Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #2000: Human IGF-I antiserum (Rabbit) for RIA

Anti-human IGF-I is an antiserum specific for human IGF-I, derived from rabbits immunized with a recombinant immunogenic analog of human IGF-I conjugated to ovalbumin. Supplied in lyophilized form, each vial contains the equivalent of 25 µl of serum. The lyophilized antiserum is stable for at least 2 years at 2-4°C.

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

For radioimmunoassay we recommend the antiserum be used at a final dilution of 1/60,000. At this dilution the antiserum binds 30-40 % of fresh [¹²⁵I]-labelled human IGF-I tracer. Half-maximal inhibition (ED₅₀) of radioligand binding is produced by 250-300 pg of human IGF-I and the limit of detection is approximately 10-20 pg.

Procedure:

Dissolve the vial contents in 1.25 ml of IGF-I 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 give an antiserum stock of 1/50. This 1/50 stock solution may be aliquoted and stored at -20°C. 50 µl of this 1/50 stock was further diluted in 10 ml of the same buffer to yield a 1/10,000 working solution. Working solutions are stable for 5 days at 4°C.

The 1/10 000 working solution was added to each RIA tube (except the antibody-negative "blanks") in a final incubation volume of 0.4 ml giving a final dilution of antiserum of 1/80,000. Each tube also contained approximately 20,000 cpm of fresh I-hIGF-I (80 Ci/g). Bound radioactivity was precipitated using conventional centrifugation procedure after addition of anti-rabbit IgG, rabbit IgG and chilled PEG.

See Protocols:
#3001 Procedure for the Iodination of IGFs.

#3002 Determination of IGF-I or IGF-II in a Range of Species by Radioimmunoassay (RIA).

Results:

The relative potencies of the following peptides at inhibiting the binding of [¹²⁵I] hIGF-I to this antiserum were found to be:

100% human, *bovine, *porcine IGF-I
70% chicken IGF-I
25% rat IGF-I
20% LONG®R³IGF-I
1% human IGF-II

* Bovine and porcine were not tested since they have the same amino acid sequence as human IGF-I

* NOT FOR USE IN HUMANS


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