Protein A-IgG complexes

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

To deplete 25 p,l of mitotic cytosol (at 12 mg/ml) of BARS, 10 pi of the p50-2 antiserum is coupled to 10 pi of protein A-Sepharose beads (Amersham Pharmacia Biotech, NJ, USA) in PBS for 1 h at 4°. To deplete 25 pi of rat brain cytosol (at 12 mg/ml) of BARS, 20 pi of the p50-2 antiserum is coupled to 20 pi of protein A-Sepharose beads. The antibody-protein A-Sepharose complex is washed (3 x) with KHM permeabi-lization buffer and incubated with mitotic or rat brain cytosol. After 30 min, the beads are pelleted and the cytosol is incubated with fresh antibody-protein A complex for another 30 min. Mock incubations are performed by binding protein-A Sepharose beads to pre-immune IgGs. Thirty-five pg of total protein is run on a 10% SDS-PAGE and the depletion efficiency is monitored by immnno-blotting (Hidalgo Carcedo et al., 2004). 

Fig. 13a-e. The increase of the signal intensities by the addition of the dendritic complexes composed of IgGs and protein A. The hapten was immobilized to the surface of the SPR sensor chip. The increase of the signal intensities on the complex formation of hapten with the antibodies were monitored. The addition of mouse IgG specific for hapten (Abl) (a), the complex of the Abl with protein A (b), one to one complex of Abl with anti-mouse IgG (Fc) antibody (Ab2) (c), two to one complex of Abl with Ab2 (d), and two to one complex of Abl with Ab2 in the presence of protein A (e)... 

Direct and indirect competition formats, illustrated in Figure 1, are widely used for both qualitative and quantitative immunoassays. Direct competition immunoassays employ wells, tubes, beads, or membranes (supports) on to which antibodies have been coated and in which proteins such as bovine semm albumin, fish gelatin, or powdered milk have blocked nonspecific binding sites. Solutions containing analyte (test solution) and an analyte-enzyme conjugate are added, and the analyte and antibody are allowed to compete for the antibody binding sites. The system is washed, and enzyme substrates that are converted to a chromophore or fluorophore by the enzyme-tracer complex are added. Subsequent color or fluorescence development is inversely proportionate to the analyte concentration in the test solution. For this assay format, the proper orientation of the coated antibody is important, and anti-host IgG or protein A or protein G has been utilized to orient the antibody. Immunoassays developed for commercial purposes generally employ direct competition formats because of their simplicity and short assay times. The price for simplicity and short assay time is more complex development needed for a satisfactory incorporation of the label into the antibody or analyte without loss of sensitivity. 

The use of protein A and protein G in immunohistochemistry is based on the same principle as that using secondary anti-IgG antibodies in the indirect two-step approach. In the first step, an antigen IgG complex is formed, which is then revealed in the second step by incubation with labeled protein A or protein G. 

Even greater flexibility was achieved by genetic fusion of streptavidin with protein A [153,154]. Protein A specifically binds the Fc domain of IgG immunoglobulins of almost all mammals without inhibiting the antigen binding activity of the antibody. The streptavidin-protein A fusion construct was used for the assembly of complexes of biotinylated P-galactosidase and different monoclonal antibodies specific for tumour cell receptors. As a result these complexes were efficiently delivered into several cancer cell lines [154]. 

Although this treatment has been available for idiopathic thrombocytopenic purpura for several years, its mechanism of action is not understood. Removal of IgG and IgG-containing immune complexes does not explain its effects in rheumatoid arthritis. The most recent hypothesis for this treatment s mechanism of action is down-regulation of B cell function through the release of small amounts of staphylococcal protein A complexed with immunoglobulins. 

Post-separation labeling was also achieved for separation of four human serum proteins (IgG, transferrin, al-antitrypsin, and albumin) using 0.2 mM of TNS. This is a virtually non-fluorescent reagent which, upon non-covalent association with proteins, produces a fluorescent complex (A= 325 nm, A = 450... 

F2. Farrell, C., Sogaard, H., and Svehag, S.-E., Detection of IgG aggregates or immune complexes using solid-phase Clq and protein A-rich Staphylococcus aureus as an indicator system. Scand. J. Immunol. 4, 673-680 (1975). 

Protein A or Protein G affinity chromatography Immunoglobulins show specific affinity for these proteins which can be obtained complexed to Sepharose (Pharmacia). It is a simple matter so apply antiserum diluted in 20 mM phosphate buffer to a small column and subsequently elute the pure IgG using a glycine buffer pH 2.7. 

A high-resolution structure has been determined for the BI IgG-binding domain of protein G. The structure comprises of four stranded /3-sheet made up of two antiparallel j8-hairpins connected by an a-helix. The two central strands of the sheet are parallel and comprise the N- and C-terminal residues. Comparison of the protein A and protein G IgG-binding domain architectures reveals no immediately obvious region that could take the place of the two interacting helices of protein A and protein G complex. 

Another peptide was also described for its ability to interact with immunoglobulins and was used as an affinity chromatography ligand.201 This peptide structure resulted from a combinational peptide library screening and was identified by its ability to make complexes with IgG that were displaced by the addition of protein A. 

---