Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3131: Determination of rat IGFBP-4 by Radioimmunoassay (RIA)

Introduction

The following procedures and materials are required to perform a specific rat IGFBP-4 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 rat IGFBP-4 bound to the specific antibody is inversely proportional to the concentration of unlabelled rat IGFBP-I present. The separation of free and bound rat IGFBP-4 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 rat IGFBP-4 is removed from the assay tube, and the tube counted in a gamma counter.

Materials and Reagents

A. RIA Buffer

B. Rat IGFBP-4 reference standards

C. Primary Antibody

D. Secondary Antibody

E. Rabbit gamma globulin (IgG)

F. [¹²⁵I]-labelled rat IGFBP-4

G. Polyethylene glycol solution


A. RIA buffer

50 mM Tris, 50 mM NaCl, 0.1% EDTA, 1 % BSA (Fraction V, (Sigma A-7888) and 0.05 % (v/v) Tween-20, pH 8.0)

To prepare 0.5 litre of RIA buffer:

1. In a 500 ml beaker, add 3.025 g Tris base, 1.46 g NaCl, 0.5 g EDTA and 5 g BSA (Fraction V Sigma A-7888).

2. Add approximately 400 mls of distilled or Milli-Q water. Mix until dissolved.

3. Adjust pH to 8.0 with 1M NaOH.

4. Pour solution into a 500 ml volumetric flask, and adjust volume to 500 mls with distilled or Milli-Q water. Add 0.25 ml of Tween-20. Mix gently. The buffer is now ready for use.


B. Rat IGFBP-4 Reference Standards

1. Reconstitute a 15 µg vial of Novozymes GroPep's rat IGFBP-4 (Catalog Code BP4CU015) in 150 µl 10 mM HCl to a final concentration of 0.1 µg/µl. Ensure complete reconstitution before proceeding.

2. Place 20 µl aliquots of the 0.1 µg/µl peptide solution into 5 ml tubes and add 1,980 µl of RIA buffer to each tube. Mix thoroughly. This 1 µg/ml stock solution in RIA buffer is stable for at least 12 months at -20oC.

3. Place 20 µl of the 1 µg/ml stock solution into a 5 ml tube and add 1,980 µl of RIA buffer to give a working standard solution of 2 ng/200 µl or 2,000 pg/200 µl (10 ng/ml). Mix thoroughly.

4. In order to produce the series of rat IGFBP-4 reference standards, add an equal volume of a standard and RIA 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 in the sequence ***

Ref. Std.# Conc. of standard

Std 1 800 µl of working standard solution 2,000 pg/200 µl
Std 2 800 µl of working standard sol. plus 800 µl of RIA buffer 1,000 pg/200 µl
Std 3 800 µl of reference standard 2 plus 800 µl of RIA buffer 500 pg/200 µl
Std 4 800 µl of reference standard 3 plus 800 µl of RIA buffer 250 pg/200 µl
Std 5 800 µl of reference standard 4 plus 800 µl of RIA buffer 125 pg/200 µl
Std 6 800 µl of reference standard 5 plus 800 µl of RIA buffer 62.5 pg/200 µl


C. Preparation of Primary Antibody

The primary antibody used in this method is Novozymes GroPep's anti-rat IGFBP-4 polyclonal antiserum (Catalog Code PAAP1). It is recommended that the anti-rat IGFBP-4 polyclonal antiserum be used at a final assay concentration of approximately 1/40,000.

Quantity required:

200 µl of the working solution per assay tube (approximately a total of 5 ml of the working solution of antibody) are required for completing a standard curve.

Preparation:

1. Reconstitute the antiserum in 100 µl sterile filtered water. Ensure that the lyophilized pellet is completely dissolved before proceeding.

2. Aliquot out 9 x 10 µl and store at -20^oC. Avoid freeze-thaw cycles.

3. Add 490 µl of RIA buffer to the remaining 10 µl of Novozymes GroPep's anti-rat IGFBP-4 polyclonal antiserum (rabbit) to give an antiserum stock solution of 1/50 dilution.

