7S IgG Research Articles

Systemic and ocular antibody responses to inactivated acute hemorrhagic conjunctivitis (AHC) virus; enterovirus 70 (EV70)

Purpose: Evaluate the immune response in rabbits injected with EV70, the agent of acute hemorrhagic conjunctivitis (AHC) and AHC associated neuropathy. Methods: Rabbits were injected intramuscularly with uv-light inactivated EV70 isolate J670/71. Neutralizing activity against EV70 was quantified in serum and tear samples and the immunoglobulin (Ig) classes of the neutralizing activity in serum identified by sucrose gradient ultra-centrifugation. Adjuvant muramyl dipeptide (MDP) was applied topically to assess the role of ocular inflammation on levels of neutralizing antibody, proteins and Ig in tears. The protective effects of human and rabbit sera and interferon-a (IFN-a) against EV70 were compared in human conjunctival and lens cells. Results: Sera collected at 6 and 13 d contained 19S IgM anti-EV70 neutralizing antibody, while serum collected 21 d post injection contained 19S IgM and 7S IgG anti-EV70 neutralizing antibody. Low titers of anti-EV70 activity (=30 U/ml) were detected in tears of seropositive rabbits. MDPinduction of conjunctivitis in seropositive rabbits increased tear IgG concentration (3-fold) and anti-EV70 neutralizing antibody titers (=10-fold). The protective effect of the rabbit and human sera against EV70 infection in conjunctival, but not lens epithelial cells, was enhanced by the addition of IFN-a. Conclusions: Immunization with uv-light inactivated EV70 elicits a classical humoral immune response in rabbits. The protective activity of serum in EV70- infected human conjunctival cells, but not lens cells, was increased by IFN-a. Adjuvant MDPinduced conjunctivitis, increased bloodconjunctival barrier (BCB) permeability and anti-EV70 neutralizing activity in tear of seropositive rabbits. The results suggest immunization with inactivated EV70 could provide systemic as well as ocular protection during natural EV70 infection.

Prevention of radiogenous mucositis and dermatitis in patients with advanced cancer of the maxillo facial region with intravenous application of 7S IGG

Prevention of radiogenous mucositis and dermatitis in patients with advanced cancer of the maxillo facial region with intravenous application of 7S IGG

Comparative quantitation of anti-viral titers of polyclonal human IgG and (Fab′) 2 preparations by an enzyme-linked immunosorbent assay (ELISA): Pitfalls of using appropriate second antibodies

When comparing anti-viral antibody titers of intravenous immunoglobulins (i.v.-Igs) comprising two 7S IgG preparations and one corresponding (Fab′) 2 product, the specificity of the second ab-peroxidase conjugate employed in the ELISA was found to be decisive for the experimental results. Abs specific for the whole IgG molecule, in contrast to the (Fab′) 2 specific ones, obviously contain populations recognizing epitopes localized on Cγ2 and Cγ3 domains, and, therefore, cause an underestimation of titers of the (Fab′) 2 preparation.

Immunoglobulins from the sera of immunologically activated persons with pairs of electrophoretic restricted bands show a greater tendency to aggregate than normal.

From in vitro data, it has been speculated that pairs of endogenous restricted bands migrating in close proximity in the gamma region upon high resolution serum electrophoresis (HRE) represent circulating immune complexes (CIC). Using a polyethylene glycol (PEG) method to separate CIC, we found a very high correlation between the presence of such band pairs and elevated levels of CIC (CHI2 = 25.7, p less than 0.001) in 51 sera. HRE appears to be a good screening technique to identify, with a high degree of certainty, samples with elevated levels of CIC for delineation by more specific methods. Yet, examination by gel-filtration chromatography and precipitation with PEG indicated that the molecules comprising the band patterns were not CIC, but polyclonal 7S IgG. These bands are usually found in patients with chronic activation of the immune system. Fractionation of the sera from 5 such patients with various cuts of PEG indicated that the average IgG concentration in the 2.5-5%, and 5-7.5% cuts from patients was 3.99 g/l and 2.2 g/l, while from healthy subjects the concentrations were 0.68 g/l and 2.88 g/l. This reversed precipitation pattern was seen both for absolute levels of IgG and for percent of total IgG. On the average the amount of precipitation of IgG in the 2.5-5% fraction of patients was about 5-fold above that seen in the healthy subjects. The endogenous bands were not associated with any specific cut of PEG, but appeared to be proportionally distributed in accord with the levels of IgG. The data is consistent with the idea that immunologically activated patients exhibit a greater tendency for immunoglobulins to associate than normal. This propensity to aggregate may cause CIC to form in situ in local compartments even though CIC do not appear to be present upon analysis by biochemical techniques.

