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.     

Quantitative Precipitin Analysis of Free and Hidden RF IgM

Quantitative precipitin analysis using rabbit IgG anti-human Fc mu was performed with 16 RF IgM, six macroglobulinaemic IgM and four normal IgM. Abnormal precipitin curves were obtained for all RF IgM, even when the latter were not readily demonstrated with conventional serological tests for RF, and for macroglobulinaemic IgM with sedimentation rates greater than 19S. These IgM formed significantly more precipitates with IgG anti-Fcmu in the antigen excess zone than did normal IgM, but the precipitin curves for the other zones were similar for all IgM. The underlying mechanisms of some of the reactions were studied and discussed. Because the divergence in the precipitin reaction for normal IgM and RF IgM was so pronounced, a model precipitin curve was constructed. This could be used to detect RF IgM, even when not readily demonstrable with conventional serological tests for RF, by direct analysis of serum. The results obtained for RF IgM suggested that the method might be applied to RF IgG and intermediate complexes comprised of IgG. The mechanisms demonstrated here might be used to develop immunological methods for routine use.     

Binding of IgA to Protein-A-Containing Staphylococci: Relationship to Subclasses

Among 54 human monoclonal IgA proteins, 5 inhibited the binding of labeled human monoclonal IgM (IgM Se and IgM Ba) to protein-A-containing staphylococci, whereas 48 did not have this ability. One protein (IpA Ha) showed discordant findings in the IgM Se and IgM Ba inhibition assays. Among the 54 IgA proteins, 49 were of the IgA1 and 5 of the IgA2 subclass All of the 5 IgA2 proteins inhibited the binding of IgM Se and IgM Ba to staphylococci, whereas the 48 proteins that reacted negatively in the two inhibition belonged to the IgA1 subclass. These findings show that the ability to react with staphylococci is a properly of the IgA2 subclass. IgM proteins may also be divided into two groups based on different reactivities with protein A. It is proposed to call the two IgM subclasses IgM1 and IgM2, where the latter is defined by ability to react with protein A.     

Transport defect of IgM into luminal space in selective IgA deficiency

This study explored the pathogenesis of a transport defect of IgM into the lumen in a patient with selective IgA deficiency. In addition to the absence of IgM in the saliva, no IgM was localized on the luminal surface of colonic mucosa from the patient despite the presence of J chain-positive IgM cells. On tissue sections, IgM cells did not bind secretory component. The serum IgM also showed a negligible capacity to bind secretory component in vitro. Such abnormalities of IgM molecules as stated above seem to be clinicopathologically linked with IgA deficiency or its associated Sj?gren's syndrome.     

Primary Selective IgM Deficiency: An Ignored Immunodeficiency

Immunoglobulin M (IgM) provides the initial response to foreign antigen and plays a regulatory role in subsequent immune response development, accelerating the production of high-affinity IgG. Though selective IgM deficiency was described more than 45 years ago in children with fulminant meningococcal septicemia, it has been largely an ignored primary immunodeficiency. It appears to be more common than originally realized. Selective IgM deficiency is observed in both children and adults with no gender bias. The most common clinical manifestation of selective IgM deficiency is infections with extracellular and intracellular bacteria, viruses, and fungi. Allergic diatheses are the second commonest presentation of selective IgM deficiency. There is an increased prevalence of autoimmune diseases, which in both humans and mice appear to be secondary to selective IgM deficiency rather IgM deficiency secondary to autoimmune diseases. Selective IgM deficiency, in some cases, is associated with 22q11.2 chromosome deletion and few familial cases of selective IgM deficiency have been reported. Innate immunity is relatively intact. T cells, T cell subsets, and T cell functions are normal. However, several patients with selective IgM deficiency and T cell and NK cell defects with Mycobacterium avium intracellulare infections have been reported. In a subset of patients with selective IgM deficiency circulating IgM+ B cells are decreased or completely lacking. Specific IgG antibody responses against pneumococcus polysaccharides are impaired in a subset of patients with selective IgM deficiency. The pathogenesis of selective IgM deficiency is unclear; decreased T helper activity, increased isotype-specific suppressor T cell activity, and intrinsic B cell defects have been reported. Patients with selective IgM deficiency and impaired pneumococcal antibody responses appear to respond to immunoglobulin therapy.     

