Antibody Production In Mice Research Articles

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice

Mutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear. We sought to define the functional consequences of the C104R mutation on B-cell function. We performed in vitro studies of TACI C104R expression and signaling. Aknock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge. C104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis. These data show that the C104R mutation and itsmurine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturbB-cell function with lymphoproliferation and immunoglobulin production defects.

Immunogenicity of a truncated enterovirus 71 VP1 protein fused to a Newcastle disease virus nucleocapsid protein fragment in mice

Enterovirus 71 (EV71) is one of the viruses that cause hand, foot and mouth disease. Its viral capsid protein 1 (VP1), which contains many neutralization epitopes, is an ideal target for vaccine development. Recently, we reported the induction of a strong immune response in rabbits to a truncated VP1 fragment (Nt-VP1t) displayed on a recombinant Newcastle disease virus (NDV) capsid protein. Protective efficacy of this vaccine, however, can only be tested in mice, since all EV71 animal models thus far were developed in mouse systems. In this study, we evaluated the type of immune responses against the protein developed by adult BALB/c mice. Nt-VP1t protein induced high levels of VP1 IgG antibody production in mice. Purified VP1 antigen stimulated activation, proliferation and differentiation of splenocytes harvested from these mice. They also produced significant levels of IFN-γ, a Th1-related cytokine. Taken together, Nt-VP1t protein is a potent immunogen in adult mice and our findings provide the data needed for testing of its protective efficacy in mouse models of EV71 infections.

Evaluation of a Recombinant Measles Virus as the Expression Vector of Hepatitis C Virus Envelope Proteins

Measles virus (MV) is a negative strand RNA virus of the family Paramyxoviridae, and the attenuated Edmonston-B strain can be engineered by the reverse genetics system. Here we constructed the recombinant Edmonston strain of measles virus (MV-Ed) that expressed hepatitis C virus (HCV) envelope proteins (rMV-E1E2). The rMV-E1E2 successfully expressed HCV E1 and E2 proteins. To evaluate its immunogenicity, NOD/Scid/Jak3null mice that were engrafted with human peripheral blood mononuclear cells (huPBMC-NOJ) were infected with this rMV-E1E2. Although human lymphocytes could be isolated from the spleens of mock-infected mice during the 2-weeks-long experiment, the proportion of mice that were infected with MV or rMV-E1E2 was decreased in a viral dose-dependent manner. Over 103 PFU of virus infection decreased the human PBL to less than 5%. Significant decrease of B cell population in human PBL from rMV-E1E2 infected NOD-SCID mice and decrease of T cell population in those from MV infected mice were observed. Human antibody production in these mice was also examined. Thus, the results in this study may contribute for future improvement of recombinant vaccine using measles virus vector.

Lactoferrin increases both resistance to Salmonella typhimurium infection and the production of antibodies in mice

Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein with antibacterial and immunomodulatory activities. The antibacterial influence of orally administered bovine Lf (bLf) against murine infection caused by Salmonella typhimurium ( S. typhimurium) has scarcely been explored. In the current study, Balb/c mice were treated orally for 7 days with either 5 or 100 mg of bovine lactoferrin (bLf). On day 7 of treatment, mice were intragastrically infected with a lethal or sublethal dose of colony forming units (CFU) of S. typhimurium. During treatment with bLf, feces from mice sublethally infected were harvested daily to prepare fecal suspensions, which were serially diluted and plated onto Salmonella Shigella agar to estimate CFU/g of feces. After sacrificing the animals on day 7, 14 or 21 post-infection, samples of intestinal fluid, Peyer's patches (PP), liver and spleen were collected to count the number of CFU by plate dilution. Intestinal secretions were also employed, along with serum samples, to evaluate total IgA, IgG and IgM antibodies, and those against Salmonella surface proteins and bLf by ELISA assay. In lethally infected mice both bLf doses decreased mortality. In sublethally infected mice, both bLf doses decreased bacterial shedding in feces and intestinal fluid, and also reduced bacterial colonization at PP and bacterial translocation in the liver and spleen. Levels of total and those IgG and IgM in serum and IgA in intestinal secretions against Salmonella surface proteins and bLf were enhanced with both doses of bLf. These findings suggest that the effect of bLf against the infection by S. typhimurium in mice may be the result of an antimicrobial activity linked with its modulatory effect on immunocompetent cells (from intestinal and peripheral organs) involved in antibody production.

