Biotechnology Reagents (IGFs) >> Product Support >> Protocols >> #3403 Procedure for Radioimmunoassay (RIA) of Bream (Acanthopagrus butcheri) Growth Hormone

For radioimmunoassay, Novozymes GroPep's Bream Growth Hormone antiserum (Catalog Code PAG1) is used at a final dilution of 1/20,000. At this dilution the antiserum binds 20-30% of [¹²⁵I] bream GH tracer (80 Ci/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 7.0 ng/ml of bream GH. Each vial contains sufficient antiserum for approximately 1000 RIA assay tubes.

See Protocol:
#3400 Procedure for Iodination of Growth Hormones.

Procedure:

The vial contents are dissolved by the addition of 1485 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 1:33 with RIA buffer to give a 1/3,300 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 1/3,300,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 1/20,000. Each tube also contains approximately 20,000 cpm of [¹²⁵I] bream 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.


Results:

The relative potencies of the following peptides at inhibiting the binding of [¹²⁵I] bream (Acanthopagrus butcheri) Growth Hormone to this antiserum were found to be:

recombinant goldfish (Carassius auratus) GH < 0.1%
recombinant human GH < 0.1%
recombinant porcine GH < 0.1%
recombinant rat GH < 0.1%


© 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