Specific binding and release of cells from beads using cleavable tetrameric antibody complexes

A two-step separation procedure is described for the positive selection of cells based on their reactivity with mouse monoclonal antibodies. In the first step cells are specifically cross-linked to hapten-modified glass beads using tetrameric monoclonal antibody complexes. In the second step bound cells are selectively eluted by reductive cleavage of the tetrameric antibody complexes. The latter are comprised of two mouse IgG1 monoclonal antibodies (one recognizing a cell surface antigen on target cells and the other a hapten coupled to the glass beads) bound together by two F(ab′)₂ fragments of rat anti-mouse IgG1 monoclonal antibody. The complexes provide a specific cleavable cross-link between cell and bead because the disulfide bonds between the two Fab′ arms of the F(ab′)₂ fragments can be broken under relatively mild conditions using dithiothreitol. This specific cleavage of the cross-linker allows elution of the specifically adsorbed cells without co-elution of non-specifically bound cells. This is shown in the purification of CD3⁺ T cells from human peripheral blood, where the removed fractions were over 90% pure and approximately 50% of the positive cells were recovered. Separation of cells labelled with limiting amounts of tetrameric antibody complexes demonstrated that this separation technique was also effective for the purification of cells expressing low amounts of antigens. This was confirmed by the purification of CD34-positive cells from human bone marrow. With this approach, colony-forming cells were enriched 15–24-fold over density separated marrow.     

Composition of immune deposits present in glomeruli of NZB/W F1 mice

This paper describes the results of experiments designed to investigate the composition of immune complexes present, in the form of immune deposits, in glomeruli of NZB/NZW F1 mice. Granular deposits of mouse IgG were present along the glomerular capillary walls of 6- to 12-month-old mice. Disappearance of mouse IgG from glomerular deposits, indicating a dissociation of immune complexes, was observed following incubation of kidney sections with an excess of mouse IgG, mouse Fc fragments, rat IgG, and rat Fc fragments, but not with human and rabbit Cohn fraction-II (FII), DNA, nucleohistone, and PBS. Antinuclear antibody activity in mouse sera or in glomerular eluates was removed by absorption with mouse IgG or mouse Fc fragments, rat IgG or rat Fc fragments, DNA, and nucleo-histone, but not by absorption with human or rabbit FII. These results suggest that the IgG antinuclear antibodies present in the sera and in glomerular deposits possess rheumatoid factor (RF) activity. In other experiments, kidney sections were incubated with various concentrations of pepsin, which digests the Fc portion of the IgG. After digestion, the sections were washed and stained for mouse IgG, IgG F(ab')2, and IgG Fc. At concentration of 10 micrograms/ml, pepsin completely removed IgG and IgG Fc, whereas faint IgG F(ab')2 deposits persisted in glomerular deposits. At the concentration of 1 microgram/ml, deposits of mouse IgG, F(ab')2, and Fc persisted, while F(ab')2 was observed bound to nuclei of glomerular cells. At the pepsin concentration of 0.1 microgram/ml or 0.01 microgram/ml, IgG F(ab')2 was bound to the nuclei of glomerular and tubular cells, indicating that the digestion of the Fc portion of IgG had released F(ab')2 with nuclear reactivity from glomerular deposits. The solubilization of mouse IgG from glomerular immune deposits with mouse IgG and the demonstration that pepsin digestion releases mouse F(ab')2 with nuclear reactivity are consistent with the interpretation that the immune deposits present in glomeruli of NZB/NZW F1 mice contain complexes formed by antinuclear IgG and IgG RF. These two antibodies probably cross-react and form multilayer aggregates which contribute to the formation of immune deposits.     

Demonstration of anti-idiotypic antibodies directed against IgM rheumatoid factor in the serum of rheumatoid arthritis patients.

