Polyacrylamide gel electrophoresis (PAGE).
S.3.2.4 Sequence analysis and CDR alignment
3.4.2 Affinity panning
Affinity panning is the process by which phage expressing antigen specific ScFv fragments are “fished out" of a sea of non-specific binders. The panning conditions are usually derived from those determined as the most efficient in a previously optimised assay system. This is because the pool of reactants that are “panned” should be representative of those that would be picked up by the assay system for
panning procedure is the conditions used to infect bacteria with the now specifically isolated phage. These conditions were changed very dramatically over the course of the thesis. Initially a system was employed whereby log phase bacteria were added to a flask that contained ScFv expressing phage that were still bound to the surface antigen after extensive washing. This method did not generate any specific phage. After discussions with other scientists working in the field the methodology of infection was changed such that an “elution step" was included. In this step specifically bound phage were eluted off using a low pH wash again after washing off any non-specific reactants. The phage removed from the antigen by this method were then transferred to another vessel where neutral pH was re-established with the addition of Tris base. The phage are returned to neutrality after transferring them since if neutralised in the flask most would re-bind. This elution step greatly increases the rate of infection since, although the phage bound to the antigen in the first method are functionally infective the binding of the expressed antibody may well sterically hinder the interaction of the N1-N2 domains of the gp III protein which is responsible for attachment and infection (Krebber etal., 1997), via either a bacterial pilus dependent or independent mechanism. This change in panning procedure was performed on an library that had already been panned by the previous method since there was no un-panned library or un transformed vector remaining. Although the new panning method should have improved infectivity rates the library by this time was probably already partially depleted of it's higher binders, since the bacteria would have only been infected by those phage that were not sterically hindered of eluted off in the bacterial broth. Panning using solid phase antigen is the most popular and established method of selecting for specific binders although a number of alternative methods are being developed. The BIAcore™ is now a well respected method for the analysis of specific molecular interactions and the determination of affinity constants. A modified BIAcore™ biosensor has been used to select phage expressing antibody ScFv fragments (Lasonder et a/., 1994, Malmborg et al., 1996) and has the advantage that affinity constants of clones can be determined in addition to the real-time binding/panning of the phage library. “In vivo veritas" or “live phage display panning” is a novel method developed by Pasqualini and Rouslahti (1996) in which phage are recovered from mice that have been previously injected with
natural conformation and may be useful in aiding the targeting of cells, drugs and genes into tissues by virtue of identification of selective markers.
3.4.3 Analysis of phage expressed and soluble antl-human Vila ScFv
The binding of the phage expressed ScFv antibody to human Vila was assessed in comparison to it's binding to an anti E-tag antibody as well as the blocked plate without antigen. This was performed since not only is it possible to have a population of phage that bind to plastic (Adey et al., 1995) but also the screening showed that some of the phage bound to the human Vila but did not bind to the anti E-tag antibody. Since in the protein translation of the ScFv construct the ScFv antibody is translated before the E-tag then it would suggest that any phage that bind the antigen and not the E-tag are doing so in a non-specific manner since the antibody protein construct is unlikely to be conformationally correct. This is probably due to the fact that the start codon is upstream of the ScFv antibody construct (position 201) and any sequence change in the ScFv antibody that causes a frame shift or stop codon to prevent the correct translation of the E-tag is likely to have an effect on the ScFv antibody itself. Only one phage expressing a conformationally correct and antigen specific ScFv antibody was thus isolated and this is probably due to culmination of both the reduced library size generated and the initially methodologically impaired panning step.The soluble ScFv antibody was analysed by ELISA, B I A c o r e a n d western blot to assess it’s specificity towards human Vila. Although each techniques can detect specificity of binding the ELISA is needed to determine the dilution factor to use in the BIAcore’'"'^ analysis and the western blot provides some information as to the antigen epitope. The western blot analysis suggests that the antibody epitope is near or at the inter-chain disulphide bridge and this is supported by the fact that there is absolutely no binding to either of the separate chains. Although this clear evidence of binding to the intact protein it must be born in mind that binding of human tissue factor to human Vila is calcium dependent (Head et a/., 1997) and that this may have an effect on the antibody binding to the separated forms. Work by Higashi etal. (1990) in which a mab to a calcium dependent epitope of bovine factor VII was produced shows that antibody binding can be calcium dependent,
Chain separation. Although an initial indication as to the location of the epitope analysis of the ScFv antibody presented in this thesis has been demonstrated further characterisation of the binding with respect to calcium dependence could be instrumental in demonstrating the precise epitope.
The original feature of the BIAcore™ was the analysis of protein/protein interactions with the subsequent calculation of the binding constant using both the association and disassociation rates. This is very useful for determining the affinity of antibodies and is now a scientifically accepted method of analysis. Although it has been used to measure the binding and selection of phage displayed libraries (Lasonder et al., 1994) it is now being used to characterise and epitope map ScFv antibody fragments (Malmborg and Borrebaeck, 1995) as well as a tool in the measurement of the effect of mutation of various amino acid residues in the CDR3 region of a previously generated antibody (Schier and Marks, 1996). Although only one specific anti-human Vila ScFv antibody was generated during this thesis, it's binding affinity is comparable with those generated by other groups (Meng et a!., 1995). This suggests that if the modifications made to the method were employed on an unpanned library of sufficient size then wider range of antibodies with lower and possibly higher affinities might be isolated.