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protein, and dissolved in O.IM NaHCOg pH 9.6 at a concentration of 2mg KLH/ml. Glutaraldehyde (Sigma, Grade 1) was then added to the peptide-carrier solution to a final concentration of 0.05%. The solution was mixed in a glass tube, and stirred with a magnetic flea at room temperature overnight in the dark. The following day, each conjugated peptide solution was dialysed against PBS (phosphate-saline buffer) for 12 hours. The final solutions were aliquoted, and stored at -20®C.
Immunisation.
(Hancock and Evan, 1990). Prior to priming, all rabbits were bled (from the ear) and the sera were collected as preimmune controls. For the priming, 400pg of conjugated peptide (2QGpl) was mixed with PBS 0.8ml and 1ml Freund's complete adjuvant (FCA, Difco) in two 5ml glass syringes connected by a disposable 3-way tap. The oil-water mixture was pumped between the two syringes until it became stiffer and stable as an emulsion. The emulsion was then injected subcutaneously into the backs of two rabbits (each 1ml) at multiple sites. After 3 weeks, rabbits were immunised again with 50pg (25|ol) conjugate in Freund's incomplete adjuvant (FIA, Difco) (975pl PBS, 1ml Freund's incomplete adjuvant). The rabbits were then immunised repeatly with FIA conjugate at 3 week intervals and were bled 10 days after the 3rd and 5th immunisation. The collected sera were tested by enzyme linked immunoabsorbance assay (FLISA). The rabbits were killed after a further 1-4 immunisations when the maximal anti-peptide titres had been reached. The sera were collected and tested.ELISA for anti-trk peptide antibodies.
The original free peptides were diluted to a final concentration of lOpg/ml in PBS. 50pl of diluted peptide was coated to each well of 96- well multidishes (Nunc) overnight at 4®C, (a control plate which had no coated peptide was prepared in parallel). The following day, plates were washed twice in 0.1% Bovine Haemoglobin (Sigma) in PBS (Hb/PBS). Then 200pl of blocking buffer (1% Hb/PBS) were added per well and left at room temperature for 1 hour. After 1 hour, plates were washed twice in 0.1% Hb/PBS. 50pl of antibody diluted in 0.1% Hb/PBS was then added to each well in triplicates. (Serially diluted antibody in doubling dilutions, starting from 1/100, 8 dilutions), and incubated overnight at 4®C. The following day, plates were washed 3 times in 0.1% Hb/PBS as before. 50pl of 1/1000 peroxidase conjugated anti rabbit Ig (Amersham) diluted in 0.1% Hb/PBS were added per well, and left 2 hours at room temperature. Plates were then washed 4 times as before in 0.1% Hb/PBS and finally, washed with PBS. lOOpl of substrate solution were added to each well, the substrate was made of as follows: one lOmg tablet of 2, 2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (Sigma) was dissolved in 18.2 ml PBS, pH to 4.3, and 1/1500 volume of 30% Hydrogen Peroxide (Sigma) was added. The optical density o f the solution in the wells was determined on an ELISA plate reader after 5-30 minutes incubation at room temperature, using a 412 nm filter.
Affinity Purification of anti-peptide antibody.
(Hancock and Evan, 1990; Harlow and Lane, 1988) Preparation of peptide-Sepharose: 1.5 g CNBr-Sepharose (Pharmacia Fine Chemicals) was swollen in 200ml ImM HCl and left 15 minutes at room temperature. The slurry was poured into a sintered glass funnel, and drained to a moist cake. The cake (5ml) was added to 5ml PBS (pH 7.5-8) containing 500pg peptide. The peptide- Sepharose solution was agitated gently for 2 hours at room temperature. After 2 hours, the slurry was poured onto a sintered support, washed with 20ml of PBS, followed by 20ml lOOmM Na acetate pH 4, then followed by 20ml 2M NaCl in PBS, and finally, washed with 20ml Tris buffered saline (TBS) (25mM TrisHCl/144mM NaCl pH 8.2). The slurry was stored in TBS at a final concentration of 50%, with 0.1% sodium azide at4 0 c .
Serum was clarified with ammonium sulphate: 5mM EDTA was added to serum, then 0.82 volumes of saturated ammonium sulphate was added and the mixture was stirred and left for 15 minutes at room temperature. After centrifugation at 10,000 rpm for 10 minutes at 4°C, the pellet was collected. The pellet was then redissolved to the original volume with TBS. 10% NP40 was added to a final concentration of 0.1% and samples were spun in a microfuge to clarify.
Affinity chromatography: 2ml of peptide-Sepharose was packed into a Pharmacia reversible column in running buffer (TBS/0.1% NP40). The column was then washed with 20ml of running buffer over 20 minutes. After 20 minutes, 2ml o f the clarified serum was run into the column at a 2ml/min flow rate. Then the column was washed with 20ml o f running buffer. The flow through was collected and reapplied to the column 3 times. The final flow through was collected and stored at -20®C. Then the direction of
flow was reversed, and the column was washed with 2 0 ml of running buffer over 10
minutes, followed by 10 ml of TBS, then followed by 10ml of 0.9 % of NaCl.
Elution of antibody: 1). Low pH elution. Bound antibody was eluted with 8ml of lOOmM sodium citrate (BDH) pH 2.5 over 10 minutes. The eluate was collected and immediately neutralised to pH 6 with 2M Tris base. Then 8ml of saturated ammonium sulphate was added to the eluate and left for 10 minutes at room temperature. The precipitated antibody was collected by centrifugation at 1 0 ,0 0 0 g for 10 minutes at 4®C and resuspended in 1ml water, then dialysed against PBS/0.1% sodium azide. 2). High pH elution. Bound antibody was eluted with 8ml lOOmM triethylamine (Sigma) (pH 11.5, freshly prepared). The remaining procedures were followed as 1). 3). Elution with 4M M gCl2. Bound antibody was eluted with 8ml of 4M M gCl2 (Sigma). The eluate was diluted 10 times with distilled water, and an equal volume of saturated ammonium sulphate was added. The remaining procedures were followed as 1). 4). Elution with 5M LiCl (Sigma). The eluate was diluted 10 times with distilled water. 5) Elution with 50% ethylene glycol (Sigma). 6) with 10% dioxane (Sigma). 7) with 3M thiocyanate (Sigma).
8) with combination of lOOmM triethy lamine and 10% dioxane, to a final volume of 8ml. 9) with combination of lOOmM sodium citrate and 50% ethylene glycol, to a final volume of 8ml. 10) with combination of lOOmM sodium citrate, 3M thiocyanate and 50% ethylene glycol, to a final volume of 8ml. All the procedures were followed as 1) or 3), depending on whether acid, base, salt or organic solvent were used.
The titres of antibody from each preparation and original serum, ammonium sulphate clarified serum, flow through, were tested by ELISA.