By Antibody-Antigen Complexes

Activation of the Complement System by Antibody-Antigen Complexes The Classical Pathway R. R. Porter and K. B. M. Reid... 

SLE is a multisystem, idiopathic, autoimmime disease characterized by infiltration of capillaries of collagen-vascular tissue by antibody-antigen complexes.The optic disc in SLE can be elevated, and there may be a painless reduction in visual acuity. SLE optic neittopathy may present as either a retrobulbar optic neittopathy or as an anterior ischemic optic neuropathy (AION). 

The process of activation of neutrophils is essentially similar. They are activated, via specific receptors, by interaction with bacteria, binding of chemotactic factors, or antibody-antigen complexes. The resultant rise in intracellular Ca affects many processes in neutrophils, such as assembly of micrombules and the actin-myosin system. These processes are respectively involved in secretion of contents of granules and in motility, which enables neutrophils to seek out the invaders. The activated neutrophils are now ready to destroy the invaders by mechanisms that include production of active derivatives of oxygen. 

Antibody binding to many tumour antigens triggers the immediate loss of the antibody-antigen complex from the transformed cell surface, either by endocytosis or extracellular shedding. 

Activation of complement via the alternative pathway requires no antibody-antigen complexes. It may be activated by endotoxin (LPS) on the surface of many Gram-negative organisms, by zymosan of the cell walls of many yeasts or by aggregated IgA. 

Immunoprecipitation of the antibody-antigen complexes is performed by incubation at 4°C overnight with proteinA-Sepharose or Cammabind sepharose. 

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

Weak bonds, generally non-covalent ones, are involved in formation of the complex, so that the reaction is readily reversed. The rate of the forward reaction is given by the concentration of substrate multiplied by the rate of constant ku and rate of the reverse reaction is given by the concentration of the product multiplied by the rate constant 2- The dissociation constant for the ES complex is 2/ 1- This is analogous to the formation of other complexes for example receptor-hormone complex receptor-neurotransmitter complex antibody-antigen complex. 

Classical pathway, activated by binding to an antibody-antigen complex. 

RG Nielsen, EC Rickard, PF Santa, DA Shakarnas, GS Sittampalam. Separation of antibody-antigen complexes by capillary zone electrophoresis, isoelectric focusing and high-performance size-exclusion chromatography. J Chromatogr 539 177-185, 1991. 

Figure 1. Schematic representations of significant biological functions displayed by host-guest complexation in homogeneous solutions or at membrane surfaces, (a) Separation (e.g., antibody-antigen complex formation), (b) Transformation (e.g., enzymatic reaction), (c) Translocation (e.g., carrier- or channel-mediated transport), (d) Transduction (e.g., receptor-mediated transmembrane signaling).

An important application of fluorescence is in immunoassays, which employ antibodies to detect analyte. An antibody is a protein produced by the immune system of an animal in response to a foreign molecule called an antigen. The antibody recognizes the antigen that stimulated synthesis of the antibody. The formation constant for the antibody-antigen complex is very large, whereas the binding of the antibody to other molecules is weak. 

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen, which are in direct contact with the antibody binding site. X-ray crystallography of antibody-antigen complexes can identify contact residues directly and unequivocally, though not surprisingly in view of the effort required, this method is not in routine use. At the other extreme, demonstration by competition enzyme-linked immunosorbent assay (ELISA) methods that two antibodies bind to different sites on... 

Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer durations and higher temperatures may result in undesirable changes in antibody concentration and molarity of the salts and pH. After heat treatment, the sections should be kept at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded thin sections for electron microscopy follows (Rangell and Keller, 2000) ... 

Gold labeling may be used in the recently introduced catalyzed reporter deposition-immunogold technique where biotinylated tyramide molecules are attached the antibody-antigen complex site the biotinylated sites are visualized by interaction with streptavidin-gold (46,47). 

Fixation of proteins to the membrane can be used to prevent their elution during washing and incubation steps. Some methods have been tried (35,80), but epitopes are sensitive to this treatment and may no longer be detectable by antibody. Another method is to fix the antibody-antigen complexes to the nitrocellulose membrane with glutaraldehyde, after they had been formed (81). 

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