V. Gheţie

Characterization of the soluble complex formed by reacting rabbit IgG with protein a ofS. aureus

Abstract By reacting rabbit IgG with Protein A of Staphylococcus aureus (SpA) in a wide range of molar ratio only one type of complex was formed with a mol. wt of 350,000 daltons and a molar composition of IgG 2 -SpA 1 . The IgG 2 -SpA 1 complex is not able to bind SpA pointing out that the Fc region binding sites of IgG are saturated by SpA. The inability of IgG 2 -SpA 1 complex to bind rabbit IgG as compared with its ability to react with rabbit IgG when fixed to sheep erythrocytes or to Sepharose beads, and to precipitate human IgG suggests that on SpA molecule there are four combining sites with different affinities for IgG. On the basis of these results a hypothetical model of IgG 2 -SpA 1 complex is presented.

Protein A as a molecular probe for the detection of antigen induced conformational change in Fc region of rabbit antibody

Abstract Rabbit IgG antibody which reacts with protein A of Staphylococcus aureus (SpA) and forms a soluble complex with molar composition (IgG2-SpA1)2 is not able to further bind SpA or to attach to SpA-Sepharose 4B thus proving that the unengaged SpA reactive sites of IgG are shielded by the already bound SpA. On the contrary the (IgG2-SpA1)2 complex was able to bind a small fragment of SpA (fSpA) clearly showing that SpA-reactive sites are present in the rabbit IgG molecules of the complex but that they are not available for the intact SpA molecule. Immune complexes contaning (IgGaFER2-SpA1)2 and ferritin attach to an SpA-Sepharose 4B column showing that SpA binding sites exist on the IgG molecules and became exposed after the conformational change of the Fcγ region induced by the antigen. Therefore SpA can be used for the direct detection of conformational changes induced by antigen in the Fcγ region of rabbit antibody.

Multivalent hybrid antibody

Abstract Hybrid antibody complexes with dual specificity were prepared by combining two antibodies with different specificities by protein A of Staphylococcus aureus (SpA). ∗ In the first step by the reaction of rabbit IgG antibody (anti-A) with an excess of SpA, a soluble complex consisting of one IgG molecule linked to one SpA molecule (IgG anti-A/SpA) was obtained (mol. wt 190,000 daltons). In the second step to the IgG anti-A/SpA complex, an equivalent amount of rabbit IgG antibody (anti-B) was added and a soluble complex with molecular formula (IgG anti-A/SpA/anti-B) 2 was obtained (mol. wt 669,000 daltons). Using this method, several hybrid SpA-antibody complexes were prepared. These hybrid SpA-antibody complexes with dual specificity are multivalent in respect with both antigens and are able to draw them together, as demonstrated by hemagglmination, hemolysis, double diffusion, immunoadsorption and rosette formation. Multivalent hybrid SpA-antibody complex with double specificity is a reagent easy to prepare, specific and sensitive for cross-linking any soluble or particulate antigens provided rabbit antibodies are available.

Crosslinkage of antibodies to staphylococcal protein A matrices

Crosslinkage of anti-human albumin (anti-HSA) with varying concentrations of glutaraldehyde to Staphylococcus aureus Cowan 1 (SpA-Staph) and to staphylococcal protein A-Sepharose (SpA-Sepharose) was tested. A concentration of 0.0075% glutaraldehyde was found efficient for an almost complete covalent binding of IgG to the matrices. The antibody activity of crosslinked anti-HSA SpA-Staph and anti-HSA SpA-Sepharose was more than 60 and 90% respectively compared with the corresponding noncrosslinked immunosorbents. Antigen was recovered with intact antigenic properties by elution with 3.5 M MgCl2.

Mutual inhibition of the binding of Clq and protein A to rabbit IgG immune complexes.

A complex of rabbit IgG antibody with horseradish peroxidase covalently linked to Sepharose 4B was used as an insoluble immune complex for studying the binding of complement factor C1q protein A from Staphylococcus aureus, and its IgG-binding fragments AB and B, to rabbit IgG. It was shown that protein A (mol. wt approx. 42,000) and fragments AB and B (mol. wts approx. 14,000 and 7000, respectively) inhibited the binding of C1q to insoluble immune complex at 4 degrees C. However, at 37 degrees C fragment B did not inhibit this binding. On the other hand, C1q, when bound to an insoluble immune complex, almost completely blocked the binding of protein A and fragment B at both temps. The higher affinity of C1q for its CH2-binding site than of fragment B for its CH2-binding site may explain the displacement of the latter from the CH2 domain. The mutual inhibition of the binding of C1q and protein A (and its smaller fragments) indicates that the binding sites for C1q and protein A are closely located in the CH2 domain.

