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Published on: March 23, 2018
Direct detection of insulin-like growth factor II (IGF-II) by chemiluminescence without interference by IGF binding
D D De León1, C Terry, S P Nissley
1National Institutes of Health, National Cancer Institute, Metabolism Branch, Bethesda, MD 20892.
Insights
A novel dot blot assay detects picogram levels of insulin-like growth factor II (IGF-II) in cell culture media. This method avoids interference from IGF binding proteins (IGFBPs), simplifying cell screening for IGF-II production.
Area of Science:
- Biochemistry
- Cell Biology
- Assay Development
Background:
- Insulin-like Growth Factor II (IGF-II) plays crucial roles in growth and development.
- Accurate detection of IGF-II in cell culture is essential for research.
- IGF binding proteins (IGFBPs) can interfere with standard IGF-II immunoassays.
Purpose of the Study:
- To describe a new dot blot method for detecting human and rat IGF-II.
- To evaluate the assay's specificity and interference from IGF-I and IGFBPs.
- To demonstrate the method's utility for screening cell cultures for IGF-II production.
Main Methods:
- Development of a dot blot assay for IGF-II detection in serum-free conditioned media.
- Assessment of cross-reactivity with human recombinant IGF-I.
- Evaluation of interference from IGF binding proteins (IGFBP 1-6) compared to radioimmunoassay.
Main Results:
- The dot blot assay detected picogram quantities of IGF-II.
- Cross-reactivity with IGF-I was less than 10%.
- IGFBPs did not diminish the IGF-II signal in the dot blot assay, unlike in radioimmunoassay.
Conclusions:
- The dot blot method effectively detects IGF-II in serum-free media without IGFBP interference.
- This assay is suitable for screening cells for IGF-II production without prior separation of IGFBPs.
- The mechanism of IGFBP interference avoidance in the dot blot assay warrants further investigation.
Abstract:
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.