Evidence for the presence of autoantibodies to the collagen-like portion of C1q in systemic lupus erythematosus.

We investigated the connection between the C1q solid-phase binding assay (C1q SPBA) and double-stranded DNA antibodies, and analyzed the immune complex material in systemic lupus erythematosus (SLE) sera. Comparison with a new monoclonal assay for C1q-bearing immune complexes (the 242G3 assay) revealed that the immune complexes in SLE bind specifically to solid-phase C1q, and not to fluid-phase C1q. The C1q solid-phase binding activity sedimented as 7S IgG, was insensitive to DNase treatment, and could be selectively absorbed by C1q-coupled beads and by bovine serum albumin-anti-bovine serum albumin C1q beads, but not by DNA. Thus, antibodies to double-stranded DNA do not interfere in the C1q SPBA. Isolated IgG from SLE serum precipitated the collagen-like portions, and not the globular, Fc-recognizing portions, of C1q. F(ab')2 fragments of IgG from SLE patient serum were able to bind C1q. These data show that in SLE sera, especially in those with low levels of CH50 and C1q, autoantibodies that react with the collagen-like part of C1q are detectable. Since in the C1q SPBA, the C1q molecule is randomly fixed to the solid phase, we can detect not only immune complexes, but also antibodies that react with the collagen part of C1q; this may explain the high percentage of positive results for SLE sera in the C1q SPBA, in contrast to results of other immune complex assays.

19S IgM rheumatoid factor-7S IgG rheumatoid factor immune complexes isolated in sera of patients with juvenile rheumatoid arthritis.

19S IgM rheumatoid factors (RF) and hidden 19S IgM RF have been associated with increased disease activity in juvenile rheumatoid arthritis (JRA). Recently, immune complexes (IC) were isolated from JRA sera by several methods which demonstrated the presence of 19S IgM RF. The present study evaluates 25 JRA patients' sera by separation on a Sepharose 4B column to which were bound F(ab')2 fragments of goat IgG antihuman IgM antibody to separate IgM-containing IC. The columns were sequentially eluted with 1 M ammonia and 0.1 M glycine-HCl buffer, pH 3.0. The isolated fractions were assayed for 19S IgM RF and 7S IgM RF by ELISA, IgG levels by immunodiffusion, and by preparative isoelectric focusing. The ammonia eluate from the alpha HIgM column revealed IgG, 19S IgM RF in six patients, and IgM RF in four patients. All were polyarticular-onset JRA patients. In the glycine-HCl eluate of sera, 19S IgM RF and IgG were also detected in 15 patients, all six seropositive, polyarticular-onset, six seronegative, polyarticular-onset, and three pauciarticular-onset patients. Significant 7S IgG RF titers were demonstrated in the glycine-HCl eluates of six patients, five seropositive, polyarticular-onset patients, and one seronegative, polyarticular-onset patient. Analysis by preparative isoelectric focusing of the IgM RF and IgG RF positive ammonia and glycine-HCl eluates showed IgM RF throughout the pH range (4-10), but the highest amount of IgM RF was in the pH range 4.0-5.5. Significant IgG RF titers were detected only in this restricted spectrotypic area of pH 4.0-5.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombocytopenic purpura in narcotics addicts.