A critical study of the use of staphylococci containing protein A for separation of IgG and IgM antibodies

This study was to determine the best conditions for using staphylococci bearing protein A to separate IgG from IgM. The validity of the technique was evaluated for detection of IgM with antimicrobial activity and for typing monoclonal IgM. The results indicate that separation of IgG and IgM is not entirely satisfactory in normal sera and worse in hyperglobulinemic sera. The detection and titration of IgM antimicrobial antibodies (rubella and hepatitis B core (HBc) specific IgM) was unreliable because IgG was only partially absorbed by staphylococcal cells, while a significant portion of IgM was bound. The use of higher concentrations of staphylococci did not improve the results because the more IgG was absorbed, the more IgM was also bound. It is shown that with anti-HBc specific IgM the risk of misinterpretation is very high with a sensitive radioimmunoassay technique allowing detection of trace amounts of nonabsorbed IgG. In contrast staphylococcal protein A proved useful in typing monoclonal IgM.     

Appearance of low molecular weight IgM during course of infective endocarditis.

Low molecular weight (LMW) IgM is the monomeric subunit of pentameric IgM. It is not found in healthy adults but occurs in a number of autoimmune, lymphoproliferative and infectious conditions. It has not been described before in infective endocarditis (IE). Eighteen patients with IE were studied; 16 with subacute bacterial endocarditis (SBE) and two with acute endocarditis. LMW IgM was detected in the sera of six patients, all having SBE in association with circulating rheumatoid factor (RF). Of the remaining 12 patients without LMW IgM only three had RF in low quantities. Sequential studies revealed that LMW IgM appeared during the later stages of the illness at or following the peak RF and IgM response. LMW IgM was not detected in any of 20 control sera. Immunoblot analysis of sera containing LMW IgM revealed the presence of small quantities of dimeric and oligomeric IgM in addition to monomeric IgM. We conclude that LMW IgM occurs predominantly in those patients with IE who have associated RF. Immunoblot analysis suggests that the presence of monomeric and oligomeric LMW IgM reflects a disorder of IgM polymerization occurring in those patients.     

Enzyme-linked immunosorbent assay for detection of immunoglobulin M antibody to hepatitis B core antigen.

An enzyme-linked immunosorbent assay for detection of specific immunoglobulin M (IgM) antibodies against the core antigen of the hepatitis B virus (anti-HBc IgM) is described. The interference of IgM rheumatoid factor was evaluated quantitatively. In the anti-HBc IgM test, the rheumatoid factor gave false-positive results when the concentration exceeded 20 IU/ml. The rheumatoid-positive sera were disclosed by a control and retested for anti-HBc IgM after absorption of rheumatoid factor with latex particles aggregated with human IgG. In five of seven selected patients with acute hepatitis B followed to biochemical and clinical recovery, anti-HBc IgM was present transiently until antibodies against hepatitis B surface antigen (anti-HBs) appeared. Two patients had persistent anti-HBc IgM during the follow-up period. Four patients with hepatitis B surface antigenemia and progression to chronic liver disease did not clear their anti-HBc IgM in the period of observation (11 to 24 months). Anti-HBc IgM could not be demonstrated in 223 of 225 Danish blood donors. The two donors found positive for anti-HBc IgM also had anti-HBs. Twenty patients with acute A or non-A non-B hepatitis were negative for anti-HBc IgM. The enzyme-linked immunosorbent assay for anti-HBc IgM described here has a high specificity and sensitivity. The diagnostic relevance needs further evaluation, including quantitation of anti-HBc IgM, but the results presented indicate that anti-HBc IgM may be helpful in differentiating between prior and recent or ongoing hepatitis B infection.     

Recognition of Two Distinct Groups of Human IgM and IgA Based on Different Binding to Staphylococci

Labeled monoclonal IgM was bound to the Cowan I strain of Staphylococcus aureus in the absence of anti-IgM antibody This binding was inhibited by 11 of 33 monoclonal IgM proteins; 22 failed to inhibit Agammaglobulinemic serum alto failed to inhibit High concentrations of IgG inhibited the binding of IgM Se to staphylococci, but IgG and IgM probably react with different receptors on tin bacterial surface. Polyclonal IgM from all of 17 individuals tested inhibited the binding of IgM Se to staphylococci, which indicates that the distinction corresponds to an IgM subclass rather than to an allotype. Two of seven monoclonal IgA proteins also inhibited the binding of IgM Se to staphylococci.     

Immunoglobulin M responses to the Norwalk virus of gastroenteritis.

