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#3501: Determination of EGF / Betacellulin by Radioimmunoassay (RIA)
Introduction
The following procedures and materials are required to perform a specific EGF radioimmunoassay. The procedure follows the basic principle of radioimmunoassay where there is competition between a radioactive and a non-radioactive antigen for a fixed number of specific antibody binding sites. The amount of [125I]-labelled EGF bound to the specific antibody is inversely related to the concentration of unlabelled EGF present. The separation of free and bound EGF is achieved by using a second antibody and a precipitating reagent. The mixture is centrifuged so that the precipitated antigen-antibody complex forms a semi-solid pellet; the supernatant containing the unbound labelled and unlabelled EGF is removed from the assay tube, and the tube counted in a gamma counter.
Materials and Reagents
A. RIA Buffer
B. EGF reference standards
C. Primary Antibody
D. Secondary Antibody
E. Rabbit gamma globulin (IgG)
F. [125I]-labelled EGF
G. Polyethylene glycol solution
A. RIA buffer
(30 mM NaH2PO4, 0.02% protamine sulphate, 10 mM EDTA, 0.025 % NaN3, 0.05 % (v/v) Tween-20, pH 7.5)
Preparation of 1 litre of buffer:
1. In a 1 litre volumetric flask, add approximately 500 ml distilled or Milli-Q water
2. Add 4.68 g NaH2PO4.2H2O (Analytical Grade, AnalaR, BDH Chemicals, Australia P/L, Vic., Australia)
3. Add 200 mg protamine sulphate (Sigma Chemical Co., St. Louis, MO, U.S.A., Grade X from salmon)
4. Add 3.72 g EDTA di-sodium salt (Analytical Grade, Univar, Ajax Chemical P/L, Australia)
5. Add 250 mg sodium azide, NaN3, (Analytical Grade, Sigma Chemical Co., St. Louis, MO, U.S.A.)
6. Mix to completely dissolve solids in the distilled or Milli-Q water; adjust to pH 7.5.
7. Adjust volume to 1 litre with distilled or Milli-Q water.
8. Add 0.5 ml Tween-20 (# P1379, Sigma Chemical Co., St. Louis, MO, U.S.A.)
B. EGF Reference Standards
1. Reconstitute a 20 µg vial of Novozymes GroPep EGF peptide in 200 µl of 10 mM HCl . Ensure the EGF is completely dissolved before diluting with RIA buffer to a final concentration of 10 ng/µl. This 10 ng/µl stock solution is stable for at least 12 months at -20°C.
2. Place 24 µl of the 10 ng/µl peptide solution into a 5 ml tube and add 2,976 µl of RIA buffer. Mix thoroughly. This gives a working standard solution of 80 ng/ml.
3. In order to produce the series of EGF reference standards, add an equal volume of a standard with buffer to produce the next standard in the dilution set. i.e., to a series of 10 x 2 ml Eppendorf tubes, add the following solutions:
*** Ensure that each standard is mixed thoroughly before using to produce the following standard sequence ***
Ref. Std.# Conc.of standard
Std 1 500 µl of working standard solution 8,000 pg/100 µl
Std 2 500 µl of working standard soln plus 500µl of RIA buffer 4,000 pg/100 µl Std 3 500 µl of reference standard 2 plus 500 µl of RIA buffer 2,000 pg/100 µl
Std 4 500 µl of reference standard 3 plus 500 µl of RIA buffer 1,000 pg/100 µl
Std 5 500 µl of reference standard 4 plus 500 µl of RIA buffer 500 pg/100 µl
Std 6 500 µl of reference standard 5 plus 500 µl of RIA buffer 250 pg/100 µl
Std 7 500 µl of reference standard 6 plus 500 µl of RIA buffer 125 pg/100 µl
Std 8 500 µl of reference standard 7 plus 500 µl of RIA buffer 62.5 pg/100 µl
Std 9 500 µl of reference standard 8 plus 500 µl of RIA buffer 31.25 pg/100 µl
Std10 500 µl of reference standard 9 plus 500 µl of RIA buffer 15.625 pg/100 µl
C. Preparation of Primary Antibody
The primary antibody used in this method is Novozymes GroPep’s human EGF polyclonal antiserum (Catalog Code PAD1). It is recommended that the anti-EGF polyclonal antiserum be used at a final assay concentration of approximately 1/100,000.
Quantity required:
50µl of the working solution per assay tube (approximately 2 mls of the working solution of antibody are required for completing a standard curve)
Preparation:
1. Add 500 µl of RIA buffer to one vial of Novozymes GroPep’s anti-EGF polyclonal antiserum (rabbit) to give an antiserum stock solution of 1/100 dilution; ensure that the lyophilized pellet is completely dissolved before proceeding.
2. Place 10 µl of the 1/100 stock solution into a 10 ml polypropylene tube. The remaining 490 µl of the 1/100 stock solution may then be aliquoted and stored at -20°C.
3. Add 9,990 µl RIA buffer to the 10 ml polypropylene tube and mix thoroughly. This is the working antibody solution (1/100,000 dilution). Note that working solutions are stable for 5 days if stored at 4°C.
D. Preparation of Secondary Antibody
The secondary antibody used was Novozymes GroPep’s Goat anti-Rabbit gamma globulin (IgG) (Catalog Code PSA1). Please note that alternative sources of this reagent may be used but the amount required for optimum precipitation will have to be determined before use.
Quantity required:
50 µl of a working solution is required for each assay tube (approximately 2 ml of the working solution of antibody are required for completing one standard curve performed in triplicate).
Preparation of a working solution of secondary antibody:
1. To a vial of Novozymes GroPep’s Goat anti-Rabbit antisera add 2.5 ml RIA buffer. Mix thoroughly to dissolve. Add vial contents to a further 10 ml of RIA buffer. Mix thoroughly.