Since November 1982 we have seen an association of thrombocytopenic purpura with chronic narcotic addiction in 70 patients with a mean platelet count of 53000 +/- 4000 (SE); 33 had stopped taking intravenous drugs for an average of 21.2 +/- 4.7 months; 13 of 15 had elevated antibody titers for a virus related to the acquired immunodeficiency syndrome. Platelet-bound IgG, IgM, and complement levels were 16.7-, 5.6-, and 3.1-fold greater than control values, respectively, and 2.6-, 1.9-, and 2.4-fold greater than values in 25 patients with classic autoimmune thrombocytopenic purpura studied at the same time. Thirty-three of thirty-six addicts had elevated circulating immune complexes, whereas 8 patients with autoimmune thrombocytopenia had no elevation. Eleven of eighteen addicts had positive serum platelet-reactive IgG titers, compared to 5 of 19 patients with classic autoimmune thrombocytopenia. The platelet-reactive IgG in sera of addicts was composed of 7S IgG antibody as well as high molecular weight (immune complex) IgG. Thus, chronic addicts appear to have a new immunologic platelet disorder associated with the presence of 7S IgG antiplatelet antibody, like patients with classic autoimmune thrombocytopenic purpura, and immune complex associated nonspecific platelet IgG, like male homosexual patients with thrombocytopenia.

Isolation of Fab and Fc Fragments from a Plasmin-Treated Human Immunoglobulin by High-Speed Gel Filtration on TSK G3000SW and G3000SWG

A method is described for isolating intact 7S IgG, Fab and Fc fragments from a plasmin-treated immunoglobulin by high-speed gel filtration on a TSK G3000SW or G3000SWG column. The isolated 7S IgG, Fab and Fc fragments reacted with antihuman IgG, Fab and Fc antiserum, respectively, in both Ouchterlony’s double immunodiffusion and immunoelectrophoresis. Each of the Fab and Fc fragments formed a single precipitation line, demonstrating their homogeneity. Anticomplementary activities of intact 7S IgG, Fab and Fc fragments were 2.5, ≤10 and 2.5 mg protein/ml to inhibit 2 units of CH(50), respectively, and the diphtheria antitoxin contents were 2.1, 6.0 and 1.5 IU/150 mg protein, respectively. The molecular composition of plasmin-treated immunoglobulin as determined by gel filtration on a TSKG3000SW column was as follows: 7S IgG 38.0 ± 0.2%; Fab 42.6 ± 0.1% and Fc fragment 19.3 ± 0.1%.

Isolation of Fab and Fc fragments from a plasmin-treated human immunoglobulin by high-speed gel filtration on TSK G3000SW and G3000SWG.

A method is described for isolating intact 7S IgG, Fab and Fc fragments from a plasmin-treated immunoglobulin by high-speed gel filtration on a TSK G3000SW or G3000SWG column. The isolated 7S IgG, Fab and Fc fragments reacted with antihuman IgG, Fab and Fc antiserum, respectively, in both Ouchterlony's double immunodiffusion and immunoelectrophoresis. Each of the Fab and Fc fragments formed a single precipitation line, demonstrating their homogeneity. Anticomplementary activities of intact 7S IgG, Fab and Fc fragments were 2.5, less than or equal to 10 and 2.5 mg protein/ml to inhibit 2 units of CH50, respectively, and the diphtheria antitoxin contents were 2.1, 6.0 and 1.5 IU/150 mg protein, respectively. The molecular composition of plasmin-treated immunoglobulin as determined by gel filtration on a TSK G3000SW column was as follows: 7S IgG 38.0 +/- 0.2%; Fab 42.6 +/- 0.1% and Fc fragment 19.3 +/- 0.1%.

IgG rheumatoid factors and cryoglobulins in mice bearing the mutant gene lpr (lymphoproliferation).

