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Plate Layouts - Liquid Phase
ELISA
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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.
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 to 1 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 +4ºC for no longer than 1 week.
Conjugate
Stock conjugate is diluted 1:5 in Diluent buffer B, mixed with an equal volume of glycerol and stored in 1 ml aliquots at -20ºC. New batches of conjugate must be titrated to determine working strength, usually of the order of 1/200.
Phosphate-buffered saline (PBS) (Dulbecco PBS) 0.01M
NaCl 8.0 g
KCl 0.2 g
MgCl2.6H2O 0.1 g
KH2PO4 0.2 g
Na2HPO4 1.14 g
CaCl2.2H22O 0.1 g
Add de-ionized water to 1 litre and check pH is 7.3-7.4
ELISA wash fluid
1 part PBS to 4 parts distilled water.
Disinfectant solutions 0.2% (w/v) citric acid
Dissolve 2 g of citric acid in 1 litre of water.
or 0.4% FAM
Dilute stock solution to 0.4% in tap water. Make up fresh each day.
Diluent buffer A 'PBST'
0.01 M 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 theTween is fully dispersed). Label and store at +4ºC
Diluent buffer B
0.01M 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.
Acid stopper solution (1.25M 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 make up to 100 ml
[CARE. SODIUM AZIDE IS EXTREMELY TOXIC. KEEP AWAY FROM ACID AND HANDLE IN A FUME CUPBOARD]
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
or Dissolve 1 OPD tablet in 50 ml 0.05M phosphate-citrate buffer (pH 5.0)
Store at -20ºC 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]
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