IgM Fractions Research Articles

Serodiagnosis of acute toxoplasmosis in macropods.

The sera of 34 Australian macropods, the brains of which had been bioassayed for Toxoplasma gondii, were used to establish that a titre greater than 1/32 was significant for a direct agglutination test against toxoplasmosis. In addition, the concentration of 2-mercaptoethanol required to destroy the IgM fraction of macropod serum was confirmed in a modified direct agglutination test. To further validate the tests, the serological responses of three eastern grey kangaroos (Macropus giganteus) dosed orally with T. gondii oocysts and one M. giganteus injected with T. gondii cysts were studied. The tests were then used to investigate a diagnosis of acute toxoplasmosis in four Tasmanian pademelons (Thylogale billardierii) clinically suspected of acquiring toxoplasmosis naturally. One hundred and fifty-one Bennett's wallabies (Macropus rufogriseus rufogriseus) and 85 T. billardierii were also tested to determine the prevalence of acute toxoplasmosis of macropods in the wild. Four percent of M. r. rufogriseus and 1.2% of T. billardierii possessed T. gondii-specific IgM in their sera.

Isolation of a natural cytotoxic IgM "antibody" in human serum sensitizing L cells to complement.

An IgM fraction of human serum was isolated and purified. A portion of this fraction firmly attaches to L cells' surfaces, which sensitizes these cells to the lytic action of low concentrations of serum C. It contains the natural cytotoxic "antibody" to L cells.

CIRCULATING MONOCYTE (MACROPHAGE)‐SPECIFIC ANTIBODIES IN PATIENTS WITH AUTOIMMUNE THYROID DISEASES

We investigated the presence of circulating monocyte-specific antibodies (monocytotoxic activities) by a complement-dependent cytotoxicity test and the relations between these monocytotoxic activities and other immunological indices in patients with autoimmune thyroid diseases. Antibodies reactive for monocytes (macrophages) were found in the sera from patients with autoimmune thyroid diseases. These antibodies were present in both IgG and IgM fractions and specific for monocytes since they were absorbed by monocytes but not by lymphocytes or granulocytes; furthermore, lymphocytotoxic and granulocytotoxic activities were not changed after the absorption of the sample sera by monocytes. Also, these antibodies did not have cross-reactivity to thyroid-specific antigens demonstrated by absorption tests and their specificity was different from anti HLA-DR antibody demonstrated by a flow cytofluorometric analysis. Monocyte-specific antibodies are reactive for autologous monocytes as well as allogenic monocytes. Patients who had positive monocytotoxic activities had high levels of TSH receptor antibodies (TRAb) and antimicrosomal antibodies in Graves' disease, and monocytotoxic activities were significantly correlated with the levels of TRAb in Graves' disease. These results suggest that the monocyte-specific antibodies (monocytotoxic activities) were significantly correlated with the immunological activities in Graves' disease.

Studies of the protective effect of different fractions of sera from pigs immune to Streptococcus suis type 2 infection

Inoculation of mice with high doses of Streptococcus suis type 2 caused depression, anorexia, lethargy and sometimes death. This was prevented by prior inoculation of whole immune pig serum, the IgM and IgG fractions of immune pig serum and serum enriched with antibody to surface antigens. Clinical signs were not prevented by prior inoculation of pre-immune serum or serum depleted of antibody to surface antigens. Serum fractions that were protective in vivo were also opsonic in vitro. Western blot analysis identified two antigens of 44 and 78 kd that were recognized strongly by protective sera and a further four of 86, 94, 130 and 136 kd that were recognized less intensely.

Tonsillectomy-induced changes in anti-keratin antibodies in patients with pustulosis palmaris et plantaris: a clinical correlation

Titers of anti-keratin antibodies (AKA) involving IgG and IgM fractions were determined by a solid-phase enzyme-linked immunsorbent assay (ELISA) and were found to be significantly higher on average in patients with pustulosis palmaris et plantaris (PPP) than in normal controls. Positive rates of IgG-AKA and IgM-AKA in sera of PPP patients were 50% and 66%, respectively. Following tonsillectomies, patients with high IgM-AKA titers that decreased after surgery were found to benefit by a marked improvement in their skin lesions. There was a close relationship between the degree of this improvement in the skin lesions and the changes in IgM-AKA titers after the operation. These results suggest that PPP may be a tonsil-related immunological skin disease.

