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Diagnostic Procedure
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GENERAL NOTE ON BUFFERS
It is important that buffers and stock solutions described below are within the pH ranges indicated. The pH of each solution should therefore be tested. If the pH does not fall within the specified ranges, steps should be taken to correct the problem. Poor water quality and unclean glassware are most frequently implicated. Should any buffer or stock solution show signs of deterioration (i.e., formation of a precipitate) or contamination (i.e., evidence of bacterial, fungal or algal growth), discard immediately and prepare a fresh batch in clean glassware
Acid stopper solution (1.25 M sulphuric acid)
68 ml of concentrated sulphuric acid is added to 1 litre de-ionized/distilled water (exact volume of acid required will vary with the purity of the preparation used).
[WARNING: ADD SULPHURIC ACID TO WATER - NEVER ADD WATER TO SULPHURIC ACID]
Azide solution (1 M)
NaN3 6.5 g
Add de-ionized/distilled water to 100 ml
[WARNING: HANDLE WITH CARE ]
Chromogen
Ortho-phenylene diamine (OPD) in phosphate-citrate buffer pH 5.0.
Citric acid monohydrate 5.11 g
Na2HPO4 anhydrous 7.3 g
Add de-ionized/distilled water to 1 litre
OPD 400 mg
Alternatively, dissolve one tablet of phosphate-citrate (Sigma P4809) in 100 ml of de-ionized water to obtain a 0.05 M phosphate-citrate buffer, pH 5.0. Dissolve one OPD tablet (Sigma P8412, 30 mg) in 50 ml of 0.05 M phosphate-citrate buffer (pH 5.0).
Store at -20°C in 10, 15, 20 and 25 ml aliquots in the dark. (Stock solution should be colourless, with a pH between 5 and 6, and if coloured should be discarded and a fresh batch prepared). Thaw at room temperature in the dark, and immediately before use activate with a 1/2000 dilution of the substrate hydrogen peroxide solution (30-33% concentration), i.e., 12.5 µl H202 per 25 ml OPD.
[WARNING: Handle OPD with care as it is a potential carcinogen; HARMFUL if swallowed, inhaled or adsorbed through skin. WEAR GLOVES]
Coating buffer
0.05 M Carbonate/bicarbonate, pH 9.6 +/- 0.05
Normal strength solution
contains : x 20 strength stock :
Na2CO3 1.59 g Na2CO3 31.8 g
NaHCO3 2.93 g NaHCO3 58.6 g
per litre Add de-ionized/distilled water to1 litre.
Normal strength coating buffer solution is prepared by dilution of x 20 strength stock solution, i.e., 5 ml of x 20 strength solution plus 95 ml sterile de-ionized water.
Alternatively, dissolve one capsule of 0.05 M carbonate/bicarbonate (Sigma C-3041) per 100 ml sterile de-ionized water.
Label and store coating buffer at +4°C for no longer than 1 week.
Diluent buffer A (for dilution of antigens)
0.01 M phosphate buffered saline (PBS), pH 7.4 +/- 0.20, plus 0.05% Tween 20. (Note that Tween 20 is very viscous and care must be taken to ensure that the delivery pipette is filled with the correct volume of this detergent and properly rinsed with the buffer to which it is being added and that the Tween is fully dispersed).
Label and store at +4°C.
Diluent buffer B (for dilution of detecting antibody and conjugate)
0.01 M PBS, pH 7.4 +/- 0.20 plus 0.05% Tween 20 plus 5% (w/v) Skimmed Milk Powder.
On the day of testing, add skimmed milk powder to Diluent buffer A to make the amount of Diluent buffer B required, i.e., add 5 g skimmed milk powder to 100 ml Diluent buffer A.
Do not store diluent buffer containing skimmed milk powder after completion of the test.
Disinfectant solution 0.2% (w/v) citric acid
Dissolve 2 g of citric acid in 1 litre of water.
ELISA wash fluid
1 part PBS to 4 parts distilled water.
Phenol red solution (0.2%)
Phenol red powder 2.0 g
1 M sodium hydroxide solution 29.2 ml
Grind the powder with the sodium hydroxide solution using a pestle and mortar, make to 1 litre with de-ionized/distilled water, and filter.
Phosphate buffered saline (PBS) (Dulbecco PBS) 0.01 M
NaCl 8.0 g
KCl 0.2 g
MgCl2.6H2O 0.1 g
KH2PO4 0.2 g
Na2HPO4 1.14 g
CaCl2.2H2O 0.1 g
Add de-ionized water to 1 litre, and check pH is 7.3-7.4.
Sample buffer
0.04 M phosphate buffer, pH 7.4 +/-0.05
NaH2PO4.2H2O 6.1 g
KH2PO4 0.78 g
make up to 1 litre with sterile distilled water
Preparation of test samples
Place tissue sample, after removal of as much glycerol/phosphate buffer as possible, in the mortar.
Grind the tissue with a little sterile sand and add sufficient sample buffer to make a 10% (w/v) suspension of epithelium. Transfer ground suspension to a small bottle and centrifuge at approximately 1000g for 10 minutes. Harvest the clarified supernatant fluid into a small glass bottle.
If a cell culture sample is to be tested, clarify the sample by centrifugation at approximately 1000g for 10 minutes to remove cell debris.
NOTE:
Samples may be stored in 0.04 M PBS at -70°C for prolonged periods. Alternatively, they may be stored at -20°C after the addition of an equal volume of sterile glycerol (50% v/v).
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