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Interest in the potential of monoclonal antibodies (mAbs) to serve as therapeutic agents has surged in the past decade with a major emphasis on human viral diseases. There has been much attention in this area directed towards the human immunodeficiency virus type-1 (HIV-1) and promising research developments have emerged on the inhibition of HIV-1 infection by mAbs and the identification of several highly conserved neutralizing epitopes. More recently, potent fully-human neutralizing mAbs have been developed against a variety of important human viral disease agents including the paramyxoviruses Hendra virus and Nipah virus, and human or humanized mAbs have been developed against severe acute respiratory syndrome coronavirus (SARS CoV), and West Nile virus, among others. Most of these more recently developed antiviral mAbs have come from the use of antibody phage-display technologies and the implementation of simplified, inexpensive yet efficient methods, for expressing and purifying the initially selected fragment antibodies is of prime importance in further facilitating this area of research.
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, 2) 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, 10). 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.
This work was supported in part by Middle Atlantic Regional Center of Excellence (MARCE) for Biodefense and Emerging Infectious Disease Research, NIH AI057168 and AI054715 grants C.C.B.
1For methods of selecting and purification of the Fab clone plasmid DNA please refer to the corresponding chapters in this book. In the present protocol, the Fab clones were encoded into the phagemid vector pComp 3X (13) that has 6-His and FLAG tags attached to the Fab C-terminus.
2Transformation of the bacteria can be alternatively performed by electroporation according to the protocol by Stratagene. However, if there is no available electroporator, prepare competent cells as follows: Inoculate TG1 cells in 10 mL 2YT (see 2.1) and grow overnight. Dilute the overnight culture of wild type bacteria with 100 mL fresh 2YT medium. Grow until bacteria reach an optical density at 600 nm of 0.35–0.45, then keep bacteria on ice for a minimum of 15 min before centrifugation. Centrifuge at 3000g for 15 min at 4°C. Discard the supernatant. Resuspend the bacterial pellet into 50 mL ice-cold CaCl2 and keep on ice for 20 min. Cells must remain cold at all times and be treated gently in the presence of CaCl2. Centrifuge at 3000g for 10 min at 4°C. Discard the supernatant and gently resuspend the pellet in 2 mL ice-cold CaCl2. Incubate on ice for at least one hour before using for transformation, or store in 30 μL aliquots at −80°C for later use.
3Measure the OD280 of prepared phage stock, usual dilution for this measurement is 20 times. OD280 ~ 1.0 corresponds to 2.33 ×1012 phages.
4The volume of medium inoculated in the expression step can be increased up to 300 mL per column used in the purification step. If inoculating a larger volume of medium, increase the volume of Ni-NTA agarose placed in the column by 100 μL per 50 mL increase in culture used.
5Alternatively, instead of manually resuspending and then shaking/rotating for 30 min at room temperature, one can shake/rotate for 1 h at room temperature. If using an orbital rotator, be sure to stabilize the tubes properly so that a rotation speed of at least 150 rpm can be used. At the end of the shaking or rotating, the pellet should be completely resuspended.
6The first portion usually yields a slightly higher concentration, and it may be useful to elute the two portions into separate tubes in order to see the difference. However, the concentrations are seldom very different, and the two portions may be eluted together instead.