anti-F Ab Research Articles

Pre-existing canine anti-IgG antibodies: implications for immunotherapy, immunogenicity testing and immunoassay analysis

One of the most enigmatic features of humoral immunity is the prevalent presence of circulating autoantibodies against IgG. These autoantibodies consist of several subsets, including rheumatoid factors, anti-Fab/anti-F(ab′)2-autoantibodies, and anti-idiotypic antibodies. Anti-IgG autoantibodies can impair the safety and efficacy of therapeutic antibodies and interfere with immunogenicity tests in clinical trials. They can also cross-react with allospecific IgG, presenting as heterophilic antibodies that interfere with diagnostic immunoassays. Owing to these factors, recent years have seen a resurgent interest in anti-IgG autoantibodies, but their underlying clinical significance, as well as biological roles and origins, remain opaque. Increased knowledge about canine anti-IgG autoantibodies could facilitate the development of canine immunotherapies and help in understanding and counteracting immunoassay interference. This study investigated the clinical significance and interconnection of heterophilic antibodies, anti-Fab, and anti-F(ab′)2-autoantibodies in dogs. We performed a 2-year prospective follow-up of dogs with heterophilic antibodies and analyzed serum for anti-Fab and anti-F(ab′)2-autoantibodies. Canine heterophilic antibodies can persist for at least 2 years in serum. A widespread occurrence of anti-Fab and anti-F(ab′)2-autoantibodies was found, with reactivity to cryptic epitopes in the IgG hinge region and sporadic cross-reactivity with mouse IgG. Canine anti-Fab and anti-F(ab′)2-autoantibodies are thus potential sources of clinical immunogenicity and immunoassay interference.

Human IgG anti-F(ab′)2 antibodies possess rheumatoid factor activity

Individual preparations of affinity purified anti-F(ab')2 antibodies and anti-Fc antibodies isolated from the sera of patients with rheumatoid arthritis (RA), were examined for reactivity with the Fab and Fc fragments of human IgG. Western blot assays demonstrated specific interaction of affinity-purified anti-Fab antibodies with both Fab and Fc molecules. Approximately one-half of the anti-Fab antibody preparations studied contained IgG antibodies reactive with Fab and Fc fragments in ELISA, suggesting the existence of naturally occurring epibody-like autoantibodies in these patients. Thirteen of 14 affinity-purified anti-Fc antibody preparations contained IgG cross-reactive with Fab molecules in ELISA. Double-adsorption assays on affinity columns demonstrated that a minimum of 14%, and possibly as much as 50%, of the IgG anti-Fab antibodies reacted with the Fc of IgG. Conversely, a minimum of 12%, and possibly as much as 70%, of the IgG anti-Fc antibodies reacted with IgG Fab molecules. Anti-Fab antibodies isolated from non-RA individuals also exhibited anti-Fc reactivity in ELISA, demonstrating the presence of these dual-reactive antibodies in other autoimmune and normal individuals. These studies establish the presence of naturally occurring IgG autoantibodies reactive with both the Fab and Fc fragments of human IgG. Their existence emphasizes the potential of anti-immunoglobulin antibodies to recognize a multiplicity of antigens, possibly including other members of the immunoglobulin supergene family.

Pretransplant serum IgA concentration and IgA–anti–fab autoantibody activity in living donor renal transplant recipients: a pilot study

Pretransplant serum IgA concentration and IgA–anti–fab autoantibody activity in living donor renal transplant recipients: a pilot study

Evidence That Tumor Necrosis Factor α (TNF)-Induced Activation of Neutrophil Respiratory Burst on Biologic Surfaces Is Mediated by the p55 TNF Receptor

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF-induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF-induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr-9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin-coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.

