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Objective:This study aimed to explore the feasibility and clinical value of monitoring the progression of early kidney injury in type 2 diabetic patients by assessment of the urinary C-terminal agrin fragment (uCAF) with enzymatic chemiluminescence immunoassay.Methods:A total of 251 patients with type 2 diabetes, who attended the Second Affiliated Hospital of Wenzhou Medical University from October 2018 to March 2020, were included in this retrospective analysis. One hundred and fifty-six participants undergoing health check-up at the Second Affiliated Hospital of Zhejiang University School of Medicine in February 2021 served as controls. Basic clinical information, glycosylated hemoglobin type A 1c and serum creatinine values were recorded, and urine specimens were collected for urinary creatinine, urinary α 1 microglobulin(uα 1M), urinary immunoglobulin G (uIgG), urinary albumin, urinary N-Acetyl-B-D-glycosaminidase (uNAG) and uCAF measurements. Based on the estimated glomerular filtration rate (eGFR), 251 patients were classified into G1~G5 stage groups with 116, 22, 28, 55 and 30 patients in each group. One hundred and sixty-six patients with early diabetic kidney disease (stage G1-G3) were divided into subgroups A1 (79), A2 (48) and A3 (39) according to the urinary albumin/creatinine ratio (UACR), the uα1M levels were divided into uα1M subgroup 1 (83 cases), uα1M subgroup 2 (42 cases), and uα1M subgroup 3 (41 cases), and uIgG subgroup 1 (83 cases), uIgG subgroup 2 (42 cases), and uIgG subgroup 3 (41 cases) according to uIgG levels. The Spearman method was used to analyze the correlation between uCAF levels and eGFR, UACR, uα1M and uIgG levels. Results:(1) The linear range of the uCAF detected by enzymatic chemiluminescence immunoassay was 3.97-2 000.00 ng/ml, with a detection limit of 2.28 ng/ml, intra-batch coefficients of variation of 1.15% and 1.57%, inter-batch coefficients of variation of 1.63% and 5.78%, and a biological reference interval of <95.35 μg/g Cr. (2) The uCAF level and positive rate (UACR≥30 mg/g) increased with the decrease of eGFR from G1-G3, uCAF level was negatively correlated with eGFR value ( r=-0.543, P<0.000 1), and the positive rate increased from 24.14% (28/116) to 85.71% (24/28) from G1-G3. The uCAF level and positivity rate decreased with the decrease of eGFR from G4 to G5. uCAF level was positively correlated with eGFR value ( r=0.495, P<0.001), and the positivity rate decreased from 30.91% (17/55) to 23.33% (7/30) from G4 to G5. (3) In patients with early diabetic kidney disease, uCAF levels and positivity rates increased gradually with the increase of UACR. uCAF levels were positively correlated with UACR values ( r=0.602, P<0.001), and the uCAF positivity rate reached 21.52% (17/79) in the A1 subgroup. (4) uCAF level was positively correlated with uα1M and uIgG levels in patients with early diabetic kidney disease ( r=0.757, 0.596, both P<0.001). Conclusion:Analytical performance of enzyme chemiluminescence immunoassay for the detection of CAF is satisfactory and could be used a biomarker for monitoring damage and progression of early diabetic kidney disease in patients with type 2 diabetes.
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Objective To prepare and identify monoclonal antibodies (mAb) against histidine-tag (His-tag ) .Methods Balb/c mice were immunized with polypeptides containing 15 histindine(His)-coupled BSA and this fusion was prepared according to con-ventional methods .Indirect ELISA was used to screen the positive clones and limited dilution for further cloning .After purified with Protein G affinity chromatography ,these antibodies for His-tag were detected for antibody titer ,relative affinity as well as subtypes by using ELISA .The specificity of these mAb was identified by ELISA and Western blotting analysis .Double-antibody sandwich ELISA was composed of these mAb paired with the corresponding recombinant protein of specific antibody ,which was used to de-tect the recombinant protein quantitatively .Results 5 hybridoma cell lines stably secreting anti-His-tag IgG1(κ) were screened .A-mong these antibodies ,1-G2 was of the highest affinity ,reaching 1 .27 × 1010 ,which could combine with different recombinant pro-teins containing His-tag in the experiment of ELISA and Western blotting .1-G2 also could be used to detect recombinant PCT pro-tein containing His-tag quantitatively by using double-antibody sandwich ELISA with antibody of PCT ,the sensitivity of detection reached 4 .6 ng/mL .Conclusion The mAb against for His-tag with high specificity ,affinity and secreted stably are successfully prepared .This prepared antibody could be used in ELISA and Western blotting to detect a variety of His-tag .
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BACKGROUND: Advanced oxidation protein products (AOPPs) are a crucial pathogenic link to such long-term uremic complications in hemodialysis patients as immune system dysregulation, accelerated atherosclerosis, dialysis-related amyloldosis and so on. However, basic studies on AOPPs are relatively few, and one of the main reasons is the fact that it is difficult to obtain AOPPs with high pudty and biological activity.OBJECTIVE: To prepare, pudfy and indentify AOPPs, with the hope of searching for a method of preparing AOPPs with high purity and biological activity.DESIGN, TIME AND SETTING: A single sample observation was completed in the Clinical Biochemistry Section of Ecsomatics Department, Third Military Medical University of Chinese PLA from September to November in 2008. MATERIALS: Human serum albumin (HSA) was provided by Chengdu Rongsheng Company Ltd. Hitrap 26/60 sephacryl S-300 was purchased from GE Healthcare.METHODS: Hypochloric acid was used in the oxidation of purified HSA to prepare in vitro the AOPPs-modified HSA (AOPPs-HSA), which was then isolated by Hitrap 26/60 sephacryl S-300. Relative molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE), native polyacrylamide gel electrophoresis (PAGE) and molecular weight standards. Structural features and biological activities were identified in the experiment of tumor necrosis factor α (TNF-α) release from monocytes.MAIN OUTCOME MEASURES: ①The purification and gel electrophoresis results of HSA. ②The purification and gel electrophoresis results of AOPPs. ③The dose-effect relationship between AOPPs-HSA and TNF-α release from monocytes. RESULTS: The relative molecular mass of AOPPs-HSA was 670 000 according to SDS-PAGE, native PAGE and molecular weight standards. Moreover, AOPPs-HSA could encourage the release of TNF-α from monocytes. The time effects showed that TNF-α release volume significantly increased after 6 hours of stimulation by AOPPs-HSA (1 g/L) and reached a peak at hour 12. CONCLUSION: Highly purified and bioactive AOPPs can be successfully prepared by the above-mentioned method, which builds a basis for further study on AOPPs.