4.1 ZONA A: RINCONADA
4.2.1 MARCO GEOLOGICO REGIONAL
A sandwich type enzyme linked immunosorbant assay (ELISA) technique is a method of calculating the concentration of a specific substance in a given sample. The principle of this technique is that the wells of a polystyrene microplate are coated with a “capture” antibody specific to the substance under investigation. A sample containing the investigation substance is then added to the wells, allowed to bind to the capture antibody and then any unbound substance washed off. An enzyme bound detection antibody is then added which binds to the sample-‐capture antibody complex. After an
incubation period unbound detection antibody is washed off. A substrate solution containing hydrogen peroxide and tetramethylbenzidine is then added. This solution is converted to a blue colour by the enzyme bound to the detection antibody. The reaction is stopped by the addition of a solution of sulphuric acid, which turns the solution yellow. The intensity of this colour is proportional to the concentration of the investigation substance in the sample.202
3.12.1
HMGB-‐1 ELISA
High mobility group box 1 (HMGB-‐1) concentrations were measured in plasma and BALF using a commercially available ELISA kit (Shino-‐Test Corporation, Kanagawa, Japan). The kit contained a 96-‐well assay plate that is pre-‐coated with an anti-‐HMGB-‐1 capture antibody. To this plate were added 100ml of assay diluent and 10ml of sample and standards. The standard range of concentrations was created by serial dilution to of an 80ng/ml concentration of HMGB-‐1 to produce a range of 80 to 2.5ng/ml, with each concentration half that of its predecessor. These were also added to the assay plate. A blank sample was also measured, using sample diluent from the ELISA kit. Duplicates of each sample and standard were measured. The addition of reagents and samples was completed within 15 minutes, after which the assay plate was covered with an adhesive strip, and incubated overnight at 37oc. The assay plate was washed 5 times on an automated plate washer, each time using 400ml of the assay kit wash buffer. 100ml of the assay kit anti-‐HMGB-‐1 detection-‐antibody solution was then added to each well. The assay plate was re-‐covered and incubated at room temperature for two hours before being washed 5 times with 400ml of the assay kit wash buffer as before. 200ml of a hydrogen peroxide and tetramethylbenzidine mixture was then added to each well, the plate covered again, protected from light and incubated for 30 minutes at room
temperature. 100ml of the assay kit stop-‐solution was then added to each well and the plate shaken briefly. The optical density of each well was measured at 450nm using a Synergy-‐2 microplate spectrophotometer (Biotek, Winooski, Vermont, USA). Mean absorbance of the blank wells was subtracted from the absorbance of standards and samples, and regression analysis of the standard absorbance curve performed. The result equation was used to calculate the HMGB-‐1 concentration of each sample.
3.12.1.1 Validation of the assay
The lower limit of detection was determined by adding two standard deviations of the mean optical density of ten zero standard replicates and calculated the corresponding HMGB-‐1 concentration. Intra-‐assay CV was assessed by the HMGB-‐1 concentration in one sample being measured four times on a single plate, divided by the standard deviation, and then multiplied by 100. The inter-‐assay CV was measured in a single sample across three consecutive plates. The percentage recovery was measured by adding a known quantity of HMGB-‐1 to a sample and calculating the resulting concentration. This was then compared to the predicted value to obtain the proportion recovered. The lower limit of detection, Intra-‐assay and inter-‐assay CV, and the percentage recovery are shown in table 3.6 with an example standard curve in figure 3.4
Table 3.6 HMGB-‐1 ELISA validation statistics Lower limit of
detection (ng/ml) Intra-‐assay CV (%) Inter-‐assay CV (%) Recovery (%) 1.0ng/ml 6.2% (n=4) 9.0% (n=3) 98.57%
Figure 3.5 Sample standard curve for HMGB-‐1 ELISA
3.12.2
RAGE ELISA
The receptor for advanced glycation end products (RAGE) concentrations in plasma and BALF were measured using a commercially available ELISA kit (R&D systems, Minneapolis, Minnesota, USA). This kit contains a 96-‐well assay plate that is pre-‐coated with an anti-‐RAGE capture antibody. To this plate were added 100ml of the assay diluent and 50ml of standards and sample that had been diluted to an appropriate degree for the range of the assay. The standard range of concentrations was created by serial dilution of a 5000pg/ml concentration of HMGB-‐1 to produce a range of 5000 to 78pg/ml, with each concentration half that of its predecessor. These concentrations were also added to the assay plate. A blank sample was measured, using sample diluent from the ELISA kit. Duplicates of each sample and standard were measured. The addition of reagents and samples was completed within 15 minutes, after which the assay plate was covered with an adhesive strip and incubated for 2 hours at room temperature. The
0 10 20 30 40 50 60 70 80 90 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 co nc en tr aw on o f HMG B-‐ 1 (n g/ m l) opwcal density
assay plate was then washed 4 times on an automated plate washer, each time using 400ml of the assay kit wash buffer, ensuring complete removal of liquid after each wash. 100ml of the assay kit anti-‐RAGE detection-‐antibody solution was then added to each well. The assay plate was re-‐covered and incubated at room temperature for two hours before being washed 4 times with 400ml of the assay kit wash buffer as before. 200ml of a substrate mixture of hydrogen peroxide and tetramethylbenzidine was then added to each well, the plate covered again, protected from light and incubated for 30 minutes at room temperature. 50ml of the assay kit stop-‐solution was then added to each well and the plate shaken briefly. The optical density of each well was measured at 450nm using a Synergy-‐2 microplate spectrophotometer (Biotek, Winooski, Vermont, USA). Corrections were made at 540nm absorbance. Mean absorbance of the blank wells was subtracted from the absorbance of standards and samples, and regression analysis of the standard absorbance curve performed. The result equation was used to calculate the RAGE concentration of each sample.
3.12.2.1 Validation of the assay
The lower limit of detection, inter-‐assay CV, intra-‐assay CV and percentage recovery were calculated as described for HMGB-‐1 above. The results of these calculations are shown in table 3.7 and a sample standard curve in figure 3.5
Table 3.7 RAGE ELISA validation statistics Lower limit of
detection (ng/ml) Intra-‐assay CV (%) Inter-‐assay CV (%) Recovery (%) 95pg/ml 4.1% (n=4) 10.6% (n=3) 96.25%
Figure 3.6 Sample standard curve for RAGE ELISA