China
Africa
Explore chapters and articles related to this topic
Antibodies and Antisera
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
It is evident that the size of the antigen-combining site will be smaller than that of most antigens. Consequently, it will bind only to a part of the antigen. It is a common misconception that the immunologist by his immunization scheme “instructs” the immunized animal to produce antibodies against a particular chosen antigen. Rather, according to modern immunological theory, introduction of a foreign substance stimulates the proliferation of, and antibody formation by, a particular subset of lymphocytes producing antibodies with the best fit against the substance. In a mouse, the total antibody repertoire is estimated to exceed more than 108 (see Reference 29). Nevertheless, this figure indicates that many antigenic determinants (or epitopes) in antigens are similar. Recent data obtained by Westhof et al.83 and Tainer et al.71 have indicated that antibodies preferentially bind to protein regions with a high degree of mobility or flexibility. Westhof et al.83 suggested that the mobility of the antigenic region may make it easier for an antigen to bind to an antibody not preconceived by nature to bind it. Thus, the antigen does not instruct the production of a perfectly fitting antibody, but selects clones of antibody-forming cells with the best possible fit. It seems reasonable that highly mobile regions of antigens stand a better chance of selecting a clone producing antibodies with a good fit. Together, these data indicate that the interactions between antigens and antibodies are dynamic and not static (as in the classical key-lock model). Nevertheless, during successful immunizations antibodies with very high binding energies (109 to 1012 ℓ/mol) are produced in large quantity. Such binding energies are strong enough to withstand dissociation under most nondenaturing conditions. This is one of the reasons why antibody elution techniques and affinity purification schemes work only with low- to medium-avidity antibodies. In polyclonal antisera, antibody subpopulations of different specificities and different avidities occur. During antigen-antibody incubation, low-avidity antibodies may bind to and block epitopes that otherwise would have bound high-avidity antibodies. During subsequent washings, low-avidity antibodies become dislodged and do not contribute to staining. By repeating the antiserum incubation, epitopes freed of low-avidity antibodies during washings stand a renewed chance of binding high-avidity antibodies. In immunocytochemistry, such repeated application of antibodies has been shown to result in increased detection efficiency and also in improved absolute sensitivity.26,60 Simple as it may be, this observation has proven very important both for pressing sensitivity and for increasing dilutions (and decreasing cost) of antibodies. As expected from the above considerations, prolonged and/or repeated antibody incubation is beneficial to a variable degree with different primary antibodies but is of little help with monoclonal antibodies which already consist of only one antibody species having only one avidity.
Automated high throughput microscale antibody purification workflows for accelerating antibody discovery
Published in mAbs, 2018
Peng Luan, Sophia Lee, Tia A. Arena, Maciej Paluch, Joe Kansopon, Sharon Viajar, Zahira Begum, Nancy Chiang, Gerald Nakamura, Philip E. Hass, Athena W. Wong, Greg A. Lazar, Avinash Gill
A typical purification protocol for human IgG1 from MabSelect SuRe tip column was reported previously,4 and other buffers with pH 3.0 or lower have been used for elution of antibodies from Protein A based resins.5 In general, a less acidic buffer is preferred for antibody elution due to the propensity for antibody instability and the need for neutralization in acidic buffers. We evaluated elution of human IgGs (excluding IgG3) with 50 mM phosphoric acid at pH 3.0, pH 3.2, pH 3.4, and pH 3.6 and found that pH 3.0 always gave satisfactory elution (73±3%), while buffer with pH 3.4 and higher gave lower yields and inconsistent elution (Fig. 3b). Other buffers such as 10 mM citrate, 100 mM glycine, 50 mM acetic acid, at pH 3.0 or lower could effectively elute IgG bound on MabSelect SuRe resin. For our routine microscale purification of human IgGs, we decided to use 50 mM phosphoric acid pH 3.0 for elution and neutralized with 1/15 volume of 200 mM sodium phosphate, 300 mM sodium chloride, pH 11.0, resulting in a final formulation with pH ∼6.0. This elution-neutralization scheme avoided some buffers such as glycine or Tris which were often incompatible with some downstream assays and screening applications.
Multi-functional antibody profiling for malaria vaccine development and evaluation
Published in Expert Review of Vaccines, 2021
D. Herbert Opi, Liriye Kurtovic, Jo-Anne Chan, Jessica L. Horton, Gaoqian Feng, James G. Beeson
Tier 1 – Standard high-throughput immunoassays. This includes quantification of immunoglobulin isotypes (IgG, IgM, IgA) and IgG subclasses to the vaccine immunogen or target antigen. It also includes quantification of antibody avidity using antibody elution with chaotropic agents (e.g. thiocyanate). Assays can also quantify antibodies to specific regions or epitopes of target antigens or use competition approaches to assess epitope-specific or allele-specific antibodies [82,83,147,218]. Typically, these assays are performed as plate-based ELISA, and throughput and reproducibility can be greatly enhanced by using automated liquid handling [219,220]. Suspension bead arrays can also be used and are particularly suitable when antibodies to multiple antigens need to be quantified.
Anti-α-galactoside and Anti-β-glucoside Antibodies are Partially Occupied by Either of Two Albumin-bound O-glycoproteins and Circulate as Ligand-binding Triplets
Published in Immunological Investigations, 2019
Karthi Sreedevi, Sabarinath P Subramanian, Geetha Mandagini, Padinjaradath S Appukuttan
Binding of O-glycoproteins and albumin along with anti-Gal or ABG to matrix holding ligand exclusive for the respective antibody, elution of all the above components from the matrix by antibody-specific sugars, binding of O-glycoproteins and antibodies along with albumin to a matrix exclusive for albumin, and lack of affinity of O-glycoproteins to any of the above matrices could be explained only by the existence of antibody-O-glycoprotein-albumin triplets in human plasma. Presence of such a construct was supported by the recognition of purified O-glycoproteins by antibodies through the latter’s binding sites in ELISA and fluorescence enhancement assay. Presence of antibody-free albumin-O-glycoprotein binary complex but not free antibodies in normal plasma and liberation of the above binary complex from antibodies following treatment of normal plasma with antibody-specific sugars also supported existence of this structure. O-Glycoproteins AOP1 and AOP2 might have escaped detection so far partly due to their existence in plasma exclusively in albumin-bound form and partly due to their remarkably high carbohydrate content. The latter trait is likely to render them poorer acceptors of dye molecules used for detection and estimation of proteins in comparison with nonglycosylated proteins. Further proof for albumin-O-glycoprotein interaction is that this event altered the surface charge and/or hydrophobicity of albumin so that triplet albumin could be eluted with much lower concentration of NaCl from blue Sephadex than required for elution of free albumin (Figure 1). Later work has shown that strength of anti-albumin antibody binding to albumin was also substantially quenched in presence of AOP1 or AOP2 (our unpublished data). O-Glycoprotein-induced changes in albumin suggested that values of total serum albumin concentration, determined on the basis of dye or antibody binding to this protein, demand a reappraisal. Composition of middle layer after DGUC of untreated normoglycemic plasma (Figure S1 inset, supplemental material) indicated that AOP1 and AOP2 are the major proteins other than albumin in this layer. The observations that these O-glycoproteins are albumin-bound (Figure 3) and that almost an equal amount of albumin-bound O-glycoproteins are in the bottom layer as triplets formed by both the antibodies together (Table 1) suggest the extent to which O-glycoproteins interact with albumin.