The autosomal recessive mutant gene, lpr (lymphoproliferation), produces a massive proliferation of T cells in autoimmune MRL mice. Since the MRL strain bearing the lpr mutation is the only strain that spontaneously develops high titers of rheumatoid factors (RF), we have investigated whether non-autoimmune mice (C57BL/6, C3H/HeJ and AKR) bearing the lpr gene could produce RF. Serum levels of RF activity were assessed by a new radioimmunoassay, in which serum samples pretreated with acetate buffer were incubated with 125I-mouse IgG and precipitated with 7% polyethylene glycol. The majority of serum from 4- to 6-month-old non-autoimmune strains of mice bearing the lpr gene exhibited significant RF activity, as did MRL-lpr/lpr mice. Sucrose density-gradient analysis has revealed that all the IgG RF activity was present in a form of immune complexes, sedimenting in the intermediate position (7-19S) and fully dissociable into 7S IgG under an acid condition. This indicates that the production of IgG RF does not require a particular background genome of the MRL strain. In addition, mice bearing the lpr mutation developed extremely large amounts of cryoglobulins, which paralleled the production of RF. Analysis of components of their cryoglobulins revealed a marked enrichment of IgG3 subclass as compared to other IgG subclasses and IgM. These results suggest that IgG3-containing immune complexes represent the major source of cryoglobulins occurring in mice bearing the lpr gene.

Efficacy of intravenous immunoglobulin preparations against viral and bacterial infections in mouse protection tests.

Our investigation indicates that pretreatment of human immunoglobulin for the elimination of the anticomplementary activity is associated with a loss of activity, the extent of which depends on the type of treatment applied. Laboratory preparations of human IgG were tested in a mouse protection assay using influenza A2-Taiwan virus, tetanus toxin and Salmonella typhimurium as the challenge. There was a 7-28% reduction in efficacy in an intravenous 7S preparation in comparison with an untreated 7S IgG. F(ab')2 fragments showed a 24-65% and Fab fragments an 80-100% reduction in efficacy. Two commercial human 7S products showed approximately 90% efficacy in the Salmonella assay; a commercial, pepsin-treated preparation showed 65-74% efficacy when compared with untreated 7S IgG.

Rheumatoid factors

Human rheumatoid factors are antibodies of IgG, IgA, or IgM class that show reactions with antigenic determinants present on other immunoglobulin molecules. The most commonly measured rheumatoid factor relates to the 19S IgM type, which reacts by agglutination of latex particles coated with 7S IgG and is often measured in the standard latex fixation test. Approximately 65 to 70 per cent of patients with rheumatoid arthritis show positive serologic tests for rheumatoid factor; however, a number of other chronic disease conditions are also associated with positive rheumatoid factor reactions, including infective endocarditis, sarcoidosis, leprosy, and other hyperglobulinemic conditions. Although extensive serologic and immunochemical studies have identified a number of specific antigenic structural sites on immunoglobulin molecules that react with rheumatoid factors, recent studies have shown that a certain proportion of such antibodies may show cross-reactivity with DNA-histone complexes as well. It is still not entirely clear how rheumatoid factors fit into the pathogenesis of rheumatoid arthritis itself.

Selective action of D-penicillamine on guinea pig peritoneal macrophage Fc γ receptors for homologous, monomeric IgG 1

Guinea pig oil-induced peritoneal exudates were pretreated with D-penicillamine. The binding of homologous, monomeric IgG 2 and IgG 1 to normal and pretreated exudates was examined. Whereas the 7S IgG 2-IgG 2 Fc γ receptor interaction remains unaffected by the pretreatment, the binding of IgG 1 to the IgG Fc γ receptor population was affected. The significance of the selective effect of D-penicillamine is discussed.

C1q and C3bi binding activities of the components of a plasmin-treated human immunoglobulin preparation.