Evidence that tumor necrosis factor alpha (TNF)-induced activation of neutrophil respiratory burst on biologic surfaces is mediated by the p55 TNF receptor

Polymorphonuclear leukocytes (PMN) residing on biologic surfaces respond with a vigorous respiratory burst when exposed to tumor necrosis factor alpha (TNF). PMN possess both the p55 and the p75 TNF receptors, but their role in the elicitation of the respiratory burst is not known. We addressed this problem by studying the effect of monoclonal antibodies (MoAbs) directed against the p55 TNF receptor (MoAb H398 and MoAb htr-9) and the p75 TNF receptor (MoAb utr-1) on TNF- induced production of O2- by PMN residing on fibronectin-coated surfaces. Neither the anti-p55 nor the anti-p75 MoAbs affected TNF- induced O2- production despite their known ability to competitively inhibit TNF binding to the corresponding receptor. Experiments with the antibodies alone showed that the anti-p55 MoAbs directly triggered PMN O2- production, whereas no response was elicited by the anti-p75 MoAb. PMN unresponsiveness to the anti-p75 MoAb could not be ascribed to low expression of p75 receptor, because binding of the anti-p75 MoAb utr-1 to PMN was, indeed, even higher than binding of the anti-p55 MoAb htr- 9. The agonistic activity of the anti-p55 MoAbs was comparable with that of TNF and was not or only minimally modified by the simultaneous presence of TNF. Triggering of the respiratory burst by TNF was completely prevented by Fab fragments of the anti-p55 MoAb H398. Moreover, the monovalent Fab fragments, which lacked any stimulatory effect on PMN O2- production, acquired strong agonistic activity on cross-linking with anti Fab antibodies, suggesting that the ability of the anti-p55 antibodies to stimulate PMN O2- production depends on their ability to cross-link the TNF receptors. The agonistic effect of the anti-p55 MoAbs was only observed with cells residing on fibronectin- coated surfaces and not with cells in suspension, and in terms of kinetics, dependence on beta 2 integrin-mediated adherence, microfilament integrity, and sensitivity to elevations of intracellular levels of cAMP, it was virtually indistinguishable from the agonistic effect of TNF. Taken together, these results suggest that the p55 receptor is responsible for TNF-induced triggering of the respiratory burst of PMN residing on biologic surfaces.

Initiation of DNA synthesis in murine B cells is independent of early changes in the cytosolic free calcium

The relationship between changes in the intracellular free Ca 2+ concentration, [Ca 2+] i, and the initiation of proliferation of murine B cells after the addition of mitogens and activators was studied. The effects of lipopolysaccharide (LPS), 12- O-tetradecanoyl phorbol-13-acetate (TPA), rabbit IgG antimouse Fab (IgG RAM Fab), and its F(ab′) 2 fragment (F(ab′) 2 anti-Fab) on the [Ca 2+] i were measured using the fluorescent calcium indicator Fura-2. In parallel experiments, DNA and/or RNA synthesis were measured by assaying [ 3H]thymidine and/or [ 3H]uridine uptake. LPS stimulated a 20–120 × increase in the [ 3H]thymidine uptake, and a 3–7 × increase in [ 3H]uridine uptake without inducing any change in the [Ca 2+] i. TPA induced a marginal increase in [ 3H]thymidine and [ 3H]uridine uptake, without effecting any change in the [Ca 2+] i. In contrast, low doses of IgG RAM Fab produced a triphasic change in the [Ca 2+] i, but had no effect on the [ 3H]thymidine or [ 3H]uridine uptake, even at much higher concentrations. Similarly, low doses of the F(ab′) 2 fragment induced sizable increases in the [Ca 2+] i without affecting the [ 3H]nucleoside uptake. However, higher concentrations of F(ab′) 2 anti Fab increased the [ 3H]thymidine uptake and [ 3H]uridine uptake, while also increasing the [Ca 2+] i. Significantly, pretreating the cells with TPA for 3 min virtually abolished the [Ca 2+] i increase induced by IgG RAM Fab while simultaneously potentiating an increase in the IgG RAM Fab-induced [ 3H]thymidine uptake 85-fold. In the presence of TPA, IgG RAM Fab also induced a 2- to 30-fold increase in [ 3H]uridine uptake. Similarly, TPA virtually abolished the [Ca 2+] i increase induced by the F(ab′) 2 anti-Fab fragment, yet it stimulated a F(ab′) 2 anti-Fab-induced uptake of [ 3H]thymidine and [ 3H]uridine by 120 and 10 times, respectively.

