Using The Phage-display Technology To Discover That Whether Antigen-binding Changes The Construction And Function Of Antibody Effector

Through the disulfide bonds in chains,two same immunoglobulin heavy chain covalently linked to the Fc fragment of the antibody ,which can bind to immune cells FcR and C1q complement to activate antibody dependent cell-mediated cytotoxicity (ADCC)and activate complement cascade reaction to remove antigen invasion. The binding site of Fc fragment with the FcR and C1q is called antibody effector (effector), which plays a major locus in the biological function of antibodies.Studies have shown that,in addition to combined with the FcR and C1q, the Fc fragment of antibody can also bind to other activated proteins,such as immunoglobulin-binding protein of bacterial Protein A (Staphylococcus aureus protein A) and Protein G (Streptococcus protein G) ,and the binding sites between Fc fragment and these different protein are concentrated in a common region that is at the junction of CH2-CH3 [1], that specific region is the main site of the antibody effector.These two structures of antibody are connected by a high flexibility short peptide (hinge region, hinge), allowing them to maintain their relative independence.The specific combination of antibody and antigen to stimulate the functions of effector is one of the important ways of antibody to carry out its biological function. After the specific binding of antibody and antigen,the combination between its Fc fragment and Fc receptor of immune cells&C1q complement would be activated and strengthened. Why the binding of antigen and the Fab fragment,which is relatively independent in the structure,would stimulate the binding of Fc fragment and FcR, C1q? Which is the mechanism about them? At present, the most supported and accepted explanation is that: the specific binding between antibody and antigen can form immune complexes, which make the antibody aggregated and then,the effector aggregated and activated to play its function.However, some foreign scholars'study[2~10] shows that,the effect of these two reactions associated mechanism that there is an alternative possibility that,it is the specific binding itself between antibody and antigen which cause changes in the structure of the antibody effector, thus affecting the combination of characteristics between Fc receptor of immune cells and C1q Complement. Among the many study of the effects of the antigen-binding induced the conformational change, The report about the semi-antigen and antibody's binding changed the binding of its Fc segment and Protein A and Protein G by Sagawa and other scholors [9] is the most direct evidence ,which make a strong certiffication that the antigen-antibody binding has led to changes in the conformation of effector when no antigen-antibody complex is formed.Using phage-display technology combined with in vitro molecular directed evolutional selection, we screened a phage-displayed Ig-binding molecules combinatorial library with antibodies both binding and non-binding to antigen. Through comparing the representative sequences and combinative characteristics of different selections, we aimed to discover that whether antigen-binding changes the construction of antibody effector and furthermore affects its combinative characteristic. The study can help interpret a mechanism of antigen-binding influence antibody function.Two parts:1. Selection of a combinatorial phage library displaying immunoglobulin-binding molecules with rabbit IgG both binding and non-binding to antigen.In this study, we chose a phage-displayed random combinatorial library of Ig-binding molecules'single domains which containing the D,A domain of effector-binding Protein A(PA-D,PA-A),the B2 domain of Protein G and the B3 domain of Protein L. The molecular directed evolutional affinity selections in vitro of the library were processed using rabbit IgG antibody molecules both binding and non-binding to antigen Tat as panning targets to compare the homogeny and dissimilarity of representative sequences in each selection libraries. Then,analyzing sequence and picking representative positive monoclones to determine combinative characteristics with rabbit IgG both binding and non-binding to antigen by ELISA. Make a judgement whether different representative sequences′combinative ability screened by different state of antibodies have a discriminating tendency. Finally,both of them were evolved into new structure that doesn't exist in bacteria Ig-binding molecules in nature:during the process of affinity screening leaded by rabbit IgG antibody molecules non-binding to antigenTat, the displayed sequences were evolved into a common form: PA-A—PA-A. However, in selection library of antigen-binding to rabbit IgG, the structure all formed PA-A—PG . Different screen consequences existed a notable distinction. The affinity selection of rabbit IgG antibody molecules both binding and non-binding to antigen Tat also obtained different dominant sequences. ELISA results of combinative ability indicated that representative monoclones were prone to combine the antibodies of identical state more forcefully.2. Selection of a combinatorial phage library displaying immunoglobulin-binding molecules with mouse IgG both binding and non-binding to antigen.Using mouse IgG antibody molecules both binding and non-binding to antigenβ-HCG as panning targets to screen the same phage-displayed random combinatorial library, to compare the homogeny and dissimilarity of representative sequences in each selection libraries. Make a judgement whether different representative sequences′combinative ability screened by different state of antibodies have a discriminating tendency. During the process of affinity screening leaded by mouse IgG antibody molecules non-binding to antigenβ-HCG,most of the displayed sequences were evolved into a common form: PA-A—PA-A—PG. However, in selection library of antigen-binding to mouse IgG, the sequences were quite different and have no obviously regularity.More experiment is needed for the research.Using different species'IgG to study the influence of antigen-binding to antibody effector's construction and characteristic by using phage display technology. It confirmed a conclusion that antigen-binding may indeed change antibody effector's construction, and futher affect its combinative characteristic. The conclusion can help interpret a new influence mechanism of antigen-binding to antibody function. At the same time, it also provides a new path of research on the structure and function of antibody.