gp96 is a major member of the heat shock protein 90 (HSP90) family and an important molecular chaperone. Under physiological con- ditions, gp96 binds to and hydrolyzes adenos- ine triphosphate (ATP) in the endoplasmic re- ticulum, thereby assisting the correct folding and assembly of newly synthesized proteins and promoting the degradation of misfolded proteins . In addition, gp96 contributes to the expression of approximately 20 proteins, such as Ig heavy chain and integrins. Recent studies have found that gp96 is highly expre- ssed in tumor cells and is closely related to the development and progression of tumors and to a poor prognosis. Wu et al. have shown that gp96 is highly expressed in liver cancer and is related to tumor type and stage . It is likely that gp96 participates in tumor devel- opment and progression by promoting the deg- radation of p53. Liu et al. found that gp96 is highly expressed in oral tumors and is correlat- ed with radiotherapy resistance . Moreover, gp96 participates in tumor immune response by activating macrophages and T cells. Acti- vated macrophages and T cells secrete tumor necrosis factor and destroy tumor cells . However, the gp96-mediated immune response has a dichotomous nature; gp96 fails to elicit protective immune responses when the dose of gp96 exceeds or is below optimum levels . Igs are also involved in tumor immunity and are likely to be related to gp96 activity. In the present study, a nude mouse model of Hep-2 tumor xenografts was established, and anti-human IgM antibody was injected intratu- morally as an interventional drug. The expres- sion levels of IgM and gp96 in the xenograft tumors were significantly lower in the experi- mental group compared with the 2 control groups, indicating that anti-IgM treatment in- hibits the expression of gp96 in laryngeal car- cinoma.
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Immunoblotting. For determination of different antibody patterns, the recomBlot CMV IgG or IgM test from Mikrogen Diagnostik was used. This qualitative test allows assignment of an antibody response to specific antigens in human serum and plasma by using immunoblot strips. These immunoblot strips contain recombinant polypeptides from IE1 (53 kDa), tegument protein pp150 (50 kDa), processivity factor pUL44 and single- stranded binding protein pUL57 (45 kDa; CM2), tegument protein pp65 (31 kDa), and two epitopes of glycoprotein B (gB1, 25 kDa; gB2, 18 kDa). The strips were incubated with 2 ml of washing buffer prior to addition of 20 l patient serum or the corresponding weak positive control. The cov- ered incubation tray was incubated for 1 h at room temperature (RT) under gentle shaking. Unbound antibodies were removed by three wash- ing steps for 5 min each at RT. Incubation with the secondary peroxidase- conjugated antibody (either anti-human IgM or anti-human IgG) was performed for 45 min at RT. The conjugate solution was removed, and the
Western blotting. Western blotting was performed using standard procedures. Briefly, 225 g of B. burgdorferi 50772 protein was loaded into the preparative wells of 0.1% sodium dodecyl sulfate-12% polyacrylamide gels, and the proteins were separated by running in an electrophoresis unit (PROTEAN IIxi; Bio-Rad Laboratories, Hercules, Calif.) at 24 mA for 4 h. The proteins were transferred from the gels to polyvinylidene difluoride membrane (Perkin-Elmer Life Sci- ences Inc., Boston, Mass.) by electrophoresing overnight at 10 V. The polyvi- nylidene difluoride was cut into strips and blocked with 1% bovine serum albu- min in PBS (pH 7.2)-0.1% Tween 20 for 1 h at 22°C. Strips were incubated for 1 h at 22°C with serum diluted to a ratio of 1:100 and washed four times with PBS-0.1% Tween 20. Horseradish peroxidase-labeled anti-human IgM or IgG heavy and light chains (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) were added, and the strips were incubated for 1 h at 22°C. Strips were washed and developed using the TMB membrane peroxidase substrate system (Kirke- gaard & Perry).