Indirect enzyme-linked immunosorbent assay for immunoglobulin G and four immunoassays for immunoglobulin M to Toxoplasma gondii in a series of heart transplant recipients.

Toxoplasma gondii infections in heart transplant recipients were monitored by indirect enzyme-linked immunosorbent assay for immunoglobulin G (ELISA-IgG), indirect ELISA-IgM in serum IgM fractions, antibody capture ELISA-IgM, IgM-immunosorbent agglutination assay (ISAGA), and IgM immunoblotting. Basic immunosuppression consisted of cyclosporine and low-dose steroids. Before transplantation, 26 of 43 recipients showed serological evidence of infection. In serum samples from 15 (35%) recipients, specific antibodies were not detected. Approximately 50% of the heart donors, were toxoplasma seropositive. Eight of the fifteen seronegative recipients received hearts from toxoplasma-seropositive donors. In four of the eight recipients, seroconversion could be demonstrated with all tests used. In three of these four patients, clinical disease developed. One patient with strong serological evidence of toxoplasmosis died, but toxoplasma parasites and antigens were not detected at autopsy. In two patients, toxoplasma cysts were found in cardiac biopsies. Seroconversion was not prevented by the use of spiramycin prophylaxis in two recipients. Reactivations of latent infections or reinfections were detected by indirect ELISA in six (23%) seropositive recipients, but symptoms and signs of active T. gondii infection were not seen. Seroconversion and reactivation of infection were readily found by a combined use of indirect ELISA-IgG and ELISA-IgM and antibody capture ELISA-IgM. Discrepancies in results could be examined by immunoblotting. IgM-ISAGA retained stable positive values longer than IgM-ELISAs did. Cyclosporine treatment did not hamper detection of seroconversion but could cause antibody levels to remain relatively low in primary infections. Seronegative recipients should receive antitoxoplasma treatment on seroconversion.

Differential glycosylation of polymeric and monomeric IGM

A small fraction of normal IgM is secreted as monomers rather than polymers. We show here that the μ chains of monomeric IgM are glycosylated differently from the μ chains of polymeric IgM and are comparable in their glycosylation to the μ chains from mutant hybridoma cell lines which produce predominantly monomeric IgM. The difference in glycosylation between monomer and polymer μ chains is due to differences in the terminal processing of their oligosaccharides. The glycosylation of the mutant μ chains is not itself responsible for the block in IgM polymer formation.

Solid phase enzyme-linked immunosorbent assay (ELISA) for anti-sheep erythrocyte antibody in mouse serum

The solid phase enzyme-linked immunosorbent assay (ELISA) was developed to measure IgM and IgG anti-SRBC antibody titers in mouse serum. Sheep erythrocytes, which have a surface negative charge, were attached directly to the bottom of an aminoplate well (96-well flat bottom, Sumitomo Bakelite Co.) which is charged positively to provide a solid phase for the ELISA antigen. Serum samples titrated were obtained from mice 5 and 10 days after SRBC immunization. Alkaline phosphatase-labeled goat anti-mouse IgM and/or IgG preparations were used as second antibody. Alkaline phosphatase activity in a well was measured by the Kind and King method [Kind, P.R.N. & King, E.J. (1954). J. clin. Path., 7, 322–326]; the optical density (OD) value of quinone as a final product was measured at 492 nm. (1) A linear relationship was observed between the antiserum concentration and OD value over a wide enough dilution range to assay antibody titers of serum samples; (2) IgM and IgG fractions from antiserum showed almost the same antibody titer as did the reconstituted samples; (3) a good correlation was observed between the serum IgM titer and the number of IgM hemolytic plaque-forming cells (PFCs) in spleen 5 days after the immunization, and was also observed between serum IgG antibody titer and the number of IgG PFCs 5 and 10 days after. Therefore, the ELISA described here requires no fixative for preparation of cell-coated plates, and serum IgM and IgG anti-SRBC antibody titers can be measured without any fractionation technique.

