A 0.05 mol/L carbonate/bicarbonate buffer solution was made up by dissolving 1.59 g of sodium carbonate (Na2CO3) and 2.93 g of sodium bicarbonate (Na2HCO3) in 1 litre of distilled water, and then the solution was adjusted to reach a pH of 9.6. The PBS/Tween20 solution was made up by dissolving 0.5ml Tween20 reagent in l00ml of PBS.
2.1.2 Phosphate Buffer saline (20x); Washing Solution for ELISA
320 g of sodium chloride (NaCl), 13.6 of potassium dihydrogen orthophosphate (KHPO4) and 48.4g of dipotassium hydrogen orthophosphate (K2HPO4) were all dissolved in 2 litres of distilled water. The stock 20x PBS solution was further diluted by a factor of 1: 20 prior to use.
2.1.3 PBS Tween
0.5 ml of Tween was dissolved in 1000 ml of PBS.
2.1.4 1% Bovine Serum Albumin (BSA)
A 1% BSA solution was prepared by dissolving 1 g of BSA in l00 ml of PBS
2.1.5 Substrate Solution (O-Phenylenediamine hydrochloride) OPD
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The substrate solution for ELISA assay was freshly prepared as follows:
a) Firstly, a one phosphate-citrate buffer tablet (Sigma P4922) was dissolved in 100 ml of distilled water.
b) After that, 10 mg of o-phenylenediamine hydrochloride tablet (Sigma P8287) was dissolved in 20 ml of phosphate-citrate buffer solution.
2.1.6 Sulphuric acid
A solution of 4 M concentration of sulphuric acid (H2SO4) was prepared by diluting 22 ml of concentrated sulphuric acid (100%) to l00 ml of distilled water, in a drop-wise approach. This solution was used to stop the reaction at the end of assay.
2.2.1 Collection and Preparation of PM10 and Dust
Airborne dust was collected within and proximal to Park Slip West opencast coal mine using mobile Negretti PM10 heads and polycarbonate Millipore collection filters in a cooperation with the operators, Celtic Energy. For instillation, particles were suspended in 0.15 M saline and wetted (suspended in solution) by sonication for 15 minutes.
2.2.2 Animal and Experimental Protocol
All animals were treated humanely under guidelines provided by Cardiff University and the local ethical committee. Study approval was obtained from both local ethical committee and home office animal project licence. The animal and experimental protocols used were as previously reported in Evans et al., (2006). Briefly, male Sprague Dawley rats (200 g, n=10 per group) were purchased from Charles River (UK) and were acclimatised within the animal holding facility for one week prior to instillation. The animals were kept on wire-bottom cages with pelleted food and tap water ad libitum. After that, the rats were lightly anaesthetised with Halothane before receiving intratracheal instillations. Control animals were instilled with normal saline only, while test animals were instilled with saline containing 2.5-10 mg of PM10. Subsequently, the blood samples were harvested 3 days and 6 weeks after dust instillation.
2.2.3 Blood Sample collection
Blood samples were collected by cardiac puncture, into tubes containing sufficient tri-potassium EDTA to give a final concentration of 1.5 mg/ml, and then the samples were centrifuged at 1500 g for 10 minutes. Afterwards, the plasma supernatants were collected and stored at -80°C until assayed.
2.2.4 ELISA Assay for von Willebrand Factor
1- The ELISA assay was measured using commercial polyclonal antisera. It was performed in duplicates because of the variable results obtained.
2- 40 µl of anti-von Willebrand Factor (DAkO Code A082) was diluted in universal tubes with 20 ml of carbonate-bicarbonate buffer (Dilution Factor 1/500).
3- Subsequently, 100 µl of the diluted anti-von Willebrand Factor was dispensed into a 96 well (NUNC Maxisorp) and then it left for 45 minutes.
4- After that, the wells were washed three times with PBS/Tween20 solution using a plate washer.
5- Next, the samples were diluted at 1 in 40 (20 µl of plasma and 780µl of PBS), and then 100 µl standards and samples were added into each corresponding well and incubated for 45 minutes. Plasma samples were diluted prior to assay as follows 0, 1:20, 1:40 and 1:80.
6- The plates were washed 3 times using a plate washer with PBS/Tween20 solution.
7- Following the washing step, the secondary antibody was diluted by adding 48 µl of secondary antibody (polyclonal antihuman vWF /HRP) to 25 ml of PBS Tween, and then it was added.
8- Afterwards, 100 µl of this solution was added to each well and the plate was incubated for 45 minutes.
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9- Following this, the plate was washed three times with PBS/Tween 20 solution.
10- Next, 100 µl of substrate solution (OPD) was added to each well and the plate was incubated for 3 -10 minutes (a lemon-Yellow colour was observed).
11- When the colour change was observed, the reaction was stopped by adding 50 µl of 2 M sulphuric acid into each well.
12- The plates were read using an Anthos absorbance reader at 490 nm.
Table (2.1) Illustrates summary of von Willbrand Factor standard series preparation, as used in the vWF ELISA tests.
2.2.5 Statistical Analysis
The Minitab Software was used to determine statistical analysis of raw data. Also, Microsoft Excel was used to prepare all graphs. The mean and standard deviation of vWF concentration in untreated and treated rats sample with MP10 after two point times; 3 days and 6 weeks from the exposure were calculated. Two-sample T-test was used to test differences between vWF concentration in control group, and both PM10 groups.