Interfacial immobilization of monoclonal antibody and detection of human prostate-specific antigen.

Antibody orientation and its antigen binding efficiency at interface are of particular interest in many immunoassays and biosensor applications. In this paper, spectroscopic ellipsometry (SE), neutron reflection (NR), and dual polarization interferometry (DPI) have been used to investigate interfacial assembly of the antibody [mouse monoclonal anti-human prostate-specific antigen (anti-hPSA)] at the silicon oxide/water interface and subsequent antigen binding. It was found that the mass density of antibody adsorbed at the interface increased with solution concentration and adsorption time while the antigen binding efficiency showed a steady decline with increasing antibody amount at the interface over the concentration range studied. The amount of antigen bound to the interfacial immobilized antibody reached a maximum when the surface-adsorbed amount of antibody was around 1.5 mg/m(2). This phenomenon is well interpreted by the interfacial structural packing or crowding. NR revealed that the Y-shaped antibody laid flat on the interface at low surface mass density with a thickness around 40 Å, equivalent to the short axial length of the antibody molecule. The loose packing of the antibody within this range resulted in better antigen binding efficiency, while the subsequent increase of surface-adsorbed amount led to the crowding or overlapping of antibody fragments, hence reducing the antigen binding due to the steric hindrance. In situ studies of antigen binding by both NR and DPI demonstrated that the antigen inserted into the antibody layer rather than forming an additional layer on the top. Stability assaying revealed that the antibody immobilized at the silica surface remained stable and active over the monitoring period of 4 months. These results are useful in forming a general understanding of antibody interfacial behavior and particularly relevant to the control of their activity and stability in biosensor development.

Expression of CD175 (Tn), CD175s (sialosyl-Tn) and CD176 (Thomsen-Friedenreich antigen) on malignant human hematopoietic cells

The expression of the histo-blood group carbohydrate structures T-nouvelle (Tn, CD175), sialylated To (CD175s) and the Thomsen-Friedenreich disaccharide (TF, CD176) on human leukemia cell lines was analyzed by their reactivity with specific monoclonal ant

Interfacial recognition of human prostate-specific antigen by immobilized monoclonal antibody: effects of solution conditions and surface chemistry.

The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.

Label-free detection of human prostate-specific antigen (hPSA) using film bulk acoustic resonators (FBARs)

Label-free detection of cancer biomarkers using low cost biosensors has promising applications in clinical diagnostics. In this work, ZnO-based thin film bulk acoustic wave resonators (FBARs) with resonant frequency of ∼1.5 GHz and mass sensitivity of 0.015 mg/m2 (1.5 ng/cm2) have been fabricated for their deployment as biosensors. Mouse monoclonal antibody, anti-human prostate-specific antigen (Anti-hPSA) has been used to bind human prostate-specific antigen (hPSA), a model cancer used in this study. Ellipsometry was used to characterize and optimise the antibody adsorption and antigen binding on gold surface. It was found that the best amount of antibody at the gold surface for effective antigen binding is around 1 mg/m2, above or below which resulted in the reduced antigen binding due to either the limited binding sites (below 1 mg/m2) or increased steric effect (above 1 mg/m2). The FBAR data were in good agreement with the data obtained from ellipsometry. Antigen binding experiments using FBAR sensors demonstrated that FBARs have the capability to precisely detect antigen binding, thereby making FBARs an attractive low cost alternative to existing cancer diagnostic sensors. © 2013 Elsevier B.V.

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

BACKGROUND: Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS: Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS: The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS: Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.

Optimal kinetics for quantification of antigen-induced cytokines in human peripheral blood mononuclear cells by real-time PCR and by ELISA.