Lymphocytotoxic autoantibodies in progressive systemic sclerosis.

In 53 patients with progressive systemic sclerosis (PSS) the lymphocytotoxic activity of their serum was measured in a microlymphocytotoxicity assay. In 21 of the 53 patients the test reacted distinctly positively in the heterologous system, and in 9 of these 21 also in the autologous system. After preparation of the immunoglobulins from these positive sera, whole cytotoxic activity was detected only in the IgM fraction but not in the IgG fraction. When using prepared T lymphocytes as target cells in the microlymphocytotoxicity test, the cytotoxic activity of the positive PSS sera showed itself to be directed against this lymphocyte population. Further analysis using the Western-blot technique showed that the IgM autoantibody in PSS sera reacted with the cell surface of CD4+ lymphocytes. The cross reactivity with extractable nuclear antigens was rather improbable. These results suggest that lymphocytotoxic autoantibodies may play a role in immunological disturbances in PSS.

Immunization with rough mutants of Salmonella minnesota: protective activity of IgM and IgG antibody to the R595 (Re chemotype) mutant.

We evaluated the immunoglobulin class responsible for the protective activity in serum obtained from humans and rabbits after immunization with the R595 (Re chemotype) mutant of Salmonella minnesota. Whole serum obtained before immunization and the IgG and IgM fractions failed to protect mice against lethal challenge with viable Klebsiella pneumoniae or Morganella morganii or with Salmonella typhi lipopolysaccharide (LPS). The protective activity of postimmunization serum resided solely in IgM antibody, whereas IgG antibody exhibited no protective activity. IgM antibody to the Re mutant was protective against bacterial challenge with both test strains of bacteria and S. typhi LPS. IgM antibody, at approximately the same concentration present in postimmunization serum, increased the LD50 of K. pneumoniae from less than 8.0 x 10(2) to greater than 2.0 x 10(4). These findings indicate that commercially prepared human IgG with high titers of antibody to antigens of the core portion of LPS would have little clinical utility.

Splenic Tissue Autotransplantation

Letters to the EditorSplenic Tissue Autotransplantation Bernd Domres, MD, Facharzt Ram N. Goel, FRCS, MD M. Heitham Schammut, and MD, Facharzt Robert R. ToussaintMD, Facharzt Bernd Domres Department of Surgery, King Khaled Hospital, P.O. Box 1852, Hail, Saudi Arabia Search for more papers by this author , Ram N. Goel Department of Surgery, King Khaled Hospital, P.O. Box 1852, Hail, Saudi Arabia Search for more papers by this author , M. Heitham Schammut Department of Surgery, King Khaled Hospital, P.O. Box 1852, Hail, Saudi Arabia Search for more papers by this author , and Robert R. Toussaint Department of Surgery, King Khaled Hospital, P.O. Box 1852, Hail, Saudi Arabia Search for more papers by this author Published Online:1 Jul 1988https://doi.org/10.5144/0256-4947.1988.297aSectionsPDF ToolsAdd to favoritesDownload citationTrack citations ShareShare onFacebookTwitterLinked InRedditEmail AboutIntroductionAsplenic patients are at an increased risk of overwhelming bacterial infection. It appears as pneumococcal sepsis in about 2% of splenectomized patients and may occur as late as 20 years after the splenectomy. Mortality in infected patients exceeds 50%, and this high mortality rate is related to the absent splenic functions. Therefore, we autograft removed ruptured spleen into an omentum pouch. This procedure does not extend the operation time more than 10 minutes.After removal of the ruptured spleen, its capsule is removed. The organ is shredded with sharp sterile device (of the type used in the kitchen to shred vegetables). The site of splenic implantation is important. It should be placed in an area that will drain into the portal venous system when neovascularization occurs. Implants function better in an omentum pouch than in a subcutaneous or peritoneal pouch. To avoid the main complications of the procedure (splenosis of the abdominal cavity or adhesions) the omental pouch should be sutured tightly so that it will not leak.We try to reimplant as much tissue of the ruptured spleen as possible. Research into splenic autotransplantation could prove that the minimum total weight of the grafted spleen should exceed 100 g, about a third of the total organ, to preserve satisfactory splenic function.Autotransplantation of ruptured spleen was performed in 49 cases. Eleven of the 49 patients died as a result of accompanying multiple injuries and not due to the splenectomy or autotransplantation.The progress of spleen regeneration in the omental pouch can be monitored by splenic scans, and postoperatively 30 patients were followed with regular thrombocyte counts, blood smears for RBC morphology such as Howell-Jolly bodies and pitted RBCs, and quantitative immunoelectrophoresis of IgM levels. After an initial increase in the numbers of thrombocytes, Howell-Jolly bodies, and pitted RBCs in the first 4 weeks, the levels returned to near normal after 8 weeks. The decreased IgM fraction returned to normal after 6 months.These results encourage us to continue autotransplantation of splenic tissue after splenectomy. Previous article Next article FiguresReferencesRelatedDetails Volume 8, Issue 4July 1988 Metrics History Published online1 July 1988 InformationCopyright © 1988, Annals of Saudi MedicinePDF download