Boosting with mycobacterial heparin-binding haemagglutinin enhances protection of Mycobacterium bovis BCG-vaccinated newborn mice against M. tuberculosis

Heterologous prime-boost regimens are a valuable strategy to improve the generation of effector-memory T cell responses against intracellular pathogens. In this study we show that newborn mice vaccinated with bacillus Calmette-Guérin (BCG) and boosted with heparin-binding haemagglutinin (HBHA) had enhanced protective immunity against intranasal or aerosol Mycobacterium tuberculosis challenge over non-boosted mice, as evidenced by a considerable reduction of mycobacterial load in spleen and lung. The route of HBHA delivery had a differential impact on cytokine and antibody production in BCG-primed mice. The prime-boost regimen induced not only HBHA-specific IFN-γ, but also other cytokines, such as IL-12 and TGF-β, which may be associated with the generation of lung Th1 effector-memory lymphocytes, responsible for the enhanced protection against M. tuberculosis challenge.

Persistence of viremia and production of neutralizing antibodies differentially regulated by polymorphic APOBEC3 and BAFF-R loci in friend virus-infected mice.

Several host genes control retroviral replication and pathogenesis through the regulation of immune responses to viral antigens. The Rfv3 gene influences the persistence of viremia and production of virus-neutralizing antibodies in mice infected with Friend mouse retrovirus complex (FV). This locus has been mapped within a narrow segment of mouse chromosome 15 harboring the APOBEC3 and BAFF-R loci, both of which show functional polymorphisms among different strains of mice. The exon 5-lacking product of the APOBEC3 allele expressed in FV-resistant C57BL/6 (B6) mice directly restricts viral replication, and mice lacking the B6-derived APOBEC3 exhibit exaggerated pathology and reduced production of neutralizing antibodies. However, the mechanisms by which the polymorphisms at the APOBEC3 locus affect the production of neutralizing antibodies remain unclear. Here we show that the APOBEC3 genotypes do not directly affect the B-cell repertoire, and mice lacking B6-derived APOBEC3 still produce FV-neutralizing antibodies in the presence of primed T helper cells. Instead, higher viral loads at a very early stage of FV infection caused by either a lack of the B6-derived APOBEC3 or a lack of the wild-type BAFF-R resulted in slower production of neutralizing antibodies. Indeed, B cells were hyperactivated soon after infection in the APOBEC3- or BAFF-R-deficient mice. In contrast to mice deficient in the B6-derived APOBEC3, which cleared viremia by 4 weeks after FV infection, mice lacking the functional BAFF-R allele exhibited sustained viremia, indicating that the polymorphisms at the BAFF-R locus may better explain the Rfv3-defining phenotype of persistent viremia.

L-selectin expression on CD4+ T cells is critical for antibody production, whereas L-selectin expression on both CD4+ and CD8+ T cells is important for dermatitis in a mouse model of atopic dermatitis (44.9)

Abstract L-selectin is a cell adhesion molecule that plays an important role in leukocyte migration especially to peripheral lymph nodes. L-selectin deficiency is known to decrease subcutaneous immune responses, however, the role of L-selectin in atopic dermatitis model is unknown. Moreover, it is not clear on which cell subset the expression of L-selectin is critical for cutaneous inflammation. Here, we showed that in mouse atopic dermatitis model elicited by epicutaneous sensitization with ovalbumin (OVA) OVA-specific antibody production and dermatitis is completely lost with L-selectin deficiency. We also found that L-selectin expression on CD4+ T cells is critical for antibody production, whereas L-selectin expression on both CD4+ and CD8+ T cells is important for dermatitis. Moreover, as few as 100 OVA-specific CD4+ T cells were able to elicit OVA-specific antibody production in L-selectin deficient mice. When wild-type CD4+ T cells were intravenously injected to L-selectin deficient mice, OVA-specific antibody-forming cells existed mostly in draining lymph nodes, suggesting that L-selectin-positive CD4+ T cells recruited to draining lymph nodes could muster antibody-forming cells independent of L-selectin. These results suggested that this mouse atopic dermatitis model is completely L-selectin dependent with L-selectin on CD4+ T cells and CD8+ T cells playing distinctive roles, and targeting L-selectin may be an option for treating atopic dermatitis.

