Development And Application Of Glycated Hemoglobin Lateral Flow Assay Strip

In 2011,the WHO released a report recommending the use of glycated hemoglobin A1c(HbA1c)to diagnose diabetes.As an indicator of diabetes testing,HbA1 c is characterized by the average blood glucose level of the patient over the past two to three months.However,glycosylated hemoglobin as a clinical diagnosis still has poor testability between different laboratories.According to the recommendations of NGSP,in order to ensure the comparability of measurement results between different laboratories,the precision of HbA1 c measurement system is required,CV<3%.Solving the differences between different laboratories is a key problem in recommending glycosylated hemoglobin as a diagnostic criterion.The advantages of the convenience of immunochromatographic lateral flow assay and their rapid diagnosis make the application more and more widely,because of its easy storage and easy transportation characteristics,it is widely used in primary medical institutions.The focus of this study is to obtain HbA1 c immunochromatographic lateral flow assay that are comparable,easy to operate,and low in cost,by selecting appropriate paired antibodies and optimizing the lateral flow assay conditions.It is convenient for diagnosis and monitoring of diabetes and can be promoted and applied in primary hospitals in China.1.The basic conditions for the establishment of immunochromatographic lateral flow assay obtained through various experiments are:(1)The labeling step is to add 100 μL of 450 nm fluorescent microspheres(solid content: 1%),add 500 μL of MES buffer(20 mM MES pH=5.6),200 μL 10 mg/mL EDC(20 mM MES pH=5.6 dissolution configuration)and 200 μL of 10 mg/mL NHS(20 mM MES pH=5.6 dissolution configuration),after shaking and mixing,add 20 μL of PB after 20 min.Resuspend the fluorescent microspheres in buffer(20 mM PB pH=7.2),add 400 μL of 10 mg/mL IHB001 antibody,react for 30 min,centrifuge to remove the supernatant,and resuspend the fluorescent microspheres with the preservation solution.Granules(preservative solution 20 mM pH=7.2 PB + 0.1% BSA + 10% sucrose).The suspended fluorescent microsphere particles were coated on a gold standard pad in an amount of 1.0 μL/cm.(2)Coating conditions IHA001 antibody diluted to 4 mg/mL with 20 mM PB(pH=7.2)buffer was coated on a nitrocellulose membrane.(3)The composition of the hemolytic agent is 0.2% NaCl + 0.1% CTAB.The above three components establish the conditions for the basis of the HbA1 c immunochromatographic lateral flow assay in this study.2.The Box-Behnken Design(BBD)method in Design-expert V8.0.6 software was used to analyze the precision factors of quality control method,and the ratio of fluorescein-BSA,BSA concentration,sample dosage and reaction time were carried out.Four factors were used to design the experiment and optimize the reaction conditions.Refer to the method for precision measurement in EP05-A3 to obtain a lateral flow assay condition with a precision of 1.61%.3.Based on the established HbA1 c immunochromatographic lateral flow assay reagent strips,a series of performance tests on quantitative were carried out.The obtained product properties are as follows: The minimum detection limit in this strip is 0.4.%,the linear range is 2.0%~14.0%.In this linear range,the correlation coefficient is r≥0.99,the precision measurement is 1.61%,and the maximum interference concentration allowed by the interfering substance: triglyceride is 25 g/L.The bilirubin is 0.1 g/L.Through the analysis of the test data of various performance indicators,the HbA1 c immunochromatographic lateral flow assay prepared in this study satisfies the trial use of the primary hospital,and the performance meets the expected requirements.