Enzyme linked immunosorbent assay kit

Plasma ApoD and TNF-α levels were determined by commercially available enzyme-linked immunosorbent assay kits (USCN Life Science Inc., USA). In brief, human plasma was collected using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Samples were centrifuged for 15 minutes at 1000 ×g. Plasma was removed and diluted 1 : 100 with Phosphate Buffered Saline (PBS) (pH = 7.0). Samples were tested in duplicates. 100 μl of each diluted standard, blank, and human samples were precoated with a monoclonal antibody specific to the related molecule (ApoD or TNF-α) and incubated for 2 hours. Subsequently, all the samples were incubated for 1 hour with 100 μl/well of a biotin-conjugated polyclonal antibody preparation, washed 3 times with a specific wash solution, and incubated for 30 min with 100 μl avidin conjugated horseradish peroxidase. Following the last 5 washes, 90 μl 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to each well. The enzyme-substrate reaction was terminated by addition of 50 μl sulphuric acid solution in each well and absorbance was measured spectrophotometrically at a wavelength of 450 nm. The sandwich ELISA used for detection of ApoD, and TNF-α had a sensitivity threshold of about 1.03 ng/ml and 0.1 pg/ml, respectively.

Serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and fasting blood glucose (FBG) levels were determined by routine automated laboratory methods in our clinical laboratory. Serum adipokines including ZAG, HMW-ADPN, leptin, and TNF-α were measured by commercially available enzyme-linked immunosorbent assay kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer’s instructions. The low limits of detection for ZAG, HMW-ADPN, leptin, and TNF-α were 1.80 ng/mL, 0.07 ng/mL, 0.06 ng/mL, and 6.50 pg/mL, respectively. The intra- and inter-assay coefficients of variation were 1.13 and 8.52% for ZAG, 0.59 and 4.60% for HMW-ADPN, 1.72 and 2.32% for leptin, and 2.07 and 2.76% for TNF-α.

Quantification of surfactant protein concentrations was performed using enzyme-linked immunosorbent assays (ELISA) according the manufacturers manual. Commercially available enzyme-linked immunosorbent assay kits (USCN, Wuhan, China) were used to quantify the amount of SP-A (E90890Hu, ELISA Kit for Surfactant Associated Protein A), SP-B (E91622Hu, ELISA Kit for Surfactant Associated Protein B), SP-C (E91623Hu, ELISA Kit for Surfactant Associated Protein C) and SP-D (E91039Hu, ELISA Kit for Surfactant-Associated Protein D) in CSF samples. The analysis was performed using a microplate spectrophotometer (ELISA-reader) at a wavelength of 450 nm and a reference wavelenght of 405 nm for measuring the absorbance. Surfactant protein concentration in ng/ml CSF was calculated by comparison between standard series and the determined values of antigen concentration (protein concentration) according to the manufacturers manual. CSF concentrations of SP-A, SP-C and SP-D lay well above the detection limit of the ELISA kits. Detection limits according the manufacturers manual were as follows; SP-A: 18.27 pg/ml (0.0183 ng/ml), SP-C: 0.126 ng/ml and SP-D: 2.55 ng/ml. CSF concentrations of SP-B of most samples were slightly below the detection limit of the ELISA kit according the manufacturers manual (SP-B: 0.62 ng/ml).