Natural antibodies targeting LPS in pleural effusions of various etiologies.

Respiratory infection, cancer, and heart failure can cause abnormal accumulation of fluid in the pleural cavity. The immune responses within the cavity are orchestrated by leucocytes that reside in the serosal-associated lymphoid tissue. Natural antibodies (NAbs) are abundant in the serum (S) having a major role in systemic and mucosal immunity; however, their occurrence in pleural fluid (PF) remains an open question. Our aim herein was to detect and measure the levels of NAbs (IgM, IgG, IgA) targeting lipopolysaccharides (LPS) in both the pleural fluid and the serum of 78 patients with pleural effusions (PEs) of various etiologies. The values of anti-LPS NAb activity were extracted through a normalization step regarding the total IgM, IgG, and IgA levels, all determined by in-house ELISA. In addition, the ratios of PF/S values were analyzed further with other critical biochemical parameters from pleural fluids. Anti-LPS NAbs of all Ig classes were detected in most of the samples, while a significant increase of anti-LPS activity was observed in infectious and noninfectious compared with malignant PEs. Multivariate linear regression confirmed a negative correlation of IgM and IgA anti-LPS PF/S ratio with malignancy. Moreover, anti-LPS NAbs PF/S measurements led to increased positive and negative predictive power in ROC curves generated for the discrimination between benign and malignant PEs. Our results highlight the role of anti-LPS NAbs in the pleural cavity and demonstrate the potential translational impact that should be further explored.NEW & NOTEWORTHY Here we describe the detection and quantification of natural antibodies (NAbs) in the human pleural cavity. We show for the first time that IgM, IgG, and IgA anti-LPS natural antibodies are detected and measured in pleural effusions of infectious, noninfectious, and malignant etiologies and provide clinical correlates to demonstrate the translational impact of our findings.

Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study.

BACKGROUND: Malignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures. METHODS: A human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with ß-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2-∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined. RESULTS: PC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells. CONCLUSIONS: These data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.

N-Glycosylation of the Ig Receptors Shapes the Antigen Reactivity in Chronic Lymphocytic Leukemia Subset #201.

Subset #201 is a clinically indolent subgroup of patients with chronic lymphocytic leukemia defined by the expression of stereotyped, mutated IGHV4-34/IGLV1-44 BCR Ig. Subset #201 is characterized by recurrent somatic hypermutations (SHMs) that frequently lead to the creation and/or disruption of N-glycosylation sites within the Ig H and L chain variable domains. To understand the relevance of this observation, using next-generation sequencing, we studied how SHM shapes the subclonal architecture of the BCR Ig repertoire in subset #201, particularly focusing on changes in N-glycosylation sites. Moreover, we profiled the Ag reactivity of the clonotypic BCR Ig expressed as rmAbs. We found that almost all analyzed cases from subset #201 carry SHMs potentially affecting N-glycosylation at the clonal and/or subclonal level and obtained evidence for N-glycan occupancy in SHM-induced novel N-glycosylation sites. These particular SHMs impact (auto)antigen recognition, as indicated by differences in Ag reactivity between the authentic rmAbs and germline revertants of SHMs introducing novel N-glycosylation sites in experiments entailing 1) flow cytometry for binding to viable cells, 2) immunohistochemistry against various human tissues, 3) ELISA against microbial Ags, and 4) protein microarrays testing reactivity against multiple autoantigens. On these grounds, N-glycosylation appears as relevant for the natural history of at least a fraction of Ig-mutated chronic lymphocytic leukemia. Moreover, subset #201 emerges as a paradigmatic case for the role of affinity maturation in the evolution of Ag reactivity of the clonotypic BCR Ig.

Natural autoimmunity in oligoarticular juvenile idiopathic arthritis.