We have identified the presence of anti-idiotypic activity against IgMRF in the sera of RA patients. Only patients seropositive for IgMRF had significant levels of anti-idiotypic activity, while seronegative patients and normal volunteers did not. When this anti-idiotypic activity was affinity-purified from a single RA patient, two separate binding activities were identified. IgG antibodies were pepsin-digested to F(ab')2 fragments before affinity-purification to remove the Fc portion capable of binding to IgMRF. Anti-idiotypic F(ab')2 fragments of IgG were eluted from an IgMRF-Sepharose 4B column. These F(ab')2 bound preferentially to IgMRF bearing an idiotype recognized by the anti-idiotypic murine monoclonal 17.109. A second anti-idiotypic F(ab')2 was affinity purified using rabbit anti-human Fc antibody bound to Sepharose 4B. These eluted antibodies behaved as the internal image of IgG, binding five out of seven IgMRF's tested. The binding of both anti-idiotypic F(ab')2 was inhibited with human IgG. The presence of both IgMRF and anti-idiotypic antibodies directed against it in the sera of RA patients suggests that anti-idiotypic antibodies alone are not capable of inhibiting the production of rheumatoid factor.     

Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors.

Fc gamma RII (CDw32) on monocytes is capable of triggering both phagocytosis and lysis of chick red blood cells (CRBC) coated with antibody of the appropriate isotype. In this report we describe the production and characterization of a mouse monoclonal IgG1 antibody specific for Fc gamma RII and compare its activity in binding studies, tissue distribution and redirected cellular cytotoxicity (RCC), with the previously identified anti-Fc gamma RII antibodies KB61 and IV.3. Immunohistochemical and flow cytometry analyses demonstrated that AT10 binds very strongly to Fc gamma RII on normal monocytes, but only weakly to that expressed on lymphocytes. This pattern does not correspond to the staining seen with either KB61 or IV.3, and appears to give an intermediate profile. The binding constant (Ka) for the Fab' fragment of AT10 was calculated at 5.3 x 10(8) M-1, four times higher than that for KB61 (1.4 x 10(8) M-1). Bispecific F(ab')2 antibodies were constructed from Fab' fragments of AT10 or KB61 thioether-linked to Fab' from an anti-CRBC monoclonal antibody. These bispecific derivatives directed monocyte cytotoxicity against CRBC as efficiently as either a monoclonal or polyclonal anti-chick erythrocyte antibody. The bispecific F(ab')2 antibodies had a distinct advantage over the conventional reagents, in that they were not blocked in the presence of human Fc gamma at 3.5 mg/ml (a concentration comparable with that provided by IgG in serum). Therefore, bispecific derivatives constructed with the high affinity anti-Fc gamma RII antibody, AT10, may be used as therapeutic reagents for targeting tumour cell lysis in vivo.     

Purification of a functional 40 kD human placental Fc gamma-receptor using a monoclonal antibody

F(ab')2-fragments of a mouse monoclonal antibody (B1D6) reacting with placental receptors for the Fc part of IgG (FcR) were used as affinity reagents for the purification of an antigen from placental extract (PE). The antigen agglutinated ovine erythrocytes (E) sensitized with rabbit antibodies (A) (EA), but not E or E sensitized with F(ab')2-fragments. It reduced the EA rosette-formation with mononuclear cells and the binding of soluble immune complexes to placental tissue. The antigen bound to aggregated IgG and Fc-fragments of IgG, but not to native IgG or F(ab')2-fragments of IgG. The data indicate that the purified antigen possesses FcR activity with low affinity for IgG. SDS-PAGE and Western blot showed one distinct band of approximately 40 kD. The electrophoretic mobility did not change after reduction and the band reacted with concanavalin A indicating that the FcR are single-chained glycoproteins.     

Murine Bispecific Antibody 1A10 Directed to Human Transferrin Receptor and a 42-kDa Tumor-Associated Glycoprotein

Previously, we observed that bispecific antibodies (“antigen forks”) that bound to certain pairs of different tumor surface antigens could inhibit cell growth. The chemically linked heteroconjugate of MAb 454A12 (murine IgG1 recognizing human transferrin receptor) and 317G5 (murine IgG1 recognizing a 42-kDa tumor-associated glycoprotein) was particularly inhibitory toward human colorectal cancer cell lines, and the iron-chelating agent deferoxamine was found to augment inhibition of tumor cell growth by this antigen fork. Further experiments revealed that an antigen fork constructed by linking Fab′ fragments instead of whole antibodies retained activity, which led us to construct a fork-secreting hybrid hybridoma. Hybridoma 454A12 was fused with hybridoma 34F2 (murine IgG1 with the same specificity as 317G5). Hybrid hybridomas whose supernatants blocked binding of both 454A12 and 34F2 probes were further tested for the ability to block growth of SW948 human colorectal cancer cells in an MTT growth assay, and were chosen for subcloning. Ascites produced by clone 1A10 was purified by affinity and cation exchange chromatography. Purified 1A10 bispecific antibody showed growth inhibitory activity comparable to that of a chemically linked heteroconjugate of its parental antibodies 34F2 and 454A12. Adding deferoxamine greatly enhanced the inhibitory activity of 1A10 and effectively prevented regrowth of tumor cellsin vitro.By heterologously crosslinking two antigens that are coexpressed on many tumor cells, this bispecific antibody is able to inhibit tumor growth with enhanced selectivity.     