Flow Cytometric Analysis of I-J Expression on Murine Bone Marrow-Derived Macrophages

Attempts to analyze bone marrow-derived macrophages (BMDM) by flow cytometry have been prohibited because of their high autofluorescence. Using an autofluorescence reduction method of Steinkamp and Stewart to reduce the autofluorescence of BMDM, we were able to examine several macrophage populations for their expression of I-A, I-J, and Mac-1 cell surface determinants. Bone marrow cells examined immediately after removal from the femur contain 50-60% Mac 1-positive cells (mainly granulocytes). During the next few days granulocytes and nonmacrophage precursor cells die, and the number of Mac 1-positive cells decrease. Once the bone marrow cells have been maintained in L cell conditioned medium (LCM) for 2 to 3 days, the number of cells expressing Mac 1 increases rapidly from 20% to 98% during the next 3 to 4 days. Bone marrow cells grown in LCM do not express I-J until these cells have been in culture 3 to 4 days, and the number of cells expressing I-J (up to 90% positive) parallels the increase in macrophages. Bone marrow cells maintained in LCM did not express detectable I-A during the 14 days these cells were examined. Two other macrophage populations often used in a variety of immunological studies were analyzed by flow cytometry. We found that the majority (up to 80%) of peritoneal cells expressed I-A, and only 20% of peritoneal cells had I-J cell surface determinants. On the other hand, peritoneal exudate cells collected 4 days after thioglycolate medium treatment were predominantly I-J positive (up to 70%), and only about 30% of these cells expressed I-A cell surface antigens. The binding of anti-I-J IgM antibody to BMDM was not to Fc receptors because pretreating these cells with up to 25 micrograms of an IgG2a myeloma protein did not block anti-I-J antibody binding. The addition of 25-200 micrograms of monoclonal anti-Fc receptor antibody was also ineffective in blocking the binding of a monoclonal anti-I-Jk antibody to BMDM. Pretreatment of BMDM with the IgM fraction of several control IgM antibody preparations did not block the specific binding of fluoresceinated anti-I-J IgM antibody. BMDM provide a pure population of macrophages that express a significant level of cell surface I-J antigens. Bone marrow cells grown in LCM are essentially devoid of other contaminating cells, and the increase in the number of I-J-positive cells parallels the increase in macrophages in these cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Low and high molecular weight cytomegalovirus-specific immunoglobulin M antibody in renal transplant patients with cytomegalovirus infections.