BACKGROUND: Oligoarticular juvenile idiopathic arthritis (oligo-JIA) is considered as an antigen-driven lymphocyte-mediated autoimmune disease. Natural antibodies (NAbs) are pre-immune antibodies produced in the absence of exogenous antigen stimulation, participating in both, innate and adaptive immunity. Considering their major immunoregulatory role in homeostasis and autoimmune pathogenesis, we designed this study to further elucidate their role in oligo-JIA pathogenesis. METHODS: Seventy children with persistent oligo-JIA and 20 healthy matched controls were enrolled in the study. Serum IgM and IgA antibodies against human G-actin, human IgG F(ab΄)2 fragments and the hapten TriNitroPhenol (TNP) as well as the total concentration of serum IgM and IgA were measured by in-house enzyme-immunoassays. Kolmogorov-Smirnov normality test, Kruskal-Wallis H and Mann-Whitney tests were used to assess data distribution, and significant differences of non-parametric data between groups of the study. Backward regression analysis was used to analyze the effect of multiple factors (age, gender, disease activity, anti-nuclear antibody positivity, presence of uveitis) on continuous dependent variables (activities and activity/ concentration ratios of IgM and IgA NAbs). RESULTS: The ratios of IgA anti-TNP, anti-actin and anti-F(ab΄)2 levels to total serum IgA concentration were found to be significantly increased in patients with oligo-JIA compared to healthy subjects. Significantly elevated levels of IgM anti-TNP antibodies were also found in children with inactive oligo-JIA compared to those of children with active disease and of healthy controls. In the presence of anterior uveitis, IgM anti-TNP levels were significantly higher than in patients without uveitis or in healthy controls. Backward regression analysis revealed that the disease activity and the presence of anterior uveitis independently affect IgM anti-TNP levels. CONCLUSUIONS: Our findings are in accordance with the hypothesis that NAbs contribute to the pathogenesis of autoimmune diseases and provide additional evidence that disturbances in natural autoimmunity may contribute to the as yet unclarified pathogenesis of oligo-JIA.

Levels of Circulating IgM and IgY Natural Antibodies in Broiler Chicks: Association with Genotype and Farming Systems.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

Combined monitoring of IgG and IgA anti-Spike and anti-Receptor binding domain long term responses following BNT162b2 mRNA vaccination in Greek healthcare workers.

Studies on the humoral response to homologous BNT162b2 mRNA-vaccination focus mainly on IgG antibody dynamics, while long-term IgA kinetics are understudied. Herein, kinetics of IgG and IgA levels against trimeric-Spike (S) and Receptor-Binding-Domain (RBD) were evaluated by in-house ELISAs in 146 two-dose vaccinated Greek healthcare workers (HCWs) in a 9-month period at six time points (up to 270 days after the first dose). The effect of a homologous booster third dose was also studied and evaluated. The peak of immune response was observed 21 days after the second dose; 100% seroconversion rate for anti-S and anti-RBD IgG, and 99.7% and 96.3% respectively for IgA. IgG antibody levels displayed higher increase compared to IgA. Declining but persistent anti-SARS-CoV-2 antibody levels were detected 9 months after vaccination; IgG and IgA anti-S levels approached those after the first dose, while a more rapid reduction rate for anti-RBD antibodies led to significantly lower levels for both classes, supporting the need for a booster dose. Indeed, a homologous booster third dose resulted in enhanced levels of anti-S of both classes, whereas anti-RBD didn't exceed the peak levels after the second dose. Previous SARS-CoV-2 infection, flu vaccination, BMI<35 and the occurrence of an adverse event upon vaccination, were associated with higher IgG antibody levels over time, which however were negatively affected by age increase and the presence of chronic diseases. Overall, after concurrently using the S and RBD target-antigens in in-house ELISAs, we report in addition to IgG, long-term persistence of IgA antibodies. Regarding antibody levels, homologous mRNA vaccination gives rise to an effective anti-viral protection up to 9 months negatively correlated to age. Considering that COVID-19 is still a matter of public concern, booster vaccine doses remain critical to vulnerable individuals.

New Analogs of Polyamine Toxins from Spiders and Wasps: Liquid Phase Fragment Synthesis and Evaluation of Antiproliferative Activity.