Multivalent hybrid antibody with double specificity as a tool for locating cell surface antigens by electron microscopy

Multivalent hybrid antibody complexes with dual specificity were prepared by combining rabbit antibody with anti-mouse immunoglobulin (mIg) and anti-peroxidase (HRP) specificity using protein A of Staphylococcus aureus. The presence of two antibody molecules with anti-mIg and anti-HRP specificity in a single molecule of hybrid complex was demonstrated by their abilities to produce hemagglutination with both HRP-coated and mIg-coated sheep red cells, to give a reaction of complete identity with mIg and HRP and to allow mIg bearing lymphocytes to form rosettes with HRP-coated sheep red blood cells. Electron microscopy of mouse lymphocytes and thymocytes (previously coated with mIg anti-Thy-1 antibody) treated with hybrid antibody complex and HRP showed strong and specific staining of the cell membrane of both cell types. The hybrid antibody complex containing anti-HRP antibody is a valuable reagent for determining various antigenic markers on cell membranes by electron microscopy.

Interspecies hybrid antibody with dual specificity

Abstract The preparation of soluble multivalent hybrid antibody by protein A of Staphylococcus aureus (SpA) is limited exclusively to rabbit IgG because other species have SpA-precipitating IgGs. Starting from a soluble complex consisting of one rabbit IgG antibody molecule linked to one SpA molecule (rabbit IgG anti-A/SpA), an interspecies hybrid antibody with dual specificity was prepared using either mouse or human IgG antibody, the molecular formula of this complex being (rabbit IgG anti-A/SpA/mouse or human IgG anti-B)2. These complexes are useful tools for the investigation of cell surface antigens against which no appropriate antibody can be raised in rabbits.

Simultaneous detection of two different cell surface antigens by electron microscopy by means of multivalent hybrid antibody with double specificity

Multivalent hybrid antibody (MHA) complexes with dual specificity were prepared by combining two antibodies of different specificities, one against ferritin (Fer), the other against horseradish peroxidase (HRP), with protein A of Staphylococcus aureus (SpA). Electron microscopy of mouse spleen lymphocytes and thymocytes (previously coated with mouse IgG anti-Thy-1 antibody) treated with IgG anti-Fer/SpA/IgG anti-mouse Ig complex and Fer gave better resolution and higher accuracy than previously obtained with IgG anti-HRP/SpA/IgG anti-mouse Ig complex and HRP (Mandache et al., 1980). Surface Thy-1 alloantigen and Fc receptor (charged with human IgG) treated with a mixture of IgG anti-Fer/SpA/IgG anti-Thy-1 and IgG anti-HRP/SpA/IgG anti-human Fab could be simultaneously detected on the thymocyte surface by either light or electron microscopy using Fer and HRP. The concomitant visualization of Thy-1 alloantigen (with Fer) and FcR (with HRP) on mouse thymocyte clearly shows that their distribution is largely independent and that the amount of Thy-1 antigen is greater. These results show that electron microscopy with a mixture of MHA is a useful technique for simultaneous location of two antigenic markers on the cell surface.

Preparation and some properties of dimeric rabbit IgG antibody

By reacting rabbit IgG with the fragment AB of protein A from S. aureus (mol. wt 14,000) an IgG dimer was formed with an approx. mol. wt of 320,000 and a molar composition of IgG2-AB1. A hybrid dimer with dual specificity consisting of IgG anti-sheep red blood cells/AB/IgG anti-bovine red blood cells was also obtained by reacting successively both rabbit antibodies with the AB fragment. The immunologic properties (affinity for antigen, complement activation and binding to Fc receptors) of the dimeric IgG were investigated in comparison with monomeric rabbit IgG and a tetrameric IgG obtained by reaction with protein A, (IgG2-protein A1)2.

Modulation of IgG effector functions by a monovalent fragment of staphylococcal protein A

The monovalent V-1 fragment of protein A (fSpA) with a mol. wt of 13,000 obtained from an u.v. mutant of Staphylococcus aureus Cowan I strain was proved to be able to modulate significantly some of the effector functions of IgG, such as complement fixation, catabolism, attachment to Fc receptors and antibody-dependent cell-mediated cytotoxicity. Moreover fSpA-like protein A obtained from the A676 strain is mitogenic and enhances NK activity of human peripheral lymphocytes. The efficiency of fSpA was found to be lower than that of protein A with regard to its ability to inhibit complement fixation, EA rosette formation and antibody-dependent cell-mediated cytotoxicity. Both protein A and fSpA had the same efficiency in activation of the complement system after reaction with human or guinea pig IgG, and in increasing the IgG catabolism. Unlike fSpA the monovalent B fragment of protein A (with mol. wt of 7000) was not able to inhibit EA rosette formation and antibody-dependent cell-mediated cytotoxicity. The results recommend fSpA, substituting for protein A, as a molecular probe for the investigation of IgG antibody and lymphocyte effector functions.