Isotypic Determinants Research Articles

Rheumatoid factor isotypes in rheumatoid arthritis diagnosis and prognosis: a systematic review and meta-analysis

ObjectiveThe first biomarker associated with the rheumatoid arthritis is rheumatoid factor (RF) and since the earliest reports a role has been proposed in the diagnosis and in the prediction of...

Monoclonal antibodies to hemagglutinin of influenza A/H7N3 virus (Orthomyxoviridae: Alphainfluenzavirus: Influenza A virus)

Variants of influenza virus A/H7 have the same high pandemic potential as A/H5. However, the information about the antigenic structure of H7 hemagglutinin (НА) is considerably inferior in quantitative terms to similar data for H5 НА.The aims of the study were development and characterization of the monoclonal antibodies (MAbs) panel for HA subtype H7 of the influenza A virus. Viruses were accumulated in 10-day-old chicken embryos. Purification and concentration of the virus, determination of protein concentration, preparation of MAbs and ascitic fluids, hemagglutination and hemagglutination inhibition (HI) tests, assessment of antibodies' activity in indirect enzyme-linked immunosorbent assay (ELISA), as well as determination of MAbs isotypes and neutralization reaction (NR) were carried out by standard methods. The obtained MAbs to А/mallard/Netherlands/12/2000 (H7N3) strain were studied in HI test with a set of strains of different years of isolation belonging to different evolutionary groups. MAbs had a reduced reactivity compared to the immunogen-virus for all the studied strains. Cross-interaction of MAbs 9E11 and 9G12 in HI test with influenza A/H15 virus has been observed. Influenza A agent with H7 HA variant could serve as a potential cause of a future pandemic. Development of the MAbs panel for subtype H7 HA is an urgent task for both veterinary medicine and public health. The obtained MAbs can be used not only for epitope mapping of the H7 HA molecule (currently insufficiently studied) and as reagents for diagnostic assays, but also for determining common («universal») epitopes in HA of different strains of this subtype.

Abstract 511: Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing

Abstract Background Traditional next generation sequencing methods for quantifying somatic hypermutation (SHM) rely on multiplex primers targeting either the framework 1 (FR1) or Leader regions of the IGH variable gene in combination with joining gene primers to amplify rearranged IGH chains from gDNA templates. Ion AmpliSeq primer panels for SHM evaluation were compared using both DNA and RNA input. Performance was compared using SHM values obtained from RNA samples amplified using FR1 variable gene primers in combination with constant gene primers to determine each IGH isotype and subtype in a single PCR reaction. Comparison of SHM frequencies measured from matched RNA and DNA samples were used to determine the feasibility for use of RNA in the study of SHM as well as comparison of the performance of Leader and FR1 gene primers in DNA studies. Methods Two multiplex primer panels were developed for DNA templates. These panels target the Leader or FR1 regions of the IGHV gene and the IGHJ gene. RNA samples were surveyed using FR1 and constant region primers. Comparisons of SHM frequency using 200ng of gDNA and 25ng of RNA from B cell lines and clinical research samples. Sequencing was accomplished using the Ion Gene Studio S5, while clonotyping, isotype determination, and somatic hypermutation analysis was performed using Ion Reporter 5.16 analysis software. Results Both RNA and DNA input assay workflows were able to correctly determine the SHM status of all rearrangements tested. IGHV SHM values were highly concordant between both RNA and DNA approaches. Additionally, SHM values derived from FR1 targeting variable gene primers delivered concordant results compared to Leader targeting variable gene primers when using DNA input across a wide range of SHM frequencies tested. Conclusions These results support the ability of highly multiplexed long-read NGS assays to accurately quantify SHM in either DNA or RNA samples. Concordant results were shown between FR1 and Leader targeting primers using DNA input. RNA based NGS methods benefit from lower sample requirements as well as the addition of isotype (and subtype) identification, opening new research areas for study of the B cell immune repertoire. Citation Format: Michelle Toro, Geoffrey Lowman, Loni Pickle, Stephanie Ostresh, Mark Andersen, Shrutii Sarda, Chenchen Yang. Clonal lineage and somatic hypermutation analysis comparing DNA and RNA as input material by long amplicon IGH chain sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 511.

