Immunoglobulin G3 antibodies specific for the 19-kilodalton carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein 1 transfer protection to mice deficient in Fc-gammaRI receptors

Merozoite surface protein 1 (MSP-1(19)) is a leading malaria vaccine candidate. Specific antibodies contribute to immunity; binding to macrophages is believed to represent the main action of malaria antibodies. We show that an MSP-1(19)-specific immunoglobulin G3 (IgG3) monoclonal antibody can passively transfer protection to mice deficient in the alpha chain of Fc-gammaRI whose macrophages cannot bind IgG3.     

Demonstration of the existence of two distinct Fc receptors for IgG isotypes on guinea-pig macrophages by the use of monoclonal antibodies

As reported in a previous paper by the authors (J. Biochem. 99, 227-235, 1986), the Fab' of a monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea-pig peritoneal macrophages with a myeloma cells line, completely inhibits the binding of ovalbumin (OA)-complexed IgG1 antibody to macrophages, but only partially the binding of OA-complexed IgG2 antibody. Based on these results, it was proposed that the cells have at least two types of Fc receptor (FcR) for homologous IgG isotypes: FcR2 for IgG2 and FcR1.2 for both IgG2 and IgG1, and also that VIA2 IgG1 is anti-FcR1.2 antibody. Thereafter, complete inhibition of the binding of OA-complexed IgG2 antibody to macrophages occurred when the Fab' of another monoclonal antibody, VIIA1 IgG1 was added to the Fab' of VIA2 IgG1, whereas the former did not affect the binding of OA-complexed IgG1 antibody. This effect of the Fab' of VIIA1 IgG1 indicates that VIIA1 IgG1 is a monoclonal antibody capable of selectively blocking the binding of OA-complexed IgG2 antibody to FcR2. When the antigen of VIIA1 IgG1 was isolated by affinity chromatography on the F(ab')2 of the antibody coupled to Sepharose, it gave a single band with a mol. wt of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It moved slightly faster than the FcR1.2 with a mol. wt of 55,000, which was isolated by the use of VIA2 IgG1, and corresponded to the fast moving portion of the broad band of FcRs isolated with OA-complexed IgG2 antibody. These results strongly suggest that VIIA1 IgG1 is a monoclonal antibody to FcR2.     

A receptor for monomeric IgG2b on rat macrophages

The binding to rat splenic and peritoneal macrophages of affinity-purified monoclonal rat IgGs, representing all IgG subclasses, was measured by the direct binding of 125I-labelled proteins using an assay that did not require the removal of unbound Ig by washing. Only rat monomeric IgGs of the subclass IgG2b bound specifically and in large amounts to rat macrophages. The binding was temperature dependent and more IgG2b bound to the cells at 4 degrees than at 37 degrees. Spleen macrophages bound approximately 10 times more IgG2b than the same number of peritoneal macrophages, although the association constants (Kas) for the binding were similar for both types of macrophage. The calculated values for the Kas, which varied slightly with each experiment and increased with decrease in temperature, fell within the range 1.3-5.3 X 10(8) M-1; the number of binding sites was estimated as about 10(5)/splenic macrophage and 10(4)/peritoneal macrophage. The binding of 125I-IgG2b to splenic macrophages was inhibited only by unlabelled proteins of the IgG2b isotype and not by IgG1, IgG2a and IgG2c proteins. Soluble IgG2b-antigen complexes also bound to the FcR for monomer but a soluble IgG2a-antigen complex did not inhibit the binding of monomeric IgG2b.     

The AC8 IgG3 monoclonal anti-cholesterol antibody modulates uptake and presentation of antigens for T cell activation