Polyamine toxins (PATs) are conjugates of polyamines (PAs) with lipophilic carboxylic acids, which have been recently shown to present antiproliferative activity. Ten analogs of the spider PATs Agel 416, HO-416b, and JSTX-3 and the wasp PAT PhTX-433 were synthesized with changes in the lipophilic head group and/or the PA chain, and their antiproliferative activity was evaluated on MCF-7 and MDA-MB-231 breast cancer cells, using Agel 416 and HO-416b as reference compounds. All five analogs of PhTX-433 were of very low activity on both cell lines, whereas the two analogs of JSTX-3 were highly active only on the MCF-7 cell line with IC50 values of 2.63-2.81 µΜ. Of the remaining three Agel 416 or HO-416b analogs, only the one with the spermidine chain was highly active on both cells with IC50 values of 3.15-12.6 µM. The two most potent compounds in this series, Agel 416 and HO-416b, with IC50 values of 0.09-3.98 µΜ for both cell lines, were found to have a very weak cytotoxic effect on the MCF-12A normal breast cells. The present study points out that the structure of both the head group and the PA chain determine the strength of the antiproliferative activity of PATs and their selectivity towards different cells.

DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.

We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

Immunological evidence and regulatory potential for cell-penetrating antibodies in intravenous immunoglobulin.

Anti-DNA cell-penetrating autoantibodies have been extensively studied in autoimmune but not in normal sera. We investigated herein the presence and properties of cell-penetrating antibodies (CPAbs) in intravenous immunoglobulin (IVIg), a blood product of pooled normal human IgG. IVIg cell penetration was observed into various cell lines, as well as cells from several organs of mice injected intravenously with IVIg therapeutic dose. In all cell types examined in vitro and in vivo, intracellular IgG localized in the cytoplasm, in contrast to the nuclear accumulation of disease-related CPAbs. IVIg was found to rapidly enter cells via an energy-independent mode. The CPAb-fraction was isolated and found to be polyreactive to nuclear and cytoplasmic components; although it corresponded to ~2% of IVIg, it accounted for its inhibitory effect on splenocyte activation. Investigation of IVIg cell penetration capacity provides insight into its mechanisms of action and may account for some of its beneficial effects in numerous diseases.

A novel pathogenic peptide of thyroglobulin (2208-2227) induces autoreactive T-cell and B-cell responses in both high and low responder mouse strains.

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208-2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A(b)), or susceptibility (A(s), E(k)) to EAT. We therefore immunized LR BALB/c (H-2(d)) and C57BL/6 (H-2(b)) strains, as well as HR CBA/J (H-2(k)) and SJL/J (H-2(s)) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B-cell and cryptic T-cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1-3), as well as in SJL/J mice (two of six mice, infiltration index 1-2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.

Fine epitope mapping within the pathogenic thyroglobulin peptide 2340-2359: minimal epitopes retaining antigenicity across various MHC haplotypes are not necessarily immunogenic.

We have previously reported that the 20-mer peptide p2340 (amino acids 2340-2359), of human thyroglobulin (Tg) has the unique feature that it causes experimental autoimmune thyroiditis (EAT) in mouse strains bearing high-responder (HR) or low-responder (LR) MHC haplotypes in Tg-induced EAT. In this study, we have employed fine epitope mapping to examine whether this property of p2340 is the result of recognition of distinct or shared minimal T-cell epitopes in the context of HR or LR MHC class II molecules. Use of overlapping peptides showed that a core minimal 9-mer epitope (LTWVQTHIR, amino acids 2344-2352) was recognized by p2340-primed T cells from both HR (H2(k,s) ) and LR (H2(b,d) ) strains, whereas a second 9-mer epitope (HIRGFGGDP, amino acids 2350-2358) was antigenic only in H2(s) hosts. Truncation analysis of LTWVQTHIR and HIRGFGGDP peptides delineated them as the minimal epitopes recognized by p2340-primed T cells from the above strains. Subcutaneous challenge of all mouse strains with the 9-mer core peptide LTWVQTHIR in adjuvant elicited specific lymph node cell proliferative responses and mild EAT only in HR hosts, highlighting this sequence as a minimal pathogenic Tg peptide in EAT. The 9-mer peptide HIRGFGGDP was not found to be immunogenic in H2(s) hosts. These data demonstrate that minimal T-cell epitopes, defined as autoantigenic in hosts of various MHC haplotypes, are not intrinsically immunogenic. Activation of naive autoreactive T cells may require contributions from flanking residues within longer peptide sequences encompassing these epitopes.