Brain antibody sequence evaluation (BASE): an easy-to-use software for complete data analysis in single cell immunoglobulin cloning

BackgroundRepertoire analysis of patient-derived recombinant monoclonal antibodies is an important tool to study the role of B cells in autoimmune diseases of the human brain and beyond. Current protocols for generation of patient-derived recombinant monoclonal antibody libraries are time-consuming and contain repetitive steps, some of which can be assisted with the help of software automation.ResultsWe developed BASE, an easy-to-use software for complete data analysis in single cell immunoglobulin cloning. BASE consists of two modules: aBASE for immunological annotations and cloning primer lookup, and cBASE for plasmid sequence identity confirmation before expression. Comparing automated BASE analysis with manual analysis we confirmed the validity of BASE output: identity between manual and automated aBASE analysis was 100% for all outputs, except for immunoglobulin isotype determination. In this case, aBASE yielded correct results in 96% of cases, whereas 4% of cases required manual confirmation. cBASE automatically concluded expression recommendations in 89.8% of cases, 91.8% of which were identical to manually derived results and none of them were false-positive.ConclusionsBASE offers an easy-to-use software solution suitable for complete Ig sequence data analysis and tracking during recombinant mAb cloning from single cells. Plasmid sequence identity confirmation by cBASE offers functionality not provided by existing software solutions in the field and will help to reduce time-consuming steps of the monoclonal antibody generation workflow. BASE can be installed locally or accessed online at Code Ocean.

Production and characterization of monoclonal antibodies against recombinant extracellular domain of CD99.

CD99/MIC2 gene product is a heavily glycosylated transmembrane protein which plays a major role in homotypic cell adhesion, apoptosis of double positive T cells and vesicular protein trafficking. It is over expressed in various cancers and has been considered as an ideal therapeutic target. The present study focused at developing monoclonal antibodies against the extracellular domain (ECD) of CD99 using hybridoma technology. In order to generate monoclonal antibodies, the recombinant ECD of CD99 was used for immunizing the mice. Resulting hybridomas were screened through indirect ELISA. Clones which gave high absorbance values were sub cloned by limiting dilution followed by isotype determination, IP, WB and FACS. The monoclonal antibody 547F2 4F12 was purified from culture supernatant using FPLC and further screened using IF. Finally, the antibodies were validated for specificity using siRNA knock-down. We were able to establish stable hybridoma clones secreting CD99 antibodies. The antibodies reacted with both the recombinant ECD as well as the wild type CD99 and their isotype's were determined as IgM. Based on these results, we propose that the purified monoclonal antibody 547F2 4F12 could be possibly used for targeting tumors which over express CD99.

Preparation of Monoclonal Antibodies With Hybridoma Techniques Against Promastigote of Leishmania infantum Antigens in Diagnosis of Visceral Leishmaniasis

Background: Since the discovery of hybridoma cells, the uses of monoclonal antibodies (mAbs) are in vogue. Such antibodies with single isotype have high specificity. The developments in the field of cell culture and technology have led to the production of improved qualities of mAbs. In general, mAbs are important reagents used in biomedical research, as well as in targeted drug delivery systems. Objective: The aim of this study was to apply different strategies to produce mAbs against proamastigote Leishmania infantum strain in Iran. Materials and Methods: At first, standard strains were cultured and antigens of L. infantum were obtained. Afterward, BALB/c mice were immunized and antibody titers were determined. For hybridoma cell formation, isolated lymphocyte cells from spleen of immunized mice and myeloma cells were fused at the ratio of 10:1 in the presence of polyethylene glycol and followed by limiting dilution method for the isolation of monoclones. Results: More than 20 positive monoclones were hybridoma, from which 3 clones had optical density over 1.5. We named these clones as 5D2 FVI6, 3G2 FV7, and 3G2 FV5 which were selected for limiting dilution. From these hybrids, anti-promastigotes L. infantum mAbs were obtained. The results of isotype determination showed IgG2b sub-class (and not IgG1, IgG2a and IgA) in 5D2 FV and 3G2 FVI monoclones. Conclusion: This study produced mAbs against promastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of promastigotes L. infantum and can be used in the diagnosis of visceral leishmaniasis.

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: implication for diagnosis of Kala-azar.

Objective(s):Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran.Materials and Methods:First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c smice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting.Results:Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones.Conclusion:This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

Isotype Determination of Rodent-Derived Monoclonal Antibodies Using Sandwich ELISA

For research groups needing to isotype a significant number of antibodies on a fairly regular basis, the protocol outlined here provides a cost-effective option. In this simple sandwich ELISA, the monoclonal antibody is first captured with antibodies that discriminate between heavy-chain isotypes and then is detected with antibodies specific for each light chain. This combination ensures that free light chain secreted by hybridomas does not yield a false-positive result, because in a true positive both the capture and detecting antibodies bind the monoclonal antibody.