Eighty-seven serum specimens from 20 human subjects experimentally inoculated one or more times with Norwalk virus were quantitatively examined for virus-specific immunoglobulin M (IgM). A sensitive and specific radioimmunoassay for anti-Norwalk virus blocking activity was applied to whole serum and to separate IgM and IgG fractions obtained by sucrose density gradient ultracentrifugation. The peak IgM response occurred at about 2 weeks after illness, but IgM was detectable at lower titers for up to 21 weeks after infection. The IgM response was seen in volunteers who became ill, whether or not prechallenge total serum antibody was present. On long-term (27 to 42 months) rechallenge, volunteers who were previously ill and had produced IgM antibody again developed illness, and a secondary IgM response greater than the first was detected. Inoculated volunteers who did not develop illness, as well as previously ill volunteers on short-term rechallenge (4 to 14 weeks), usually failed to generate an IgM response, whether or not an IgG response had occurred. In ill subjects, the rise in IgM and IgG occurred concomitantly. Virus-specific IgM is not necessarily indicative of primary infection with Norwalk agent inasmuch as reinfection produces an enhancement of the IgM response. Furthermore, Norwalk-specific IgM responses do not appear to be associated with subclinical illness.     

Serologic diagnosis of toxoplasmosis with emphasis on the detection of Toxoplasma-specific immunoglobulin M antibodies

Commercially available kits were used for the detection of Toxoplasma-specific IgM antibodies. False positive IgM results were observed in whole sera containing Toxoplasma-specific antibodies together with rheumatoid factor when tested by immunofluorescence (IFA). False negative IgM results occurred in whole sera containing competing levels of Toxoplasma-specific IgG antibodies, as indicated by the IFA:IgM-M ratio. False positive and false negative IgM results often occurred when whole sera were tested. These false reactions were eliminated by fractioning IgM from IgG using the Isolab's IgM Isolation System. All five sera in this study with an IFA titer to Toxoplasma of greater than or equal to 1:16,384 also contained Toxoplasma-specific IgM antibodies. This suggests that sera with high titers to Toxoplasma should be tested for Toxoplasma-specific IgM antibodies.     

Determination of specific cytomegalovirus IgM antibodies using infected air dried cells and isolated nuclei by immunoperoxidase assay

A simple immunoperoxidase assay (IPA), adapted for detection of serum IgM antibodies to cytomegalovirus (CMV) is described. The antigen consisted of CMV infected human embryonic fibroblasts or isolated nuclei. The sera were absorbed with aggregated gamma-globulins prior to testing. Rabbit anti-human IgM peroxidase conjugate was used to detect IgM bound to viral antigen. In parallel the enzyme linked immunosorbent assay (ELISA) technique was used to determine IgG and IgM antibodies to CMV, respectively. All patients with acute CMV infections who were tested had CMV-specific IgM antibodies by IPA, both whole cell and nuclei antigen. The maximal IgM titers were higher by ELISA than by IPA but in 3 of the CMV patients IgM was detected earlier by IPA (with both types of antigens) than by ELISA. In 3 of 5 transplant patients with recurrent CMV infection IgM was demonstrated by immunoperoxidase techniques, while by ELISA IgM was demonstrated in only 2 of them. No cross reactivity with other herpes viruses was observed. The described IPA is simple, rapid and has the potential for widespread use in routine laboratories.     

Comparison of the fluorescent treponemal antibody absorption (FTA-ABS) test with the FTA-ABS double staining test for detection of antitreponemal immunoglobulin M in the 19S fraction of human serum.

A widely used immunoglobulin M (IgM) detection assay for the diagnosis of neonatal congenital syphilis is the fluorescent treponemal antibody absorption test used with fractionated serum (FTA-ABS 19S IgM test). Reading the results of the FTA-ABS test is more cumbersome than reading those of the FTA-ABS double staining (FTA-ABS-DS) test, a confirmatory test for specific IgG. To verify that the FTA-ABS-DS test used with an anti-human IgM conjugate could detect specific IgM in fractionated serum samples (FTA-ABS-DS 19S IgM test), 164 fractionated (QUIK-SEP IgM Isolation System; ISOLAB, Inc., Akron, Ohio) serum specimens from infected neonates or adults or from IgG-seronegative subjects were tested by both techniques. The sensitivity limits of the two tests were assessed with reactive serum samples diluted to an endpoint titer. Samples nonreactive by the FTA-ABS 19S IgM test (n = 74) were either nonreactive (n = 65), minimally reactive (n = 5), or reactive (n = 4) by the FTA-ABS-DS 19S IgM test. Samples minimally reactive by the FTA-ABS 19S IgM test (n = 32) were minimally reactive (n = 1) or reactive (n = 31) by the double staining test. All samples reactive by the FTA-ABS 19S IgM test (n = 58) were also reactive by the FTA-ABS-DS 19S IgM test. There was a directly proportional linear relationship (r = 0.9794) between titers obtained by both tests. FTA-ABS-DS 19S IgM titers were constantly equal to or higher than FTA-ABS 19S IgM titers. Fluorescence intensity reading repeatability was 91.4% for the FTA-ABS-DS 19S IgM test and 81.7% for the FTA-ABS 19S IgM test (P = 0.015). Because the more easily read FTA-ABS-DS 19S IgM test is at least as sensitive as, if not more sensitive than, the FTA-ABS 19S IgM test, it is a good alternative to the latter test for the detection of specific IgM in human fractionated sera for those using fluorescence microscopes with incident light.     