Clinical evaluation of PRMT1 gene expression in breast cancer.

Methylation of arginine residues has been implicated in many cellular activities like mRNA splicing, transcription regulation, signal transduction and protein-protein interactions. Protein arginine methyltransferases are the enzymes responsible for this modification in living cells. The most commonly used methyltransferase in man is protein arginine methyltransferase 1 (PRMT1). Since methylation processes appear to interfere in the emergence of several diseases, including cancer, we investigated the localisation of the protein in cancer tissue and, for the first time, the relation that possibly exists between the expression of PRMT1 gene and breast cancer progression. We used tumour specimens from 62 breast cancer patients and semi-quantitative RT-PCR to determine the expression of PRMT1 gene and was found to be associated with patient's age (p = 0.002), menopausal status (p = 0.006), tumour grade (p = 0.03), and progesterone receptor status (p = 0.001). Survival curves revealed that PRMT1-v1 status-low expression relates to longer disease-free survival (DFS; p = 0.036). To the contrary, PRMT1-v2 status is not associated neither with the clinical or pathological parameters nor with DFS (p = 0.31). PRMT1-v3 was not statistically significantly expressed in breast cancer tissue. Selected cancer and normal breast samples were stained for PRMT1. In both normal and cancerous breast tissues, staining was in the cytoplasm and only in rare cases the cell nucleus appeared stained. Present results show a potential use for this gene as a marker of unfavourable prognosis for breast cancer patients.

Grouping of patients with common variable immunodeficiency based on immunoglobulin biosynthesis: comparison with a classification system on CD4-naïve cells.

The present study compared two different systems of classification of patients with common variable immunodeficiency (CVID); one based on in vitro immunoglobulin biosynthesis; and another on CD4-naïve cell counts. Peripheral blood mononuclear cells (PBMCs) were isolated from 35 patients with CVID and 20 healthy controls. They were stimulated for the secretion of IgM and IgG after stimulation with Staphylococcus aureus Cowan I (SAC) upon supplementation of interleukin-2 (IL-2) or with pokeweed mitogen. T cell subsets were estimated by flow cytometry. By the first system, patients were classified into group A (n=18) with secretion of neither IgG nor IgM; into group B (n=12) with detectable IgM but no IgG secretion; and into group C (n=5) with IgM and IgG secretion similar to controls. By the second system, patients were classified into group I (n=12) with less than 109 CD4-naïve cells/mul; into group II (n=12) with CD4-naïve cells within 109-225microl(-1); and into group III (n=11) with more than 225 CD4-naïve cells/mul. All groups I-III were defective for in vitro release of IgG and IgM. The likelihood ratio for splenomegaly in patients with <225 CD4-naïve cells/mul was 5.08 (p: 0.024). CD4-naïve cell counts of patients were positively correlated to serum levels of IgG and IgA of patients. The presented results revealed that the former system described adequately the function of B cells and the latter the clinical status of the patient. Our proposal is that both should be used for the characterization of patients with CVID.

Naturally occurring B-cell autoreactivity: a critical overview.

In over one century of research in immunology marked progress in the scientific knowledge and the implications derived from it has been made. At the same time several contradictory and seemingly opposing results have been obtained. The term autoimmunity is still conceived by many as a term directly related to an immunopathological state. However, strong evidence exist that not only the immune system is able to recognize self-constituents, but it appears also that this property is essential for homeostasis. Direct or indirect alterations of such self-recognition properties of the immune system may contribute to pathology. In this review, the most recent advances in the field of naturally occurring B-cell autoreactivity in health as well as in disease are presented and discussed.