Isotype Determination of Antibodies.

On identifying a new monoclonal antibody, or in characterizing antibodies in sera evoked by disease or immunization, it is particularly informative to determine the serological class of the antibodies. The serological class of the protein is determined by the structure of the antibody constant region. Several methods of class or isotype determination are outlined in this unit: sandwich ELISA, electrophoresis, and immunofixation, or use of a variety of commercially available kits. Different purification schemes and approaches for enzymatic fragmentation of the antibodies depend on the class or isotype of the antibody, so this information streamlines these processes. © 2017 by John Wiley & Sons, Inc.

Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray.

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG1/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.

The role of ISCOMATRIX bilayer composition to induce a cell mediated immunity and protection against leishmaniasis in BALB/c mice.

Development of new generation of vaccines against leishmaniasis is possible because long-term protection is usually seen after recovery from cutaneous leishmaniasis. ISCOMATRIX is particulate antigen delivery system composed of antigen, cholesterol, phospholipid and saponin. In this study, the role of ISCOMATRIX bilayer composition made by different phase transition temperature (Tc) to induce a type of immune response and protection against leishmaniasis was assessed. ISCOMATRIX formulations with different bilayer compositions consisting of EPC (Tc <0 °C), DMPC (Tc 23 °C) and DSPC (Tc 54 °C) were prepared. Different ISCOMATRIX formulations were mixed with soluble Leishmania antigens (SLA). BALB/c mice were immunized subcutaneously, three times with 2-week intervals. As criteria for protection, footpads swelling, parasite burden, determination of IgG isotypes and the level of IFN-γ and IL-4 were assessed. Although the groups of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC showed the smallest footpad swelling and least parasite burden compared with the buffer group, the difference was not significant. Moreover, the highest level of IFN- γ and IL-4 was observed in the splenocytes of mice immunized with ISCOMATRIX DMPC or ISCOMATRIX DSPC, respectively. After challenge, the mice immunized with ISCOMATRIX DSPC showed the highest elevation of IgG, IgG1 and IgG2a antibodies (P<0.01) compared with control group. However, our results indicated that ISCOMATRIX EPC, DMPC or DSPC generated a mixed Th1/Th2 response that was not protective. Our results showed that the adjuvanticity of prepared ISCOMATRIX doesn't influence with different phospholipids at least in our mice model.

Changes in the amino acid sequence of the recombinant human factor VIIa analog, vatreptacog alfa, are associated with clinical immunogenicity

Vatreptacog alfa, a recombinant human factor VIIa (rFVIIa) analog developed to improve the treatment of bleeds in hemophilia patients with inhibitors, differs from native FVIIa by three amino acid substitutions. In a randomized, double-blind, crossover, confirmatory phase III trial (adept(™) 2), 8/72 (11%) hemophilia A or B patients with inhibitors treated for acute bleeds developed anti-drug antibodies (ADAs) to vatreptacog alfa. To characterize the formation of anti-vatreptacog alfa ADAs in hemophilia patients with inhibitors. This was a post hoc analysis of adept(™) 2. Immunoglobulin isotype determination, specificity analysis of rFVIIa cross-reactive antibodies, epitope mapping of rFVIIa single mutant analogs and pharmacokinetic (PK) profiling were performed to characterize the ADAs. Immunoglobulin isotyping indicated that the ADAs were of the immunoglobulin G subtype. In epitope mapping, none of the rFVIIa single mutant analogs (V158D, E296V or M298Q) contained the complete antibody epitope, confirming that the antibodies were specific for vatreptacog alfa. In two patients, for whom PK profiling was performed both before and after the development of ADAs, vatreptacog alfa showed a prolonged elimination phase following ADA development. During the follow-up evaluation, the rFVIIa cross-reactivity disappeared after the last vatreptacog alfa exposure, despite continued exposure to rFVIIa as part of standard care. Results from the vatreptacog alfa phase III trial demonstrate that the specific changes made, albeit relatively small, to the FVIIa molecule alter its clinical immunogenicity.

