There are two different methods to obtain monoclonal antibodies: hybridoma technology and recombinant antibody technology (e.g. phage display, ribosome display and bacterial display). The hybridoma technology has contributed to major scientific advances and has become one of the most important technologies in the bio-medical laboratory, however this technology has some limitations, for example: its high cost, it requires considerable time, expertise, and specialized facilities for work with cell cultures, and relies on the use of laboratory animals. In addition, many molecules are not immunogenic in mouse or are toxic and cannot be used as antigens. In the therapeutic application of these antibodies, the standard methods of producing monoclonal antibodies yield rodent antibodies that are rejected by the human immune system. Various approaches to overcome this limitation have been attempted such as the generation of either chimeric or humanized antibodies. However, by using recombinant antibody technology (e.g. phage display, ribosome display and bacterial display) instead of the hybridoma technology to select monoclonal antibodies, the limitations above-mentioned can be avoided.
Phage display antibody libraries
One of the major advantages of phage display antibodies compared with standard hybridoma technology is that the generation of specific scFv/Fab fragments to a particular antigen can be performed within a couple of weeks. The starting point is usually an antibody library, of either naive or immune origin, comprising a population of, ideally, 10e9–10e11 clones. After usually two to, maximally, three rounds of selection, the population is enriched for a high percentage of antibody fragments specific for the target antigen. The display of human antibody fragments is of particular interest since any therapeutic derived from these antibody fragments is believed to have a minimal risk of an immune response in patients.
Many different methods have been described for antibody selection, including yeast, bacterial or ribosomal display, and picking from arrays. Of all of these methods, however, antibody phage display has developed into the most robust, versatile and thus widespread selection method during the past decade. In consequence, the obvious choice for a proteome-wide antibody project is to use such a well-established method that has already proved its adaptability to automation. Antibody phage display not only greatly extends our capacity to generate antibodies but also extends their potential applications for the direct functional analysis of epitopes.
Screening of Phage Display Libraries
Screening of phage display libraries is usually accomplished by a process which consists of affinity selection steps called panning or biopanning during which phage populations are exposed to targets in order to selectively capture binding phages. In the process involved successive rounds of binding, washing, elution and amplification. Several rounds of this process will increasingly enrich the originally very diverse phage population with phages with a propensity to bind to the target in question.
The target molecule is immobilized on a solid support by passive or active adsorption. The solid support can be polysterene tubes, MaxiSorp plates, or magnetic beads. The binding steps consist in presenting the pool of phage particles carrying the displayed peptides, proteins or antibodies to the immobilized target molecules. Knowlegde of the binding properties of the target molecules can facilitate to work out the best binding conditions in order to increase the chance to pick up positive clones. However, not only phages with affinity binding will bind to the target molecules without many phages that bind in a not specific way, in addition phages can bind to the surface of the solid support and other components in the reaction. In order to remove non-binding phages is necessary several washing steps.
The way of doing the washing need to be taken in consideration because it is required a balance between specificity and avidity of selected clones. Some clones may be strong binders with low specificities while others may be weak binders with high specificities. If washing is too stringent then highly specific, but weak binders may be lost. If washing is not stringent enough then populations of selected clones may be dominated by strong binders with low specificity. In practice this balance is achieved by adjusting washing times, detergent concentrations and using regimes in which washing stringencies are progressively increased.
Several different treatments can be used to elute bound phages from targets molecules such as dramatically lowering or increasing pH, reducing agents, enzymatic cleavage or elution by affinity. However, it is necessary to take in consideration that harsh elution conditions no affect phage integrity. The recovered phage population is commonly amplified before the next round of selection, however, it may be used directly without amplification to reduce background problems caused by non-specific phages that are carried through the panning process. In most cases, the eluted phage are used to re-infect E.coli which be streaked on plate to obtain individual colonies. A large number of single colonies are picked into 96-well plate to rescue the clones as phages. The phage are analyzed to identify positive clones.
The phage display panning consists in different steps of binding, washing and elution.
Antibody affinity maturation
Affinity maturation is a method to improve the affinity of monoclonal antibodies. This method is special useful when it needs antibodies for therapeutic applications since it requires commonly antibodies with high specificity and affinity. There are several methods to increase antibody affinity including untargeted mutagenesis and oligonucleotide-directed mutagenesis. Read more!
Example of affinity maturation by chain shuffling
Different methods have been used to re-construct and produce humanized antibodies from monoclonal antibodies originally obtained with the hybridoma system. This is possible because in the segmented structure of an antibody can be modified the domains carrying antigen- binding or effector functions. Chimeric antibodies can be created by coupling the animal antigen-binding variable domains to human constant domains. The human constant regions provide an isotype relevant to the desired biological functions, however most of the humanized antibodies on the market are of the IgG1 subtype. Sometimes the constant regions are removed to minimize T cell activation and cytokine release.
The antigen-binding site from the rodent antibody has to be changed by the antigen-binding site from the human antibody applying protein engineering. The most popular technique for antibody humanization is called CDR grafting which consist in replacing only the complementarity determining region (CDR) peptide loops in the antigen site into human variable (V) regions. The best estimated framework sequences and structures to human Variable region can be found with help of protein sequence databases to design humanized antibodies that are more likely to be stable and get expressed in an expression system. Read more!
Other services that Vrisko Limited offers are: dimerization of scFv and Fab Fragments, Phage Display peptides libraries, Recombinant IgG production, ScFv and Fab fragment construction.