Sera from 27 renal transplant patients with primary and recurrent CMV infections and which were known to contain CMV-specific IgM antibodies were investigated by indirect immunofluorescence for the presence of virus-specific high molecular weight IgM (19S IgM) and low molecular weight IgM (7S IgM). After sucrose gradient fractionation of the sera, 19S IgM was found in all 27 patients, whereas 7S IgM was present in 11 out of 19 (56%) patients with primary CMV infection and in 1 out of 8 (12%) patients with recurrent CMV infection. The presence of 7S IgM was unrelated to the titre of the virus-specific IgM in whole serum. The presence of IgM rheumatoid factor was monitored by a sensitive fluorescence assay using measles virus antigen/antibody complexes. The absorption of the serum fractions with heat-aggregated gamma globulin failed to remove the specific IgM staining indicating that it was not due to IgM rheumatoid factor. On the other hand adsorption with protein A/sepharose removed the specific IgM staining from the 7S IgM fractions but not from the 19S IgM fractions. This suggests that specific 19S and 7S IgM antibodies may belong to different subclasses of IgM.

A clinical study of immunoglobulin class specific antibody response following hepatitis A.

Anti-HAV activity was determined by radioimmunoassay for serum fractions of IgG, IgM, and IgA separated with sucrose gradient centrifugation and immunoglobulin absorption using class-specific anti-immunoglobulin. Anti-HAV following hepatitis A was detected initially in IgM and IgA within one week after onset, and also detected in IgG within the next one week. IgM anti-HAV activity reached its peak level at the 21st-30th day of illness with a mean titer of 16.4 by RIA, and then declined gradually, whereas serum IgM concentration increased and reached the peak earlier, and no correlation was seen between them. IgG anti-HAV activity increased rapidly and it exceeded a RIA titer of 80 in 80 days from onset in 12 of the 18 cases. According to the kinetics of IgM and IgG anti-HAV, four types of antibody response following the disease were observed. However, no correlation between antibody response patterns and clinical findings was seen, therefore less relationship of anti-HAV to the liver cell injury was indicated. Furthermore, it was suggested that some immunological mechanisms controlled the level of antigen-specific antibody activity, which maintained the high level of IgM antibody in case of poor response of IgG antibody.

An immunochemical assay for natural IgM antibodies with an affinity to galactose and whose titer is reduced in the sera of cancer patients

Normal sera contain an IgM fraction which binds to free galactosyl groups of agarose. This fraction causes complement-dependent lysis of Ehrlich ascites tumor cells (EATC). Cancer sera show a much reduced titer of this lytic activity. In this paper we describe an immunochemical assay for these galactophilins (GALF). Agarose columns were immunoabsorbent and bound proteins were detected by labeled antibody to human IgM. All 157 normal sera contained GALF. The serum GALF concentrations of normal sera were linearly correlated to the serum cytolytic titers. However, EATC absorbed only 15% of normal serum GALF. The titers in normal females are higher than in males. The titers in 147 nonmalignant chronic disease patients were lower than the normals only in women over 40 years of age. The sera of 138 cancer patients, taken before any therapy, were significantly lower than the corresponding normal or chronic disease controls. Lymphoproliferative neoplasms were associated with the lowest titers while melanoma sera showed normal titers. It is likely that GALF is bound to tumor associated antigens with free galactosyl groups. These are on the cell surface and/or are shed into the circulation. GALF titration may prove useful in following treatment efficacy in individual cancer patients.

Nonimmune binding of Ig to Clostridium perfringens. Preferential binding of IgM and aggregated IgG.

In an attempt to find a bacterial IgM receptor, a large number of bacterial strains of different species were screened for the ability to bind human IgM. Certain strains of the anaerobic bacterium Clostridium perfringens were found to bind a major fraction of polyclonal IgM. One bacterial strain showed a particularly high binding capacity and was studied in more detail. This strain is also able to bind a minor fraction of polyclonal IgA and IgG. Inhibition experiments indicate that the different Ig classes bind to one and the same R structure. The ability of the strain to bind polyclonal Ig is correlated to the number of subunits in the Ig. This correlation can most simply be explained by increasing avidity with increasing number of subunits. In agreement with this hypotheses, experiments with aggregated IgG show that binding ability increases with aggregate size. Experiments with Ig fragments indicate that the binding structure in Ig is located in the F(ab')2 region. The ability of this bacterial strain to bind a majority of IgM molecules as well as aggregated IgG is potentially useful in immunologic work and represents a new type of Ig binding to bacteria.