2. Place 200 µl in a 5 ml tube. Add 1,800 µl RIA buffer to the 5 ml tube.
3. Mix thoroughly.
E. Preparation of the Rabbit gamma globulin (IgG)
The Rabbit gamma globulin used in preparing this method was obtained from Dako, CA, USA. Alternative sources of this reagent may be used, but it should be noted that the amount required for optimum precipitation will have to be determined.
Quantity required:
10 µl of a 1/100 working solution is required for each assay tube (approximately 0.5 ml of the working solution of antibody (1/100 dilution) is required for completing a standard curve).
Preparation of a 1/100 working solution of rabbit gamma globulin:
1. Place 5 µl of Rabbit gamma globulin (IgG) (Dako, distributed in Australia by Jomar Diagnostics) into a 5 ml polypropylene tube.
2. Add 495 µl RIA buffer to the 5 ml tube.
3. Mix thoroughly.
F. Preparation of the [125I]-labelled EGF working solution
[125I]-labelled EGF (40 - 80 Ci/g) is prepared using a Chloramine - T method of iodination as detailed in the technical protocol “Procedure for Iodination of Insulin-like Growth Factor Polypeptides”.
Given that the half-life of 125I is 60 days, it is not advisable to use tracer beyond 60 days from the date of preparation.
Quantity required:
50 µl of a working solution of [125I]-labelled EGF is required per assay tube (approximately 2 mls of the working solution of [125I]-labelled EGF are required to complete a standard curve performed in triplicate).
Preparation:
1. Determine the number of counts per minute (cpm) per µl of undiluted [125I]-labelled EGF.
2. Place 2 mls of RIA buffer (or convenient volume) in a 5 ml (or suitably sized) polypropylene tube.
3. Add the appropriate volume of [125I]-labelled EGF into the RIA buffer such that 50 µl of this working solution of radiolabelled EGF reagent gives 20,000 cpm.
4. Mix thoroughly.
5. Place a 50 µl test sample in a suitable gamma counter tube and verify that approximately 20,000 cpm is contained in the 50 µl sample.
G. Preparation of Polyethylene glycol (PEG) solution
Quantity required:
1 ml PEG solution per assay tube (approximately 50 mls is sufficient to complete a standard curve)
Preparation:
1. Add 438 mg NaCl (Technical Grade) to a 100 ml beaker
2. Add 50 ml distilled or Milli-Q water, and mix to dissolve
3. Add 3 g polyethylene glycol 6000 (Technical Grade, Ajax Chemicals, Auburn, Australia)
4. Mix thoroughly until all the PEG has dissolved
5. Store at 4°C until required for assay. Please note that this solution will not be an effective precipitating agent for the radioimmunoassay unless it is used at 4°C.
Procedure for Radioimmunoassay
1. Number all the tubes required for completing the assay using a permanent marker pen. The tubes recommended for this application are polypropylene tubes of dimensions 12 x 75 mm.
2. Add the assay reagents in the order as depicted in the suggested assay schema (see Table 1 below). It is recommended that all reference standards, the zero reference standard, the unknown samples and the “blank” tubes be assayed in triplicate. (Note that the “blank” tubes refer to a zero reference standard assayed in the absence of primary antibody - this gives the non-specific binding of the radiolabelled EGF in the assay).
Table 1
Additions should be made in the order of reagents indicated. All assay treatments should be done in triplicate.
“Total” “Blank” “Zero std" “Ref.stds” “Samples”
Reagent:
EGF Ref. stds. - - - 100 µl of std - (1 - 10)
Unknown - - - - 100 µl
samples
[125I]-labelled EGF 50 µl 50 µl 50 µl 50 µl 50 µl
RIA Buffer - 200 µl 150 µl 50 µl 50 µl
Primary antibody - - 50 µl 50 µl 50 µl
Total Volume: 50 µl 250 µl 250 µl 250 µl 250µl
3. Once all the necessary reagent additions have been made as outlined in Table 1, mix the assay tubes thoroughly by vortexing.
4. Cover the tubes to prevent contamination of the assay by dust and other particulate matter, and incubate at 4°C for 16 - 20 hours.
The following steps should be performed at 4°C for optimal results.
5. Add the following reagents to all RIA tubes (excepting the 'Total' tubes) :
• 50 µl of anti-Rabbit gamma globulin working solution
• 10 µl of Rabbit gamma globulin working solution
6. Vortex each tube and incubate for 30 minutes at 4°C.
7. Add 1 ml of PEG solution (4°C) to all RIA tubes (excepting the “Total” tubes).
8. Vortex each tube thoroughly.
9. Incubate for a further 10 minutes at 4°C.
10. Centrifuge all tubes (excepting the “Total” tubes) at 4,000 g for 30 minutes at 4°C in a pre-cooled centrifuge
11. Aspirate the supernatants from each tube as soon as the centrifugation is completed. Note that the pellet at the bottom of each tube may be difficult to see and care should be taken when aspirating the supernatant.
12. Count the radioactivity contained in all tubes in a gamma counter. The details of the gamma counter and program used at Novozymes GroPep Ltd. to calculate the radioimmunoassay results are as follows:
Manufacturer and model number: LKB1261 Multigamma Gamma Counter
Software: RIACALC LM by Wallac
EGF Radioimmunoassay Standard Curve
© Novozymes GroPep Limited
28 Dalgleish Street
Thebarton SA 5031
PO Box 10065 BC
Adelaide SA AUSTRALIA
Telephone +61 (0)8 8354 7700
Facsimile +61 (0)8 8354 7788
Email:
AUAD-bioreagents@novozymes.com
Website: www.gropep.com.au
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