Pathogenicity of a human thyroglobulin peptide (2340-2359) in mice with high or low genetic susceptibility to thyroiditis.

We have previously identified a 20-mer peptide of human thyroglobulin (hTg), p2340 (aa2340-2359), which induced experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) and HLA-DR3 transgenic mice. In this study, we investigated the thyroiditogenic potential of p2340 in 'high responder' CBA/J (H-2(k)) and SJL/J (H-2(s)) or 'low responder' C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice. Mice were immunized subcutaneously with 100 nmol of p2340 in complete Freund's adjuvant (CFA) and both the proliferative capacity of their lymph node cells in the presence of p2340 or intact Tg and the production of peptide-specific antibodies were investigated. The p2340 peptide was found to contain B-cell and non-dominant T-cell epitope(s) in all strains tested. Moreover, it elicited EAT in CBA/J (2/6, infiltration index (I.I.) 1) and SJL/J (5/5, I.I. 1-3) mice after direct challenge and in BALB/c (4/7, I.I. 1) and C57BL/6 (1/5, I.I. 1) after adoptive transfer of p2340-primed lymph node cells. P2340 is the first Tg peptide found to be pathogenic in low as well as high responder mouse strains and thus will allow us to investigate mechanisms of EAT induction in a genetically resistant host.

Human thyroglobulin peptide p2340 induces autoimmune thyroiditis in HLA-DR3 transgenic mice.

In a previous study we demonstrated that the human thyroglobulin (hTg) peptide p2340 (aa 2340-2359) can stimulate a T cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2(k)) mice. In the present study we examined whether p2340 can induce EAT in single HLA class II DR3 transgenic mice. This peptide was found to be immunogenic at the T cell level in DR3 mice, since it induced specific proliferative responses, as well as IL-2 and IFN-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). Immunization of HLA-DR3 mice with p2340 in CFA elicited EAT (infiltration index of 1 to 2) in eight of nine mice. Peptide-primed LNC responded to intact hTg, whereas, hTg-primed LNC did not respond to p2340 in culture, suggesting that p2340 contains subdominant T cell epitope(s). P2340 was also found to be immunogenic at the B cell level, since strong p2340-specific IgG response was detected in all transgenic mice tested. Thus, we provide evidence for a pathogenic role of an hTg peptide in HLA-DR3 transgenic mice. Therefore, p2340 could be presented by DR3 molecule in patients with Hashimoto's thyroiditis and participate in the development of the disease.

Thyroxine-binding antibodies inhibit T cell recognition of a pathogenic thyroglobulin epitope.

Thyroid hormone-binding (THB) Abs are frequently detected in autoimmune thyroid disorders but it is unknown whether they can exert immunoregulatory effects. We report that a THB mAb recognizing the 5' iodine atom of the outer phenolic ring of thyroxine (T4) can block T cell recognition of the pathogenic thyroglobulin (Tg) peptide (2549-2560) that contains T4 at aa position 2553 (T4(2553)). Following peptide binding to the MHC groove, the THB mAb inhibited activation of the A(k)-restricted, T4(2553)-specific, mouse T cell hybridoma clone 3.47, which does not recognize other T4-containing epitopes or noniodinated peptide analogues. Addition of the same THB mAb to T4(2553)-pulsed splenocytes largely inhibited specific activation of T4(2553)-primed lymph node cells and significantly reduced their capacity to adoptively transfer thyroiditis to naive CBA/J mice. These data demonstrate that some THB Abs can block recognition of iodine-containing Tg epitopes by autoaggressive T cells and support the view that such Abs may influence the development or maintenance of thyroid disease.

Serological detection of hepatitis B viral infection by a panel of solid-phase enzyme-linked immunosorbent assays (ELISA).

