I. Moraru

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.

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.

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.

The Fc receptor for IgA expression and affinity on lymphocytes and macrophages

The interaction between the Fc receptor for IgA (FcR alpha) and human secretory IgA (sIgA) was studied in a heterologous system by using rabbit alveolar macrophages or splenic lymphocytes. The capacity of FcR alpha-bearing cells to interact with the ligand via the Fc region was demonstrated. The proportion of FcR alpha-bearing lymphocytes and macrophages was found to be 11 and 17%, respectively. The cell-bound radiolabelled ligand showed a release half-time of 70 min for lymphocytes and 45 min for macrophages. The equilibrium constant (K) and the maximum number of ligand molecules bound per cell (n) in mean values was 5.2 +/- 0.05 X 10(8) M-1 and 3.3 +/- 0.15 X 10(5) molecules per cell, respectively. Increased percentages of FcR alpha-bearing cells and of ligand density per cell were obtained after stimulation by lung inflammation. A significant increase of the K value was recorded with macrophages from rabbits with lung acute inflammation (mean value 7.3 +/- 0.21 + 10(8) M-1) while the increase with lymphocytes was not significant. The calculation method of K and n is discussed.

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.

Binding and cytotoxic effect of ricin toxin on multivalent hybrid antibody-coated target cells.

Multivalent hybrid antibodies with dual specificity were prepared by cross-linking with protein A two antibodies of different specificity, one against ricin toxin and the other against the H-2 antigens of murine leukemia EL4 cells. This bifunctional antibody specifically attached to EL4 cells was able to capture ricin toxin (RcA2) molecules with an affinity 20 times higher than that of the galactose-containing glycoproteins of the cell surface (nonspecific binding). The hybrid-bound RcA2 gained access into the target cell cytoplasm by endocytosis and blocked [3H]thymidine incorporation as efficiently as free RcA2 does on nontreated EL4 cells (3.3 X 10(-11) M). These results indicate that multivalent hybrid antibody, easy to prepare in purified form and endowed with high affinity for both target cell and ricin toxin, may be utilized efficiently for the specific delivery of toxins to target cells.

In vivo follow-up of the cytotoxic effect of protein A—ricin toxin conjugate, on antibody-coated target cells

Antibodies of the IgG class, specifically interacted with H-2 antigens of murine leukemia EL4 cells, were used to bind the ricin toxin covalently linked to protein A of Staphylococcus aureus. The toxin thus complexed, introduced in the cytoplasm by endocytosis, was able to kill the leukemic cells inoculated in animals. The interaction of immunotoxin with the leukemic cells was performed in vitro using one, two or three treatments and the cytotoxic effect on the target cells was followed up in vivo. The time interval between immunotoxin treatments was indicated by the membrane turn-over study of EL4 cells coated with specific antibodies in their monomeric form, complexed by protein A or interacted with protein A--ricin toxin conjugate. A proportion of 99.8% cells killed was obtained after three treatments.

In vivo follow up of the cytotoxic effect of ricin toxin vectorized by multivalent hybrid antibody on target cells

A multivalent hybrid antibody complex composed of two IgG molecules specific for ricin toxin and two specific for the H-2 antigens of murine leukemia EL4 cells, cross-linked by SpA, was used as vector of the toxin to the target cells. The high affinity of the hybrid antibody for the specific antigens achieved an efficient attachment to the EL4 cell membrane and binding of ricin toxin; this high-molecular-weight complex, introduced by endocytosis into the leukemic cell cytoplasm, was able to specifically deliver the toxin to the target cells. The effect of multivalent hybrid antibody-vectorized toxin was followed up in vivo. This method enabled determination of the proportion of killed cells (over 90% after a single treatment of leukemic cells or about 99% after double treatment). The presence of a low proportion of tumoral cells maintaining their proliferative capacity is discussed.