Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3402: Procedure for Radioimmunoassay (RIA) of Salmon / Trout (Oncorhynchus / Salmo) Growth Hormones

Anti-salmon / trout (Oncorhynchus / Salmo) growth hormone hormone is a polyclonal antiserum raised against recombinant trout (Oncorhynchus mykiss) growth hormone. There are only two mutations between the growth hormone of Pacific salmon/trout (Oncorhynchus) and Atlantic salmon/trout (Salmo), and the antiserum has been shown to have equal cross-reactivity with both growth hormones.

Supplied in lyophilized form, each vial contains 5 µl of the antiserum, sufficient for approximately 1000 RIA assay tubes. The antiserum is stable for at least 2 years at 2-4°C in a lyophilized state.

Recommended handling for use in Radioimmunoassay (RIA):

For radioimmunoassay we recommend the antiserum be used at a final dilution of 1/100,000 to 1/200,000. At this dilution the antiserum binds 20-30% of [¹²⁵I] salmon/trout GH tracer (80 Ci/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 1.0 ng/ml of salmon/trout GH.

See Protocol #3400 Procedure for Iodination of Growth Hormones

Procedure:

The vial contents are dissolved by the addition of 500 µl of RIA buffer (30 mM sodium phosphate pH 7.5 containing 10mM EDTA, 0.2 g/l protamine sulphate, 0.5 ml Tween-20 and 0.25 g/l sodium azide) to the vial to give a 1/100 serum dilution. After reconstitution, prepare a working solution by diluting the antiserum a further 1:250 with RIA buffer to give a 1/25,000 working solution. Working solutions are stable for 5 days at 4°C. The remaining 1/100 dilution should be aliquoted and stored at -20°C.

The 1/25,000 working solution (50 µl) is added to each RIA tube (except the antibody-negative “blanks”) in a final incubation volume of 0.3 ml giving a final dilution of antiserum of 1/150,000.

Each tube also contains approximately 20,000 cpm of [¹²⁵I] salmon/trout GH (80 Ci/g) (no more than 3 weeks old). Bound radioactivity is precipitated using a conventional centrifugation procedure after addition of anti-rabbit antiserum, rabbit IgG and chilled PEG.

See Protocol #3002 Determination of IGF-I or IGF-II in a Range of Species by Radioimmunoassay (RIA) for a detailed description of the RIA procedure.

Results:

The relative potencies of the following peptides at inhibiting the binding of [¹²⁵I] salmon (Oncorhynchus kisutch) / trout (Oncorhynchus mykiss) Growth Hormone to this antiserum were found to be:

Recombinant tilapia () GH < 0.1%
Recombinant human GH < 0.1%
Recombinant bovine GH < 0.1%


Procedure for Assay of Salmon/Trout GH in Serum Samples

To assay salmon/trout GH in serum samples, the antiserum can be used at a dilution of 1/100,000. The radiolabelled salmon/trout GH should NOT be added to the assay tubes until 16 hours after the addition of the standard or serum sample.

This sensitive assay allows the detection of salmon / trout GH levels as low as 45 pg/ml and the half maximal inhibition (ED₅₀) of radioligand binding is produced by 300 pg/ml of trout GH.

Serum levels of salmon / trout GH are pulsatile and are normally between 0.2 - 0.5 ng /ml under standard conditions. These values are modified by stress (take care with bleeding), by feeding / starvation, or by a change in salinity.


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