Biomedical subjects

Michael Papamichail

Publications and source records attributed to Michael Papamichail.

At least 19 recordsLinked to original sources

PubMed2006

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.

Antigens, Surface↗

PubMed2006

Ii-Key/HER-2/neu(776-790) hybrid peptides induce more effective immunological responses over the native peptide in lymphocyte cultures from patients with HER-2/neu+ tumors.

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.

Antigen Presentation↗

PubMed2006

Characterization of a novel cell penetrating peptide derived from Bag-1 protein.

A highly cationic peptide (BagP), located within the normally expressed human protein Bag-1, was tested for its capacity to act as a cell penetrating peptide. BagP was found to translocate and transport high molecular weight cargos in several cell types, in varying degrees with a preference for adherent cells. The penetration phenomenon was not found to be subject to saturation for the highest amount of peptide tested (100 microM), whereas the time needed for maximum translocation to be achieved, was cell type-dependent. Finally, BagP internalization depends on its charge, cellular metabolism and cell-surface heparan sulfate proteoglycans.

Amino Acid Sequence↗

PubMed2006

Vaccination with human HER-2/neu (435-443) CTL peptide induces effective antitumor immunity against HER-2/neu-expressing tumor cells in vivo.

HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.

Animals↗

PubMed2006

Cell culture medium composition and translational adult bone marrow-derived stem cell research.

The use of fetal calf serum (FCS) for the culture of cells to be used in clinical trials raises potential hazards that cannot be neglected, but this is a regulatory issue. However, as specifically regards the isolation and expansion of human mesenchymal stem cells (MSCs), unfortunately serum-free media have not yet been defined. The alternative of using autologous serum is feasible only for the minority of clinical protocols involving low numbers of MSCs, because a minimum concentration of 10% in the culture medium is required. Besides, because allogeneic serum results in MSC growth arrest and death, use of pooled human serum does not represent an alternative. Finally, vast numbers of MSCs cultured in FCS-containing media have already been used in many clinical trials targeting a variety of disorders, without any significant side effects, including ventricular arrhythmia.

Bone Marrow Cells↗

PubMed2005

Effect of IL-21 on NK cells derived from different umbilical cord blood populations.

IL-21 plays a role in the proliferation and maturation of NK cells developed from hematopoietic stem cells. In this study, we found that IL-21, in the presence of physiological concentration of hydrocortisone (HC), has a significant impact on the functions of NK cells derived from umbilical cord blood (CB) populations. We demonstrate that IL-21, in combination with Flt3-ligand, IL-15 and HC, induces high proliferative responses and, apart from enhancing NK-mediated cytotoxicity, it also induces a significant increase in lymphokine-activated killer activity of CB/CD34+-derived CD56+ cells. In addition, IL-21 induced changes in the CD56+ cell cytokine secretion profile. Thus, we observed increased levels of IL-10 and granulocyte macrophage colony-stimulating factor, whereas tumor necrosis factor-alpha levels decreased. IFN-gamma production was also modified by IL-21, depending on the presence or absence of IL-18. CB/CD34+ cells did not express the IL-21R ex vivo, but receptor expression was induced during their commitment to differentiation into CD56+ cells. Our data ascribe to IL-21 an essential role on NK cell development and function under conditions similar to the in vivo CB microenvironment.

Anti-Inflammatory Agents↗

PubMed2005

Immunogenic HER-2/neu peptides as tumor vaccines.

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination.

Animals↗

PubMed2005

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice.

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.

