Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #4100: Determining fish IGF-I levels using the Novozymes GroPep Fish IGF-I RIA Kit

Intended Use

This radioimmunoassay (RIA) kit provides materials required for the quantitative in vitro measurement of serum or plasma IGF-I in various fish species. This is the only intended use for the kit. Using the kit, serum IGF-I parallelism has been demonstrated for barramundi, bream, salmon, tilapia, trout and tuna (see Figure 1). Other species are under investigation.

Summary and Explanation

Insulin-like growth factor I (IGF-I) is a single chain polypeptide that stimulates growth and differentiation in many cell types.

The measurement of serum or plasma IGF-I is useful in a variety of different applications. For example, IGF-I mediates many of the properties of growth hormone and is therefore a useful indicator of some growth hormone actions.

IGF-binding proteins present in the serum actively bind IGF-I and may interfere with any assay for IGF-I by sequestering the IGF-I present in the reaction mixture.

IGF-binding proteins MUST therefore be removed from the serum prior to assay (e.g. using an acid ethanol extraction protocol).

Principle of the Test

Radioimmunoassays rely on the specific interaction between antigen and antibody.

A limited amount of radioactive antigen, together with a known amount of corresponding antibody, is combined with the assay sample. The radiolabelled and unlabelled antigens compete for the same antibody binding sites.

A second antiserum precipitates the antibody-bound antigen, separating it from the unbound antigen which remains in solution. After careful removal of the supernatant, the precipitate is counted in a gamma-counter.

The amount of radiolabel present in the precipitate is directly related to the amount of unlabelled antigen present in the original sample.

The IGF-I concentration in the samples is calculated from a calibration curve generated using the standards provided.

Reagents Supplied:

Store kits at 2-8°C. Lyophilized components, with the exception of the tracer, are stable for at least 3 months at this temperature.

Sufficient reagents are supplied to assay 100 serum samples in triplicate. For reliable data, each sample should be assayed in triplicate.

Sufficient IGF-I standard is supplied for three standard curves, so three assay runs can be performed.

a. Standard (Lyophilized): 3 vials of fish IGF-I (105 ng/vial).
See Reagent Preparation for details.
b. ¹²⁵I-Fish IGF-I tracer (Lyophilized). See Certificate of Analysis for details of radiolabel and activity date.
c. Antiserum 1 (Rabbit) (Lyophilized): 1 vial.
d. Antiserum 2 (Sheep) (1.1 ml): 1 vial.
e. IgG for precipitation: (Rabbit) 1 vial.
f. QC Serum (Lyophilized): 3 vials.
g. RIA buffer (50 ml) 10 X concentrated, pH 7.5
h. PEG solution (100 ml) 5 X concentrated.

Materials needed but not provided

• Gamma counter
• Vortex mixer
• Centrifuge refrigerated capable of 4,000 g
• Microfuge capable of 10,000 g
• Glass / plastic disposable test tubes (12 X 75 mm)
• Sterile bottles (500 ml)
• Sterile distilled or Milli-Q water
• Micropipettes 20, 100, 500 µl and 1.0 ml
• Test tube racks
• Acid ethanol solution, 0.855 M Tris and acid ethanol blank solution.

Specimen collection

1. Blood collected in tube and left overnight at 4°C.

2. Centrifuge at 10,000 g in a microfuge (13,000 rpm or maximum speed) for 15 minutes at 4°C.

3. Remove serum and store at -20°C.

Method & Protocol

Reagent Preparation

1. Standards. Three vials of lyophilized IGF-I in carrier protein are supplied for 3 standard curves.
For each assay a series of ten standards is prepared from one of the lyophilized vials. Add 1.5 ml of RIA buffer to one vial, mix well and leave overnight at 4°C to reconstitute. This will give an IGF-I concentration of 70 ng/ml. Nine serial 1/3 dilutions are then made from the starting dilution, i.e. 350 µl of standard plus 700 µl of RIA buffer to give a final volume of 1.05 ml. Mix well. Each of these dilutions is one standard. Reconstitute a new vial of lyophilized IGF-I standard for each new assay.

2. ¹²⁵I-Fish IGF-I tracer. Reconstitute in 80 µl RIA buffer. Store this stock solution at -20°C. Immediately before use, calculate the dilution in RIA buffer required to give a working solution of ~20,000 cpm/50 µl. Add 50 µl to ALL tubes including 'Totals', Blanks, Reference, Standards, QC and unknown Samples. Make up fresh working solution for each new assay.

3. Antiserum 1. Add 500 µl of RIA buffer to one vial to reconstitute. This will produce a stock solution equivalent to a 1/50 dilution. Store stock solution at -20°C.

Prepare sufficient fresh working solution immediately prior to assay and discard after use. For the working solution, the stock must be diluted 1/85.7 in RIA buffer giving a final dilution in the assay tube of 1/30,000. 50 µl is required for each tube as shown in Table 1.

4. Antiserum 2. Store stock solution (1.1 ml) at 2-8°C. Prepare sufficient fresh working solution immediately prior to assay and discard after use. The working solution is a 1/20 dilution of the stock solution in RIA buffer. 50 µl is required for each tube as shown in Table 1.

