Abstract
The p16INK4a is a protein that expressed in Liquid-based cervical cytology specimens and has been proved link to cervical cancer. The p16INK4a could be detection by piezoelectric immunosensor and the immobilization of the p16INK4a antibody influence the sensitivity of the piezoelectric immunosensor. 5μL mouse polyclonal antibody against p16INK4a was bound onto the surface of immonosensor through two methods. (directly immobilized method; protein A method). Absorb of the p16INK4a antibody on the surface of immonosensor caused a shift in the resonant frequency of the immunosensor and The frequency changes recorded showed a better reproducibility. The activity of the immobilization antibody with the directly method and protein A method was tested with p16INK4a antigen. The resonant frequency for different antibody immobilization methods were different, and the sensitivity for p16INK4a detection also different. The protein A method was found to be much more better than the directly method for the immobilization of the p16INK4A antibody on the gold electrode of the quartz crystal for cervical lesion detection. The Protein A method created more reproducible and stable immobilization antibody layers with p16INK4A antigen.
Highlights
The cervical cancer is one of the most common cancers in developing Asian countries (Razak et al, 2013)
A 5μL staphylococcal protein A solution in Phosphate-buffered saline (PBS) with concentration of the 1 mg/mL was evenly spread on the up side of the crystal for 20 min, rinse with distilled water to remove the excessive SPA solution, make the quartz crystal air drying and the frequency of quartz crystal microbalance (QCM) which record as F0’ was measured. 5μL p16INK4a antibody solution with concentration of 5.0 mg/mL was applied to the crystal surface. the quartz crystal was incubated over silica gel blue at 4°C for different time (1hour, 2hourss, 6 hours, 12hours), dip the quartz crystal to PBS(PH7.2) solution and wash with distilled water twice, making air drying and measured the resonance frequency of quartz crystal and recorded as F1’
The frequency shift caused by absorb of antibody was calculated as eqution4 and equation5:
Summary
The cervical cancer is one of the most common cancers in developing Asian countries (Razak et al, 2013). Conventional cytologic diagnosis is often hampered by high false-negative rates compared with biopsy and interobserver variability (Bergeron et al, 2010) Another question arose as to whether all women with minimally abnormal tests needed such an expensive and uncomfortable procedure. Detection of molecular alterations caused by HPV in host cells could potentially be used as an adjunct to cytological screening Among these candidate biomarkers, only p16INK4A, one of the cyclin-dependent kinase inhibitors, has sufficient evidence on which to make recommendations to apply to HPV-associated precancer lesions (Eleuterioet et al, 2009). If a substance with the weight of 1ng is homogeneously attached to the active area of crystal, a frequency change of 0.4518 Hz. Used the p16INK4a as a biomarker, the quartz crystal microbalance could be used as immonosensor for early cervical cancer detection. The effect of the immobilization methods on the resonant frequency of quartz crystal was observed and the activity of immobilized antibodies to p16INK4a antigen was tested
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