Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #4060 Bream (Acanthopagrus butcheri) Growth Hormone antiserum (Rabbit) for RIA

The antiserum was obtained from rabbits immunized with recombinant bream growth hormone conjugated to ovalbumin. Supplied in lyophilized form, each vial contains 15 µl of the antiserum. The antiserum is stable for at least 2 years at 2-4°C in a lyophilized state.

1. Recommended handling for use in Radioimmunoassay (RIA):

For radioimmunoassay we recommend the antiserum be used at a final dilution of 1/20,000. At this dilution the antiserum binds 20-30% of [¹²⁵I] bream GH tracer (80 C_i/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.

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 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.

#3403 Procedure for the Radioimmunoassay (RIA) of Bream (Acanthopagrus butcheri) Growth Hormone


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%

* NOT FOR USE IN HUMANS


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