Natural anti-cholesterol antibodies (ACHAs) exist in mammalian species, moreover their level sensitively changes in pathological situations, such as atherosclerosis or HIV infection. The conditions of their production and functional role, however, still remained ill defined. Recently we developed IgG3 type monoclonal ACHAs that selectively react with 'clustered cholesterol' of live immune cells, such as membrane microdomains (lipid rafts and caveolas). These antibodies inhibited HIV-1 infection of Th cells and macrophages by remodeling the HIV-1 receptor/coreceptor distribution in the plasma membrane of target cells. As a novel modulatory effect, here we show that the AC8 IgG3 monoclonal anti-cholesterol antibody (mACHA), but not the AC9 IgM mACHA, spontaneously bind to all professional APCs, such as murine macrophages (Mfs) or bone marrow derived dendritic cells (DCs) and B lymphocytes. Upon binding, AC8 mAb remarkably enhanced the efficiency of yeast uptake by macrophages, but not the uptake of OVA-Ig immune complexes by DCs. Binding to B lymphoma APCs, AC8 mAb remodeled their surface membrane by microclustering rafts and recruiting MHC-II and the CD80 costimulators to common microdomains. The modulated APCs induced an enhanced activation signaling (higher Ca(2+)-signals and NFAT1 activation) in Th cells conjugated with them, relative to untreated APCs. The results presented herein highlight the modulatory potential of the IgG3 type AC8 mAb on both innate and adaptive effector cell functions.     

Identification of antibody classes and Fc receptors responsible for phagocytosis of Trypanosoma musculi by mouse macrophages

The phagocytosis of Trypanosoma musculi by macrophages in the presence of specific antibodies was investigated. In 14-day-infected mice, opsonic antibodies were detected in serum, and phagocytosis of parasites by peritoneal macrophages was observed. The mechanism of T. musculi phagocytosis was analyzed. The binding of trypanosomes to peritoneal macrophages and J774 cells was observed in the presence of serum from hyperimmune mice and from mice infected 14 or 28 days earlier, but not in the presence of control mouse serum or sera from 7-day-infected mice. Binding was partially inhibited by mouse monoclonal immunoglobulins G1 (IgG1) or IgG2a and almost completely inhibited by a mixture of both. Binding was also partially inhibited by the anti-Fc gamma 1/gamma 2b receptor monoclonal antibody 2.4G2. Binding of T. musculi was also induced by fractions of serum from 28-day-infected mice obtained by protein A-Sepharose chromatography. Only the IgG1-rich fraction eluted at pH 6.0 and the IgG2a-rich fraction eluted at pH 4.5 promoted binding which could be almost completely inhibited by monoclonal IgG1 and IgG2a. These data indicate that IgG1 and IgG2a anti-T. musculi antibodies are responsible for the phagocytosis of T. musculi by mouse macrophages and both Fc gamma 2a and Fc gamma 1/gamma 2b receptors are involved. Such a mechanism is likely to account for the elimination of parasites in T. musculi-infected mice.     

Regulation of macrophage Fc receptor expression and phagocytosis by histidine-rich glycoprotein.

Regulation of macrophage Fc receptor (Fc gamma R)-mediated phagocytic function by histidine-rich glycoprotein (HRG) was investigated. Pretreatment of oil-elicited inflammatory mouse peritoneal macrophages with HRG for 1-3 hr increased their Fc gamma R-mediated binding and phagocytosis of IgG-opsonized sheep erythrocyte conjugates (EA). A significant reduction of Fc gamma R-dependent EA binding and phagocytosis occurred after pretreatment of macrophages with HRG for more than 8 hr. These results indicate that HRG is capable of modulating Fc gamma R expression in a biphasic fashion, which directly affects the overall efficiency of phagocytosis. HRG differentially regulated the functions of Fc gamma R subclasses. For example, HRG reduced the efficiency of Fc gamma RII (Fc gamma 2b/gamma 1R)-dependent phagocytosis of erythrocytes conjugated with monoclonal IgG2b or IgG1 by macrophages pretreated with HRG for 24 hr. However, when similar studies were performed using erythrocytes coated with monoclonal IgG2a, HRG was less effective in inhibiting Fc gamma RI (Fc gamma 2aR)-dependent phagocytosis. As an HRG-binding glycosaminoglycan, heparin failed to block the regulatory function of HRG on macrophages. Similarly, interferon-gamma (IFN-gamma) was not capable of blocking the functional activity of HRG. These studies suggest that HRG regulates macrophage function via a novel pathway different from that of heparin or IFN-gamma.     

