Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #2050: Human IGF-II antiserum (Rabbit) for RIA

Anti-Human IGF-II is an antiserum specific for IGF-II. The antiserum was derived from rabbits immunized with recombinant human IGF-II conjugated to ovalbumin. Supplied in lyophilized form, each vial contains ~20 µl serum. The lyophilized antiserum is stable for at least 2 years* at 2-4°C.

* Longer term stablilty is expected to be demonstrated.

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

For radioimmunoassay we suggest the antiserum be used at a final dilution of 1/5,000. At this dilution the antiserum binds 20% of [¹²⁵I]-labelled human IGF-II tracer (80 Ci/g). Half-maximal inhibition (ED₅₀) of radioligand binding is produced by ~10 ng/ml of human IGF-II. Each vial contains sufficient antiserum for ~300 assay tubes. It is recommended that each laboratory establish their own working dilutions for this reagent.

Procedure:

Stock Solution:
Reconstitute the vial contents by adding 0.5 ml 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). Mix thoroughly and allow to dissolve for 30 min at room temperature. This stock solution (1/25 dilution) may be aliquoted and store at -20°C for up to 3 months.

Working solution:

Add the total vial contents to a further 16 ml of RIA buffer to give a working solution (1/825 dilution). If using a frozen aliquot, add a further 3.2 ml RIA buffer per 0.1 ml of stock solution. Working solutions are stable for 5 days at 4°C.

The working solution (50 µl of the 1/825 dilution) was 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 ~1/5,000. Each tube also contained approximately 20,000 cpm of [¹²⁵I] hIGF-II (80 Ci/g) (< 3 weeks old). Bound radioactivity is precipitated using conventional centrifugation procedure after addition of anti-rabbit IgG antiserum, 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]-labelled human IGF-II to this antiserum were found to be:

human, porcine IGF-II
100%
chicken IGF-II
96%
rat IGF-II
91%
bovine IGF-II
85%
human, chicken, rat IGF-I
< 0.1%
ovine insulin
< 0.1%

* NOT FOR USE IN HUMANS


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