MRL dengue fever virus IgM capture ELISA. The MRL ELISA (product code EL1500M) was performed according to the manufacturer’s instructions by use of the recommended classical Centers for Disease Control and Prevention protocol. After rehydration of the anti-human IgM-coated microtiter plate with wash buffer for 5 min, 100 l of patient serum diluted 1:101 was added to each well and incubated for 60 min at room temperature (23°C). After the plate was washed three times with the buffer provided, 100 l of inactivated dengue-1 through dengue-4 was added to each well, and the plate was incubated overnight at 4°C. The antigens were provided in lyophilized form and prepared directly before use by reconstitution with the solution provided. The plate was washed as described above, and 100 l of conjugate solution (peroxidase-conjugated mouse antifla- vivirus antibody) was added to each well for 30 min at room temperature before another wash and the addition of 100 l of tetramethylbenzidine substrate per well. After 10 min at room temperature, the reaction was stopped by the addition of 100 l of stop reagent (1 M sulfuric acid) per well, and the optical density at 450 nm was read with a microtiter plate reader. Positivity was determined by comparison to the IgM cutoff calibrator serum provided. A positive sample was defined as having a sample/calibrator absorbance ratio of ⱖ1.0, and a negative sample was defined as having a ratio of ⬍1.0.
DBLMSP and DBLMSP2 bind human IgM - To gain insight into the functional role of DBLMSP and DBLMSP2 during malaria pathogenesis, we first expressed the entire coding region of both proteins from the P. falciparum 3D7 strain using a recently developed expression system based on mammalian cells, which has been shown to produce natively-folded P. falciparum proteins (20). Both proteins were expressed (Fig. 1A), and their immunoreactivity to sera from malaria- exposed and unexposed control individuals was quantified. To our surprise, while other MSP3 family members (MSP3, MSP6, H101 and MSP11) and DBL-containing merozoite surface proteins (EBA140, EBA175, EBA181) reacted, as expected, with only the exposed sera, DBLMSP and DBLMSP2 showed equally strong immunoreactivity to both the unexposed control and exposed sera (Fig. 1B). This suggested that recombinant DBLMSP and DBLMSP2 bound immunoglobulins present in normal serum from individuals without prior exposure to the malaria parasite. Indeed, when each protein was exposed to purified human immunoglobulin isotypes, strong binding was observed to IgM, but not to IgA, IgE, or IgG (Fig. 1C). This binding was specific to human IgM since they did not bind immunoglobulins from other mammalian species including goat, rabbit, guinea pig and cow, or purified mouse IgM (Fig. 1D). To confirm these observations, we showed that DBLMSP-coated beads but not control beads incubated in normal human serum purified bands with masses there were consistent with the heavy and light chain of IgM (Fig. 1E), and their identities were subsequently confirmed by mass spectrometry.
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PUU IgM m -capture assay. Human sera were analyzed for PUU-specific IgM by a modification of a procedure described previously (33). Optimal conditions for the assay were determined by box titrations of all included reagents. Goat anti-human IgM serum (Cappel), diluted 1:500 in 0.05 M carbonate buffer (pH 9.6), was incubated in microtiter wells at room temperature overnight and stored at 48C until use. Patient and control sera, diluted 1:200 in EIA buffer, were incubated for 1 h in duplicate wells. bac-PUU-N antigen (purified antigen, 1.0 mg/ml; unpurified antigen, 1:500 dilution of solubilized insect cell extract) and negative control antigen (diluent only) were incubated alone for 1 h and then with horseradish peroxidase-conjugated bank vole anti-PUU MAb 1C12, diluted 1:2,000, for 1.5 h. All incubations and washings were performed as described above. Specific antibody binding was detected by using tetramethylbenzidine substrate according to the manufacturer’s instructions (ICN Biochemicals, Cleveland, Ohio). The enzyme reaction was stopped after 15 min with 2 M H 2 SO 4 , and OD at 450 nm was measured. In all plates, one acute-phase NE
RDT sensitivity and timing of specimen collection. RDT sen- sitivity by DPO of specimen collection could only be analyzed for RMI and Yap specimens due to limited information from the other countries. For NS1, the lowest sensitivity was observed on day 0 (RMI ⫽ 53.5%) and day 5 (RMI ⫽ 50.0%; Yap ⫽ 42.0%) (Fig. 2; see also Tables S3 and S7 in the supplemental material). The sensitivity for RMI specimens increased incrementally from days 1 to 3 and then decreased from days 4 to 5; however, the sensitivity of the Yap specimens had a range from 42.0 to 53.0 throughout the first 5 days (see Table S7 in the supplemental ma- terial). Conversely, in RMI the lowest anti-DENV IgM was on days 0 and 1 (0 and 28.6%, respectively), and the highest sensitiv- ity occurred on day 5 (83.3%) (see Table S4 in the supplemental material). For specimens collected after day 5, there was also a decrease in sensitivity (47.1%), which is attributed to the wide range of DPOs (DPO 6 to 45) for this data point. For Yap, there were insufficient anti-DENV IgM-positive specimens for analysis. The anti-DENV IgG titer is negatively correlated with RDT NS1 positivity. Finally, we measured the correlation between anti-DENV IgG titers and the proportion positivity of NS1 in the true positive serum specimens as determined by rRT-PCR. Of the FIG 1 Performance of the SD BIOLINE Dengue Duo rapid diagnostic test. (A