Animals↗

PubMed2005

Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent cells defined by multilineage potential, ease to gene modification, and immunosuppressive ability, thus holding promise for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which, however, does not compensate for the very low percentage in their niches given the vast numbers of cells required for the relative studies. Taking into consideration the lack of a uniform approach for MSC isolation and expansion, we attempted in this study, by comparing various culture conditions, to identify the optimal protocol for the large-scale production of MSCs while maintaining their multilineage and immunosuppressive capacities. Our data indicate that, apart from the quality of fetal calf serum, other culture parameters, including basal medium, glucose concentration, stable glutamine, bone marrow mononuclear cell plating density, MSC passaging density, and plastic surface quality, affect the final outcome. Furthermore, the use of basic fibroblast growth factor (bFGF), the most common growth supplement in MSC culture media, greatly increases the proliferation rate but also upregulates HLA-class I and induces low HLA-DR expression. However, not only does this upregulation not elicit significant in vitro allogeneic T cell responses, but also bFGF-cultured MSCs exhibit enhanced in vivo immunosuppressive potential. Besides, addition of bFGF affects MSC multilineage differentiation capacity, favoring differentiation toward the osteogenic lineage and limiting neurogenic potential. In conclusion, in this report we define the optimal culture conditions for the successful isolation and expansion of human MSCs in high numbers for subsequent cellular therapeutic approaches.

Cell Culture Techniques↗

PubMed2005

Interactions between human mesenchymal stem cells and natural killer cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells representing an attractive therapeutic tool for regenerative medicine. They possess unique immunomodulatory properties, being capable of suppressing T-cell responses and modifying dendritic cell differentiation, maturation, and function, whereas they are not inherently immunogenic, failing to induce alloreactivity to T cells and freshly isolated natural killer (NK) cells. To clarify the generation of host immune responses to implanted MSCs in tissue engineering and their potential use as immunosuppressive elements, the effect of MSCs on NK cells was investigated. We demonstrate that at low NK-to-MSC ratios, MSCs alter the phenotype of NK cells and suppress proliferation, cytokine secretion, and cyto-toxicity against HLA-class I- expressing targets. Some of these effects require cell-to-cell contact, whereas others are mediated by soluble factors, including transforming growth factor-beta1 and prostaglandin E2, suggesting the existence of diverse mechanisms for MSC-mediated NK-cell suppression. On the other hand, MSCs are susceptible to lysis by activated NK cells. Overall, these data improve our knowledge of interactions between MSCs and NK cells and consequently of their effect on innate immune responses and their contribution to the regulation of adaptive immunity, graft rejection, and cancer immunotherapy.

Adult↗

PubMed2005

Multicentre evaluation of stable reference whole blood for enumeration of lymphocyte subsets by flow cytometry.

BACKGROUND: Clinical indications for lymphocyte subset enumeration by flow cytometry include monitoring of disease progression and timing of therapeutic intervention in infection with human immunodeficiency virus. Until recently international standardisation has not been possible due to a lack of suitable stable reference material. METHODS: This study consisted of two trials of a stabilised whole blood preparation. Eleven participants were sent two standard protocols for staining plus gating strategy and asked to report absolute counts for lymphocyte subsets. RESULTS: No significant difference was detected between the two methods when results from the two assays and all partners were pooled. Significant differences in results from the different partners were observed. However, representative mean counts were obtained for geometric means, geometric coefficient of variation, and 95% confidence interval for CD3 910 cells/mul, 9%, and 888 to 933, respectively), CD4 (495 cells/mul, 12%, and 483 to 507), and CD8 (408 cells/mul, 13%, and 393 to 422). CONCLUSION: We have introduced a stabilised blood preparation and a well-characterized biological standard. The availability of this reference material greatly simplifies the validation of new techniques for CD4(+) T-cell enumeration and the expansion of external quality assurance programmes for clinical laboratories, including those that operate in resource-restricted environments. (c) 2005 Wiley-Liss, Inc.

Journal Article↗

PubMed2005

CRD-BP/IMP1 expression characterizes cord blood CD34+ stem cells and affects c-myc and IGF-II expression in MCF-7 cancer cells.

The coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein (CRD-BP/IMP1) is an RNA-binding protein specifically recognizing c-myc, leader 3' IGF-II and tau mRNAs, and the H19 RNA. CRD-BP/IMP1 is predominantly expressed in embryonal tissues but is de novo activated and/or overexpressed in various human neoplasias. To address the question of whether CRD-BP/IMP1 expression characterizes certain cell types displaying distinct proliferation and/or differentiation properties (i.e. stem cells), we isolated cell subpopulations from human bone marrow, mobilized peripheral blood, and cord blood, all sources known to contain stem cells, and monitored for its expression. CRD-BP/IMP1 was detected only in cord blood-derived CD34(+) stem cells and not in any other cell type of either adult or cord blood origin. Adult BM CD34(+) cells cultured in the presence of 5'-azacytidine expressed de novo CRD-BP/IMP1, suggesting that epigenetic modifications may be responsible for its silencing in adult non-expressing cells. Furthermore, by applying the short interfering RNA methodology in MCF-7 cells, we observed, subsequent to knocking down CRD-BP/IMP1, decreased c-myc expression, increased IGF-II mRNA levels, and reduced cell proliferation rates. These data 1) suggest a normal role for CRD-BP/IMP1 in pluripotent stem cells with high renewal capacity, like the CB CD34(+) cells, 2) indicate that altered methylation may directly or indirectly affect its expression in adult cells, 3) imply that its de novo activation in cancer cells may affect the expression of c-Myc and insulin-like growth factor II, and 4) indicate that the inhibition of CRD-BP/IMP1 expression might affect cancer cell proliferation.

Adult↗

PubMed2005

A potential role for hydrocortisone in the positive regulation of IL-15-activated NK-cell proliferation and survival.

Although glucocorticoids (GCs) have been described as acting mainly as anti-inflammatory and immunosuppressive drugs, they may also positively influence the immune system. In the present study, we demonstrate for the first time that hydrocortisone (HC), in synergy with interleukin-15 (IL-15), induces a dramatic increase in the expansion of peripheral blood-derived CD56+ cells, favoring the preferential outgrowth of classical natural killer (CD56+CD3- NK) over CD56+CD3+ natural killer T (NKT) cells. HC plus IL-15-driven CD56+ cells exhibited an increased potential for cytokine production with no impairment in their NK- and lymphokine-activated killer (LAK) activities. Elevated levels of GC-induced leucine zipper protein (GILZ) messenger RNA (mRNA) were detected in both NK and NKT cells cultured with HC and IL-15, in comparison to IL-15 alone. Phosphorylation status of signal transducer and activator of transcription 5 (STAT5) was not affected by the presence of HC in either of the populations. On the contrary, HC differentially affected the IL-2/IL-15R beta- and gamma-chain surface expression and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) in IL-15-activated NK and NKT cells. Our data ascribe a novel role to GCs on mature NK-cell expansion and function and open new perspectives for their use in cellular adoptive cancer immunotherapy.

Adoptive Transfer↗

PubMed2005

Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human myocardium.

The aim of the study was to investigate whether a combination of mesenchymal stem cells (MSCs) capable of differentiating into cardiac myocytes and endothelial progenitors (EPCs) that mainly promote neoangiogenesis might be able to facilitate tissue repair in myocardial scars. Previous studies have shown that intracoronary transplantation of autologous bone marrow stem cells results in improvement of contractility in infracted areas of human myocardium. Eleven patients with an anteroseptal myocardial infarction (MI) underwent transcoronary transplantation of bone marrow-derived MSCs and EPCs to the infarcted area through the left anterior descending artery. Eleven age- and sex-matched patients served as controls. Wall motion score index was significantly lower at follow-up in the transplantation (P = 0.04) but not in the control group. On stress echocardiography, there was improvement of myocardial contractility in one or more previously nonviable myocardial segments in 5 out of 11 patients (all with recent infarctions) and in none of the controls (P = 0.01). Restoration of uptake of Tc(99m) sestamibi in one or more previously nonviable myocardial scars was seen in 6 out of 11 patients subjected to transplantation and in none of the controls (P = 0.02). Cell transplantation was an independent predictor of improvement of nonviable tissue. Intracoronary transplantation of MSCs and EPCs is feasible, safe, and may contribute to regional regeneration of myocardial tissue early or late following MI.

