Psicología y religión

2.4.1 Serum source, collection and storage 2.4.1.1 Source of murine serum

Peripheral blood was taken either by retro-orbital venesection from mice in the middle of studies or by cardiac puncture at the end. The blood was allowed to clot for two hours at 4°C and then microfuged at 2000g at room temperature for 10 minutes. The serum removed and stored in aliquots at -20°C until required.

2.4.1.2 Source of human serum

Peripheral blood was drawn by routine venepuncture and allowed to clot for 30 minutes at 37°C. The samples were then centrifuged at 500g at room temperature for 20 minutes and the serum removed and stored in aliquots at -20°C until required.

2.4.2 Serum IgG concentration

These assays were carried out in collaboration with K. B. Bodman. Serum IgG levels were measured using an ELISA essentially as described by Engvall (1989). Ninety-six well, flat bottomed, maxisorp microtitre plates (Life Technologies) were coated with 100 pl/well of 5 pg/ml affinity purified F(ab')2

fragment of goat anti-mouse (or human) IgG (Jackson Immunoresearch Laboratories, ME, USA) in PBS and left overnight at 4°C. After washing four times with PBS-0.05% Tween 20 (PBS-T), the plates were blocked with 200 pl/well PBS containing 1% BSA, for one hour at 37°C. Calibrated mouse (The Binding Site, Birmingham Research Park, Birmingham, UK) or human (Behring Diagnostics, London, UK) serum was diluted to a range of 0.001-10 pg/ml of IgG in PBS-T containing 1% goat serum. Supernatant samples were diluted 1:3-5 and sera diluted 1:5000-10,000. The samples and the standards were added in duplicate (100 pl/well) to the washed plates and incubated for one hour at 37°C. After washing with PBS-T, alkaline phosphatase conjugated F(ab')2 sheep anti-mouse IgG (Sigma) diluted 1:30,000 (or alkaline phosphatase conjugated F(ab')2 goat anti-human IgG [Sigma] diluted 1:1000) in PBS-T was added to the plates (100 pl/well) and incubated for one hour at 37°C. The plates were then washed four times with PBS-T. A colour reaction was produced using p-nitrophenyl phosphate tablets (Sigma) in carbonate/bicarbonate buffer pH9.6 (2 tablets/10 ml BIG buffer) containing 2 mM magnesium chloride and added at 100 pl/well. The reaction was stopped with 50 pl/well 1M sodium hydroxide and the ODs were read at 405 nm on a Dynatech MR 580 ELISA reader (Dynatech Laboratories Ltd, West Sussex, UK). The immunoglobulin concentration of each sample was interpolated from the standard CD curve plotted on four cycle one-way logarithmic paper.

2.4.3 Serum IgG galactosylation

IgG galactosylation was measured in collaboration with K. B. Bodman. Serum IgG GO in the human and murine individuals were measured as follows using a modified version of previously published assays (Thompson et al 1992,

Sumar et ai 1990 ). Ninety six well maxisorp microtiter plates were coated overnight with 50 pl/well of recombinant truncated protein G' (Sigma, P-4689) at 5.0 pg/ml in PBS at 4°C. The wells were aspirated and blocked with 100 pi 0.05% Tween 20, 1% BSA in PBS (PBS-T-BSA) for one hour at 37°C followed by three washes with 0.05% Tween 20 in PBS. Standards with known GO levels and sera diluted 1:100 in 0.1 M glycine, 0.16 M NaCI, pH 7.0 were added in triplicate (50 pl/well) to 2 identical plates and incubated for 2 hours at 37°G. After washing, 50 pl/well PBS was added and the plates floated on a waterbath at 85°C for 15 min to partially denature the IgG molecules and thus expose the oligosaccharides. The biotinylated lectin Bandeiraea simplicifolia n (BSII, Vector Laboratories Inc., Cambridgeshire, UK) was diluted 1:500 (4 pg/ml) in PBS-T-BSA containing 0.1 mM calcium chloride and biotinylated goat F(ab')2 anti-human IgG (Sigma) 1:10,000 (0.04 pg/ml) in PBS-T-BSA and each added at 50 pl/well to the cooled plates and incubated at 4°C overnight. After three washes, 50 pl/well of Straptavidin- horseradish-peroxidase (DAKO Ltd., Buckinghamshire, UK) was added and incubated at 37°C for 1 hour. A colour reaction was produced using 50 pl/well 0.1 M citrate phosphate buffer pH 4.1 containing 0.5 mg/ml 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulphonic acid) (Sigma) and 1:2000 hydrogen peroxide. After 15 minutes, the reaction was stopped with 50 pl/well sodium fluoride (2 mg/ml) and the plates were read on an automated ELISA reader (Dynatech Laboratories Ltd, Billinghurst, West Sussex, UK) at 410 nm. The results were expressed as the ratio of BSn:anti-lgG binding and quantified against the standard curve of known GO samples. The GO standards used in this study were human (both RA and control subjects) and murine (Balb/c, CBA/Ca, DBA/1 and MRL Ipr/lpr) IgG molecules whose GO values were defined by comparison in both dot blot and ELISA systems with human and murine IgG whose GO, G1 and G2 values had been determined by Dr T Rademacher and colleagues at the Department of Biochemistry, University of Oxford, using the hydrazinolysis method. In some experiments, biotinylated goat F(ab')2 anti­ human IgG was replaced by the biotinylated lectin Ricinus communis agglutinin I (RCA I), diluted 1:5000 (1 pg/ml), and the results were expressed

as the ratio of BSn : RCAI. For measuring galactosylation levels of secreted IgG in cell culture supernatants, protein G’ was diluted to 0.05 or 0.20 pg/ml prior to coating and all volumes added to the wells were 100 pi. The concentration of IgG was determined as above in each supernatant (ranged from 0.2-8.0 mg/ml) and serum standard and each diluted to 0.5 or 2.0 pg/ml in glycine buffer. The results were expressed as the ratio of BSn binding to RCAI binding.