Abstract
310
Objectives: Insulin receptor content in human breast cancer specimens was six-fold higher than that of normal breast tissues and higher than that of any other normal organs include liver. The disulfide constrained display random peptide library was employed to search the high-affinity peptides for insulin receptor and therefore the peptides are potential for diagnostic imaging for breast cancer after labeled.
Methods: Rat liver insulin receptors were immobilized on polystyrene plate by incubating in 0.1mol/L NaHCO3 overnight at 4 degrees centigrade. 2×10(11) virions of phage library were incubated with immobilized receptors 40min at room temperature in a polystyrene plate. The bound phages with the immobilized insulin receptors were eluted with 0.2mol/L glycine-HCl and then were amplified with 200μl Ecol.ER2738 and 20ml LB-Medium in a 250ml Erlenmeyer flask. After three rounds of bio panning, 11 microphage colons were sequenced according to the ELISA and the consensus peptides were identified. We performed an 8-point Scatchard analysis (3tubes/dose point) of peptide binding to the immobilized insulin receptor. Rat liver insulin receptor was coated on the inner surface of the polystyrene tube at 4 degrees centigrade overnight with 0.1mol/L NaHCO3. The control tubes were incubated at 4 degrees centigrade overnight with 0.1mol/L NaHCO3. The difference of average radioactivity between coated tubes and control tubes is the special bound of labeling peptide bind to insulin receptor at any dose-point. Table Curve 2D software was used in Scatchard analysis.
Results: Eleven ELISA positive clones (OD value range from 0.189 to 0.238 where three control clones range from 0.01 to 0.03) were picked out for DNA sequencing. Only a same insert peptide sequence (CQSKXXRHCY) was identified from all of the 11 colons. An asymptotic mathematical formula Y=0.8063-0.8926X for determining dissociation constant (Kd) has been derived from Scatchard analysis. Y is the quotient by dividing peptide-receptor complex radioactivity by free I-125-CQSKXXRHCY radioactivity. X is the concentration of I-125-CQSKXXRHCY × 10(-9) mol/L. The dissociation constant (Kd) of peptide-receptor complex is 8.926×10(-8)mol/L.
Conclusions: Peptide CQSKXXRHCY was identfied from peptide library after three rounds of selection. Scatchard analysis indicated that I-125-CQSKXXRHCY had a high affinity for insulin receptor. The dissociation constant (Kd) of peptide-receptor complex reaches nano-mol level. Peptide CQSKXXRHCY appears to be a promising scintigraphic imaging agent of breast cancer.
Research Support (if any): The study was suported by national natural science fund( 30270415)
- Society of Nuclear Medicine, Inc.