Specific point mutations in the envelope protein of Tick-borne encephalitis virus enhance non-viraemic transmission efficiency in a tick vector

Background: Tick-borne encephalitis virus (TBEV) is transmitted to humans by Ixodid ticks causing >10,000 cases of disease annually. The risk of human infection relates to the efficiency of virus transmission between infected and uninfected ticks. Here we identify specific mutations in the viral envelope protein that affect transmission efficiency of TBEV between ticks. Methods: The genomes of 4 field isolates of TBEV deficient in haemagglutination, were sequenced and recreated by site-directed mutagenesis, in a TBEV infectious clone. They were then compared with the wild-type infectious clone in mice, porcine kidney PS cells and adult and nymphal I. ricinus ticks. Results: Sequence analysis revealed unique amino acid substitutions D67G, E122G or D277A in the envelope glycoprotein. Each mutation resulted in an increase of net charge and hydrophobicity on the virion surface. When introduced individually into the TBEV infectious clone (IC), each substitution inhibited haemagglutination and reduced mouse neuroinvasiveness from 65% to 15-30%. Antibody production in infected mice was 1.5-3 times lower for IC-E122G and IC-D277A suggesting lower levels of viraemia and/or deficient immune stimulation induced by these viruses. All mutants demonstrated delayed growth in PS cells during the first 24hpi; however, mutant IC-D67G exhibited significantly better growth characteristics than IC-E122G and IC-D277A. The reproduction of IC-E122G and IC-D277A in fasting ticks was similar to that of control HA positive virus whereas the titres of IC-D67G were significantly lower (2.5-3 vs. 1-2 log10PFU/ml, respectively). In feeding ticks, the titre of IC-E122G increased approximately 1000-fold and IC-D277A and IC-D67G - approximately 300-fold, whereas for control virus the increase was about 10-fold. Non-viraemic transmission efficiency from infected to uninfected ticks was increased by each individual substitution in nymphal I. ricinus (Figure 1). Figure 1 Tick-to-tick transmission rate (clear bars) is expressed as the proportion of infected I.ricinus nymphs. Black triangles show the average virus titres in individually infected recipient nymphs as determined by plaque assay. Conclusion: We hypothesize that the mechanism of adaptation of TBEV to its host utilizes the shift of charge/hydrophobicity at several critical aminoacid residues exposed on the virion surface. This shift results in different biological consequences depending on the localisation of certain aminoacid residue. The results provide valuable information concerning the maintenance in nature and the emergence of pathogenic variants of TBEV. Abstracts for SupplementInternational Journal of Infectious DiseasesVol. 14Preview Full-Text PDF Open Archive

Combined Administration L-Cystine and L-Theanine Enhances Immune Functions and Protects against Influenza Virus Infection in Aged Mice

Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.

Erythropoietin and Cyclophosphamide Combination Treatment Additively Enhances Antibody Production in Mice

Erythropoietin and Cyclophosphamide Combination Treatment Additively Enhances Antibody Production in Mice

BAFF aids generation of IgG anti-ganglioside antibodies in response to Campylobacter jejuni lipo-oligosaccharide

Ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) can induce the production of IgG anti-ganglioside antibodies, but the generation mechanism has yet to be clarified. B-cell activating factor belonging to the TNF family (BAFF) helped murine B cells produce anti-ganglioside antibodies against C. jejuni LOS. In splenocyte culture, however, anti-ganglioside antibodies were produced in the presence of a soluble transmembrane activator and calcium-modulating and cyclophilin ligand interactor immunoadhesin (TACI-Ig), a receptor for BAFF. TACI-Ig adenoviral vectors failed to decrease production of anti-ganglioside antibodies in mice sensitized with C. jejuni LOS and did not alter IgG subclasses, evidence that BAFF aids but is not essential for the generation of IgG anti-ganglioside antibodies in response to C. jejuni LOS.

Mouse APOBEC3 affects the production of virus-neutralizing antibodies by restricting early retroviral replication, not by altering the B-cell repertoire

Recent genetic analyses have indicated that polymorphisms in the mouse APOBEC3 locus constitute the Rfv3 gene that influences the production of virus-neutralizing antibodies in mice infected with Friend leukemia retrovirus [1,2]. Mice of the resistant genotype preferentially express the exon 5-lacking transcript in higher levels, while susceptible mice express the full-length transcript in lower levels [2,3]. Mouse APOBEC3 expressed in the resistant strains restricts the replication of mouse retroviruses in vitro through a mechanism independent of its deaminase activity [2]. However, the mechanisms through which mouse APOBEC3 affects the production of virus-neutralizing antibodies remain unclear. To address this question, we analyzed retroviral replication and the production of virus-neutralizing antibodies in mice of different APOBEC3 genotypes and those lacking its expression. Strain A mice with the susceptible APOBEC3 genotype nevertheless produced high levels of virus-neutralizing antibodies when they possessed the H-2b haplotype, and class-switching to IgG was observed in the presence of virus-specific T helper cells. Further, APOBEC3-deficient mice produced virus-neutralizing antibodies when their T helper cells had been primed with the viral antigen. Friend virus-induced derangements in the hematopoiesis and resultant splenomegaly are not directly responsible for the delayed antibody responses, because higher levels of viremia and lower antibody responses were observed upon infection with nonpathogenic Friend murine leukemia helper virus in the absence of APOBEC3. In mice of the resistance-associated APOBEC3 genotype lower levels of viremia were observed even before the detection of virus-neutalizing antibodies in the blood, indicating that the polymorphisms in the APOBEC3 locus affect the production of neutralizing antibodies as a result of restricted retroviral replication.

