Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #4050: Salmon/Trout (Oncorhynchus/Salmo) Growth Hormone antiserum (Rabbit) for RIA

The antiserum was obtained from rabbits immunized with 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 all growth hormones.
Supplied in lyophilized form, each vial contains 5 µl of the antiserum. 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 C_i/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 1.0 ng/ml of salmon/trout GH. Each vial contains sufficient antiserum for approximately 1000 RIA assay tubes.

Procedure:

The vial contents are dissolved by the addition of 500 µl of RIA buffer (30 mM sodium phosphate pH 7.5 containing 10 mM 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 C_i/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 Protocols:
#3400 Procedure for the Iodination of Growth Hormones.

#3402 Procedure for Radioimmunoassay (RIA) of Salmon / Trout (Oncorhynchus / Salmo sp.) Growth Hormones

Results:

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

recombinant tilapia (Oreochromis mossambicus) 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 and 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.

* NOT FOR USE IN HUMANS


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