The media from the stimulated cells were assayed for the presence of IL-6, IL-1 p, TNF-a and IL-8. Cytokines were assayed using an ELISA apart from IL-8 release from neutrophils for which a RIA was employed.
2.7.1 EUSA for IL-6
Microtitre plates (Immulon 4, Dynatech) were coated with immuno-affinity purified goat polyclonal anti-IL-6 antibodies (Taktak et al., 1991) at 1 pg/ml, diluted in phosphate buffered saline (pH 7.2). Plates were incubated at 4° C overnight. The wells were then decanted and washed with 0.01 M phosphate/0.05 M NaCI buffer (wash/dilution buffer, pH 7.2) containing 0.1 % (v/v) Tween 20. Standards of IL-6 (100 pi human recombinant IL-6 standard, preparation 88/514: NIBSC) were added to wells over the concentration range of 0-3ng/ml and the supernatants to be tested were added to the remaining wells. Plates were incubated for 2 h at room temperature and washed three times with wash/dilution buffer. Biotinylated affinity- purified polyclonal goat anti-IL-6 antibody (100 pi of a 0.014 mg/ml solution) was added to each of the wells and incubated for a further 1 h at room temperature. The plates were washed 3 times and 100 pi of a 1:5000 dilution of avidin-HRP (Dako Ltd) were added into each well. Plates were then incubated for 15 min at room temperature before washing 3 times with wash/dilution buffer. Wells were then developed with 100 pi of 0.2 mg/ml orthophenylenediamine (OPD, Sigma) in 0.1 M citric acid-phosphate buffer pH 5.0 plus 0.4 pl/ml 30 % H2O2 (Sigma). The reaction was terminated by the addition of 150 pi of 1 M H2SO4, and the absorbance at 492 nm was measured on a Titertek Multiscan spectrophotometer. A standard curve was plotted of the absorbance versus the concentration of IL-6.
2.7.2 ELISA for IL-1 J3
The assay of IL-ip used a similar protocol to the IL-6 assay described above with the following modifications. Microtitre plates were coated with an immuno-affinity purified goat polyclonal anti-human IL-ip antibody (Rafferty et a!., 1991) diluted at
5 M.g/ml. The plates were incubated overnight at 4° C. The IL-1p standard used was the international standard for IL-1 [3 (NIBSC: 86/680) at a concentration range of 0-8 ng/ml. The detecting antibody was biotinylated immuno-affinity purified polyclonal anti-IL-1 p antibody (Rafferty et al., 1991) used at a dilution of 1:4000. A curve was plotted of the absorbance versus the concentration of IL-ip.
2.7.3 ELISA for TNF-a
Miaotitre plates were coated with a mouse monoclonal antibody to human TNF-a
at a concentration of 2 pg/ml in bicarbonate buffer at pH 9.6 (Meager, 1987). These plates were allowed to incubate overnight at 4° 0. The following day the plates were washed with wash buffer and then blocked overnight using 1 % human serum albumin (HSA) in PBS at pH 7.2-7 4. The TNF-a standard (international standard for TNF-a:NIBSC 86/659) was diluted in PBS plus 1 % human serum albumin (HSA) at a concentration range of 0-200 ng/ml. The standards and the samples were aliquoted into the washed wells and allowed to incubate for 1 h at 37° C The washed plates then received 100 pi of biotinylated goat polyclonal anti-
TNF-a antibody used at a dilution of 1:200 and were allowed to incubate for 1 h at 37° C. After this period the plates were washed, streptavidin-HRP at a dilution of 1:5000 added to each well and the plates allowed to incubate at 37° C for 30 min. The plates were then washed, the substrate OPD added and the colour allowed to develop in the dark for 15 min. The reaction was terminated by addition of 1M sulphuric acid and the absorbance for each well monitored at 492 nm. A standard curve was plotted of the absorbance versus the concentration of TNF-a.
2.7.4 ELISA for IL-8
The assay protocol was followed according to the manufacturers (R and D systems) instructions. A standard curve was plotted of the absorbance versus the concentration of IL-8.
2.7.5 Radioimmunoassay for IL-8
The samples to be tested were thawed out and an equal volume of 22 % (w/v) PEG/1 % Protamine sulphate (PEG/PS) was added to each tube and mixed well by vortexing. The tubes were then left for 1 h at 4° C. After this period, the tubes
were centrifuged for 5 min at 5000 rpm at 4° C and the supernatants transferred into labelled luckham tubes. The human IL-8 standard was diluted in doubling dilutions over the concentration range 40000 pM to 20 pM in spun sample buffer (SSB). 1.5 ml Eppendorf tubes without lids were labelled and placed in Eppendorf transfer tubes. To each sample tube the following were added: 100 pi of sample, 100 pi of anti-human IL-8 antibody at 1:50 dilution and 50 pi of ^^^l-human IL-8. To the standard tubes, all the above were added except that various standards were added instead of the sample. Total tubes or label only tubes contained 50 pi of ^^^l-human IL-8. The “O” tubes or reference binding tubes contained the following: 100 pi of SSB, 100 pi of IL-8 antibody (1:50 dilution) and 50 pi of ^^^l-human IL-8. To the non-specific-binding (NSB) tubes the following were added: 100 pi of SSB, 100 pi PBS.GP (PBS containing gelatin and protamine sulphate) and 50 pi of ^^^1- human IL-8. After an overnight incubation at 4° C, 50 pi of a 1:30 dilution of donkey anti-goat IgG was added to all the tubes. The tubes were then incubated for 8 h or overnight at 4° C. After this incubation period 1 ml of PBS/azide was added to each tube and then spun at 5420 g for 10 min. The supernatants were discarded and the pellet counted on a Canberra Packard Cobra multigamma counter (model 5005).