Anticardiolipin Antibody Isotype Determination in Amniotic Fluid of Pregnant Patients with Systemic Lupus Erythematosus and/or Antiphospholipid Syndrome

Objective: To determine the levels and isotypes of aCl, as well as anti beta 2 glycoprotein 1 (antiβ2-GP1) antibodies in serum and amniotic fluid of pregnant patients with SLE and/or APLS, and healthy pregnant women serving as a control group. Material and Methods: We analyzed serum and amniotic fluid of pregnant patients with SLE and/or APLS, and of healthy pregnant women through ELISA. Results were compared using a Student’s T test. Results: 6 of 13 patients (46.1%), 5 with SLE and one with primary APLS had antiphospholipid antibodies in amniotic fluid. Two patients had IgG aCl and 4 patients had antiβ2-GP1 (one of them also showing IgM) in amniotic fluid. In serum, 4 patients (30%) had antiphospholipid antibodies present (one IgG aCl and three anti β2-GP1) as opposed to none in the control group having antiphospholipid antibodies in amniotic fluid. Only one control had IgM aCl in serum. Antiβ2-GP1 in the amniotic fluid of patients showed a statistically significant value when compared to controls. Conclusion: aCl and antiβ2-GP1 may be present in the amniotic fluid of patients with and without a history of fetal loss. The presence of IgM aCl and antiβ2-GP1 in amniotic fluid suggests its localized production.

Determination of Anti-Factor VIIII Antibody Titers, Isotypes and Epitopes by SPR Analysis of Human Plasma

Abstract 202Development of neutralizing anti-factor VIII (FVIII) allo-antibodies (“inhibitors”) occurs in a significant proportion of congenital Hemophilia A (HA) patients receiving exogenous FVIII, and autoimmune responses to FVIII can also occur. Accurate early detection of these immune responses, including low-titer or non-neutralizing antibodies, could have prognostic value and thus assist clinicians in evaluating therapeutic options. Rapid, sensitive, high-information-content surface plasmon resonance (SPR) assays have been developed to quantify the titer, IgG subtype distribution and domain/epitope specificity of anti-FVIII antibodies by direct analysis of pre-treated plasma samples. Samples were injected directly onto the biosensor of a Biacore T-100 instrument, on which recombinant FVIII was captured via a covalently immobilized anti-FVIII monoclonal antibody (mAb), and the increase in resonance units (RU) was converted to antibody titers. The lower limit of detection was 100ng/mL (∼1nM ∼0.01 Bethesda units/mL), allowing characterization of anti-FVIII antibodies that could not be detected by the Bethesda assay. Subsequent serial injections of isotype-specific anti-human mAbs yielded quantitative measurements of the fractions of anti-FVIII antibodies with specific subtypes. Plasma samples from 200 HA subjects age 2 and above (with and without a measurable inhibitor) as well as samples from 3 autoimmune adults were analyzed. Distinct IgG subtype distributions were observed, with most inhibitor subjects exhibiting mixed IgG1 + IgG4, or IgG1 + IgG2 + IgG4 responses. Serial samples from 1 subject following initial FVIII infusions showed transient class switching with the appearance of anti-FVIII-IgG3.Epitope mapping was next carried out via competition studies utilizing (a) a panel of 11 monoclonal antibodies (mAbs) whose epitopes on the FVIII-C2 domain have been mapped at high resolution and (b) recombinant FVIII-C2 proteins. The mAb-competition assays allowed the (FVIII-C2) epitope specificity of anti-FVIII antibodies in plasma to be mapped at a similar resolution, identifying specific clusters of surface-exposed FVIII residues that comprise or overlap immunodominant epitope(s) recognized by human antibodies. Complementary solution-phase competition SPR assays utilized wild type FVIII-C2 protein and selected proteins from a panel of 60 FVIII-C2 point mutants (muteins) that were identified during epitope mapping of the mAbs by SPR. This second approach alleviated several technical issues associated with the earlier blocking studies that utilized mAbs to compete the polyclonal plasma IgG (e.g. dissociation of the blocking mAb and steric effects) and provided reproducible quantitative results. Because saturating concentrations of FVIII-C2 proteins were used, changes in the number of RUs measured before and after addition of WT-FVIII-C2 indicated the fraction of polyclonal anti-FVIII antibodies that recognized this domain. Notably, the IgG subtype distribution of all FVIII-C2 specific antibodies reflected the total IgG subtype distribution, indicating that affinity maturation of B-cell epitopes preceded antibody class switching. Interestingly, saturating amounts of either FVIII-C2 or the high-affinity anti-FVIII-C2 mAb BO2C11 completely competed binding of antibodies from 1 subject (with HA genotype A2201P) to FVIII captured on the biosensor surface, indicating that his antibody response was to a single B-cell epitope or else to closely overlapping epitopes. However, although rare subjects had a completely FVIII-C2 specific response or no binding to FVIII-C2, between 30–50% of the anti-FVIII antibodies in the vast majority of inhibitor-positive samples targeted the FVIII-C2 domain. This was true for inhibitor subjects with severe HA due to the intron 22 inversion, for subjects with other HA genotypes resulting in severe HA and for the 3 autoimmune subjects.Results of these competition SPR assays are directly translatable to our laboratory's efforts to design FVIII muteins that retain adequate pro-coagulant activity but can avoid neutralization by anti-FVIII antibodies in inhibitor patients' blood. FVIII muteins having amino acid substitutions that eliminate immunodominant B-cell epitopes are expected to have potential for development as less immunogenic/antigenic bypass therapies for patients who develop a high-titer inhibitor. Disclosures:Howard:Haplomics: Equity Ownership; Baxter, Bayer, Biogen: Research Funding; Haplomics: Patents & Royalties. Pratt:Bayer, CSL Behring, Pfizer: Research Funding; Puget Sound Blood Center: Patents & Royalties.