Monoclonal antibodies against the GP-2 subunit of laminin

Two stable rat X mouse hybridoma lines have been isolated. These hybridoma lines produce IgG antibodies directed against the polypeptide portion of the GP-2 subunit of laminin. Antibodies produced by the hybridomas have been shown to be IgG 2b (lambda) and IgG 2a (kappa), respectively. In competition ELISA assays the monoclonal antibodies exhibited different binding affinities for laminin. Furthermore, the two antibodies were partially additive in their reactivity to laminin. Preliminary results also indicate that the antibodies recognized different antigenic determinants in laminin as determined by their reactivity to basement membranes in human and mouse tissues. The monoclonal antibody designated LAM-I stained a broad spectrum of human and mouse tissues; the other monoclonal antibody LAM-II reacted with mouse, but not human tissues. The results indicate that these monoclonal antibodies could be utilized to explore the organization of laminin in basement membranes of different tissues and species.     

Lactoferrin-Lipid A-Lipopolysaccharide Interaction: Inhibition by Anti-Human Lactoferrin Monoclonal Antibody AGM 10.14

Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities. The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect. In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations. In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 [IgG1] antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated. The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A. The binding of LF to S-form LPS was markedly weaker than that to lipid A. Moreover, the rate of LF binding to R-form LPS was inversely related to core length. The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction. In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF. AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity. In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS. Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A. In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.     

Determination of the affinity of monoclonal 19 S IgM rheumatoid factor for IgG by modified immunoassay (ELISA)

In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenstr?m's macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.     

Screening the interactions between HIV-1 neutralizing antibodies and model lipid surfaces

Our work is motivated by the observation that rare, broadly neutralizing antibodies (NAbs), 4E10 and 2F5, associate with HIV-1 lipids as part of a required first step in neutralization before binding to membrane-proximal antigens. Subsequently, induction of these types of NAbs may be limited by immunologic tolerance due to autoreactivity with host cell membranes. Despite the significance of this lipid reactivity there is little experimental evidence detailing NAb-membrane interactions. Simple and efficient screening assays are needed to select antibodies that have similar lipid reactivity as known NAbs. To this end we have developed a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol self-assembled monolayers (SAMs) that replicate salient lipid surface chemistries and NAb binding to lipid surfaces. Specifically, we probed the relative importance of charge and hydrophobicity on antibody-surface interactions. We found that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies (mAbs). Furthermore, we confirmed the importance of charge-mediated antibody surface interactions, originally suggested by results from mAb interactions with conventional lipid vesicle/bilayer surfaces. Our approach, using self-assembled thiol monolayers that replicate the binding behavior of NAbs on lipid surfaces, thus provides an efficient and useful tool to screen interactions of mAbs and lipid-reactive NAbs.     

Fc receptors on mouse neutrophils and eosinophils: antigenic characteristics, isotype specificity and relative cell membrane density measured by flow cytometry.

The antigenic characteristics, isotype specificity and density of Fc receptors (FcR) on mouse neutrophils and eosinophils were studied with the aid of the rat monoclonal antibody 2.4 G2 to the mouse macrophage FcR (Unkeless, 1979). This MAb was tested for its reactivity with mouse neutrophil and eosinophil FcR, and for its ability to block the binding of sheep erythrocytes (E) coated with mouse antibodies of different isotypes to granulocytes. The use of E conjugated with fluorescein isothiocyanate (FITC) allowed an objective read-out by flow cytometry. The MAb 2.4.G2 reacted with both neutrophil and eosinophil FcR, blocking the binding of E coated with mouse IgG1, IgG2a and IgG2b in a dose-dependent manner. Blocking was specific, since it did not occur with any of several control MAb of the same rat isotype (IgG2b) as 2.4.G2. Furthermore, the binding to E through the granulocyte receptor for complement (C) was unaffected. IgG3 was unable to promote binding of E to either neutrophils or eosinophils, although it induced high levels of binding to macrophages. These results show that: (i) neutrophil, eosinophil and macrophage FcR have antigenic similarities; (ii) neutrophils and eosinophils, in contrast to macrophages, either have a common FcR for IgG1, IgG2a and IgG2b, or have different FcR for these isotypes which share the antigenic determinant recognized by 2.4.G2; (iii) in contrast to macrophages, neutrophils and eosinophils lack the FcR for IgG3. The MAb 2.4.G2 was used in an indirect immunofluorescence assay monitored by flow cytometry to measure the relative FcR density on neutrophils and eosinophils. This assay showed that neutrophils possess about 65% more FcR than eosinophils on a cell-for-cell basis, providing an explanation for the higher binding of neutrophils to IgG-coated particles at suboptimal antibody concentrations.     