Each of the three major components isolated from a commercial plasmin-treated human immunoglobulin preparation, namely, the plasmin-resistant 7S IgG fraction (PRG), Fab fragment and Fc fragment, was tested before and after heat treatment for binding C1q and fixing C3bi. In unheated state, only PRG was found to bind C1q, whereas none bound C3bi. The binding of C1q by PRG was enhanced by heat treatment which also conferred the activity of binding C3bi to PRG and to Fc fractions, From these results, anticomplementary activity of unheated PRG fraction seems to be due mainly to the complement activation via the classical pathway, whereas the activation by the heat-treated Fc fragment might be via an alternative pathway.

Isolation of human IgA utilizing protein A affinity chromatography.

Human serum IgA was isolated employing a system of G-200 column chromatography, anion exchange (DE-52) chromatography, and passage over protein A and anti-IgG Sepharose 4B columns. Polyacrylamide gel electrophoresis (PAGE) analysis of the isolated immunoglobulin revealed polypeptides corresponding to alpha and light immunoglobulin chains of IgA which were identified immunochemically as IgA. Ultracentrifugation studies revealed that the isolated immunoglobulin co-migrated with 7S IgG markers. This procedure may serve as an alternative to classical isolation procedures of IgA from human serum.

Antibody Spectrum and Anticomplementary Activity of a Plasmin-Treated Human Immunoglobulin Preparation

A commercial plasmin-treated human immunoglobulin was studied for its antibody spectrum and anticomplementary activity. Except for a decrease in anti-vaccinia antibody titers in two of three paired samples collected before and after plasmin treatment, there was practically no fall in titers of diphtheria antitoxin and of antibodies to measles, rubella and mumps viruses. Among the three major fractions isolated from the preparation, the plasmin-resistant 7S IgG fraction showed the highest anticomplementary activity when tested by our routine method described in the ‘Japanese Minimum Requirements’. The Fab fraction showed moderate anticomplementary activity which was thought to be due to the selective consumption of C3. On the contrary, the Fc fraction was not found to be anticomplementary in our test method. In some of the above-mentioned features, the preparation seems to be different from the domestic product formerly studied by ourselves.

Antibody spectrum and anticomplementary activity of a plasmin-treated human immunoglobulin preparation.

A commercial plasmin-treated human immunoglobulin was studied for its antibody spectrum and anticomplementary activity. Except for a decrease in anti-vaccinia antibody titers in two of three paired samples collected before and after plasmin treatment, there was practically no fall in titers of diphtheria antitoxin and of antibodies to measles, rubella and mumps viruses. Among the three major fractions isolated from the preparation, the plasmin-resistant 7S IgG fraction showed the highest anticomplementary activity when tested by our routine method described in the "Japanese Minimum Requirements'. The Fab fraction showed moderate anticomplementary activity which was thought to be due to the selective consumption of C3. On the contrary, the Fc fraction was not found to be anticomplementary in our test method. In some of the above-mentioned features, the preparation seems to be different from the domestic product formerly studied by ourselves.