previously (22), while the specificity was lower. This difference could be attributed to the fact that we diluted samples only 1:20 as opposed to 1:100 (22) or to the assignation of cutoff values, since Talarmin et al. (22) chose to maximize the spec- ificity of their assay whereas we placed more emphasis on sensitivity. Certainly, the behavior and duration of IgA for both secondary and primary infection warrants further investigation. The decision to compare detection of IgA in both serum and saliva to detection of IgM in serum was based on the reasoning that IgM detection is the routine diagnostic method and, as such, should be the benchmark for diagnostic techniques. However, our results strongly indicate that to better assess the specificity and sensitivity of a DEN-specific IgA assay, studies assessing the sensitivity and specificity of IgA detection in confirmed (e.g., paired) samples would be worthwhile. The kinetics of IgA response to DEN infection clearly needs addi- tional study, since previous reports suggest that DEN-specific IgA is found in serum for only about a month (22), whereas our preliminary data suggest that DEN-specific IgA may actually persist longer than IgM (Balmaseda et al., unpublished). In- terestingly, Koraka et al. (16) reported recently that titers of DEN-specific IgA were significantly higher in acute-phase se- rum from DSS patients as opposed to DF patients and thus may correlate with clinical outcome. In our study, IgA titers were not determined, so this analysis could not be performed. Nonetheless, these data suggest that detection of IgA in serum may be a new and effective tool for the diagnosis of dengue.
A related group of RA-specific autoantibodies, the so- called antikeratine antibodies (AKA), was first described in 1979 . These antibodies stain keratin-like structures in the cornified layer of esophagus cryostat sections but do not recognize cytokeratins, as is suggested by the name. AKA can be detected by indirect immunofluorescence in 36–59% of RA sera with a specificity of 88–99% (reviewed in ). Several studies have shown that the APF and AKA antibodies target the same antigen, the epithelial protein filaggrin (for references see ). Filaggrin (filament aggregating protein) is involved in the organization of cytoskeletal structures in epithelial cells and is synthesized as a large, heavily phosphorylated pre- cursor protein, profilaggrin. During differentiation of epithelial cells, profilaggrin is partly dephosphorylated and proteolytically cleaved into 10–12 filaggrin subunits. Finally, about 20% of the arginine residues are enzymati- cally deiminated to citrulline residues . Conversion of the basic amino acid arginine into the neutral residue citrulline is catalyzed by the enzyme peptidylarginine de- iminase (Fig. 1). It is this modification that has been shown to be essential for the autoantigenicity of filaggrin [46,47]. Immunoblotting assays and an enzyme-linked immunosor- bent assay (ELISA) using filaggrin purified from human skin as an antigen have been developed for the detection of antifilaggrin antibodies (AFA). Vincent et al.  could detect AFA in 41% of RA sera with 99% specificity. When combining the AFA immunoblot assay with AKA testing, a much higher sensitivity (64%), without loss of specificity, could be achieved . However, the sensitivity of the assay appears to be dependent on the method for purifi- cation of the filaggrin. Slack et al. calculated sensitivities of 12 and 16% for two different filaggrin preparations, while only one of five positive sera reacted with both
Immunoblot has been evaluated as a diagnostic method for congenital toxoplasmosis. Like enzyme-linked immunofiltration assay (ELIFA), immunoblot can be used to compare antibody patterns and to determine if the antibodies are transmitted by the mother or synthesized by the fetus or infant. Among the 48 infants tested, 27 had congenital toxoplasmosis and 21 were suspected but had none. Reproducibility, sensitivity, specificity, and positive predictive values in immunoblot for immunoglobulins (Igs) G 1 M 1 A and/or G 1 M were 90, 92.6, 89.1, and 92.4%, respectively. G 1 M immunoblot and G 1 M ELIFA have better sensitivities than the conventional IgM immunosorbent agglutination assay, IgM enzyme-linked immunosorbent assay (ELISA), IgM immunofluorescence antibody test, in vitro culture, and mouse inoculation. The novel antibodies, i.e., those synthesized by infants against Toxoplasma gondii, were of the IgG class in most cases, although a confident diagnosis could be related to the number of observed Ig classes (G 1 M, G 1 A, and G 1 M 1 A). Immunoblot has a better resolution than ELIFA. In prenatal diagnosis, immunoblot could be complementary to in vitro culture and mouse inoculation. In the other cases, early detection by immunoblot appears to give the best results when compared with the other serological methods.