Human monoclonal thyroglobulin autoantibodies of high affinity. I. Production, characterisation and interaction with murine monoclonal thyroglobulin antibodies.

Four hybridomas secreting human thyroglobulin (Tg) autoantibodies of different IgG subclasses and light chain types (IgG1 lambda, IgG1 kappa, IgG2 lambda and IgG2 kappa) were obtained by direct fusion of Hashimoto thyroid lymphocytes with the mouse myeloma X63-Ag.653. The autoantibodies were specific for human Tg and the functional affinities were high (only 2.6-3.9 log10 pM Tg required to give 50% inhibition of binding in ELISA). Using thyroid lymphocytes, 4 lines secreting Tg autoantibodies were obtained from 11 fusions compared with 1 line from 32 fusions of Epstein Barr virus infected blood lymphocytes, which emphasises the importance of using lymphocytes derived from a tissue known to be enriched in thyroid autoantibody secreting precursor B cells. These 4 human Tg autoantibodies, as well as an IgG2 lambda Tg antibody previously derived from Hashimoto blood B cells and an IgG4 kappa monoclonal Tg antibody present in a Hashimoto serum, were used in attempts to probe the interaction between human Tg autoantibodies and the Tg molecule (2 polypeptides of 330 KD). The binding to 125-I Tg by 3/7 murine monoclonal antibodies was inhibited (36-78%) by an IgG2 lambda and an IgG4 kappa human monoclonal Tg autoantibody, indicating an overlap between the epitopes recognised by these 3 murine monoclonal Tg antibodies and 2 monoclonal human Tg autoantibodies. None of the human Tg autoantibodies (or the murine monoclonal Tg antibodies) bound to Tg denatured by reduction and alkylation. Although the number of observations is limited, our study demonstrates that high affinity human monoclonal Tg autoantibodies, like polyclonal serum Tg autoantibodies, recognise non-linear B cell epitopes on conformationally intact human Tg.     

IgG immune complex-binding in macrophages from arthritis-susceptible and arthritis-resistant mice following collagen type II immunization

Occupancy of Fc gamma receptors (FcgammaR) by immune complexes (IC) induces secretion of various inflammatory mediators and cytokines. Therefore, knowledge of the FcR function is fundamental for understanding inflammatory processes. Here, we report an alteration in the FcR function in collagen-induced arthritis (CIA). The FcgammaR-binding activity of peritoneal macrophages from arthritis-susceptible DBA/1 mice following collagen type II (CII)/CFA immunization was assessed by Fc rosetting of SRBC opsonized with different IgG subclasses. A progressive reduction of IgG1 IC-binding was observed after immunization, and by the time of arthritis onset, the IgG1 IC-binding was abolished. Binding of IgG2a or IgG2b IC, however, was not affected. The blocked IgG1 IC-binding was reversed by a prior mild acid wash of the CIA macrophages, indicating receptor occupancy as the cause of the blocked binding. The impaired IgG1 IC-binding was associated with arthritis development, as macrophages from CII/CFA-immunized, arthritis-resistant SWR mice or DBA/1 mice, immunized with CFA alone, did not show this effect. Normal DBA/1 macrophages, blocked with a monoclonal antibody to FcgammaRIIB/FcgammaRIII, and macrophages from FcgammaRIII-deficient mice did not bind IgG1 IC, indicating FcgammaRIII as responsible for IgG1 IC-binding. Our data suggest that an increased degree of saturation of FcgammaRIII precedes the development of CIA, which is reflected by a reduced IgG1 IC-binding in macrophages of CII/CFA-immunized DBA/1 mice.     