Evaluating vaccine‐induced immune responses in a murine model of LCMV infection

Human immunodeficiency virus (HIV) is a retrovirus that infects vital cells of the human immune system. Currently, HIV infection is treated with antiretroviral therapy (ART), which is expensive and has severe side effects. Therapeutic vaccination using plasmid DNA has been proposed as an alternative to ART. DNA vaccines have been shown to induce immune responses against encoded proteins that may help treat or cure disease. To study novel DNA vaccine approaches against viral infection we are developing a murine model of infection that mimics HIV infection in humans. Lymphocytic choriomeningitis virus (LCMV) can establish a chronic infection in mice and is well characterized in the rodent model. We designed a DNA vaccine against LCMV expressing the viral glycoprotein and vaccine potency will be enhanced using in vivo electroporation. To measure vaccine‐specific antibody production in immunized mice, we developed an enzyme‐linked immunosorbent assay (ELISA) using plate‐bound whole LCMV virus. ELISA results are reported from a pilot study examining vaccine dose at two weeks post‐immunization. These responses will be monitored weekly until the study endpoint, at which time vaccine‐specific cellular immune responses will be assessed using murine splenocytes. This work was funded by the HHMI Program, the MARC U*STAR T34 08663, and UPenn SUIP.

Aging Exacerbates Restraint Stress-Induced Inhibition of Antigen-Specific Antibody Production in Mice

We have recently found that exposure to acute restraint stress suppresses antigen-specific antibody production, including IgE, in a murine model of allergic rhinitis. Although age-related alterations in immune responses are known, it remains unclear whether aging modulates the antibody production under stressful conditions. In this study, we set out to determine the effects of aging on antibody production under acute restraint stress in mice. Both young and aged CBA/J mice were repeatedly sensitized intranasally with phospholipase A2 (PLA2) without adjuvants. Restraint stress was applied using uniform cylinders once a week for a continuous 8h period, on 5 occasions in total. Blood samples were taken at 0, 20 and 30 days after primary sensitization, and production of PLA2-specific antibodies and levels of IL-4, IFN-gamma, IL-10 and IL-1 beta in sera were determined by ELISA. Repeated intranasal sensitization with PLA2 induced PLA2-specific IgE, IgG1 and IgG2a production in aged mice. We found that exposure to restraint stress significantly inhibited production of PLA2-specific IgE, IgG1 and IgG2a in aged mice. In addition, antibody production under restraint stress decreased significantly in aged mice when compared with young mice. No IL-4, IFN-gamma, IL-10 or IL-1 beta were detected in sera from non-stressed or stressed aged mice. Aging exacerbates the immunosuppressive role of acute restraint stress in antigen-specific antibody production in mice.

Gene gun-supported DNA immunisation of chicken for straightforward production of poxvirus-specific IgY antibodies

Orthopoxviruses code for numerous immunomodulatory proteins, the structure and function of which are clarified inadequately. Antibodies constitute a potent tool to study such proteins, enabling conclusions on protein location and time course of expression. However, common antibody production in mice or rabbits requires tedious protein expression and injection, as well as blood collection at regular intervals. To simplify this procedure, IgY antibodies specific for poxviral proteins (F1L and p28) were generated by immunisation of chickens, because antibody retrieval from eggs allows the non-invasive generation of huge amounts of antibodies. The main intentions were (i) to decrease invasiveness, (ii) to immunise with native forms of proteins and (iii) to circumvent previous protein expression and purification. Therefore, chicken were immunised with DNA expression vectors coding for conserved domains of the selected proteins delivered for the first time by a gene gun. Four weeks after initial immunisation specific antibodies were found in the egg yolk as proven by immunofluorescence staining of poxvirus-infected cells. The specific IgY titre rose to 1:80,000 and was stable for more than 120 days. With this investigation we present an universal procedure for IgY design and production that can be applied for various issues in the future.