Natural antibodies are often polyreactive: a single antibody clone may bind to diverse unrelated antigens (19, 20). One exam- ple of a polyreactive IgM is monoclonal antibody 2E4, which rec- ognizes multiple distinct antigens (21). Interestingly, we found that both 2E4 and BALB/c natural IgM could bind both to Ad5 and to an unrelated antigen: dinitrophenol coupled to bovine se- rum albumin (DNP-BSA) (Fig. 4A and B). When BALB/c natural IgM was separated by affinity chromatography on DNP-BSA, the DNP-BSA-binding pool of natural IgM was able to bind to Ad5, but DNP-BSA-nonbinding natural IgM showed decreased reac- tivity to Ad5 (Fig. 4B). Thus, at least some of the Ad5-binding natural IgM antibodies in mouse serum are polyreactive, because these antibodies can bind to both DNP-BSA and Ad5.
Protein Fv (pFv) is a recently described 175-kD gut-associated sialoprotein with a potent capacity for augmentation of antibody-dependent immune functions. To investigate the molecular basis for Fab-mediated binding of pFv, we evaluated a panel of 52 monoclonal IgM and found that approximately 40% bound pFv. Whereas the majority (> or = 75%) of V H3 and V H6 IgM strongly bound pFv, only a small minority (< 20%) of IgM from other V H families bound pFv, and these antibodies had weaker binding interactions. Inhibition studies suggested that all binding occurred at the same (or overlapping) site(s) on pFv. Surface plasmon resonance studies demonstrated binding affinity constants up to 6.7 x 10(8) M-1 for pFv. Biopanning of IgM and IgG Fab phage-display libraries with pFv
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Methods: The full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system. Results: The native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively.
Molecular profiling studies have established a common signature for sBL that distinguishes it from other types of Myc-translocated and Myc-activated DLBCL [20–22], and have also provided evidence for subtypes within sBL, e.g. those with or without activating mutations in ID3. ID3 mutations were absent in eBL compared to sBL [21, 23, 24]. Thus far, “molecular sBL” signatures were primarily derived from sBL collected in Northern European or US locations and have not distinguished between pediatric or adult patients . To arrive at the molecular expression signatures, sBL was defined as CD20+, BCL6+, CD10+, BCL2-, CD5-, Ki-67 ≥ 95% and IgH-Myc translocation [15, 16]. In the aforementioned profiling studies, 100% of “molecular sBL” carried a BCL6 translocation; yet, only 88% carried the classical IgH-Myc translocation . The “molecular sBL” category included 36 cases of “atypical” sBL because of a non-prototypical immunophenotype, and ultimately led to a 58-gene signature. Dave et al. arrived at very similar molecular signatures , and again, the molecularly defined sBL class contained cases that were inconsistent with classification based on the immunophenotypical definition of BL and of PBL [11, 15, 16, 26]. This report provides evidence that there exist additional subtypes of t(8;14) sBL that can be distinguished on the basis of IgM and CD79a expression.
