Contrastes de hipótesis

2.6.1) IL-12 p40

(ELISA, IL-12 p40 kit obtained from Cambridge biosiences, Cambridge, U.K)

Each required well on a maxisorb ™ plate (NUNC) was coated with IL-12 p40 capture antibody (lOOpl, Ipg/ml, PBS), Sealed with an adhesive cover and left overnight (4°C), NB: the plates could be left for several days. When required the plate was washed three time in wash buffer (200pl per well, PBS + 0.1% tween) and blocked with blocking

buffer (PBS + 0.5% BSA, 300pl per well) for 2 hours with gentle agitation (room temperature - RT). IL-12 p40 recombinant protein standards were prepared, the stock concentration (10,000 pg/ml) was diluted to 2000 pg/ml in complete medium and six fiirther 2.5 fold serial dilutions were made to give a seven point standard curve over a concentration range from 2000 pg/ml to 8.2 pg/ml.

After the blocking period the plate was washed three times with wash buffer (200pl) and then the standards, controls and IL-12 supernatants to be measured were added to the plate (lOOpl of each) in duplicate wells. The medium used in the IL-12 culture assays was used as a background control (usually complete medium). This was immediately followed by the addition of the IL-12 p40 detection antibody (0.4pg/ml diluted in blocking buffer + 0.1% tween 20). The plate was then sealed and incubated with vigorous agitation for 2 hours (700 rpm , RT). After incubation the plate was washed three times with wash buffer (200pl) and streptavidin-HRP (horse raddish peroxidase)

was added (lOOpl, 0.05pg/ml). The plate was re-incubated for 30 minutes (RT), washed

three times in wash buffer (200pl) and then the HRP substrate was added (Method 1). In figures 3.3.5 and 4.5. l(i) & (ii)B OPD (see buffers section) was used and in (method 2) figures 4.5.1(ii)A and figures 4.5.2: 3,3’,5,5’-Tetramethyl Benzidine (TMB, Sigma) was used as a substrate (lOOpl, either substrate).

When colour was observed in the wells of the lowest concentration in the standard curve the reaction was terminated with the addition o f stop solution (3M HCL, 50pl). The optical densities corresponding to the IL-12 p40 concentrations were immediately measured at 450rim using a spectrophotometer plate reader. A graph was drawn matching the known IL-12 p40 concentrations with the recorded optical densities and from this the unknown sample IL-12 p40 concentrations were calculated.

2.6*2) IL-6

(IL-6 kit was obtained from Genzyme U.K, West Mauling, U.K)

Each required well on a maxisorb ™ plate (NUNC) was coated with IL-6 capture antibody (lOOpl, 5 pg/ml), sealed and left overnight (4°C), NB: the plates could be left for several days. When required, the plate was washed three time in wash buffer (200pl per well, PBS + 0.1% tween) and blocked with blocking buffer (PBS + 0.5% BSA, 300pl per well) for 2 hours with gentle agitation (room temperature - RT).

IL-6 recombinant protein standards were prepared, the stock concentration (1 pg/ml) was diluted to 6ng/ml in complete medium; six further 2 fold serial dilutions were made to give a seven point standard curve over a concentration range of 6ng/ml to 81.2pg/ml. After the blocking period the plate was washed three times with wash buffer (200pl) and then the standards, controls and IL-6 supernatants to be measured were added to the plate (lOOpl of each) in duplicate wells. The medium used in the IL-6 culture assays was used as a background control (usually complete medium).

C hapter 2 - M aterials a n d M ethods

The plates were then sealed with an adhesive strip and incubated for 2 hours at 37°C with vigorous agitation (700 rpm). After the incubation period the plate was washed three times with 200pl of wash bufter. This was immediately followed by the addition the IL-6 detection antibody (1.5 pg/ml, diluted in blocking buffer + 0.1% tween 20). The plate was then sealed and incubated with vigorous agitation for 2 hours (700 rpm , 37°C). After incubation the plate was washed three times with wash buffer (200pl) and

streptavidin-HRP (horse radish peroxidase) was added (lOOpl, 0.05pg/ml). The plate was re-incubated for 15 minutes (37°C), washed three times in wash buffer (200pl) and

then the HRP substrate 3,3’,5,5’-Tetramethyl Benzidine (TMB, lOOpl) was added. When colour was observed in the wells of the lowest concentration in the standard curve the reaction was terminated with the addition of stop solution (2M HCl, lOOpl). The optical densities corresponding to the lL-6 concentrations were immediately measured at 450r|m using a spectrophotometer plate reader. A graph was drawn matching the known lL-6 concentrations with the recorded optical densities and from this the unknown sample IL- 6 concentrations were calculated.

2.6.3) TNF-a

(TNF-a kit was obtained from Genzyme U.K)

Each required well on a maxisorb plate (NUNC) was coated with TNF-a capture

antibody (lOOpl, 4 pg/ml), sealed and left overnight (4°C), NB: the plates could be left for several days. When required, the plate was washed three time in wash buffer (200pl per well, PBS + 0.1% tween) and blocked with blocking buffer (PBS + 0.5% BSA,

300pl per well) for 2 hours with gentle agitation (room temperature - RT). TNF-a recombinant protein standards were prepared, the stock concentration (70ng/ml) was diluted to 1000 pg/ml in complete medium; six fiirther 2 fold serial dilutions were made

to give a seven point standard curve over a concentration range of 1000 pg/ml to 15.6

After the blocking period the plate was washed three times with wash buffer (200pl) and

then the standards, controls and TNF-a supernatants to be measured were added to the plate (lOOpl of each) in duplicate wells. The medium used in the TNF-a culture assays was used as a background control (usually complete medium). The plates were then sealed with an adhesive strip and incubated for 2 hours at 37°C with vigorous agitation

(700 rpm). After the incubation period the plate was washed three times with 200pl of wash buffer. This was immediately followed by the addition the TNF-a detection antibody (300 ng/ml, diluted in blocking buffer + 0.1% tween 20). The plate was then sealed and incubated with vigorous agitation for 2 hours (700 rpm , 37°C). After incubation the plate was washed three times with wash buffer (200pl) and streptavidin-

HRP (horse raddish peroxidase) was added (lOOpl, 0.05pg/ml).

The plate was re-incubated for 15 minutes (37°C), washed three times in wash buffer

(200pl) and then the HRP substrate, 3,3’,5,5’-Tetramethyl Benzidine (TMB, lOOpl) was added. When colour was observed in the wells of the lowest concentration in the standard curve the reaction was terminated with the addition o f stop solution (2M HCL, lOOpl). The optical densities corresponding to the TNF-a concentrations were immediately measured at 450r|m using a spectrophotometer plate reader. A graph was drawn matching the known TNF-a concentrations with the recorded optical densities

C hapter 2 - M aterials and M ethods