CHAPTER2
2.8 KININ GENERATION ASSAYS
loaded with 1/250 anti-rabbit phosphate conjugate (diluted in 0.01 M PBS, pH 7.2) and incubated for 1 h at 37° C in a shaking water bath. The plate was washed with 200 µI 0.01 M PBS/0.5 % Tween for 3 min each at RT. Finally, the wells were all loaded with 100 µI of a 1 mg/ml chromogenic pNPP substrate (Sigma, St. Louis, 1 tablet in 5 ml of MgCh/10 % diethanolamine, pH 9.6 substrate buffer) and the colour was allowed to develop until the highest absorbances peaked at 1 to 1.5 absorbance units (Appendix 2.10). Readings were taken at RT with a Biorad Microplate Reader 3550 (Biorad, UK) using Biorad Microplate Manager software. The mean blank absorbance was subtracted from the standards, controls and samples, and a curve of absorbance versus HUK concentration was plotted, values expressed as ng/ml.
Acid-alcohol extraction The synovial fluid was mixed with an equal volume of 0.003 % HCI in absolute ethanol, vortexed and then incubated at - 20°C for 90 min. This was then centrifuged (7,000 g, 4°C, 10 min) and the supernatant decanted. The precipitate was washed with one volume of acid alcohol (diluted 1:1 in distilled water) and after recentrifugation, the corresponding supernatants pooled, evaporated to dryness at 55°C and the dry residue was reconstituted in the required volume of assay buffer, centrifuged and the clear supernatant used for measurement.
Kinin ELISA
To optimise binding of bradykinin (BK) to the plate it was necessary to first conjugate BK to a large protein. Cytochrome C was used for this purpose.
Conjugation of bradykinin to cytochrome C: Bradykinin (Sigma, UK) was conjugated to cytochrome C via a linker molecule, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Sigma, UK), according to a method modified from that of Carlsson et al. (1978).
SPDP (13 µM in 0.1 M phosphate buffer pH 8.6, containing 20 % ethanol) was mixed with an equal volume of cytochrome C (type IIS from Saccharomyces cerevisiae, Sigma, UK, IO µM in phosphate buffer pH 8.6) and incubated at 22°C for 60 min. The mixture was then filtered through ultra-free filters, (Millipore, UK) with 10,000 nominal molecular weight cut off, to remove any unconjugated SPDP. The cytochrome C-SPDP conjugate was resuspended in the two volumes of 0.3 M phosphate buffer pH 6. One volume of bradykinin (1 rnM) was then added and this was incubated at 22°C for 60 min. Before use in the ELISA, each new batch of conjugate was tested to determine the optimum concentration for coating of the ELISA plate.
ELISA procedure: The acid-alcohol extracts were reconstituted to the original volume of the synovial fluid in PBS, and kinin concentration measured by an ELISA method as follows. Nunc Immulon Maxisorp (Nunc, UK) microtitre plates were coated overnight at 4 °C with 100 µI/well of 3 to 4 µg/rnl bradykinin-cytochrome C conjugate in sodium carbonate buffer, pH 9.6. Also overnight at 4°C, the sample or standard was preincubated with an equal volume of anti-rabbit anti-bradykinin monoclonal antibody (kindly donated by M. Webb, Sandoz, London) (Phillips and Webb, 1989) at 9.4 µg/rnl. The next day, empty sites on the plate were blocked with I % bovine serum albumin (BSA< Roche, Germany) for 30 min at RT. The preincubated samples or standards (100 µI/well) were then incubated on the plates for 3 h at 37°C. In the next step, alkaline phosphatase labelled anti-mouse lgG (Sigma, UK) was added (diluted 1:250 in 0.01 M PBS, pH 7.2)) and then incubated for 2 h at 37°C. Finally, the substrate, disodium p-nitrophenyl substrate (Sigma, UK) was added, incubated at RT for approximately one hour until sufficient colour developed (i.e. maximum absorbance at 405 nm). Incubations were carried out in a humid box. Between each step the wells were washed three times for 3 min with PBS containing 0.05 % Tween (Sigma, UK). All dilutions were carried out in PBS containing 0. I % BSA, unless stated otherwise. Nonspecific binding to the plate was determined for each plate by incubation of anti-bradykinin antibody (preincubated with buffer only) in wells that had been coated with coating buffer containing no bradykinin conjugate, and subtracted from all absorbance values. The absorbance values obtained for the standard BK was used to plot a standard curve of absorbance versus log concentration from which the basal and generated kinin contents of the SF samples were determined (Bio Rad microplate manager software). The basal and generated kinin contents of control urine were also measured during each run to determine the inter- and intra-assay coefficients of variation.
An important aspect of this assay is the use of two kininase/protease inhibitor cocktails.
Firstly, aliquots of all samples and control urine were stored in inhibitor cocktail. This contained proteins that inhibited the enzymic activities of TK, PK and other trypsin-like proteases ((IO µM aprotonin and 10 µM SBTI), thereby preventing the release of kinin from endogenous k.ininogen. It also contained 10 µM captopril, an inhibitor of the KIi family of kininases, and phosphoramidon, another kininase inhibitor, to ensure that the basal kinins present in the samples were not destroyed.
A second aliquot of each sample and control urine was stored in kinin generating cocktail, which was similar to the inhibitor cocktail except that it lacked aprotonin and SBTI. The absence of these protease inhibitors ensured that kinin could be released from endogenous kininogen and once formed, was protected by inhibitors of the peptidases. The detailed stepwise method for the competitive ELISA is tabulated in appendix 2.11.