Lipoproteins and malaria. II. Immunoregulatory role of immunoglobulin-lipoprotein complexes in mice infected with Plasmodium chabaudi on antibody producing cells

Injection of lipoproteins from Plasmodium chabaudi infected mice into mice previously immunized with either human serum transferrin, bovine serum albumin, polyvinyl-pyrrolidone or tetanus toxoid, was followed by a decrease in the levels of antibodies directed against these antigens, suggesting a blockade of antibody secreting cells (Goumard et al., 1982). However, lipoproteins in P. chabaudi infected mice are complexed with immunoglobulins during the second week of infection (Demonchy et al., 1982). In this study, the effects of lipoproteins and Ig-lipoprotein complexes (Ig-Lp) on antibody secreting cells was investigated in vitro. Spleen cells from mice immunized with tetanus toxoid were cultured in microplates and the antitetanus antibodies (anti-TT Ab) were measured in the culture supernatants using a radioimmunoassay (Goumard et al., 1984). Ig-Lp purified from day-11 or day-13 P. chabaudi infected mice inhibited the secretion of anti-TT Ab when introduced into microcultures. On the contrary, lipoproteins purified from either day-5, day-7, day-21 or day-28 infected mice as well as lipoproteins from uninfected mice did not inhibit anti-TT Ab secreting cells. Ig-Lp formed in vitro with lipoproteins purified from day-7 infected mice (Lp J-7) and day-20 infected mice sera, inhibited anti-TT Ab secreting cells. IgG purified from day-20 sera and incubated with Lp J-7, inhibited anti-TT Ab secreting cells but no inhibitory effect was observed with the F(ab')2 fragments of these Ig. Pre-incubation of anti-TT Ab secreting cells with Fc fragments of mouse IgG blocked the inhibitory effect of Ig-Lp purified from infected mouse sera or formed in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the immune response: V. An analysis of the function of the Fc portion of antibody in suppression of an immune response with respect to interaction with components of the lymphoid system

Pepsin digested, F(ab')₂ antibody has less ability to inhibit an antibody response than has intact IgG antibody, when the antibodies were given one day after antigen. F(ab')₂ antibody has to be given with antigen to attain maximal suppression, while IgG antibody, administered after antigen, is still highly immunosuppressive. The IgG antibody was able to terminate established immune responses, whereas F(ab')₂ antibody could not do so. We interpret these findings to indicate that F(ab')₂ antibody suppresses immune responses by simple masking of antigen, whereas IgG antibody alters the immune response through a further activity which takes place after antibody has combined with antigen. This further activity involves the Fc portion of antibody. Two alterations in immune mechanism are suggested: (1) increased destruction of antigen and (2) inactivation of the antibody forming cell precursor population by antigen—antibody complexes. This latter possibility is considered in detail. The tripartite inactivation model has been constructed to explain the presently known observations concerning immunosuppression by antibody and to make a prediction which has been verified. A further prediction concerning the affinities of antibodies produced under IgG or F(ab')₂ antibody-mediated immunosuppression is put forward.

Thymus-bone marrow cell synergism does not give a simple thymus cell dose-response relationship but a multi-phasic relationship where the response increases once the dose of thymus cells is decreased to a sufficiently low level. Such a dose-response relationship is not explainable in terms of the usual mechanisms proposed for thymus-bone marrow cell interaction and this deviation from a simple dose-response relationship is interpreted in terms of the proposed function of thymus-derived cells in controlling antibody feedback regulation.

     

Fc-mediated immune precipitation. III. Visualization by electron microscopy.