4. Place 40 µl of the 1/50 stock solution into a 15 ml polypropylene tube. The remaining 460 µl of the
1/50 stock solution may then be aliquoted and stored at -20^oC for subsequent use.

5. Add 11.16 ml RIA buffer to the 15 ml polypropylene tube and mix thoroughly. This antibody solution is the working antibody solution (1/14,000 dilution). Note that working solutions are stable for 5 days if stored at 4^oC.

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:

100 µl of a working solution is required for each assay tube (approximately 4 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 12.5 ml RIA buffer. Mix thoroughly.


E. Preparation of the Rabbit gamma globulin (IgG)

The Rabbit gamma globulin used in preparing this method was obtained from Antibodies Inc, 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:

100 µl of a 1/200 working solution is required for each assay tube (approximately 4 ml of the working solution of antibody (1/200 dilution) are required for completing a standard curve).

Preparation of a 1/200 working solution of Rabbit gamma globulin:

1. Place 20 µl of Rabbit gamma globulin (IgG) (Antibodies Inc. USA) into a 10 ml polypropylene tube.

2. Add 3980 µl RIA buffer to the 5 ml tube.

3. Mix thoroughly.


F. Preparation of the [¹²⁵I]-labelled IGFBP-4 working solution

[¹²⁵I]-labelled IGFBP-4 (70 - 90 Ci/g) is prepared using a chloramine - T method of iodination as detailed in the technical bulletin 'Procedure for Iodination of Insulin-like Growth Factor Binding Proteins'. 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:

200 µl of a working solution of [¹²⁵I]-labelled IGFBP-4 is required per assay tube (approximately 8 mls of the working solution of [¹²⁵I]-labelled IGFBP-4 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 IGFBP-4.

2. Place 8 mls of RIA buffer (or convenient volume) in a 10 ml (or suitably sized) polypropylene tube.

3. Add the appropriate volume of [¹²⁵I]-labelled IGFBP-4 into the RIA buffer such that 200 µl of this working solution of radiolabelled IGFBP-4 reagent gives 15,000 cpm.

4. Mix thoroughly.

5. Place a 20 µl test sample in a suitable gamma counter tube and verify that approximately 1,500 cpm is contained in the 20 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 0.234 g sodium monophosphate dihydrate (NaH₂PO₄.2H₂O) to a 100 ml beaker.

2. Add 40 ml distilled or Milli-Q water and mix to dissolve. Adjust the pH to 7.3 with 1 M NaOH.

3. Add 5 mg sodium azide. When dissolved, adjust the volume to 50 ml.

4. Add 3 g Sigma polyethylene glycol-8000.

5. Mix thoroughly until all the PEG has dissolved.

6. 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 IGFBP-4 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:

IGFBP-4 Ref. stds. - - - 200 µl of std -
(1 - 10)

Unknown - - - - 200 µl
samples

RIA Buffer - 400 µl 200 µl - -

Primary antibody - - 200 µl 200 µl 200 µl

[¹²⁵I]-labelled IGBPF-4 200 µl 200 µl 200 µl 200 µl 200 µl


Total Volume: 200 µl 600 µl 600 µl 600 µl 600 µl

3. Once all 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 18 - 24 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) :

• 100 µl of Rabbit gamma globulin working solution
• 100 µl of anti-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 4000 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: LKB 1261 Multigamma Gamma Counter
Software: RIACALC LM by Wallac


IGFBP-4 Radioimmunoassay Standard Curve



Figure:

Rat IGFBP-4 radioimmunoassay
using Novozymes GroPep's anti-rat IGFBP-4
polyclonal antiserum (Catalog Code
PAAP1).
The assay can detect the very low
concentrations (200 ng/ml) of
IGFBP-4 in rat serum and can detect
samples containing 1 ng rat IGFBP-4.


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