Effects of Alcohol Consumption on Humoral Immunity in Mice

Nwaogu, I.C., Ezeibe, M.C.O. and Ifeduba, O.F. 2008. Effects of alcohol consumption on humoral immunity in mice. J. Appl. Anim. Res., 34: 181–183. To study the effect of alcohol consumption on spleen and antibody production to Peste des Petit Ruminantes (PPR) vaccine, 28-3 week old inbred albino mice were divided into 2 equal groups. The first group was given 10% ethanol (v/v) in water for 1 week, 20% (v/v) for another 2 weeks and 30% for the remaining period of the experiment. The second group served as control. At the 6th week the mice were given 0.1 ml of reconstituted PPR vaccine orally. At d 14 post vaccination, the animals were sacrificed and sera samples collected. The mean body weights, spleen weights, spleen body mass indices and antibody titre of the alcohol exposed mice were (P<0.01) lower than those of the control mice. These results suggest that alcohol consumption adversely affects spleen and reduces the production of antibodies in mice.

Investigating the Mechanisms Underlying FVIII Antibody Production in Hemophilic Mice Following Recombinant and Plasma-Derived FVIII Infusion

The formation of Factor VIII (FVIII) antibodies after FVIII treatment in hemophilic patients is currently the major treatment-related complication faced by hemophilic patients. It has been reported that the use of plasma-derived FVIII (pdFVIII) in patients and animal models of hemophilia A will result in reduced anti-FVIII antibody formation compared to recombinant FVIII (rFVIII). In an attempt to explain this phenomenon, an initial hypothesis relating to the uptake of FVIII by antigen presenting cells has been formulated. In contrast, here we examine the hypothesis that the cytokine microenvironment induced by pdFVIII has a critical influence on the reduction of anti-FVIII antibody titers in hemophilia A mice. Micro-array and confirmatory quantitative RT-PCR experiments performed on CD11c+ dendritic cells, isolated 24 hours after rFVIII or pdFVIII infusion, show that pdFVIII causes a different immune gene expression profile than rFVIII administration. Both treatments caused the up regulation of pro-inflammatory gene expression. However, the rFVIII and pdFVIII treatments caused the up regulation of genes that induce Th1 and Th2 responses, respectively. Moreover, after administering rFVIII or pdFVIII concentrates to mice, we observed that each treatment induced a distinct T cell splenic cytokine micro-environment. Recombinant FVIII induced the release of Th1 cytokines and IL10, while pdFVIII induced the release of Th2 cytokines and TGF-α. In this mouse model, we observed the formation of high titers of anti-human VWF antibodies in the pdFVIII-treated mice and we propose that in these animals, VWF competes with FVIII for antigenic presentation. We further investigated the potential of antigenic competition by treating mice with FVIII and increasing concentrations of another unrelated protein, recombinant human factor IX (FIX). Our studies have shown an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies allude to new and additional mechanisms that may contribute to the reduction of anti-FVIII antibody development in hemophilia A mice treated with pdFVIII.

Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Inter‐laboratory comparisons of assessment of the allergenic potential of proteins in mice

Assessment of the potential allergenicity of novel proteins, including those expressed in genetically modified plants, is an important issue. In previous studies, we have shown that the IgE measurement induced by systemic exposure of BALB/c mice to a range of proteins correlates broadly with what is known of their allergenic potential in humans. The approach used a homologous passive cutaneous anaphylaxis (PCA) assay that reflects IgE-dependent biological activity and is of sufficient sensitivity to detect IgE production in the absence of adjuvant. In previous studies, the immunization phase was conducted independently in two separate facilities, and the subsequent analytical work (PCA) conducted in a single facility. The purpose here was to further evaluate the transferability of this approach. To this end, BALB/c mice were exposed to a range of doses of peanut agglutinin or ovalbumin, allergenic proteins of peanut and hen's egg, respectively, in two independent laboratories. Serial doubling dilutions of serum pooled for each treatment group were analyzed for specific IgE. At higher doses of allergen very similar, or identical, IgE titers were achieved in both laboratories, although at lower doses, responses were somewhat more variable. These data demonstrate that, although technically demanding, the measurement of protein allergen-induced IgE antibody production in mice using PCA is relatively robust and is transferable and reproducible between laboratories. This approach may provide a useful tool for the safety assessment of novel proteins and suggests that continued evaluation of the approach with a wider range of protein allergens and non-sensitising proteins is justified.

Synthesis of QS‐21‐Xylose: Establishment of the Immunopotentiating Activity of Synthetic QS‐21 Adjuvant with a Melanoma Vaccine

A helping hand: The adjuvant QS-21-xylose (see structure; the xylose residue is shown in red) was obtained in pure form by chemical synthesis. When combined with its synthetic apiose isomer to furnish synthetic QS-21 (sQS-21), the saponin combination was shown to aid the production of antibodies in mice when injected with a melanoma vaccine. The preparation of sQS-21 provides access to pure, functional saponin adjuvants of defined composition.