Anti-filamin and -vinculin antibodies in sera from patients with myasthenia gravis and polymyositis.

The activity of anti-filamin (F) and anti-vinculin (V) antibodies (Ab) was determined in sera from patients with myasthenia gravis (MG), polymyositis (PM) and Duchenne muscular dystrophy (DMD). Compared with normal controls, activities of both antibodies were significantly (p<0.005) higher in sera from patients with MG and PM, especially with a marked increase in anti-F Ab in ocular myasthenia. In sera from patients with DMD, both activities were slightly higher. Filamin and vinculin are known to be enriched at the periphery of the Z-discs and in the postsynaptic structures of the neuromuscular junction in skeletal muscle. This suggests that these autoantibodies may play some role in the pathogenesis of MG and PM.

Antigenic and structural characteristics of an immunoglobulin-related peptide purified from marmoset T-lymphoma cells

A 7800 M r peptide, apparently derived from a 70,000 M r immunoglobulin-related protein synthesized by a marmoset T-lymphoma cell line, bears immunoglobulin-related determinants apparently contained in a disulfide-bonded loop comparable to that of the V H fragment of classical H chain. A membrane-enriched, crude particulate fraction was prepared from marmoset T-lymphoma cells, and peptides generated by papain digestion of the particulate fraction were selectively purified by affinity chromatography using IgG purified from serum of a goat immunized with the Fab fragment of a human Waldenstrom macroglobulin. The smallest of the affinity-purified peptides was particularly reactive with unfractionated goat anti Fab (GαFab) serum, purified IgG from GαFab serum, and with antibodies affinity purified from GαFab serum using Waldenstrom macroglobulins. Partial specificity of GαFab antibodies was determined by competition radioimmunoassay and radioimmunoprecipitation assays. High-performance reverse-phase liquid chromatography was used for final purification of the smallest peptide which retained its reactivity with GαFab serum and affinity-purified GαFab antibodies. The apparent molecular weight of the peptide, determined by gel filtration in 6 M guanidine, was estimated at 3900 in its native state but increased to 7800 after reduction and alkylation, indicating a role for a disulfide bond in the tertiary structure of the peptide. The amino acid composition of the peptide was presented, and its possible relationship to immunoglobulin-like T-cell surface proteins was discussed.

Antigen-, anti-F(ab')2- and anti-IgE-induced histamine release from rat mast cells.

The existence of IgE antibody molecules on peritoneal mast cells from rats immunized with dinitrophenylated ascaris extract was demonstrated by double antibody immunofluorescence staining and by histamine release induced by anti-IgE antibodies. Cross-linking of the IgE molecules, which are fixed to mast cells, by interaction with either the homologous antigen or with antibodies to the Fab or Fc regions of IgE, triggered in the presence of calcium ions a chain of intracellular reactions which involve cyclic nucleotide modulation and energy-requiring processes leading ultimately to the release of histamine from the granules of these cells. Although there was no apparent difference in the mechanism(s) underlying the reactions triggered by any of these three agents, the extent of histamine release caused by antibodies to the Fc region of the IgE was significantly lower than that induced by cross-linking of IgE molecules through their Fab regions by reactions with either anti-Fab antibodies or antigens.

The Demonstration of Plasmin Agglutinators in Human Sera

Plasmin agglutinators are natural IgG anti Fab antibodies. These antibodies bind to erythrocytes which are coated with the Fab fragments of anti-Rh antibodies that have been hydrolyzed with plasmin. The plasmin agglutinators are demonstrable in most human sera albeit in low titer. These agglutinators can be inhibited with Fab fragments that have been hydrolyzed with subtilisin, bromelin, trypsin, and plasmin but cannot be inhibited by Fab fragments from pepsin, ficin, chymotrypsin, papain or elastase digests. The Fab fragments from naturally fragmented immune serum globulin gave identical inhibition reactions to those produced by plasmin hydrolysis.