| ?-hydroxybutyrate dehydrogenase is one of the lactate dehydrogenase isoenzymes,which is ubiquitous in humans and can be used for early diagnosis and screening of various diseases.The determination of ?-hydroxybutyrate dehydrogenase in human blood plays an important role in the auxiliary diagnosis of nasopharyngeal carcinoma,lung cancer,intrahepatic cholestasis of pregnancy,sickle cell disease and other diseases.At present,the method for detecting ?-hydroxybutyrate dehydrogenase in clinical practice is liquid biochemical method,but this method requires large-scale automated biochemical instruments.which is expensive and requires a skilled technician to operate.At present,there is no simple,rapid and convenient POCT product to detect ?-hydroxybutyrate dehydrogenase in the clinic.In this study,a dry chemical test paper for the rapid detection of ?-hydroxybutyrate dehydrogenase in human body was developed by using the principle and technology of dry chemisrty.At the same time,a small bench-top reflectance photometer developed by ourselves can quantitatively detect a-hydroxybutyrate dehydrogenase in human blood.Clinical trials had demonstrated that the performance of ?-hydroxybutyrate dehydrogenase can meet clinical needs,and the formed product had obtained the CFDA registration number,which can be put into application of clinical practice.The product filled the blank of dry chemical products at home and abroad which had the characteristics of simple operation,short time of detection and low cost of reagent,and had wide prospects of application in domestic and foreign markets.In this experiment,the design of single-factor and full-factor experiments was used to optimize formulations of reagent and materials of test strip.The results showed:test wavelength of the substrate was 550nm,reaction time was 120s,and sample loading was 15?L of the test paper.The concentration of the substrate,pH and ion concentration of the buffer,the concentration of developer,the type of surfactant and,the concentration of stabilizer,the concentration of coenzyme and the concentration of protective agent of main components involved in the test paper reaction were optimized.The optimum formulation of the main raw materials of the test paper:the concentration of ?-hydroxybutyric acid was 15mmol/L,and the ion concentration of the buffer was 100mmol/L,pH was 8.2,the concentration of developer was 50mmol/L,the concentration of Tween-20 is 0.5%,the concentration of stabilizer was 4%,the concentration of coenzyme was 10%,and the concentration of protective agent was 1%.The test paper materials were screened,the reagent layer was CNPC-SS12 of a pore size of 15?m,the diffusion layer was PES30/24,and the middle layer was 6613The optimized formula was applied to the reagent layer,the selected membrane was assembled into a test paper to verify the precision,accuracy,linear range and anti-interference of the test paper.The results showed that the intra-assay precision of the test paper was 6.8%?7.1%,the inter-batch precision was 4.0%?4.9%,and the total precision was 7.8%?8.6%,all less than 10%.the deviation of the high and low accuracy of the test Randox calibration were respectively 4.9%and 5.3%,both less than 15%.and the linear range of the test paper was 24U/L?953U/L,the correlation equation is y=0.970x+1.971,the correlation coefficient is R2=0.9997.The test paper was opened for 21 days,the relative deviation of the results was within ± 10%,and the test paper remained stable.When stored at 37? for 14 days,the relative deviation of the test paper results was within ± 10%.It indicates that dry chemical test paper of a-HBDH can be stored for 12 months under 2?8? refrigeration conditions.Common interfering substances were below the following concentrations:hemoglobin 5g/L,bilirubin 342?mol/L,triglyceride 37mmol/L,ascorbic acid 170?mol/L,which had no significant interference with detection of a-HBDHThe dry chemical test paper was compared with reference method of the liquid.The correlation equation was y=0.995x+20.934(r=0.995),the slope of the Passing-Bablok regression equation was 0.995,which was close to the theoretical value of 1.The Bland-Altman analysis showed that 4.16%of the points were beyond the limit of the consistency.It shows that dry chemistry had good correlation with liquid reagents and both of them were comparable.The effects of different anticoagulants were compared,test results of the heparin sodium plasma and serum showed the best homology,the relative bias of heparin sodium plasma was 5.7%.The difference between the concentration of a-HBDH of cerebral infarction patients and normal people determined by test strips was obvious,it indicated that the test strip was suitable for the auxiliary diagnosis of patients with cerebral infarction.The clinical results showed that the prepared test paper could be applied to the detection of a-HBDH,which could be applied and promoted in primary hospitals. |
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