Secretory Component-Binding Properties of Normal Serum IgM

Our aim was to investigate why serum IgM is poorly transferred into secretions in normal subjects. Indeed, the low IgM level in secretions contrasts with the capacity of monoclonal IgM to bind to secretory component (SC), but it is not well established to what extent normal serum IgM can do so. The mean SC affinity was studied with a polyclonal IgM preparation from 250 normal subjects and with a representative pool of 100 different monoclonal IgM. The SC-binding percentages varied as a function of the IgM/SC molar ratio according to a common hyperbolic curve, with similar association constants: Ka = 4.19 +/- 2.61 x 10(7) M-1 (polyclonal pool) and Ka = 5.80 +/- 2.73 x 10(7) (monoclonal pool). It thus appears that the large difference in IgM concentrations between blood and secretions cannot be due to an SC-binding defect of serum IgM, but is probably explained by its low diffusion from blood to the extravascular compartment.     

Regulation of IgG Antibody-dependent Cellular Cytotoxicity in Vitro by IgM Antibodies

IgM antibodies have previously been reported to either inhibit or induce antibody-dependent lymphocyte cytotoxicity (ADCC). Here we show that human lymphocytes lyse bovine erythrocytes (E_b) in the presence of either IgM or IgG anti-E_b from rabbits. Seven out of 20 IgM preparations (Sephadex G-200) were ADCC-active. IgG-dependent ADCC was inhibited by human IgG but not by IgM. In contrast, IgM ADCC was inhibited by both IgG and IgM. The effector cells in IgM ADCC were a subpopulation of lymphocytes with distinct Fc receptors for both IgG and IgM. Most of them also had sheep erythrocyte receptors. Extensive purification of the ADCC-active IgM antibody preparations indicated that very small amounts of contaminating IgG anti-E_b were responsible for ADCC induction. When purified and ADCC-inactive IgM antibodies were mixed with suboptimal concentrations of IgG antibodies, a strong enhancement of ADCC was found. To achieve enhancement, the two antibody isotypes had to be present on the surface of the same target cells, and the IgM effect was not due to the release of soluble ADCC-enhancing factors. Thus, in this system, IgM antibodies are not capable of inducing ADCC on their own. However, they enhance ADCC by improving the contactual interaction between target cells and a special subset of effector cells.     

Differential IgM response to conformational and linear epitopes of parvovirus B19 VP1 and VP2 structural proteins

The IgM immune response against conformational and linear epitopes of B19 structural proteins VP1 and VP2 was examined in serum samples with a suspect B19 infection to determine the most suitable antigen for use in IgM detection and also to evaluate a possible relationship between the course of B19 infection and the presence of epitope type-specific IgM. The detection of IgM against conformational epitopes was performed by ELISA using undenatured VP1 and VP2 antigens whereas the detection of IgM against linear epitopes was performed by Western blot assays using denatured VP1 and VP2. IgM immune response against VP1 conformational epitopes appeared dominant, being detected in all serum samples positive for specific IgM, whereas IgM against VP2 linear antigen were found less frequently, being identified in less than half of the B19 IgM positive sera. In the examination of the course of infection, IgM against VP1 conformational epitopes appeared in the active phase of B19 infection at the same time and with the same frequency as IgM anti VP2 conformational epitopes and anti linear VP1 epitopes. IgM against VP1 conformational epitopes were seen to be long-lasting because in the recent phase of infection they were still present when other specific IgM were absent. During the active phase of B19 infection, IgM against VP2 linear epitopes were less frequently found than other specific IgM and in the recent phase they underwent a rapid temporal diminution. The data demonstrate that a sensitive B19 IgM test needs to be performed in diagnostic laboratories by ELISA using conformational B19 antigens; Western blot assays can be used only as confirmatory tests using VP1 linear antigens.     