Immunoassays for the detection of hepatitis B virus (HBV) in biological samples were developed. Using recombinant HBV antigens (Ags) and HBV-specific antibodies (Abs), we designed and evaluated a panel of enzyme-linked immunosorbent assays (ELISAs) detecting the main hepatitis B-related viral markers, namely HBV surface Ag (HBsAg), HBV e Ag (HBeAg), Abs to HBsAg (anti-HBs), Abs to HBV core Ag (anti-HBc) and Abs to HBeAg (anti-HBe), in blood serum. The ELISAs were validated using a panel of prescreened, by commercial tests, serum samples. In principle, HBV Ags or anti-HBV monoclonal antibodies (mAbs) were immobilised on microplate wells. Horseradish peroxidase (HRP) or biotin were used to prepare labeled Abs. Specifically for the determination of HBsAg and HBeAg, two-site sandwich immunoenzymometric assays were developed. The useful range was estimated at 20-500 ng/ml and human serum samples assayed were diluted 10- and 4-fold for HBsAg and HBeAg, respectively, with phosphate buffered saline (PBS) containing Tween 20 and gelatin. For the detection of Abs to HBs an indirect ELISA was formulated. Sera were similarly 4-fold diluted in the same buffer. Finally, competitive ELISAs were used for detecting anti-HBc and anti-HBe and sera tested were diluted 20- and 5-fold, respectively. All selected dilutions resulted in the accurate and reliable determination of HBV Ags and anti-HBV Abs. Taken altogether, these ELISAs are highly specific and equally sensitive to the circulating tests. However, their design could be very useful for research and/or preclinical studies of selected HBV-infected individuals.

Induction of murine thyroiditis by a non dominant E(k)-restricted peptide of human thyroglobulin.

We have previously shown that the human thyroglobulin (hTg) 20-mer peptide p2340 (aa 2340-2359) contains an epitope recognized by Tg-reactive B cells in patients with Graves' disease. The presence of several Ek-binding motifs within p2340 prompted us to examine whether this peptide can stimulate a T-cell response and elicit experimental autoimmune thyroiditis (EAT) in AKR/J (H-2k) mice. The peptide was found to be immunogenic at the T-cell level since it induced specific proliferative responses as well as interleukin-2 and interferon-gamma secretion in secondary cultures of peptide-primed lymph node cells (LNC). The p2340-specific proliferation was blocked almost completely by an Ek-specific monoclonal antibody (mAb) but was unaffected by a control Ak-specific mAb. Peptide-primed LNC did not respond to intact hTg and conversely, LNC primed in vivo with hTg did not respond to p2340 in culture, suggesting that p2340 contains non-dominant T-cell epitope(s). Direct subcutanaeous challenge of AKR/J mice (n = 9) with p2340 in adjuvant, elicited mild to moderate EAT (infiltration index of 1-2) and strong p2340-specific immunoglobulin G responses in all mice tested. These data delineate a new thyroiditogenic sequence within the carboxyl terminal region of hTg.

A skeletal muscle troponin T specific ELISA based on the use of an antibody against the soluble troponin T (16-31) fragment.

Proteolytic degradation of muscle, which occurs post-mortem as part of the meat-ageing process, results in the production of protein fragments. In beef, degradation of skeletal muscle troponin T (TnT) results in the generation of a 16-residue long peptide (TnT (16-31)), identified in trichloroacetic acid (TCA) soluble extracts. We report the development of a competitive enzyme-linked immunosorbent assay (ELISA) for the quantification of TnT (16-31), using polyclonal antibodies raised against synthetic TnT (16-31). The ELISA procedure is based on inhibition of binding of the antibodies to immobilised TnT (16-31) by TnT (16-31) present in solution. Its useful range is 30 pmol to 2 nmol TnT (16-31)/ml. Quantification of TnT (16-31) in TCA muscle extracts showed that its concentration was enhanced with ageing. Moreover, a correlation between TnT (16-31) levels and meat tenderness was observed. The ELISA developed herein may prove advantageous for future use at the research and industrial level.