Determinants of Susceptibility and Resistance to Feline Leukemia Virus Infection. II. Susceptibility of Feline Lymphocytes to Productive Feline Leukemia Virus Infection<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The interplay between feline leukemia virus (FeLV) and feline lymphocytes (lc) infected in vitro or in vivo was investigated. Surface marker analysis and viral infectivity (VI) assays of lc populations were used to determine susceptibility of lc subsets to FeLV. The principal FeLV-replicating cell in the mesenteric lymph node of persistently infected, preleukemic cats was a nonadherent, complement receptor (CR)-bearing lc (B-cell). The lymph nodes of preleukemic cats also had increased numbers of uninfected T-cells [cells forming rosettes with guinea pig erythrocytes (GPE)] and cells with receptors for the Fc portion of 7S IgG (Fc gamma R cells) as compared with lymph nodes of age-matched specific-pathogen-free (SPF) cats. The induction of productive infection of feline peripheral blood mononuclear leukocytes (PBL) in vitro depended on a 48-hour in vitro preincubation period before virus exposure. The equivalent susceptibility of whole and adherent cell-depleted PBL to productive infection and the failure of hydrocortisone to enhance viral infection were compatible with identification of the FeLV-replicating cell as an lc. Furthermore, lc from susceptible SPF kittens replicated 50 times as much FeLV as did lc from resistant adult SPF cats. The Ic productively infected with FeLV after in vitro exposure were more precisely identified with the use of Ficoll-Isopaque density gradient separations of rosetted and nonrosetted lc. Whole PBL, GPE rosette-positive PBL (T-cells), and CR-positive PBL (B-cells) were permissive to FeLV infection, and maximal VI was evident at 14 days after exposure. The substantial (1,325-fold) increment in VI found in the Fc gamma R-depleted PBL suggested a role for Fc gamma R cells in the containment of FeLV infection. Unstimulated mononuclear leukocytes from blood, spleen, lymph node, thymus, and marrow were susceptible to productive FeLV infection after in vitro exposure. The degree of spontaneous DNA synthesis in marrow, thymus, and spleen but not lymph node or PBL was inversely related to permissiveness to viral replication. Mitogen activation of lc was associated with decreased viral replication when either T-cell mitogens (concanavalin A, phytohemagglutinin, or pokeweed mitogen) or a B-cell mitogen (dextran sulfate) was used. Virus production by spleen cells and PBL was enhanced twofold to tenfold by prior lc stimulation by the B-cell mitogen, lipopolysaccharide, or protein A-bearing Staphylococcus aureus, a mitogen for feline T-cells with Fc gamma R. Both productively infected (preincubated) and nonproductively infected (freshly isolated) PBL transferred infectious FeLV to autochthonous peritoneal macrophages (M theta); most of the virus in PBL-peritoneal M theta cocultures was produced by adherent cells, irrespective of whether the adherent or nonadherent cell population was inoculated originally.

Colony formation by subpopulations of human T lymphocytes. II. Characteristics of colony cells and colony suppressor cells.

Phytohaemagglutinin-induced human T-lymphocyte colony formation in semisolid agar culture is the property of erythrocyte rosette-forming cells (E-RFC) negative for the 7S IgG receptor (FcR-). E-RFC positive for the 7S IgG receptor (FcR+), on the other hand, exhibit a limited capacity for colony formation and suppress colonies formed by FcR- E-RFC. T colonies are composed of small lymphocytes and lymphoblasts, the vast majority being negative for the Fc receptor. Most colony cells (86%) carry the Leu 3a antigen, suggesting that they belong to the inducer/helper T-cell subset. FcR+ colony suppressor cells are small, slowly sedimenting cells (sedimentation velocity less than 3.8 mm/h) and are strongly adherent to plastic, and their activity depends on the ability to synthesize DNA.

Sequential changes in serum thyroglobulin (Tg) and its autoantibodies (TgAb) following subtotal thyroidectomy of patients with preoperatively detectable TgAb.

The changes in serum concentrations of thyroglobulin antibodies (TgAb) were investigated in eight patients with various thyroid disorders before, during and after thyroid surgery. TgAb was measured by a radioassay using 125I-thyroglobulin. In six patients the TgAb decreased rapidly during operation. Of these patients four had low concentrations pre-operatively, and the decrease in TgAb resulted in undetectable concentrations for more than 2 days, Serum thyroglobulin (Tg), measured in the specimens with undetectable TgAb, i.e immediately following surgery, showed concentration vs time curves resembling curves from patients with no antibody present. Furthermore, two of these patients had such a high concentration of TgAb pre-operatively that the initial Tg release was not sufficient to remove the TgAb, resulting in detectable TgAb throughout the investigation period. Serum samples drawn during and after operation were chromatographed according to molecular size, showing measureable Tg corresponding to 19S Tg. TgAb was only measureable in fractions corresponding to 7S IgG. The expected presence of complexed Tg and TgAb could not be demonstrated, but the analytical methods might have been unable to reveal this. The remaining two patients with thyroiditis showed no decrease in TgAb. This was compatible with a low content of Tg in the throid measured in one of them.