Fc-mediated interactions between immune complexes are of major importance for the precipitin reaction. In the present study these interactions were investigated by means of electron microscopy. Keyhole limpet haemocyanin (KLH) was adsorbed to a thin glow charged carbon supporting film and reacted with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2 fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes and ferritin-anti-ferritin complexes could be demonstrated.     

Certain Polyclonal Antinuclear Antibodies Cross-React with the Surface Membrane of Human Lymphocytes and Granulocytes

Five out of 24 human sera with antinuclear antibody (ANA) titers of 1250 or more contained ANA that bound in vitro to normal viable human mononuclear blood cells and granulocytes, but not to erythrocytes. The antibodies can be eluted off from the cell membranes and shown to possess ANA activity. Antinative DNA antibodies and lupus erythematosus factor were not recovered in eluates, indicating that they did not react with the cells. The cells absorbed 75%-87% of the ANA activity from three sera. ANA reacted with both T-lymphocyte-depleted and -enriched mononuclear cells. No or minimal amounts of ANA bound to mouse spleen cells in suspension; in contrast, the ANA eluted from human cells reacted with nuclei of smeared mouse spleen cells. The cross-reacting antibodies were predominantly IgG that bound well at 37 degrees C, and F(ab')2 fragments carried both activities. The ANA-binding plasma membrane antigen was resistant to trypsin and RNAse but was completely inactivated by glutaraldehyde. The data indicate that human leukocyte plasma membranes and cell nuclei from many species contain a cross-specific antigen. Alternatively, the antigen may be produced in the nucleus and somehow attach to the plasma membrane.     

IgG on Human Blood Lymphocytes Studied by Immunofluorescence

Surface immunoglobulins (sIg) on human blood lymphocytes were identified by immunofluorescence (IFL) after staining with conjugated F(ab'2) fragments of the anti-Ig antibodies. A large fraction (approximately 20%) of freshly isolated lymphocytes was found to carry sIgG. Polyclocal IgG, which was present on Fc-receptor-bearing lymphocytes, could be removed by incubation and repeated washings only at 37 degrees C. Lymphocytes treated at 37 degrees C expressed the same percentage of sIg+ cells in direct IFL when F(ab')2 fragments of the antibody was used as when undigested aggregate-free IgG antibody was used. Indirect IFL using F(ab')2 fragments in both steps yielded similar sIg+ values. However, much higher percentage of cells carried sIg when undigested antibody was included in one of the steps. The results suggest that incubation and washing at 37 degrees C and the use of F(ab')2 fragments of the antibodies are important to eliminate absorbed sIgG and to avoid absorption of IgG during the staining procedure, thus preventing overestimation of the number of sIg+ B lymphocytes identified by IFL.     

Fc-Dependent IgG-Mediated Suppression of the Antibody Response: Fact or Artefact?

IgG antibodies have been shown to suppress the antibody response to all epitopes of their specific antigen as well as those the IgG do not bind to, so-called 'non-epitope-specific suppression'. The present study was undertaken to clarify whether there is a true IgG-mediated Fc-dependent suppression of the antibody response. This question is of fundamental importance to the understanding of the mechanism behind this phenomenon. It is demonstrated that F(ab')2 fragments of a monoclonal TNP (trinitrophenyl)-specific IgG2a antibody are unable to suppress the murine in vitro non-epitope-specific plaque-forming cell response against SRBC (sheep erythrocytes) when SRBC-TNP is used as antigen. The same monoclonal IgG antibody, when administered in intact form, is able to induce up to 98% suppression of the SRBC-specific antibody response. The lack of suppression is not due to mitogenic effects of pepsin in the F(ab')2 fractions or increased breakdown of F(ab')2 fragments, as compared with intact antibody, in the cultures. These data clearly demonstrate that there is indeed a highly efficient, Fc-dependent, non-epitope-specific suppressive mechanism mediated by IgG antibodies and support a hypothesis involving binding of the antigen-antibody complexes to Fc receptors as a step in the effector mechanism.     