Real-time polymerase chain reaction (PCR) has recently been described as a new tool to measure and accurately quantify mRNA levels. In this study, we have applied this technique to evaluate cytokine mRNA synthesis induced by antigenic stimulation with purified protein derivative (PPD) or heparin-binding haemagglutinin (HBHA) in human peripheral blood mononuclear cells (PBMC) from Mycobacterium tuberculosis-infected individuals. Whereas PPD and HBHA optimally induced IL-2 mRNA after respectively 8 and 16 to 24 h of in vitro stimulation, longer in vitro stimulation times were necessary for optimal induction of interferon-gamma (IFN-gamma) mRNA, respectively 16 to 24 h for PPD and 24 to 96 h for HBHA. IL-13 mRNA was optimally induced by in vitro stimulation after 16-48 h for PPD and after 48 to 96 h for HBHA. Comparison of antigen-induced Th1 and Th2 cytokines appears, therefore, valuable only if both cytokine types are analysed at their optimal time point of production, which, for a given cytokine, may differ for each antigen tested. Results obtained by real-time PCR for IFN-gamma and IL-13 mRNA correlated well with those obtained by measuring the cytokine concentrations in cell culture supernatants, provided they were high enough to be detected. We conclude that real-time PCR can be successfully applied to the quantification of antigen-induced cytokine mRNA and to the evaluation of the Th1/Th2 balance, only if the kinetics of cytokine mRNA appearance are taken into account and evaluated for each cytokine measured and each antigen analysed.

Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1

We have cloned and studied the expression in Escherichia coli K-12 of chromosomal rfb genes determining the biosynthesis of the O7 lipopolysaccharide (LPS) antigen from E. coli K1 strain VW187. Two E. coli K-12 strains carrying recombinant cosmids gave positive coagglutination reactions with protein A-rich staphylococcal particles bearing an O7-specific rabbit polyclonal antiserum. Silver-stained polyacrylamide gels of total membranes extracted with hot phenol showed O side chain material which had O7 specificity as determined by immunoblotting experiments. However, the amount of O7 LPS expressed in E. coli K-12 was considerably lower than that produced by the wild-type strain VW187. Deletion and transposition experiments identified a region of about 17 kilobase pairs which is essential for the expression of O7 LPS. The existence of homologies between the O7 LPS genes and other E. coli O side chain genes was investigated by Southern blot hybridization experiments. An O7-specific probe fragment of 15 kilobase pairs did not hybridize to genomic DNA digests of E. coli strains belonging to several different O types, demonstrating that the O7 LPS genes are unique.

The histamine H4 receptor is a potent inhibitor of adhesion-dependent degranulation in human neutrophils

The histamine H4 receptor regulates the inflammatory response. However, it is not known whether this receptor has a functional role in human neutrophils. We found that fMLP (1 μM), but not histamine (0.1-1 μM), induced Mac-1-dependent adhesion, polarization, and degranulation (release of lactoferrin). A pretreatment of neutrophils with histamine (0.001-1 μM) or JNJ 28610244 (0.1-10 μM), a specific H4 receptor agonist, led to inhibition of degranulation. Total inhibition of degranulation was obtained with 0.1 μM histamine and 10 μM JNJ 28610244. Furthermore, such inhibition by histamine of degranulation was reversed by JNJ 7777120 and JNJ 28307474, two selective H4 receptor antagonists. However, neither histamine nor the H4 receptor agonist JNJ 28610244 prevented fMLP-induced, Mac-1-dependent adhesion, indicating that the H4 receptor may block signals emanating from Mac-1-controlling degranulation. Likewise, engagement of the H4 receptor by the selective agonist JNJ 28610244 blocked Mac-1-dependent activation of p38 MAPK, the kinase that controls neutrophil degranulation. We also show expression of the H4 receptor at the mRNA level in ultrapure human neutrophils and myeloid leukemia PLB-985 cells. We concluded that engagement of this receptor by selective H4 receptor agonists may represent a good, therapeutic approach to accelerate resolution of inflammation.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses.

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.

