Direct detection of insulin-like growth factor II (IGF-II) by chemiluminescence without interference by IGF binding proteins.
A dot blot method for the detection of picogram quantities of human and rat insulin-like growth factor II (IGF-II) in serum-free conditioned media is described. The crossreactivity of human recombinant IGF-I in the assay was < 10%. None of the IGF binding proteins (IGFBP 1-6) diminished the IGF-II signal. In contrast, significant interference by the IGFBPs was observed when the same concentrations of IGFBPs and 125I-IGF-II were used in a radioimmunoassay which utilized the same antibody. Why IGF-II is detected in the dot blot assay without IGFBP interference is not understood. We speculate that the conformation of the IGF-II/binding protein complex may be altered by binding to the nitrocellulose, exposing the IGF-II epitope that is recognized by the antibody. IGF-II was detected in 1 microliter of serum-free conditioned media from BRL 3A cells (which secrete IGF-II) while no signal was generated by 50 microliters of BRL 3A2 conditioned media (which do not secrete IGF-II). In summary, this method is ideal for screening cells in serum free-culture for production of IGF-II without the need for separation of IGF-II from cell derived IGFBPs.
De León
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DD
National Institutes of Health, National Cancer Institute, Metabolism Branch, Bethesda, MD 20892.
Terry
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C
Nissley
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SP
D000818:Animals; D016477:Artifacts; D001345:Autoradiography; D002352:Carrier Proteins / Q000378:metabolism*; D016895:Culture Media, Serum-Free; D006801:Humans; D015151:Immunoblotting; D018969:Insulin-Like Growth Factor Binding Proteins; D007335:Insulin-Like Growth Factor II / Q000032:analysis*; D008163:Luminescent Measurements; D011863:Radioimmunoassay; D051381:Rats; D011994:Recombinant Proteins; D013002:Somatomedins / Q000378:metabolism
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D013002:Somatomedins; D007335:Insulin-Like Growth Factor II