Immunoelectron microscopic localization of thyroglobulin in the human thyroid gland

The microidentification of the organelles containing thyroglobulin (TG) in the follicular cells of human thyroid glands were studied by the immunoelectron microscopic method. Fresh human thyroid glands were used for the purification of TG. TG was purified by differential salt fractionation and DEAE-cellulose chromatography. Anti-human TG rabbit IgG antibody was obtained by immunization of this purified TG. F(ab')2 fragments of anti-TG rabbit IgG antibody were prepared by pepsin digestion. The specificity of the antibody was tested on an Ouchterlony immunodiffusion plate. Conjugation of the purified F(ab')2 fragments of anti-TG rabbit IgG antibody to horse radish peroxidase was performed in order to use the direct peroxidase labelled antibody method. Under immunohistochemical light microscopy, the luminal colloid and the follicular cells appeared heavily stained. Under immunoelectron microscopy, positive reactions were observed in the rough endoplasmic reticulum, the Golgi apparatus, and the apical cell border. To date, no clear direct evidence of the presence of TG in the rough endoplasmic reticulum and the Golgi apparatus of human thyroid cells has ever been reported in immunoelectron microscopic studies. This study indicates that the use of F(ab')2 fragments of anti-TG rabbit IgG antibody in immunoelectron microscopic study is useful for the identification of TG in organelles of human thyroid follicular cells.     

Cyclic tetramolecular complexes of monoclonal antibodies: a new type of cross-linking reagent

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies, when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1, yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody, stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens, and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine.     

Relationship between histocompatibility antigens, other surface determinants and the IgE receptor on rat mast cells.

Rat alloantibodies recognizing classical transplantation antigens (CTA) or non-H-1 determinants were able to compete effectively with monomeric IgE or IgG-coated sheep erythrocytes for receptor sites on the rat mast cell surface. Inhibitory capacity, however, was entirely confined to anti-CTA antibodies of the IgG2a subclass, whereas IgG1 antibodies lacked this ability. Analogously, F(ab')2 fragments of anti-CTA antibody consistently failed to affect IgE binding, but exposure of cell-bound F(ab')2 to anti-rat IgG restored its inhibitory capacity. From these results it was concluded that receptor sites recognizing the Fc portion of the anti-CTA molecule are involved in the inhibition process. Based on a cytotoxicity assay and on comparative absorption studies on alloantisera, the existence and relative amount of CTA and I region-associated antigens on purified rat mast cells and lymph node cells were analyzed. Whereas the CTA concentration per unit surface area on both cell types was very similar, rat mast cells consistently lacked Ia antigens.     

Bispecific F (ab')2 monomer prepared with anti-CD3 and anti-tumor monoclonal antibodies is most potent in induction of cytolysis of human T cells

Induction of cytolytic activity of peripheral blood mononuclear cells towards target cells was studied by preparing bispecific F(ab')2 which was composed of two Fab' fragments, one of which was derived from anti-CD3 monoclonal antibody and the other from anti-tumor monoclonal antibody. After reduction of the interchange disulfide bonds of these fragments by dithiothreitol, a thiol-disulfide interchain reagent, 5,5'-dithiobis-2-nitrobenzoic acid, was added to convert the free SH groups of one of the Fab' fragments to mixed disulfide derivatives. These were then coupled with the other Fab' fragment bearing free SH groups, producing a bispecific hybrid F(ab')2 monomer of high yield. The bispecific F(ab')2 monomers were able to render nonactivated peripheral blood mononuclear cells cytotoxic against natural killer-resistant tumor cell lines at doses as low as 1 microgram/ml. The polymeric forms of the F(ab')2 fragments prepared by use of cross-linking reagents such as N-succinimidyl-3-(2-pyridyldithiol)-propionate (SPDP) or S-acetylmercaptosuccinic acid anhydride (SAMSA) were less efficient for induction of cytolytic activity than the monomeric one. It may be feasible to use bispecific F(ab')2 monomers in cancer immunotherapy because of the ease of preparation, as well as the efficiency in inducing cytolytic activity and their high tissue permeability due to their small size (100-110 kDa). In addition, redirected cytolytic activity towards peripheral blood mononuclear cells by reticuloendothelial system cells, resulting from linking these two types of cells through Fc-Fc receptor interactions, does not occur.     

Fc-dependent and Fc-independent opsonization of Cryptococcus neoformans by anticapsular monoclonal antibodies: importance of epitope specificity

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab')(2) fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab')(2) fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab')(2) fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab')(2) fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.     

