Triggering of the superoxide generation of macrophages by crosslinking of Fc gamma receptor

Crosslinking of monomeric IgG2 molecules bound to the Fc gamma receptors on the cell surface of guinea pig macrophages generated the triggering signal for the superoxide-generating system. A binding experiment indicated that macrophages have saturable binding sites for monomeric IgG2. Scatchard analysis of the binding data showed that macrophages have an average of 4 X 10(5) binding sites per cell and the association constant for the binding was 4.2 X 10(6) M-1. Binding of monomeric IgG2 to macrophages could be detected by subsequent reaction with the 125I-labeled F(ab')2 fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG2 monomer to Fc receptor did not stimulate superoxide release, further addition of the F(ab')2 fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide, and the amount released was dependent on the dose of cell-bound IgG2. When macrophages were bound with a constant dose of IgG2 monomer in the first step, the superoxide release triggered by the addition of the F(ab')2 of anti-guinea pig Fab was dependent on the dose of the F(ab')2 fragment added. These results show that crosslinking of Fc receptors triggers the superoxide generation.     

The kinetics of antibody binding to membrane antigens in solution and at the cell surface.

The reaction kinetics of 125I-labelled mouse monoclonal antibodies binding to three cell-surface antigens of rat thymocytes (Thy-1.1, W3/25) were studied. The differences between bivalent and univalent interactions were determined by using antibody in the F(ab')2 or Fab' form and by using antigen in polymeric or monomeric forms. Association rate constants (k+1), dissociation rate constants (k-1) and equilibrium constants were determined. Also, the dissociation kinetics of rabbit antibodies against rat Thy-1 antigen were studied. The major findings were as follows. (i) With F(ab')2 antibody there was no simple relationship between antigen density at the cell surface and extent of bivalent binding. Extensive univalent binding was observed unless the antibody had a high k-1 for the univalent interaction, in which case all binding was bivalent. (ii) k+1 values were similar for F(ab')2 or Fab' antibody, and for the different antibodies were in the range 0.8 x 10(5)--1.1 x 10(6) M-1.s-1. These differences were sufficient to affect the interpretation of serological assays with the different antibodies. (iii) Antibody bound bivalently dissociated much more slowly than that bound univalently. However, the k-1 values for the univalently bound antibody were sufficiently low in most cases that the lifetime of the univalent complex was similar to or greater than the time needed for the assay. Thus the results could be interpreted on the basis of irreversible reactions. The overall conclusion from the study is that for an understanding of the binding of antibody to cell-surface antigens the kinetics of the interaction are of major importance and theories based on equilibrium binding are inappropriate.     

The binding of monoclonal antibodies to cell-surface molecules. A quantitative analysis with immunoglobulin G against two alloantigenic determinants of the human transplantation antigen HLA-A2.

Monoclonal IgG1 (immunoglobulin G1) PA2.1 and MA2.1 antibodies recognize polymorphic sites of the human transplantation antigen HLA-A2. They are distinguishable because MA2.1 binds HLA-A2 and HLA-B17, whereas PA2.1 binds HLA-A2 and HLA-A28. The affinities of PA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-A28 are similar and relatively low (1.9 X 10(7) M-1). The affinities of MA2.1-Fab for HLA-A2, three HLA-A2 variants and HLA-B17 are similar and high (1.2 X 10(9) M-1). The difference in affinity is due to the rates of dissociation, which give half-times of dissociation of 290 min for MA2.1-Fab and 4 min for PA2.1-Fab. For both Fab, equilibrium measurements and kinetic determinations gave consistent estimates for affinity. When PA2.1-F(ab)2 or IgG is incubated with cells it reaches equilibrium within 3 h, with most molecules bound bivalently to the cell. Under similar conditions, MA2.1-F(ab)2 does not reach equilibrium and a significant proportion of molecules bound with one and two sites are found. For the lower-affinity antibody (PA2.1), estimates of the binding constants for one- and two-site interactions could be made. By simple Scatchard analysis the avidity of F(ab)2 or IgG is 1.3 X 10(9) M-1, giving an enhancement factor of 68 between bivalent and univalent binding. This is a measure of the equilibrium constant for the interchange between bivalent and univalent binding. Analysis of the results with more realistic models indicates that the actual value is larger (10(3)-10(4) M-1) than 68 M-1. The avidities of F(ab)2 and IgG for HLA-A2 are identical, showing the Fc does not interfere with bivalent binding to cells.     

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.     

