Interest in the potential of monoclonal antibodies (mAbs) as therapeutic agents has surged in the past decade. Currently, however, the main therapeutic targets of antibodies remain cancer and autoimmune diseases (1
) and only one antibody targeting viral infection, Synagis (palivizumab), has been approved by FDA for prophylaxis against respiratory syncytial virus (3
). Recently, potent neutralizing human monoclonal antibodies have been selected against the emerging paramyxoviruses Hendra virus and Nipah virus (4
) which are now being evaluated in vivo
for efficacy in a ferret model. Newly discovered human mAbs against the human immunodeficiency virus type-1 (HIV-1) have been developed which target highly conserved epitopes on the HIV-1 envelope glycoprotein and have not demonstrated any affinity to human proteins or lipids (5
). In addition, humanized or fully human mAbs have been selected against severe acute respiratory syndrome coronavirus (SARS CoV) (6
) and West Nile virus (7
), among others (8
). Perhaps among the most significant advances in facilitating the development of specific antiviral mAbs has been the implementation of bacterial phage display platforms using combinatorial antibody libraries (9
). Such phage libraries can be prepared to encode human antibodies as fragment antibodies (Fabs) containing the light chain and the first two domains of the heavy chain. To meet increasing research needs, the implementation of new methods for expressing and purifying these recombinant antibodies is of primary importance to facilitate their efficient production while decreasing production costs.
The recombinant antibody production methods usually consist of three major steps: transformation, expression, and purification. In the transformation step, plasmid DNA coding for a His-tagged Fab is transformed into HB2151 bacterial cells, and successfully transformed cells are selected using ampicillin. During this step, Fab expression is suppressed by a high glucose concentration in the medium. In the next step, isopropyl β-D-1-thiogalactopyranoside (IPTG) in the absence of glucose is used to induce Fab expression. In the purification step, Polymyxin B is used to release the Fab from the bacterial periplasm. Finally, Ni-NTA affinity chromatography is used to specifically purify the His-tagged Fab. In addition to the use of such mAbs as antivirals, these Fabs can serve as important tools as specific detection reagents in various other techniques, such as enzyme-linked immunosorbent assays (ELISA), Western blots, immunoprecipitation, and flow cytometry analysis.
For research purposes it is often convenient to express and purify these Fabs in smaller amounts. However, quite often the quantities of these Fabs are insufficient for other purposes and there is necessity of rapid methods for their expression and purification. Here, we have devised such a rapid method, in which at first a stock of monoclonal recombinant phage harboring the plasmid (phagemid) encoding the desired Fab is prepared and then this phage is used for a multiple rapid Fab expression and purification procedure.