Capture antibody

A REVIEW OF ANTIBODY CAPTURE AND BACTERIOPHAGE AMPLIFICATION IN CONNECTION WITH THE DIRECT ANALYSIS OF WHOLE-CELL BACTERIA BY MALDI-TOF MS... 

Some arrays used in proteomics contain antibodies covalently bound onto the array surface for immobilization. Then these antibodies capture corresponding antigens from a complex mixture. Afterwards, a series of analysis are carried out. For instance, bound receptors can reveal ligands. With this information in hand, binding domains for protein-protein interactions can be detected. The main problem in using microarray methods for proteomics is that protein molecules must show folding with the array in the correct conformation during the preparation and incubation. Otherwise, protein-protein interactions do not take place. 

Plates for antibody-capture assays Where a specific antibody is available, this may be a better assay to use than step 8 above, because the antigen is less likely to be subject to denaturation... 

Key Words ELISA antibodies capture antibody sandwich detection antibody substrate dual antibody. 

Antibody capture of viruses can be used as a preparatory step in nucleic acid amplification techniques. Immunocapture of virus particles can be used to streamline and/or optimize the concentration, purification and specificity requirements of polymerase chain reaction assays. 

Surface chemistry is a key technology for protein microarray development. The supports used for protein immobilization have to fulfil some important requirements they must provide good quality spots, low background, simplicity of manipulation and compatibility with detection systems. An ideal surface or immobilization procedure for all proteins and applications does not exist however current methods are more than adequate for many applications. Basic strategies for protein immobilization consider covalent versus non-covalent and oriented versus random attachment, as well as the nature of the surface itself [106]. It has been demonstrated that the specific orientation of immobilized antibody ( capture agents ) consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvement over surfaces with randomly oriented capture agents [107]. 

Fig. 3. Schematic of staining process of SARS-CoV immunochip. (1) Spotting A high-precision robot transfers the samples, SARS-CoV proteins, and glycans of various complexities, from 96-well plate to nitrocellulose-coated glass slides. (2) Staining Before staining, the slides are rinsed with IX phosphate-buffered saline (PBS), and blocked with 1% bovine serum albumin (BSA)-PBS containing 0.05% NaN3 and 0.05% Tween-20. They are subsequently incubated with horse anti-SARS sera. The primary antibodies captured by microarrays are detected using biotinated anti-horse immunoglobulin (Ig)G, and visualized by Cy3-streptavidin.

Cussac D, Newman-Tancredi A, Duqueyroix D, Pasteau V, Millan MJ. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays influence of receptor reserve and relationship to agonist-directed trafficking. Mol Pharmacol 2002 62 578-589. 

Newman-Tancredi A, Cussac D, Marini L, Millan MJ. Antibody capture assay reveals bell-shaped concentration-response isotherms for h5-HTlA receptor-mediated G alpha(i3) activation conformational selection by high-efficacy agonists, and relationship to trafficking of receptor signaling. Mol Pharmacol 2002 62 590-601. 

Monoclonal antibodies, regardless of whether they are of high or low affinity, do not form a lattice with antigen, and, hence only rarely form insoluble precipitates. However, in immunohistochemistry, the capability of a primary antibody to form precipitating immune complexes is of little importance because reaction with immobilized tissue antigen entails antibody capture onto tissue rather than precipitation. 

The antibody capture affinity assay samples the free MAb binding sites by the addition of FITC-labeled antigen to the incubation mixture for 1 min, and the antigen-antibody complexes are immobilized on anti-IgG particles. The plates then are treated as above. 

This principle requires two different antibodies (capture and tracer antibody). 

Although most of the principles of antibody capture in packed bed mode are applicable to fluidized-bed technology, today applications are hindered by the very limited availability of sorbents specifically designed for fluidized beds. For instance, the potential applicability of a protein A affinity capture in a fluid bed seems very attractive and may become a useful operation with appropriate dense solid phases. Collected fractions rich in antibody obtained at the issue of a capture step in fluidized-bed mode can be further repurified or polished by other types of packed-bed chromatography, as described in Section V.I. 

---