Development of Two Murine Monoclonal Antibodies Recognizing Human nG1m(a)-Like Isoallotypic Markers

Antigenic determinants of immunoglobulin molecules are categorized into three groups: idiotypes, isotypes, and allotypes. An isoallotype is defined as an isotypic determinant in one or more subclasses and an allotype in another subclass of a given isotype. The isoallotype nG1m(a), formerly called non-a, is an allotype located on human IgG1 molecules lacking the G1m(a) allotype. This marker, however, is also detectable on all human IgG2 and IgG3 molecules. Anti-isoallotypic antibodies are useful tools for structural studies of immunoglobulins, forensic science, and epidemiological demographic investigations. In this study, two murine monoclonal antibodies (MAbs) recognizing nG1m(a)-like epitope(s) were generated against a human IgG myeloma protein. These MAbs are produced by hybridoma clones (4F18B12 and 6F18D1) obtained by fusion of SP2/0 myeloma cells with splenocytes from BALB/c mice immunized with heavy chain of a human IgG3 myeloma protein. These MAbs reacted with Fc, but not Fab fragment of the immunizing IgG3 paraprotein. Specificity of the MAbs was further analyzed using a panel of purified myeloma proteins and polyclonal IgG3, including IgG1 (n = 9), IgG2 (n = 4), IgG3 (n = 8), and IgG4 (n = 6) subclasses. Our results demonstrated that these MAbs reacted with linear epitope(s) located on heavy chain of all IgG2 and IgG3 molecules tested and some paraproteins of IgG1 subclass, but none of the IgG4 molecules. So these MAbs seem to recognize nG1m(a)-like isoallotypic marker. These MAbs showed no cross-reactivity with serum of other species tested. Both MAbs belonged to IgG1 subclass with affinity constants of 4.5 x 10(9) mol(-1) (4F18B12) and 3.46 x 10(9) mol(-1) (6F18D1), respectively. These MAbs might be used as a tool to detect nG1m(a) + IgG molecules.

Immunizations and enzyme-linked immunosorbent assay (ELISA) for antibody isotype determination

Immunizations and enzyme-linked immunosorbent assay (ELISA) for antibody isotype determination

Mechanisms of isotype determination during immunoglobulin class switch recombination in human B-cells

Mechanisms of isotype determination during immunoglobulin class switch recombination in human B-cells

Mangifera indica L. extract (Vimang) and mangiferin modulate mouse humoral immune responses.

The present study investigated the effects of orally administered Vimang (an aqueous extract of Mangifera indica) and mangiferin (the major polyphenol present in Vimang) on mouse antibody responses induced by inoculation with spores of microsporidian parasites. Inoculation induced specific antibody production with an exponential timecourse, peaking after about one month. Vimang significantly inhibited this antibody production from about three weeks post-inoculation, and most markedly by four weeks post-inoculation; by contrast, mangiferin had no significant effect. Determination of Ig isotypes showed that the IgM to IgG switch began about four weeks post-inoculation, with IgG2a predominating. Vimang significantly inhibited IgG production, but had no effect on IgM. Mangiferin did no affect either IgM or IgG2a, but significantly enhanced production of IgG1 and IgG2b. Neither Vimang nor mangiferin enhanced specific antibody secretion by splenic plasma cells from mice inoculated with microsporidian spores, whether administered in vivo before serum extraction or in vitro to the culture medium. Inoculation with spores induced splenomegaly, which was significantly reduced by Vimang and significantly enhanced by mangiferin. These results suggest that components of Mangifera indica extracts may be of potential value for modulating the humoral response in different immunopathological disorders.