Cross-linking of B lymphocyte Fc gamma receptors and membrane immunoglobulin inhibits anti-immunoglobulin-induced blastogenesis

The Fc portion of rabbit anti-mouse immunoglobulin (Ig) antibodies interferes with anti-Ig-induced B lymphocyte activation as measured by DNA synthesis on day 3 of culture or maturation to Ig-secreting cells in the presence of soluble helper factors on day 4 or 5. To investigate this Fc-dependent effect at an earlier stage in B cell activation, rabbit IgG anti-mouse mu-chain- or delta-chain-specific antibodies were compared with their F(ab')2 fragments for the ability to induce mouse B cells to undergo blast transformation, as defined by an increase in cell volume during the first 24 hr of culture. Both F(ab')2 anti-Ig reagents induce blast transformation, although F(ab')2 anti-mu antibodies induce a greater size change than F(ab')2 anti-delta antibodies. Whole anti-mu or anti-delta antibodies do not induce blast transformation; however, in the presence of a monoclonal anti-mouse Fc gamma receptor antibody that blocks IgG binding to Fc gamma receptors (Fc gamma R), whole anti-mu or anti-delta antibodies induce blast transformation as well as their F(ab')2 fragments. Because the anti-Fc gamma R antibody alone has no effect on blast transformation, it appears that the simultaneous binding of membrane IgM (or IgD) and Fc gamma R by whole anti-Ig antibodies prevents this early event in membrane Ig-induced B cell activation.     

Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

The Gradiflow, a preparative electrophoresis instrument designed to separate molecules on the basis of their size and charge, was used to purify antibody Fab and F(ab')2 fragments. The method described is charge based, utilizing the difference in the pI between the antibody Fab/F(ab')2 fragments and antibody Fc fragments that occur after enzyme digestion of whole antibody molecules. This method of purification was successful across a range of monoclonal and polyclonal antibodies. In particular, F(ab')2 fragments were purified from a number of mouse monoclonal antibodies (both IgG1 and IgG2a isotypes) and Fab fragments were purified from egg yolk IgY polyclonal antibodies. This is a rapid purification method which has advantages over alternative methods that usually comprise ion exchange and gel filtration chromatography. This method may be applicable to most antibody digest preparations.     

Equilibrium theory for the binding of bivalent antibodies to regularly spaced sites on a DNA molecule

In the autoimmune disease, Systemic Lupus Erythematosus, an individual produces antibodies that bind to his or her own DNA. In this paper we consider a single, long DNA-like molecule in a solution containing bivalent antibodies that can bind to the DNA molecule at regularly spaced sites. The antibody can be attached to DNA by either one or two binding sites. We assume that, when an antibody molecule binds through both its sites, it spans a fixed number of free sites that remain accessible to antibody binding. In this model, antibody molecules can interdigitate along the DNA molecule. We allow steric hindrance within such interdigitating clusters of bound antibodies. We derive analytical expressions for the average number of free, monovalently bound and bivalently bound antibodies, and see how this distribution is influenced by steric hindrance and by the relative binding strengths of the singly and doubly bound antibody.     

The Binding of Bispecific Monoclonal Antibodies to the Solid Phase-Adsorbed Antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-human IgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (K _a) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than K _afor Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90–95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5–10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Postabsorption neutralization of poliovirus.

Nineteen neutralizing murine monoclonal antibodies against poliovirus type 1, including representatives reacting with each of the antigenic sites on the virion, were tested for their abilities to neutralize the virus either before or after attachment to susceptible cells. All antibodies neutralized unattached virus; six had reasonable titers of postabsorption neutralization (PAN). Experiments with antibodies lacking PAN activity showed that Fc-specific rabbit anti-mouse antibodies could confer PAN activity. PAN was shown to involve the prevention of the cell-mediated conversion of virus to 135S and 80S particles. Evidence that one of the PAN-positive antibodies probably bound bivalently to preabsorbed virions, whereas a PAN-negative antibody bound monovalently, is presented. Two PAN-positive antibodies were added to an excess of virus in suspension, and only one antibody caused the virus to aggregate.     

Modeling bispecific monoclonal antibody interaction with two cell membrane targets indicates the importance of surface diffusion

We have developed a mathematical framework for describing a bispecific monoclonal antibody interaction with two independent membrane-bound targets that are expressed on the same cell surface. The bispecific antibody in solution binds either of the two targets first, and then cross-links with the second one while on the cell surface, subject to rate-limiting lateral diffusion step within the lifetime of the monovalently engaged antibody-antigen complex. At experimental densities, only a small fraction of the free targets is expected to lie within the reach of the antibody binding sites at any time. Using ordinary differential equation and Monte Carlo simulation-based models, we validated this approach against an independently published anti-CD4/CD70 DuetMab experimental data set. As a result of dimensional reduction, the cell surface reaction is expected to be so rapid that, in agreement with the experimental data, no monovalently bound bispecific antibody binary complexes accumulate until cross-linking is complete. The dissociation of the bispecific antibody from the ternary cross-linked complex is expected to be significantly slower than that from either of the monovalently bound variants. We estimate that the effective affinity of the bivalently bound bispecific antibody is enhanced for about 4 orders of magnitude over that of the monovalently bound species. This avidity enhancement allows for the highly specific binding of anti-CD4/CD70 DuetMab to the cells that are positive for both target antigens over those that express only one or the other We suggest that the lateral diffusion of target antigens in the cell membrane also plays a key role in the avidity effect of natural antibodies and other bivalent ligands in their interactions with their respective cell surface receptors.     