Anti-idiotypic activity against anti-myeloperoxidase antibodies in pooled human immunoglobulin.

We investigated the ability of six different pooled human immunoglobulin (PHIG) preparations to inhibit the binding of anti-myeloperoxidase (MPO) antibodies to MPO. All six PHIG preparations inhibited the binding of anti-MPO antibodies from six sera to MPO in a concentration-dependent manner in the concentration range 0.016-10 mg/ml. There was considerable variation in the ability of each PHIG preparation to inhibit the binding of anti-MPO antibody in a given serum. Further differences were seen in the ability of a given PHIG to inhibit anti-MPO binding in different sera. F(ab')2 fragments from two PHIG preparations also inhibited in a concentration-dependent manner anti-MPO binding to MPO in all six sera in the concentration range 0.002-2.65 mg/ml, with a maximum inhibition of 42%. Little inhibition was seen with F(ab')2 of normal human IgG from individual donors (1.8-12.2% at the maximum concentration of 2 mg/ml). F(ab')2 fragments from three anti-MPO containing sera and two affinity-purified anti-MPO antibodies were eluted by affinity chromatography from Sepharose-bound PHIG F(ab')2 and showed anti-MPO antibody activity. We have shown that PHIG and F(ab')2 fragments of PHIG inhibit anti-MPO binding to MPO, and further that F(ab')2 fragments of PHIG bind to F(ab')2 fragments of anti-MPO antibodies. These observations indicate binding between the variable regions of PHIG and the antigen binding site of anti-MPO antibodies, and are consistent with an anti-idiotypic reaction. The variability seen in the inhibitory effect of the different PHIG preparations in anti-MPO-positive sera implies differences in their anti-idiotype content, while the variability of the inhibitory effect of a particular PHIG preparation between different sera suggests heterogeneity in the idiotypic repertoire of anti-MPO antibodies. Such variations in the inhibitory effect of different PHIG preparations on antibody binding may be an important determinant of their therapeutic effect.     

Binding of the human complement subcomponent C1q to hybrid mouse monoclonal antibodies

Activation of the classical pathway of the complement system is initiated by the binding of C1q to antibody complexes. Here we evaluated the C1q binding capacity of series of monospecific and bispecific hybrid mouse monoclonal antibodies (mAb) and compared them with parental (conventional) mAb. The hierarchy in C1q binding capacity of the bispecific anti-HuIgA1/HRP mAb with homologous H-H chain combinations (IgG2a-2a, IgG2b-2b and IgG1-1) and the parental anti-HuIgA1 or anti-HRP mAb was identical; IgG2a greater than IgG2b much greater than IgG1. Hybrid IgG1-2a mAb bind intermediate amounts of C1q when compared with the IgG1 and IgG2a parental antibodies. IgG1-2b and IgG1-1 hybrid mAb did not bind any C1q, like the IgG1 mAb. We could not observe any difference in C1q binding efficiency between monovalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 HRP mAb and the bivalently bound IgG1-2a, IgG2a-2a and IgG2b-2b anti-HuIgA1 mAb, respectively. Furthermore, these hybrid ms anti-HuIgA1 and bs anti-HRP/HuIgA1 mAb were able to lyse HuIgA1-coated erythrocytes, in the presence of 50% human serum, as efficiently as their parental counterparts. These data indicate that a simultaneous binding of both F(ab') fragment to antigen is not a necessary prerequisite for binding and activation of C1q.     

Generation of Monoclonal Rheumatoid Factors after Immunization with Collagen II-Anti-Collagen II Immune Complexes

Two monoclonal IgG rheumatoid factors were obtained after hybridization of spleen cells from DBA/1 mice immunized with immune complexes containing native collagen type II and a monoclonal anti-collagen II antibody. One of these rheumatoid factors reacted not only with purified murine Fc fragments, but also with Fab fragments of the anti-collagen II antibody used for immunization, whereas no reactivity was seen with Fab fragments from normal mouse IgG. The findings demonstrate the ability of immune complexes encompassing native collagen type II to induce production of IgG rheumatoid factors, and suggest that an idiotypic relationship may exist between certain rheumatoid factors and anti-collagen II antibodies.