One of the gene products involved in MDR is P-glycoprotein (P-gp). P-gp is a 170kD plasma membrane efflux pump protein and belongs to the ATP-binding cassette superfamily of transport proteins . Comparative inhibitor Wortmannin sequence analysis of P-gp with other ABC family members shows that it consists of two transmembrane (TM) domains, each containing six putative TM segments followed by an ATP-binding consensus motif and two cytoplasmic nucleotide-binding domains (NBDs). The TMs of P-gp are found mainly responsible for binding to and transporting a broad spectrum of drugs. Photolabeling and mutational studies indicate that the drug binding domain is within the TM domains of P-gp, and drug transport is powered by hydrolysis of ATP at the two cytoplasmic NBDs [1, 2].
Structure-function analyses of P-gp suggested that two subunits of P-glycoprotein are involved in the selection of its drug substrate and/or specificity of MDR modulator. Crucial amino acid residues of P-gp are localized within or near the TM region [3, 4]. P-gp functions as an efflux pump expelling drugs out of tumor cells, resulting in a decrement intracellular concentration of cytotoxic drugs. Overexpression of P-gp is associated with poor clinical outcome [5�C7]. Identification of the tumor cells sensitive to antineoplastic drugs may help for the selection of the antineoplastic agents . Although methyl thiazolyl tetrazolium (MTT) can provide direct observation on the tumor cells sensitive to antitumor drugs, it is a time-consuming process.
Methods used extensively for detection of the P-gp protein expression, such as ELISA, immunohistochemical method (IHCA), and flow cytometry (FCM), depend on the reaction of the P-gp protein to its corresponding monoclonal antibody. Nevertheless, use of the intact monoclonal antibodies may provide false positive signals in detection of the tumor cells since the Fc fragments of IgG may bind to the Fc receptors on the surface of normal cells as well. Thus, use of the Fab antibody may be superior to reducing the false signal resulting from the intact antibody [9, 10]. Meanwhile, Fab is smaller than IgG in size, facilitating trafficking of the antigen across cell membrane and may provide more information than the intact antibody during the ICHA process [11, 12].In this communication, we constructed and characterized the phage-displayed mouse Fab against human MDR1/P-gp.
2. Materials and Methods2.1. AnimalsThe study was conducted in accordance with national guidelines for care and use of animals. All mice were purchased by the Dalian Medical University Laboratory Animal Center (approval number LA 2009-008). The animals used in the study were age-matched, mature BALB/c female mouse weighing approximately 18�C22g.2.2. Construction of Phage-Displayed Mouse Fab Antibody Library against Human Colorectal Cancer MDR1/P-gpSix BALBC female mice were injected subcutaneously (0.2mL/10g) Brefeldin_A with 0.