Análisis Factorial Exploratorio

Two methods were used to detect protein concentration in the release medium. For BSA the Bradford method was used to bind a dye to the protein and the colour change was distinguished by measuring the absorbance with UV-vis spectroscopy. For Serp-1 a sandwich ELISA was performed using antibodies to capture and detect the active form of Serp-1.

3.4.1 UV-Visible Spectroscopy

The Bradford method was used to add an acidic dye reagent to the BSA release samples. In 15 mL tubes, one part dye reagent concentrate was added to four parts of the BSA release samples. If need be, the samples were diluted with water and the appropriate amount of dye added. The linear region of the assay is 1.2 to 10.0 µg/mL. The tubes were gently shaken to mix and left to incubate at room temperature for 15 min. Samples were then transferred to a 1 cm cuvette. The absorbance of each sample was measured at 595 nm on a Beckman Coulter, DU520, UV-visible spectrophotometer. Three readings for each sample were taken. The absorbance was compared to a calibration curve to determine protein concentration.

3.4.2 Enzyme-Linked Immunosorbent Assay (ELISA)

A sandwich ELISA was used to detect Serp-1 in the release samples. The protocol used was inherited from Viron Therapeutics standard operating procedures and details can been seen in Appendix A.

Briefly, samples and standards were added to a polypropylene 96 well plate for dilutions. The assay range is 2 to 24 ng/mL. A standard curve was generated n the first two columns of the 96 well plate by diluting purified Serp-1, 200 ng/mL, in assay buffer

from 0 to 24 ng/mL. In the remaining columns, the samples were serially diluted into the assay range. All samples and standards were run in duplicate.

For the assay, a polystyrene 96 well plate was used. Each well was first coated with the coat antibody in calcium carbonate buffer and the plate was incubated in the fridge at 4 °C overnight. The coat buffer was removed and blocking buffer was added to the wells and allowed to incubate at room temperature for one hour. The plate was washed three times with wash buffer. Then the standards and samples were transferred from the dilution plate to the assay plate and allowed to incubate at room temperature for one hour. The plate was washed three times with wash buffer. The biotinylated detection antibody was diluted in assay buffer and added to each well, incubating at room temperature for one hour. The plate was washed three times with wash buffer. HRP streptavidin was diluted in assay buffer and added to each well, incubating at room temperature for one hour. The plate was washed three times with wash buffer. Equal portions of substrate A and substrate B were mixed from the TMB substrate kit. This mixture was dispensed into the wells and incubated at room temperature for only four

minutes. At this time a stop solution of 2 N H2SO4 was added to each well. The plate

was then immediately placed in the plate reader and the optical density for each well was read at 450 nm using a Bio-Tek Instruments EL307C manual microplate reader. The data was transferred to an Excel spreadsheet and the concentration of each sample was determined using the standard curve generated on each plate. Only those values in the linear region of the standard curve were used.

3.4.2.1 Serp-1 Activity

The activity of Serp-1 is also measured by ELISA. The ELISA uses antibodies that are specific to the active form of Serp-1. Therefore, for studies where the activity was of interest, an ELISA was performed and the resulting concentration of each sample compared to a control. Similar concentrations are representative of similar activity levels. This was performed to determine if Serp-1 maintains its activity through the freeze-thaw cycles used for PVA processing and also to compare Serp-1 activity in different buffers.

3.4.3 SDS-PAGE and Western Blot

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were used to determine the amount of Serp-1 remaining in the PVA matrix. The PVA hydrogels were collected after selected time points (12 h and 48 h) from a controlled release study and a fully loaded matrix (0 h) was also prepared. The hydrogels were melted back into liquid form by heating above 70 °C in a round bottom flask equipped with a mechanical mixer. Once in the liquid form, the PVA samples were diluted with water and prepared for the SDS-PAGE.

The 0 h sample was diluted 16X and 64X and the 12 h and 48 h samples were

diluted 8X and 32X in water. 40 μL of each dilution was added to 15 μL of 4X SDS-

PAGE sample buffer. The samples, controls (Serp-1, 5 μg/mL) and standard marker

were loaded into the gels as shown in TABLE 3.1 for electrophoresis. Further detailed

methods can be seen in Appendix B.

T

3.1.

Gel 1

Lane 1 2 3 4 5 6 7 8 9 10 Sample _Standard Bio-Rad _(64X) 0 h _(32X) 12 h _(32X) 48 h Serp-1 Serp-1 Serp-1 Serp-1 Serp-1 Serp-1