Systemic lupus erythematosus sera inhibit antigen presentation by macrophages to T cells

Several reports have demonstrated that systemic lupus erythematosus (SLE) patients have a decreased response to exogenous antigens both in vivo and in vitro. We examined the effects of SLE sera on macrophage (Mφ) antigen-presenting functions. Mφ from normal donors were pulsed with tetanus toxoid antigen in the presence of SLE or normal human serum (NHS), fixed in paraformaldehyde, and incubated with autologous T cells. Of 16 SLE sera tested, 11 inhibited the T-cell proliferative response (measured by [ 3H]thymidine uptake) compared to control NHS; mean percentage inhibition was 53 ± 23%. This inhibition did not result from interference with antigen uptake by Mφ and was found in both IgM and IgG fractions of the sera. There was a positive correlation between the amount of inhibition and the cytotoxic reactivity of the SLE sera against Mφ as measured by Terasaki assay ( r = 0.659, P < 0.01). However, the presence and the amount of the inhibition did not correlate with serum immune complexes by Clq ELISA, serum anti-DR antibodies, or clinical disease activity of the SLE patients. We conclude that some SLE sera possess IgM and IgG antibodies reactive with Mφ which affect Mφ antigen-presenting functions, and might relate to decreased antigenic response in SLE patients.

細菌細胞膜に含まれるカルジオリピンの抗原性とその抗体の細菌に対する反応性

I investigated the possibility of production of antibody to bacterial membrane-cardiolipin using cardiolipin micelles, Staphylococcus aureus (S. aureus) and L-form of S. aureus. I also investigated and the reactivity of anti-cardiolipin (CL) antibody with bacteria and DNA.I confirmed that the bacterial membrane-cardiolipin induced the production of anti-CL antibody, which was present mainly in the IgM fraction, but also in the IgG fraction.The anti-CL antibody reacted with L-form of bacteria and bacterial membrane fractions, but it did not react with the vegetative form of bacteria other than S. aureus nor with DNA. On the basis of these findings, I considered that the L-form of bacteria or fragments of bacterial membranes can be detected by immunofluorescence antibody technique or nitrocellulose membrane-enzyme immunoassay using the anti-CL antibody.

Characterization of immune complexes detected by the 125I-C1q binding assay in breast cancer

The aim of the present study was to isolate and characterize immune complexes measured by the 125I-C1q binding assay in breast cancer sera. The C1q binding assay detects immune complexes by their binding to 125I-C1q and subsequent precipitation with PEG. We purified the C1q binding material from these precipitates by superose 6 gel filtration and cation exchange chromatography. The isolation steps were monitored by the C1q binding assay and the purified material was analyzed by SDS-PAGE and immunodiffusion. The C1q binding material isolated from two breast cancer sera contained complexes of IgM, C4b binding protein (C4-bp), and C1q. The C4-bp fraction showed significant C1q binding activity which increased after the addition of IgM. This C4-bp was not different from C4-bp isolated from normal serum. The IgM fraction, however, differed from normal IgM by its binding to C4-bp and C1q and its stronger affinity to the cation exchange column. These unique properties were not due to rheumatoid factor activity. They might be caused by a different glycosylation pattern or by complex formation with an as yet unknown polysaccharide antigen.

Binding of swine IgM immunoglobulins to peripheral nerve myelin sheaths in electron microscopic immunocytochemistry.

The IgM fraction of normal swine serum (NSS) was found to adhere to human peripheral nerve myelin sheaths from randomly selected neuropathies and control nerves in electron microscopic immunocytochemical assays. The reaction could be blocked by preincubation with undiluted fetal calf serum (FCS), whereas endoneurial background staining was already abolished by preincubation with 10% FCS. Only the IgM, but not the IgG or the IgA fraction of NSS adhered to peripheral nerve tissue. Such strong adherence of one of the components in an immunocytochemical assay may be a source of misinterpretation. Binding of swine IgM to human peripheral nerve myelin has not been described before and is of interest in the discussion about IgM binding to myelin-associated glycoprotein in IgM gammopathies.