Study On Colloidal Gold Immunochromatographic Assay For Quantitative Detection Of 25(OH)VD

Vitamin D,also known as anti-rickets vitamin,plays an important role in maintaining the balance of calcium in human body.Vitamin D and calcium are key nutrients to support bone development in children and maintain bone strength in adults.The 25-hydroxyvitamin D（25（OH）VD）is considered as the main indicator of the nutritional status of vitamin D.Clinically,the quantitative detection of 25（OH）VD in human serum can timely respond to the body’s vitamin D nutritional status.It’s great significant for patients who lack vitamin D for prevention and treatment.The immunochromatographic test strip has advantages of simple operation,rapid detection,high sensitivity,and strong specificity.It has been rapidly developed in clinical immunology detection.A colloidal gold immunochromatographic assay for rapid and quantitative detection of 25（OH）VD was preliminarily established in the study.Detailed research contents were as follows:25（OH）VD was used as the raw material,the full-antigens（25（OH）VD-BSA and 25（OH）VD-OVA）were prepared by the method of DCC/NHS active ester.The reaction process was monitored by Thin layer chromatography,and UV-visible spectrophotometry was used to characterize 25（OH）VD-BSA and 25（OH）VD-OVA.The mice were immunized with 25（OH）VD-BSA.The anti-serum titer of the mice after the second immunization was monitored by enzyme-linked immunosorbent assay（ELISA）.After the fourth immunization,the booster immunization was performed.The spleen cells of the positive mice were fused with mouse myeloma cells.After a series of operations such as“screening-clone-screening”toward the fused cells,39 kinds of hybridoma cell lines excreting monoclonal antibody against25（OH）VD were obtained.11 kinds of anti-25（OH）VD monoclonal antibody ascites were prepared by the method of vivo of mouse.The ascites was purified by method of octanoic acid-ammonium sulfate.The concentration,affinity and inhibition rate of the obtained antibody were measured by UV spectrophotometry and indirect ELISA.The results showed that these antibodies had good affinity for 25（OH）VD and titers were all over 8000.Five antibodies were used to detect the sample in which 25（OH）VD was spiked at 100 ng/m L,the inhibition rates were all higher than 70%.In this study,a colloidal gold immunochromatographic assay for the quantitative detection of 25（OH）VD was developed based on the prepared anti-25（OH）VD monoclonal antibody.The optimum parameters for the test strips were as follows:the labeling p H of the antibody was 6.5;the concentration of the labeled antibody was 3μg/m L;and the volume of the immune-probe used for each test was 3.0μL.The equailibration time of the related immune dynamic assay was 15 min.When25（OH）VD was detected in PBS solution,the relevant linear regression equation was y=-1.2251 log（x）+2.4593（R²=0.9877）,the linear range was 5-60 ng/m L,and the sensitivity was 3.65 ng/m L.The cross-reaction rates of the test strip with 25（OH）VD₂,25（OH）VD₃,1,25（OH）₂VD₃,1,25（OH）₂VD₂,VD₂,and VD₃were 100%,99%,10%,22%,98%,and 8%,respectively.The recovery rates for intra-and inter-assays of test strips were 85.6%-90.76%and 95.83%-106.7%,respectively.The coefficient of variation for intra-and inter-assays of test strips were between 7.0%to 23.72%and7.59%to 8.74%,respectively.This test strip was used to detect 25（OH）VD in the actual human serum.It was found that the matrix effect was obvious,and the method to reduce the matrix effect was preliminarily explored using different sample pretreatment methods.