A quantitative fluorometric assay for detection and characterization of Fc receptors

A new quantitative fluorometric binding assay that uses fluoresceinated aggregated IgG is proposed for the study of Fc receptors. The method was compared with a radiolabeling binding assay on three well characterized murine cell lines (38C-13, EL4, and BW). The apparent association constant of the binding and the amount of aggregated IgG bound per cell at saturation were calculated. The fluorometric assay enables the detection of 5 X 10(-10) M bound aggregated IgG. Inhibition studies with monomeric IgG, reduced and alkylated aggregated IgG, and aggregated F(ab')2 fragments of IgG confirmed the specificity of the assay. Staphylococcal protein A inhibited the binding of the aggregated IgG to Fc receptors.     

Idiotype vaccines against human T cell acute lymphoblastic leukemia. I. Generation and characterization of biologically active monoclonal anti-idiotopes

A murine monoclonal anti-tumor antibody termed SN2 (Ab1), isotype IgG1-kappa, that defines a unique human T cell leukemia-associated cell-surface glycoprotein, gp37 (m.w. 37,000), was used to generate monoclonal anti-idiotype antibodies (Ab2) in syngeneic BALB/c mice. The Ab2 were screened on the basis of their binding to the F(ab')2 fragments of SN2 and not to the F(ab')2 of pooled normal BALB/c mice sera IgG1 or to an unrelated BALB/c monoclonal antibody of the same isotype. Fifteen Ab2, obtained from two fusions, were specific for the SN2 idiotope and not against isotype or allotype determinants. To find out whether these Ab2 are directed against the paratope of SN2, the binding of radiolabeled SN2 to leukemic MOLT-4 and JM cells which contain gp37 as a surface constituent was studied in the presence of these anti-idiotopes. Clone 4EA2 inhibited the binding 100% at a concentration of 50 ng and 4DC6 inhibited 90% at a concentration of 250 ng. A third clone 4DD6 gave about 50% inhibition. Similar was the inhibition of SN2 binding to insolubilized MOLT-4 antigen or cell membrane preparation. The binding of SN2 (Ab1) to 4EA2 and 4DC6 was also inhibited by semipurified preparation of gp37 antigen. These results demonstrate that at least two of the anti-idiotope antibodies are binding either at or near the binding site idiotope of SN2. Next, the purified Ab2 was used to immunize syngeneic mice to induce antibody binding to MOLT-4 cells or gp37. Sera from mice immunized with 4EA2 and 4DC6 coupled to keyhole limpet hemocyanin contained antibodies which bind to semipurified gp37 antigen and MOLT-4 cells. Immune sera inhibited the binding of iodinated Ab2 and Ab1 indicating that an anti-anti-idiotopic antibody (Ab3) in mice shares idiotopes with Ab1 (SN2). Also, the binding of iodinated Ab2 to Ab1 was inhibited by rabbit antisera specific for gp37. Collectively, these data suggest that anti-idiotype antibodies 4EA2 and 4DC6 may be useful in the generation of idiotype vaccines against human T cell leukemia.     

The binding of bispecific monoclonal antibodies to the solid phase-adsorbed antigens

The ability of bispecific antibodies (Babs) formed by fusion of hybridomas and parent monoclonal antibodies (Mabs) to interact with the solid phase-adsorbed antigens was studied. Mabs specific to the three different antigens [horseradish peroxidase (HRP), human IgG (hIgG), and human myoglobin (Mb)] as well as Babs with the double specificity [antimyoglobin/antiperoxidase (anti-Mb/HRP) and anti-hIgG/antiperoxidase (anti-hIgG/HRP)] were used. It was shown by radioimmunological and immunoenzyme assays that parent Mabs bind to solid phase-adsorbed antigens considerably more effectively than Babs. The observed equilibrium binding constant (Ka) of antiperoxidase parental Mabs to immobilized HRP is 21 and 38 times higher than Ka for Babs binding sites (anti-Mb/HRP and anti-hIgG/HRP, respectively) to peroxidase. It was calculated that about 90-95% of all bound parental antiperoxidase Mabs were associated with immobilized HRP bivalently, and only about 5-10% were bound monovalently. On the contrary, parental Mabs against hIgG bind to the sorbed antigen essentially only monovalently. It was also shown that the avidity of anti-Mb/HRP Babs significantly increased when two antigens, Mb and HRP, were simultaneously adsorbed on the solid phase. These data imply that Babs bearing an enzyme-binding site (for example, binding to HRP) cannot be more effective than standard conjugates (e.g., enzyme-conjugated antibodies) in heterogeneous noncompetitive immunoassays.     

Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.     

Analysis of the interaction of antibodies with immunoglobulin idiotypes on neoplastic B lymphocytes: implications for immunotherapy

We have examined the reactions of a panel of nine monoclonal anti-idiotype antibodies with the surface immunoglobulin in situ on guinea pig L2C leukemic lymphocytes. Equilibrium binding constants were shown to range between 10(7) and 10(8) M-1 for univalent Fab' gamma fragments and between 10(8) and 10(9) M-1 for intact IgG. Saturation of the cell surface binding sites was achieved with 2.9 X 10(5) Fab' gamma molecules/cell and 1.2 X 10(5) IgG molecules/cell for each antibody, a result that is consistent with a bivalent mode of interaction for the IgG. Despite these overall similarities in binding characteristics antibodies showed striking differences in their ability to clear Ig from the cell surface by antigenic modulation in vitro. This suggested differences in the readiness with which the antibodies cross-linked neighboring surface Ig molecules. Such an interpretation was supported by differences in the times required to achieve bivalent binding at 0 degree C, and in the rates at which labeled antibody dissociated from the cell surface in the presence or absence of an excess of unlabeled antibody. The data are consistent with there being two functionally distinct types of anti-idiotype antibody: those that form predominantly intra-Ig bridges, with each antibody Fab being linked to an Fab on one target molecule ("monogamous" binding) and not favoring modulation; and those that form predominantly inter-Ig bridges ("bigamous" binding) and favor modulation. The nature of interaction is presumably dictated by the orientation of the particular idiotope concerned. This distinction could be of great importance in the therapeutic use of anti-idiotype to ablate B cell neoplasms.     

Immunoglobulin G3 from polyclonal human immunodeficiency virus (HIV) immune globulin is more potent than other subclasses in neutralizing HIV type 1.

Passive antibody prophylaxis against human immunodeficiency virus type 1 (HIV-1) has been accomplished in primates, suggesting that this strategy may prove useful in humans. While antibody specificity is crucial for neutralization, other antibody characteristics, such as subclass, have not been explored. Our objective was to compare the efficiencies of immunoglobulin G (IgG) subclasses from polyclonal human HIV immune globulin (HIVIG) in the neutralization of HIV-1 strains differing in coreceptor tropism. IgG1, IgG2, and IgG3 were enriched from HIVIG by using protein A-Sepharose. All three subclasses bound major HIV-1 proteins, as shown by Western blot assay and enzyme-linked immunosorbent assay. In HIV-1 fusion assays using X4, R5, or X4R5 envelope-expressing effector cells, IgG3 more efficiently blocked fusion. In neutralization assays with cell-free viruses using X4 (LAI, IIIB), R5 (BaL), and X4R5 (DH123), a similar hierarchy of neutralization was found: IgG3 > IgG1 > IgG2. IgG3 has a longer, more flexible hinge region than the other subclasses. To test whether this is important, IgG1 and IgG3 were digested with pepsin to generate F(ab')(2) fragments or with papain to generate Fab fragments. IgG3 F(ab')(2) fragments were still more efficient in neutralization than F(ab')(2) of IgG1. However, Fab fragments of IgG3 and IgG1 demonstrated equivalent neutralization capacities and the IgG3 advantage was lost. These results suggest that the IgG3 hinge region confers enhanced HIV-neutralizing ability. Enrichment and stabilization of IgG3 may therefore lead to improved HIVIG preparations. The results of this study have implications for the improvement of passive immunization with polyclonal or monoclonal antibodies and suggest that HIV-1 vaccines which induce high-titer IgG3 responses could be advantageous.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

A monoclonal anti-type II collagen antibody with cross-reactive anti-Ig activity specific for the F(ab')2 fragment

An IgG2a hybridoma antibody (BC-10) was obtained by a myeloma fusion with lymphocytes from B10.RIII mice immunized against native bovine type II collagen. This anti-collagen monoclonal exhibited extensive cross-reactivity with several type II collagen species. BC-10 was found to have self-associating properties, but not the specificity of a typical IgG rheumatoid factor, inasmuch as this mAb bound to F(ab')2 fragments of itself and of normal mouse IgG. Self binding was inhibited by the association of BC-10 with type II collagen, and inhibition assays indicated that antibodies with the capacity to inhibit BC-10 binding to collagen were present in the sera from B10.RIII arthritic mice, but not from DBA/1 LacJ arthritic mice. Joint inflammation and histopathologic features consistent with arthritis were observed in mice injected with the BC-10 hybridoma.