Monoclonal anti-human IgG antibodies for quantitation of allergen-specific IgG in human sera

Eight monoclonal anti-human IgG antibodies were fully characterized and evaluated as possible reagents in solid phase radioimmunoassay for quantitating allergen-specific IgG antibody. Four monoclonal antibodies (HG24D, HG2-14, HG2-18, and HG2-25) recognize CH2 domain of human IgG and bind to human IgG fixed to microtiter plate with high affinities. These monoclonal antibodies were more suitable than polyclonal rabbit anti-human IgG antibody in Phadebas RAST for honey bee venom-specific IgG antibody. Nonspecific binding was much lower, and the slopes of standard curves were much steeper. In contrast to polyclonal antibody, the standard curve was hardly influenced by human serum IgG in sample diluent. These advantages of monoclonal antibodies that recognize CH2 domain of human IgG made it possible to quantitate egg white- and Dermatophagoides pteronyssinus-specific IgG antibodies with use of allergen disks prepared for IgE RAST. This property allows a single system to be used for measurement of IgG and IgE antibodies against clinically relevant allergens.     

Normal Human Cord Blood B Cells can Produce High Affinity IgG Antibodies to dsDNA that are Recognized by Cord Blood-Derived Anti-Idiotypic Antibodies

It is possible to identify, in Epstein-Barr virus-transformed normal human cord blood B cell populations, cells present at a low frequency that produce IgG antibodies specific for dsDNA. By cloning out these B cells as immortalized monoclonal cell lines, it could be shown that the antibodies were the products of CD5 positive B cells. Two monoclonal anti-dsDNA antibodies were derived from cell lines T52 and A7 and these were further characterized as anionic (pI ～ 6.4) IgG4k antibodies that bound with affinities of 7.18 × 10⁹ 1/mol and 3.28 × 10⁹ 1/mol, respectively, to dsDNA but did not bind to ssDNA. These affinities were similar to those of polyclonal IgG anti-dsDNA antibodies from lupus patients, which ranged from 1 × 10⁹-8.9 × 10¹⁰ 1/mol. Both T52 and A7 monoclonal anti-dsDNA antibodies were recognized by cord blood-derived IgM antibodies. These IgM antibodies were not rheumatoid factors but bound to the F(ab')2 of A7 and T52 while failing to recognize T50, which is an autologous IgG4k monoclonal antibody without specificity for dsDNA. A cloned B cell line A24 generated from the same cord blood sample as A7 produced an IgM monoclonal antibody that bound to the heavy chains of T52 and A7, but not T50 on Western blot and inhibited the binding of these antibodies to dsDNA. A7 and T52 competitively inhibited each other in their binding to the anti-idiotype A24, and A24 inhibited the binding to dsDNA of some polyclonal IgG anti-dsDNA antibodies purified from sera of lupus patients. The level of inhibition of binding of these antibodies to dsDNA was directly proportional to the levels of expression of the idiotype recognized by A24 on these antibodies. The normal human cord blood, therefore, may contain cells that form an idiotype/anti-idiotype network in which the idiotype is expressed on IgG antibodies with specificity for dsDNA and the anti-idiotype is an IgM antibody that binds to a heavy chain idiotope in such a way as to interfere with its interaction with dsDNA. The presence of a similar idiotype on some polyclonal anti-dsDNA antibodies in lupus that are similarly inhibitable by the cord blood-derived anti-idiotype raises the possibility that this network may persist in later life and perhaps become dysfunctional in systemic lupus erythematosus.     

Enhancing effect of C1q on IgG monoclonal antibody binding to hapten

We have previously demonstrated that IgG antibody binding to microfilariae of Dirofilaria immitis increased in the presence of purified C1q. The present study was designed to examine the mechanism of the C1q effect using a system with an antihapten monoclonal antibody (MoAb) and a hapten as an antigen. Microtiter plates were coated with 4-hydroxy-3-nitrophenyl-acetyl (NP)-bovine serum albumin (BSA), and mouse anti-NP MoAb (IgG) was added in the presence of C1q. The amount of IgG which bound to NP-BSA increased with the addition of C1q (p less than 0.01) when the antibody had both specificity to the antigen and ability to fix C1q. The C1q effect, examined using two anti-NP MoAbs with different affinities, was more apparent with the low-affinity antibody (LAMoAb) than with the high-affinity (HAMoAb; percent enhancement of IgG binding was 19 vs. 12%). The C1q effect on LAMoAb binding was doubled when a small amount of HAMoAb was incubated with LAMoAb. The C1q effect on IgG binding might be operative in the early phase of infection, where a small amount of high-affinity antibody and a relatively large amount of low-affinity antibody are produced in the host.     

