lp-PLA2 rapid test reagent was developed using double sandwich method and quantum dots-based immunochromatographic technology,and then its performance was evaluated.The reagent used quantum dots as tracer material,which was easy to handle and has good accuracy.At last,the eating habits of randomly selected residents in Nanjing district were collected and their Lp-PLA2 level were tested using the rapid test kit.The relationship between eating habit and Lp-PLA2 level was analyzed using statistic methods.The major results are as follows.(1)UCOE was chosen as expression plasmid of Lp-PLA2.Lp-PLA2 protein was successfully expressed in CHO-K1 cells with 20 ?g/mL yield.The purified protein had 90%purity and good immunoreactivity.Purified Lp-PLA2 protein was used as coating protein in control line and the key material of calibrators.(2)The preparing conditions of the membranes composing the test kit was optimized to enhance the signal and lower background interference.The concentration of antibody PLA5 and antigen Lp-PLA2 coated on the nitrocellulose membrane were 1.0 mg/mL and 0.75 mg/mL respectively.The antibody PLA1 and QDs were chemically conjugated,and the complex was diluted with the buffer containing 2%sucrose and 0.2%BSA at the ratio of 1:15.Blocking reagent contained 5%trehalose and 1%casein.(3)The LOD of the kit was 15.44 ng/mL.The linear range was 15.70-2000 ng/mL,which was better than reference reagent.The coefficient of variation was less than 5%.The results were not interfered by physiological concentration lipoprotein,which was better than reference reagent.Compared with the stability at 4?,the test kit could be saved at 37? for 30 days stably.In summary,the performance of the test kit for Lp-PLA2 was qualified.(4)200 people were selected in Nanjing randomly.We collected their dietary habits and detected their Lp-PLA2 levels using rapid test reagent and reference reagent.There was good correlation between two reagents,which indicates that the rapid test reagent was reliable in measuring Lp-PLA2.By analyzing the relationship between Lp-PLA2 levels and dietary structure,the personalized dietary instruction could be established according to Lp-PLA2 concentration.In conclusion,recombinant Lp-PLA2 protein was obtained and the rapid test kit for Lp-PLA2 was developed.The relationship between Lp-PLA2 levels and dietary structure was also established.Personalized dietary guidelines can be given to different people for preventing atherosclerosis according to the Lp-PLA2 level.