5. IgG. Add 225 µl of RIA buffer to the vial to reconstitute. Store stock solution at 2-8°C.
Prepare sufficient fresh working solution immediately prior to assay and discard after use. The working solution is a 1/20 dilution in RIA buffer. 10 µl is required for each tube as listed in Table 1.

6. RIA Buffer. Pour the 10 X concentrate into a sterile 500 ml bottle. Wash the concentrate from the bottle with sterile distilled or Milli-Q water and make up to a final volume of 500 ml with sterile distilled or Milli-Q water. Check pH of solution and adjust to pH 7.5 if necessary with 5 M HCl. Store at 4°C.

7. PEG solution. Pour the 5 X concentrate into a sterile 500 ml bottle. Wash the concentrate from the bottle with sterile distilled or Milli-Q water and make up to a final volume of 500 ml with sterile distilled or Milli-Q water. Store at 4°C.

8. Acid/Ethanol Extraction Mix.

Carefully add 62.5 ml of 2 M HCl to 437.5 ml of 100 % Ethanol. Mix gently and when cool transfer to a sterile 500 ml bottle and store at -20“°C.

9. Acid/Ethanol Blank Solution. Combine:
1 ml RIA buffer
4 ml Acid/Ethanol Extraction Mix
2 ml 0.855 M Tris
Make up fresh for each standard curve.

10. QC Serum. Three vials of fish serum (lyophilized) are provided. Reconstitute overnight at 4“°C in 40 µl RIA buffer. Vortex vigorously prior to the acid ethanol extraction step. Use a new vial for each acid/ethanol extraction and assay. This QC sample contains approximately 30-50 ng/ml of IGF-I.

11. 0.855 M Tris. Dissolve 51.8 g of Tris base (MWt 121.14) in 350 ml sterile distilled or Milli-Q water in a sterile 500 ml bottle. Make up to a final volume of 500 ml with sterile distilled or Milli-Q water. Store at 4°C.

Acid Ethanol Extraction of Serum Samples

1. Extract the QC serum samples provided in parallel with the unknown samples to be assayed.

2. To 40 µl of plasma/serum (and QC samples), add 160 µl of acid-ethanol (a/e) extraction mix.

3. Vortex and incubate at room temperature for 30 minutes.

4. Add 80 µl of 0.855 M Tris and vortex.

5. Spin in a microfuge at 10,000 g (~13,000 rpm or maximum speed) for 10 minutes at 4“°C.

6. Collect supernatant and assay 50 µl (in triplicate).

Radioimmunoassay Procedure

For optimal results ensure that assay is set up on ice.

1. Set up and label required number of tubes, in
triplicate, for the “Totals”, Blanks, Reference,
Standards, QC and Samples to be assayed.
Tube contents are detailed in Table 1.

2. Add 50 µl of acid ethanol (A/e) extracted
sample to the sample tubes, 50 µl of A/e blank
solution to standard, reference and blank tubes,
and 50 µl of a/e extracted QC serum to QC
tubes.

3. Add 200 µl of RIA buffer to the sample,
reference and QC tubes.

4. Add 250 µl of RIA Buffer to the blank tubes.

5. Add 200 µl of the appropriate standard to the
standard tubes.

6. Add 50 µl of diluted antiserum-1 to all tubes
except the Blanks and “Totals” (tubes to
determine the total amount of radioactivity
added to the assay).

7. Add 50 µl of diluted tracer to all tubes.

8. Cap tubes for “Totals” to prevent adding any further solution.

9. Vortex all tubes and incubate overnight at 4°C.

Table 1: Assay tube contents.
All volumes in µl.

NEXT DAY

For optimal results ensure that all steps are performed on ice.

1. Add 50 µl of diluted antiserum-2 to all tubes except 'Totals'.
2. Add 10 µl of diluted IgG to all tubes except 'Totals'.
3. Vortex all tubes and incubate for 30 minutes at 4“°C.
4. Add 1 ml of cold PEG solution (4“°C) to all tubes except 'Totals'.
5. Vortex all tubes.
6. Centrifuge all tubes (except 'Totals') at 4000 rpm for 20 minutes at 4“°C.
7. Carefully remove supernatant and count all tubes.
8. Process data using RIA software packages or manually using the appropriate methods.

Serum Parallelism

Supplied standards have been tested across a variety of
different fish sera for parallelism of assay. (See Figure 1).

If you are using this kit for a species other than those described in 'intended use' you are strongly advised to test for parallelism before adopting this methodology.

Performance Characteristics:

Sensitivity
The limit of detection for this assay is typically 0.15 ng/ml IGF-I.

Precision and reproducibility

The intra-assay precision was determined from the mean of nine triplicates from fish serum samples.
Overall coefficient of variation was 3.0%.
The inter-assay precision was determined from the mean of three triplicates of fish serum samples.
Overall coefficient of variation was 16.0%.

Recovery

Four different fish serum samples were spiked with known amounts of IGF-I and extracted and assayed. Recoveries of spiked IGF-I are as shown in Table 2.

Table 2: IGF-I recovery in various fish species

Performance Characteristics:

Specificity (cross-reactivity)

Recombinant human IGF-I < 0.5%
Recombinant human IGF-II < 1%
Recombinant salmon insulin ND

Figure 1: Assay Specificity and Performance


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