Partial elucidation of an anti-hapten repertoire in BALB/c mice: Comparative characterization of several monoclonal anti-fluorescyl antibodies

The anti-fluorescyl repertoire in BALB/c mice was examined by producing nine hybridomas secreting antibodies with fluorescein specificity. All nine purified monoclonal anti-fluorescyl antibodies contained kappa light chains and gamma heavy chains (IgG1 and IgG2). Isoelectric focusing profiles of reduced and alkylated Ig preparations demonstrated restricted, yet relatively different spectrotypes. Collectively, the hybridomas provided a diverse range of antibody affinities as determined by several methods, including dissociation rate and ligand inhibition studies. Homogeneity of purified preparations was confirmed by dissociation rate experiments which showed that each monoclonal anti-fluorescyl preparation exhibited a single first-order off-rate. Competitive inhibition experiments with structurally related ligands indicated a high degree of fine specificity. Absorption profiles of bound fluorescein revealed distinct relative differences within the active sites of the molecules studied, and suggested the lack of any apparent correlation between affinity and wavelength maximum. Characteristics of the clones are discussed in terms of the range and prevalence of anti-fluorescyl antibody affinities and the diversity of possible binding mechanisms.     

Differences between the activities of human monoclonal IgG1 and IgG3 subclasses of anti-D(Rh) antibody in their ability to mediate red cell-binding to macrophages

Four IgG1 and three IgG3 human monoclonal antibodies specific for the blood group D(Rh) antigen were tested for their ability to mediate red cell-binding to macrophages in vitro. The IgG3 monoclonals were found to opsonize at a density of approximately 100 molecules per red cell, whereas the IgG1 antibodies were only active at a level of 10,000 molecules per cell. There was no substantial difference between the two IgG subclasses in their ability to bind to Fc receptors on macrophages and it is suggested that the more potent opsonic activity of IgG3 is the result of the relatively long hinge, leading to greater accessibility to the Fc receptor binding site on the Fc piece.     

Characterization of a novel high-affinity monoclonal immunoglobulin G antibody against the ricin B subunit

There is an urgent need for the development of a passive immunotherapy against the category B select agent ricin, a lethal ribosome-inactivating toxin composed of an enzymatic A subunit (RTA) and a single binding B subunit (RTB). To date, only one monoclonal antibody (MAb), a mouse immunoglobulin G (IgG1) against RTA called R70, has been deemed sufficiently potent in animal models to warrant further testing in humans. In this study, we have identified and characterized MAb 24B11, a murine IgG1 directed against RTB. In a Vero cell cytotoxicity assay, 24B11 was approximately two times more effective at neutralizing ricin than was R70. The equilibrium dissociation constants of 24B11 (KD = 4.2 x 10(-9) M) and R70 (KD = 3.2 x 10(-9) M) were virtually identical, suggesting that the difference in neutralization activity between the two MAbs was not due to differing affinities for the toxin. 24B11 blocked ricin attachment to galactoside receptors on primary mouse splenocytes and on the apical surfaces of human mucosal epithelial cell monolayers. Surprisingly, R70 also effectively interfered with ricin attachment to receptors on cell surfaces. Using a phage-displayed peptide library, we determined that 24B11 binds an epitope on RTB adjacent to, but not within, one of the two galactose binding domains. Finally, we demonstrate that R70 and 24B11, when combined, function synergistically to neutralize ricin in vitro, raising the possibility that these two MAbs could serve as a novel immunotherapeutic in vivo.     

Sedimentation equilibrium analysis of recombinant mouse FcRn with murine IgG1

The interaction of mouse IgG1 or IgG1-derived Fc fragment with recombinant, insect cell expressed mouse FcRn has been analyzed using sedimentation equilibrium. This results in a model for the interaction in which the two binding sites for FcRn on Fc or IgG1 have significantly different affinities with macroscopic binding constants of < 130 nM and 6 microM. This data indicates the formation of an asymmetric FcRn:Fc (or IgG1):FcRn complex which is consistent with earlier suggestions that for this form of recombinant FcRn, binding to IgG1 or Fc does not result in a symmetric 2:1 complex in which both binding sites are equivalent.