1.Establishment of quantificative ELISA for determination of human decoy receptor 3
Bing WU ; Wei CHEN ; Zeng WANG ; Long CHEN ; Jingrong CHEN ; Haiping WU ; Shangyi WU ; Kuanfu WANG ; Mei ZHANG ; Zhenhuan ZHANG ; Lurong ZHANG ; Hengshan ZHANG ; Jianhua LIN
Chinese Journal of Clinical Laboratory Science 2018;36(1):5-8
Objective To establish an ELISA for quantitative determination of decoy receptor 3 (DcR3) in human plasma.Methods A solid phase double antibody sandwich method was established for quantitative determination of DcR3.The anti-DcR3 antibody was immobilized onto ELISA plate.DcR3 in samples was captured by anti-DcR3 on ELISA plate and then detected by biotin-anti-DcR3 and subsequent peroxidase-labeled streptavidin,and the color was developed by adding substrate.The standard DcR3 samples on the same plate were detected simultaneously to calculate the DcR3 concentrations in unknown samples.The sensitivity,specificity,precision,recovery,linearity and DcR3 range in normal human adults were assessed.Results The sensitivity of the developed assay was 0.051 ng/mL.The intra-coefficient of variation (CV) was less than 10％ and inter-CV was less than 15％.The average recovery rate was 90.50％.When 2-fold amount of anti-TNF-α was added into the coated antibodies,10-fold amount of biotin-labeled anti-LIGHT,antiFAS or anti-TNF-α was added into the detection antibodies,or 10 fold amount of purified LIGHT protein was added into the standard DcR3 samples as competitor,no disturbing effects on standard curve were found.The linear range of the assay was from 0.25 to 16 ng/mL (r≥0.98).The concentration of DcR3 tested in 128 plasma samples from healthy adults was (0.21 ± 0.05) ng/mL with 95％ CI ranged from 0.14 to 0.28 ng/mL and no difference of age and sex was found.Conclusion The established ELiSA for determining plasma DcR3 exhibited high specificity,sensitivity,precision,fine linearity and wide detecting range.This method could be used for quantification of DcR3 in plasma.
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