Simultaneous and label-free determination of wild-type and mutant p53 at a single surface plasmon resonance chip preimmobilized with consensus DNA and monoclonal antibody.
Simultaneous determination of wild-type and total p53 proteins (wild-type and mutant combined) present in cancer cell lysates has been performed with a dual-channel surface plasmon resonance (SPR) instrument. To achieve specificity, each channel of the SPR chip was modified with a consensus double-stranded (ds-) DNA and a monoclonal antibody. The high affinity of the consensus ds-DNA to the wild-type p53 and the antibody to total p53 results in remarkably low detection levels (10.6 and 1.06 pM for the wild-type and total p53, respectively). The difference between the SPR signals reveals the extent of p53 mutation, which is indicative of cancer development. The SPR signals increase with the p53 concentration across a wide range (from low picomolar to nanomolar levels) that amply encompasses the typical cellular p53 concentrations. The applicability of the method to real sample analysis has been demonstrated with the comparative analyses of normal and cancer cell lysates. The normal cell samples all displayed significantly higher levels of wild-type p53. In contrast, elevated levels of mutant p53 were observed from the cancer cell lysates. In comparison with enzyme-linked immunosorbant assay (ELISA), SPR obviates the need of a second antibody labeled with an enzyme in the "sandwich enzyme immunoassay" format and is capable of real-time monitoring of the binding events. Thus, SPR could potentially serve as an attractive technique for rapid, sensitive, reliable, and label-free cancer diagnoses.
Wang
Yongcan
Y
College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, People's Republic of China 410083.
Zhu
Xu
X
Wu
Minghua
M
Xia
Ning
N
Wang
Jianxiu
J
Zhou
Feimeng
F
D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D045744:Cell Line, Tumor; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D003911:Dextrans / Q000737:chemistry; D006801:Humans; D050505:Mutant Proteins / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D013194:Staining and Labeling; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000032:analysis* / Q000235:genetics / Q000276:immunology
D055910:Antibodies, Immobilized; D000911:Antibodies, Monoclonal; D003911:Dextrans; D050505:Mutant Proteins; D016159:Tumor Suppressor Protein p53; D004247:DNA