Adult↗

PubMed2005

Beneficial effect of short-term exposure of human NK cells to IL15/IL12 and IL15/IL18 on cell apoptosis and function.

Monokines IL12, IL15, and IL18 have been shown to activate NK cell function, however with high apoptosis induced by their combination within 48 h. Here, we demonstrate for the first time that CD56+ cells incubated for only 18 h with the combination of IL15/IL12 or IL15/IL18, then washed, and further cultured in plain medium, exhibit low levels of apoptosis. These shortly activated CD56+ cells show high killer activity against NK- and LAK-sensitive tumor targets that persists over a culture period of 18 days after two additional 6 h cycles of exposure to the monokines applied every 8 days and also retain their ability for high cytokine production during each exposure. Moreover, these repetitive short-term exposures of CD56+ cells to the monokine combinations result in long-lived CD56+ cells with slower rates of FcgammaRIII receptor (CD16) decline, therefore exhibiting higher antibody depended cytotoxicity, as opposed to the continuous incubation with the monokine combinations. In conclusion, short-term exposure of CD56+ cells to IL15/IL12 or IL15/IL18 at 8-day intervals may hold a promise for improved clinical results in cellular adoptive cancer immunotherapy and for the in vivo injections of the monokines.

Apoptosis↗

PubMed2004

Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes.

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.

Amino Acid Motifs↗

PubMed2004

Generation of human tumor-specific CTLs in HLA-A2.1-transgenic mice using unfractionated peptides from eluates of human primary breast and ovarian tumors.

HER-2/neu oncoprotein is overexpressed in a variety of human tumors and is associated with malignant transformation and aggressive disease. Due to its overexpression in tumor cells and because it has been shown to be immunogenic, this protein represents an excellent target for T-cell immunotherapy. Peptide extracts derived from primary HLA-A*0201-positive (+) HER-2/neu+ human tumors by acid elution (acid cell extracts (ACEs)) were tested for their capacity to elicit in HLA-A*0201 transgenic mice, cytotoxic T lymphocytes (CTLs) lysing HLA-A*0201+ HER-2/neu+ tumor cells. Injections of ACE in transgenic mice induced CTLs capable of specifically lysing HER-2/neu+ tumor cell lines (also including the original HER-2/neu+ primary tumor cells from which the ACEs were derived) in an HLA-A*0201-restricted fashion. Adoptive transfer of ACE-induced CTLs was sufficient to significantly prolong survival of SCID mice inoculated with HLA-A*0201+ HER-2/ neu+ human tumor cell lines. Cytotoxicity of such ACE-induced CTL lines was directed, at least as detected herein, also against the HER-2/ neu peptides HER-2 (9(369)) and HER-2 (9(435)) demonstrating the immunodominance of these epitopes. HER-2 peptide-specific CTLs generated in the HLA-A*0201-transgenic mice, upon peptide immunization, lysed in vitro HER-2/neu+ human tumor cell lines in an HLA-A*0201-restricted manner and, when adoptively transferred, conferred sufficient protection in SCID mice inoculated with the same human tumor cell lines as above. However, CTLs induced by ACEs displayed enhanced efficacy in the therapy of xenografted SCID mice compared with the HER-2 peptide-specific CTLs (i.e., HER-2 [9(369)] or HER-2 [9(435)]). Even by administering mixtures of CTLs specific for each of these peptides, the prolongation of survival achieved was still inferior compared with that obtained with ACE-induced CTLs. This suggested that additional epitopes may contribute to the immunogenicity of such tumor-derived ACEs. Thus, immunization with ACEs from HER-2/neu+ primary tumor cells appears to be an effective approach to generate multiple and potent CTL-mediated immune responses against HER-2/neu+ tumors expressing the appropriate HLA allele(s). By screening ACE-induced CTL lines with synthetic peptides encompassing the HER-2/neu sequence, it is feasible to identify immunodominant epitopes which may be used in mixtures as vaccines with enhanced efficacy in both the prevention and therapy of HER-2/neu+ malignancies.

Animals↗

PubMed2004

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.

Animals↗