Expression of a 4-(hydroxy-3-nitro-phenyl) acetyl (NP) specific equi-murine IgE antibody that mediates histamine release in vitro and a type I skin reaction in vivo.

Due to characteristic clinical signs, immunoglobulins of isotype E (IgE) are believed to be involved in several allergic diseases of the horse. To date, closer investigations have been hampered by the fact that neither purified equine IgE nor anti-equine IgE monoclonal antibodies were available for IgE isotype determination. As an approach to solve this problem, we constructed a stable cell line (EqE6) that expresses recombinant equi-murine IgE specific for 4-(hydroxy-3-nitro-phenyl) acetyl (NP). Biochemical analysis of the purified protein revealed a highly glycosilated IgE monomer of approximately 230,000 Da. The biological ability of the NP-IgE to mediate histamine release after crosslinking with antigen was demonstrated in vitro using equine blood leucocytes. In vivo, the intradermal application of NP-IgE followed by antigen crosslinking induced a type I hypersensitivity skin reaction in horses. Both results indicate that the recombinant NP-IgE contains an intact and functional Fc(epsilon) RI binding site and mediates effector functions in equine basophils and cutaneous mast cells. This equi-murine IgE can be used for the production of IgE-specific polyclonal and monoclonal antibodies. In addition, the NP specificity allows the antigen-specific activation of equine Fc(epsilon)-receptor-expressing cells, such as mast cells and basophils. This property could be used to investigate IgE-mediated mechanisms for a better understanding of equine type I allergic diseases.

Detection of IgA and IgG but Not IgE Antibody to Respiratory Syncytial Virus in Nasal Washes and Sera from Infants with Wheezing

Purpose of the Study. The role of respiratory syncytial virus (RSV) in stimulating an immunoglobulin E (IgE) antibody response and enhancing the development of asthma remains controversial. The aim of this study was to measure IgE, immunoglobulin A (IgA), and immunoglobulin G (IgG) antibody responses to immunodominant RSV antigens in nasal washes and serum samples from infants with and without respiratory symptoms.Study Population. Forty infants aged 6 weeks to 2 years (20 with wheezing, 9 with rhinitis, and 11 without respiratory tract symptoms) were included in the investigation.Methods. The children were enrolled in an emergency department during the mid-winter months and seen again at follow-up when they were asymptomatic. Nasal washes were obtained by standard methods and were evaluated for RSV antigen. Moreover, determination of antibody isotypes (IgE, IgA, and IgG) to RSV antigens was performed in nasal washes and serum samples by using an enzyme-linked immunosorbent assay. In a subset of nasal washes, IgE to RSV was also evaluated by using a monoclonal anti-FcE antibody-based assay.Results. At enrollment, 15 patients with wheezing, 2 with rhinitis, and 1 control subject tested positive for RSV antigen. Thirteen patients with wheezing were <6 months old, and most (77%) were experiencing their first attack. Among the children with positive test results for RSV antigen, an increase in both nasal wash and serum IgA antibody to RSV-Fa and Ga was observed at the follow-up visit. There was no evidence for an IgE antibody response to either antigen.Conclusions. Both IgA and IgG antibodies to the immunodominant RSV-Fa and Ga antigens were readily detected in the nasal washes and serum samples from patients in this study. The investigators were unable to demonstrate specific IgE antibody to these antigens and concluded that the production of IgE as a manifestation of a Th2 lymphocyte response to RSV is unlikely.Reviewer’s Comments. There have been numerous investigations suggesting that RSV bronchiolitis may be a risk factor for recurrent wheezing and the development of asthma during childhood. In some cases, investigators have suggested that a possible mechanism for this association begins with the ability of RSV to stimulate IgE antibody production. The current publication by de Alarcon et al summarizes a well-designed, focused investigation that could not detect the presence of specific IgE antibody to immunodominant RSV antigens during or after an infection in infants with this common respiratory virus. Data from this investigation directly questions IgE antibody production as the primary mechanism of RSV-induced bronchiolitis and subsequent wheezing and should help spark continued interest and debate in this research area.