Detection of IgM antibodies to delta antigen after coinfection and superinfection with the delta virus

A sensitive microtitre radioimmunoassay was developed for detection of IgM antibodies to delta antigen. The assay was based on the selective binding of IgM from test sera to antihuman IgM (u-chain specific) fixed to wells of a microtitre plate, and utilized delta antigen extracted from the liver of an experimentally infected chimpanzee. This test proved to be useful in distinguishing between coinfection and superinfection with the hepatitis delta virus (HDV). Transient anti-delta IgM responses were observed in patients coinfected with HDV, while prolonged elevated IgM levels were found in HBsAg carriers with chronic liver disease superinfected with HDV. Two distinct serological patterns were observed in both coinfection and superinfection. In coinfection, only 50% of patients with detectable anti-delta IgM went on to develop a long-lasting antibody response. Following superinfection with HDV either stationary or fluctuating levels of IgM antibody were demonstrated. In patients with fluctuating antibody levels, the presence or absence of IgM antibody related to the level of viral replication.     

2005年-2008年宁波地区孕妇TORCH-IgM检测结果分析

目的了解宁波地区孕妇中TORCH-IgM的阳性率,探讨选择性TORCH-IgM筛查的临床价值,为有效预防TORCH感染提供科学依据。方法采用酶联免疫法(ELISA)检测9385例孕妇血清的TORCH-IgM抗体水平,根据有无高危因素比较不同孕妇的血清抗体阳性率。结果高危因素组的孕妇TORCH-IgM阳性率分别为:TOX-IgM 1.02%、RV-IgM 1.86%、CMV-IgM 3.76%、HSVⅠ-IgM 7.75%、HSVⅡ-IgM 3.85%;无高危因素组的孕妇TORCH-IgM阳性率分别为:TOX-IgM 0.35%、RV-IgM 1.23%、CMV-IgM 2.26%、HSVⅠ-IgM 5.23%、HSVⅡ-IgM 2.64%;两组比较差异有显著性(P0.05)。结论对具有高危因素的孕妇应常规进行TORCH-IgM筛查,以便及早发现宫内感染,干预不良妊娠。     

J chain deficiency in human IgM monoclonal antibodies produced by Epstein-Barr virus-transformed B lymphocytes

Six human IgM monoclonal antibodies against Pseudomonas aeruginosa were purified and characterized. On agarose-acrylamide sodium dodecyl sulfate (SDS) gels run under nonreducing conditions, IgM monoclonal antibodies showed variable amounts of a slower migrating form of IgM in addition to the one co-migrating with plasma IgM. Protein blotting with anti-J chain antibody showed that the slower migrating form did not contain J chain. Analysis of one of the monoclonal antibodies by sucrose gradient centrifugation showed that the J chain-deficient form sedimented faster than the complete IgM. It is known that IgM preparations lacking J chain sediment faster by sucrose gradient centrifugation analysis and tend to form hexamers. The slower migrating form of IgM we observed on SDS gels under nonreducing conditions could be hexameric IgM. Further evaluation of this monoclonal antibody demonstrated that both forms of IgM had the same antigen-binding activity. Glycosylation of the light chain was demonstrated in two of the monoclonal antibodies.     

Diagnostic significance of immunoglobulin M antibodies to Toxoplasma gondii detected after separation of immunoglobulin M from immunoglobulin G antibodies.

Failure to demonstrate immunoglobulin M (IgM) antibodies by indirect immunofluorescence (IgM-IFA) in sera from some patients with acute acquired toxoplasmosis has recently been attributed to an inhibitory effect of high titers of IgG antibodies in these sera (Pyndiah et al. J. Clin. Microbiol. 9:170-174, 1979). To confirm these findings and define their importance for diagnosis, we used gel filtration to separate IgM from IgG antibodies in a series of sera that were negative in the IgM-IFA test. A total of 68 sera were from patients with acquired toxoplasmosis, 13 were from uninfected adults, 13 were from infants with congenital toxoplasmosis, and 7 were from uninfected neonates. Of the 68 sera from patients with acquired toxoplasmosis, IgM preparations (from the separated sera) were positive in the IgM-IFA test in 36 (53%). There was a significant (P = 0.00003) association between high titers of IgM-IFA antibodies in the IgM preparations (corrected for dilution of IgM antibodies by the gel filtration procedure) and recent acquisition of infection. IgM antibodies were also detected in 5 (38%) of the IgM preparations of 13 sera from congenitally infected infants but not in any of the IgM preparations of sera from uninfected neonates. IgG antibodies to Toxoplasma gondii were shown to interfere with demonstration of IgM antibodies in the IgM-IFA test. Treatment of sera with protein A resulted in greater dilution of IgM antibodies and less efficient separation of IgM from IgG antibodies than did separation of sera by gel filtration. Treatment of sera with protein A did not result in increased detection of IgM antibodies to T. gondii. Testing of IgM preparations (obtained by gel filtration) resulted in a significant increase in sensitivity of the IgM-IFA test for the diagnosis of recently acquired and congenital toxoplasmosis.