Macrophage migration inhibitory factor is up-regulated in human first-trimester placenta stimulated by soluble antigen of Toxoplasma gondii, resulting in increased monocyte adhesion on villous explants

Considering the potential role of macrophage migration inhibitory factor (MIF) in the inflammation process in placenta when infected by pathogens, we investigated the production of this cytokine in chorionic villous explants obtained from human first-trimester placentas stimulated with soluble antigen from Toxoplasma gondii (STAg). Parallel cultures were performed with villous explants stimulated with STAB, interferon-gamma (IFN-gamma), or STAB plus IFN-gamma. To assess the role of placental MIF on monocyte adhesiveness to human trophoblast, explants were co-cultured with human myelomonocytic THP-1 cells in the presence or absence of supernatant from cultures treated with STAB (SPN), SPN plus anti-MIF antibodies, or recombinant MIF. A significantly higher concentration of MIF was produced and secreted by villous explants treated with STAB or STAB plus IFN-gamma after 24-hour culture. Addition of SPN or recombinant MIF was able to increase THP-1 adhesion, which was inhibited after treatment with anti-MIF antibodies. This phenomenon was associated with intercellular adhesion molecule expression by villous explants. Considering that the processes leading to vertical dissemination of T. gondii remain widely unknown, our results demonstrate that MIF production by human first-trimester placenta is up-regulated by parasite antigen and may play an essential role as an autocrine/paracrine mediator in placental infection by T. gondii.

Fungicidal activity of human monocyte-derived multinucleated giant cells induced in vitro by Paracoccidioides brasiliensis antigen

Multinucleated giant cells (MGC) are characteristic cells in granulomatous disorders such as paracoccidioidomycosis (PCM) and also are formed in vitro from peripheral blood mononuclear cells by several stimuli. In this study, the authors investigated in vitro formation of MGC derived from monocytes of healthy individuals, stimulated with Paracoccidioides brasiliensis antigen (PbAg), compared with other stimuli such as IFN-gamma and supernatant of Con-A-stimulated peripheral blood mononuclear cells (CM-ConA). Besides, the fungicidal activity of monocytes and monocyte-derived MGC challenged with P. brasiliensis were compared, at a ratio of one fungus per 50 monocytes. Results demonstrated that PbAg, IFN-gamma, and CM-ConA stimuli were able to induce MGC generation, with fusion indices significantly higher than control cultures. Striking results were observed when MGC induced by PbAg and IFN-gamma presented higher fungicidal activity than monocytes, submitted to the same stimuli, showing a better capacity of these cells to kill P. brasiliensis. In summary, the results suggest that PbAg is able to induce MGC generation, and these cells presented higher fungicidal activity against P. brasiliensis than monocytes.

Antigen distribution in mucocutaneous biopsies of human paracoccidiomycosis

The authors studied the distribution of Paracoccidioides brasiliensis antigen(s) in human skin and oral mucosa. In biopsies obtained from untreated patients showing the chronic form of the disease, the authors demonstrated the P. brasiliensis antigen using two polyclonal immune sera raised in rabbits, one against the exoantigens of P. brasiliensis and the other against a 43-kDa glycoprotein. Langerhans' cells were detected through double immunolabeling using an anti-S100 protein monoclonal antibody. Double labeling immunohistochemistry showed that both of the immune sera labeled the yeast cells in the center of the granuloma and those transmigrating through the epithelial layer equally well. Granulomas exhibited the P. brasiliensis antigen permeating cells, mainly at the periphery of the granulomatous inflammation. The P. brasiliensis antigen(s) accumulated in the macrophages but not in the Langerhans' cells. P. brasiliensis antigens, detected by antiserum against parasite exoantigens, were also deposited between basal keratinocytes, but not in the granular cells, in 47% of the biopsies. P. brasiliensis antigens, as assessed by immunoelectron microscopic techniques, are present in the cytoplasm of the yeast cells in the host tissues. Antigens are transported to the cell membrane and later excreted through the cell wall. Antigenic deposits are also seen at the fungus-host interface.