Recombinant antibody piezoimmunosensors for the detection of cytochrome P450 1B1.
The phase I enzyme known as cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of many endogenous and exogenous compounds, including carcinogens. CYP1B1 is overexpressed in a wide variety of human diseases ranging from diabetes to malignancies, such as invasive breast cancer. Because of its microsomal location in the cell, CYP1B1 could not be measured directly by existing methods but only assessed indirectly via the determination of the catalytic products. We report here a rapid, sensitive piezoimmunosensor for detection of CYP1B1 using single-chain fragment variable antibodies (scFv) as recognition elements and a quartz crystal microbalance (QCM) as the transducer. Three anti-CYP1B1 scFvs (designated B-66, D-23, and L-21) were biotinylated and used to capture and specifically detect CYP1B1 from samples in solution. ScFvs are smaller than most commonly used antibodies and can be coated onto QCM surfaces at much higher density to improve sensor sensitivity and specificity. The scFv-QCM biosensors showed excellent sensitivity (detection limit, 2.2 +/- 0.9 nM) and specificity with a dissociation constant K(d) = (1.54 +/- 0.59) x 10(-7) M. CYP1B1 were quantitatively detected in normal and malignant cell lysates (e.g., human T47D breast cancer cell microsomes). Results demonstrate that an anti-CYP1B1 scFv-QCM immunosensor could be used to detect P450 enzymes in biological samples.
Shen
Zhihong
Z
Department of Chemistry, Oakland University, Rochester, Michigan 48309, USA.
Yan
Heping
H
Parl
Fritz F
FF
Mernaugh
Raymond L
RL
Zeng
Xiangqun
X
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis*; D001665:Binding Sites; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D065633:Cytochrome P-450 CYP1B1; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007128:Immunoglobulin Fragments / Q000032:analysis; D051379:Mice; D011791:Quartz / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013499:Surface Properties; D013997:Time Factors
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D011791:Quartz; D006046:Gold; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; C585498:Cyp1b1 protein, mouse; D065633:Cytochrome P-450 CYP1B1