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demonstated by Sephadex G200 chromatography with 125 I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the
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other reasons have been reported [25-27]. In one study that reported IgM anti-CMV in 14% of SLE cases, 3/12 were con- sidered an artifact after a RF neutralization assay . Other studies interpreted IgM anti-CMV as false positive results based on negative PCR [25,26]. In contrast, one study detected CMV DNA by PCR in whole blood from 100% of SLE patients versus 72% in controls (P = 0.02) , suggest- ing a significant difference in sensitivity of the PCR assay. A recent study using immunostaining of CMV pp65 in peripheral blood leukocytes has suggested that reactivation of CMV is common among IgM anti-CMV(+) SLE patients under inten- sive immunosuppressive therapy . The RF neutralization assay or PCR to detect CMV DNA was not performed in the present study; however, IgM anti-CMV was not associated with RF by laser nephelometry or ELISA (Figure 1), suggesting it was not directly due to RF. Furthermore, none of the IgM anti-CMV(+) group was positive for IgM antibodies to EBV– viral capsid antigen (data not shown). Also, the IgM anti- CMV(+) group did not have higher levels of IgM class autoan- tibodies to snRNPs, U1–70 k, dsDNA, chromatin, β 2 -glyco-
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developed a monoclonal IgM antibody, ROC 129.1, to a human desmosomal calcmodulin- binding protein. This antibody reacts with a submembranous 250-kD protein from human keratinocytes and stains human epidermis in a "cell-surface pattern". Permeability studies indicated that the epitope with which this monoclonal reacts is on the inner surface of the cell membrane. Immunoelectronmicroscopy localized the antigen to the desmosome. The epitope is restricted to stratified squamous epithelia and arises between 8-12 wk of fetal development. This desmosomal calmodulin-binding protein, which we have termed keratocalmin, may be involved in the calcium-regulated assembly of desmosomes.
Human and animal secretory fluids. We obtained serum samples from two laboratory volunteers (blood types A and B). We obtained colostrum from a nursing laboratory colleague and milk samples from the same colleague, one of us, and four anonymous healthy donors (3). Vaginal washes from three anony- mous healthy donors were the kind gift of G. Brooks, University of California San Francisco. A human bile sample was collected intraoperatively from a pa- tient undergoing a wedge resection of the liver for carcinoma and an incidental cholecystectomy. This patient had no gallstones and a normal gallbladder pa- thology. This bile sample was a kind gift from L. Way, Veterans Administration Medical Center, San Francisco, Calif. We collected saliva from two laboratory volunteers. We bought pooled human colostral IgA from Sigma Chemical Co. (St. Louis, Mo.).
significantly diminished in the productive rearrangements of CD5(-) B cells. 3-23/DP-47 was the most frequently used VH gene segment and was found significantly more often than expected from random usage in productive rearrangements of both CD5(+) and CD5(-) B cells. Evidence for selection based on the D segment and the JH gene usage was noted in CD5(+) B cells. No differences were found between the B cell subsets in CDR3 length, the number of N-nucleotides or evidence of exonuclease activity. Somatically hypermutated VHDJH rearrangements were significantly more frequent and extensive in CD5(-) compared to CD5(+) IgM+ B cells, indicating […]
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Hantaan virus is the causative agent of severe hemorrhagic fever with renal syndrome. Clinical surveillance for Hantaan virus infection is unreliable, and laboratory verification is essential. The detection of virus-specific immunoglobulin M (IgM) and IgG in serum is most commonly used for the diagnosis of hantavirus infection. Testing of oral fluid samples instead of serum offers many advantages for surveillance. However, commercial tests for hantavirus-specific antibodies are unavailable. For the detection of Hantaan virus in the oral fluid of humans, we have developed a monoclonal antibody-based capture enzyme-linked immunosorbent IgM assay (IgM capture ELISA) and indirect enzyme-linked immunosorbent IgG and IgM assays (indirect IgG and IgM ELISAs) for paired serum and oral fluid samples using the Saccharomyces cerevisiae yeast-expressed nucleo- capsid protein of the Hantaan-Fojnica virus. The sensitivity and specificity of the oral fluid IgM capture ELISA in comparison with the results of the serum Hantaan virus IgM assay were 96.7% and of 94.9%, respectively. Thus, data on the overall performance of the oral fluid IgM capture ELISA are in close agreement with those of the serum IgM assay, and the method exhibits the potential to serve as an easily transferable tool for large-scale epidemiological studies. Data on the indirect IgM ELISA also showed close agreement with the serum IgM assay data; however, the indirect IgG ELISA displayed a lower sensitivity and a lower specificity. In conclusion, the IgM capture ELISA can be used with oral fluid instead of serum samples for the diagnosis of Hantaan virus infection.