A signal amplification probe enhances sensitivity of antibodies and aptamers based Immuno-diagnostic assays.

One major unmet need is improving the sensitivity of immune-diagnostic assays. This is particularly important in the field of biomarker discoveries and monitoring. We have established a novel signal amplification probe system enabling a highly sensitive target detection platform to be used in immuno-assays. The probe consists of a double stranded DNA that can carry a large number of signaling elements such as biotin or fluorescent molecules. The DNA probe anchors to the recognition unit, whether an antibody or an aptamer, by covalent conjugation or by a simple and rapid molecular association process. Binding curves obtained by using the DNA amplification probe are dose dependent and linear over a wide range of antigen concentration. The optimal slopes are characterized by high signals and low background increasing the assay sensitivity and reducing the limit of detection by up to 10-fold compared to biotinylated antibodies commonly used in ELISA systems. When using aptamers in combination with the amplification probe for antigen recognition, the limit of detection is comparable to that obtained by biotinylated antibodies. Biotin labeled aptamers practically cannot be used for detection of low target levels. The DNA amplification probe system enables to expand the range of diagnostic assays including clinical samples and meet research needs.

Novel mechanism of antibodies to hepatitis B virus in blocking viral particle release from cells.

UNLABELLED: Antibodies are thought to exert antiviral activities by blocking viral entry into cells and/or accelerating viral clearance from circulation. In particular, antibodies to hepatitis B virus (HBV) surface antigen (HBsAg) confer protection, by binding circulating virus. Here, we used mathematical modeling to gain information about viral dynamics during and after single or multiple infusions of a combination of two human monoclonal anti-HBs (HepeX-B) antibodies in patients with chronic hepatitis B. The antibody HBV-17 recognizes a conformational epitope, whereas antibody HBV-19 recognizes a linear epitope on the HBsAg. The kinetic profiles of the decline of serum HBV DNA and HBsAg revealed partial blocking of virion release from infected cells as a new antiviral mechanism, in addition to acceleration of HBV clearance from the circulation. We then replicated this approach in vitro, using cells secreting HBsAg, and compared the prediction of the mathematical modeling obtained from the in vivo kinetics. In vitro, HepeX-B treatment of HBsAg-producing cells showed cellular uptake of antibodies, resulting in intracellular accumulation of viral particles. Blocking of HBsAg secretion also continued after HepeX-B was removed from the cell culture supernatants. CONCLUSION: These results identify a novel antiviral mechanism of antibodies to HBsAg (anti-HBs) involving prolonged blocking of the HBV and HBsAg subviral particles release from infected cells. This may have implications in designing new therapies for patients with chronic HBV infection and may also be relevant in other viral infections.

Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa flagellin for treating infections in a murine burn wound model.

BACKGROUND: In an era of increasing drug resistance, immunotherapy is a desirable treatment against Pseudomonas aeruginosa infections. The flagellum, which is an important pseudomonal virulence factor, was targeted for immunotherapy. The aim of the study was to evaluate the efficacy of polyclonal immunotherapy targeted against the N-terminal of flagellin (anti-N'-fla-b) for treating severe P. aeruginosa infection in a murine burn wound model. METHODS: Groups of 12 mice were infected (subeschar) with P. aeruginosa strain PA01, and were treated either with systemic anti-N'-fla-b immunoglobulin G (IgG), nonspecific IgG, or imipenem. The control groups included mice with burn alone, mice with untreated infected burn, and mice without burn infected with P. aeruginosa. Three separate regimens were examined: prophylaxis (preinfection), therapeutic (postinfection), and combined. The efficacy of anti-N'-fla-b was evaluated by monitoring the mortality and morbidity (relative weight loss) during a period of 2 weeks. RESULTS: Anti-N'-fla-b IgG immunotherapy significantly decreased the mortality rate of infected burned mice followed by severe P. aeruginosa infection. The mortality rate in the anti-N'-fla-b-treated groups ranged from 0 to 17 percent compared with 58 to 83 percent in nontreated groups infected with 2 to 5 x 10(6) colony-forming units of P. aeruginosa (p < 0.05). The mortality rate in the anti-N'-fla-b-treated groups was similar to that of groups treated with imipenem. The three tested regimens yielded similar results. Morbidity paralleled survival results. Histopathologic examination revealed an earlier reepithelialization of the infected wound in the anti-N'-fla-b-treated mice compared with untreated mice. CONCLUSION: Immunotherapy with anti-N'-fla-b IgG, given either as prophylaxis or therapeutically, effectively reduced mortality and morbidity and improved wound healing in a severely P. aeruginosa-infected murine burn model.

Preclinical evaluation of two neutralizing human monoclonal antibodies against hepatitis C virus (HCV): a potential treatment to prevent HCV reinfection in liver transplant patients.

Passive immunotherapy is potentially effective in preventing reinfection of liver grafts in hepatitis C virus (HCV)-associated liver transplant patients. A combination of monoclonal antibodies directed against different epitopes may be advantageous against a highly mutating virus such as HCV. Two human monoclonal antibodies (HumAbs) against the E2 envelope protein of HCV were developed and tested for the ability to neutralize the virus and prevent human liver infection. These antibodies, designated HCV-AB 68 and HCV-AB 65, recognize different conformational epitopes on E2. They were characterized in vitro biochemically and functionally. Both HumAbs are immunoglobulin G1 and have affinity constants to recombinant E2 constructs in the range of 10(-10) M. They are able to immunoprecipitate HCV particles from infected patients' sera from diverse genotypes and to stain HCV-infected human liver tissue. Both antibodies can fix complement and form immune complexes, but they do not activate complement-dependent or antibody-dependent cytotoxicity. Upon complement fixation, the monoclonal antibodies induce phagocytosis of the immune complexes by neutrophils, suggesting that the mechanism of viral clearance includes endocytosis. In vivo, in the HCV-Trimera model, both HumAbs were capable of inhibiting HCV infection of human liver fragments and of reducing the mean viral load in HCV-positive animals. The demonstrated neutralizing activities of HCV-AB 68 and HCV-AB 65 suggest that they have the potential to prevent reinfection in liver transplant patients and to serve as prophylactic treatment in postexposure events.

Antibodies raised against N'-terminal Pseudomonas aeruginosa flagellin prevent mortality in lethal murine models of infection.

The goal of this study was to investigate if antibodies raised against N'-terminal Pseudomonas aeruginosa (Pa) flagellin could afford protection in two lethal mouse models of Pa infection. To that end, rabbit polyclonal antibodies were generated against the N'-terminal domains (amino acids 1-156) of recombinant Pa01 or Salmonella muenchen flagellins, termed anti-N'-fla-b and anti-N'-fla-Sm, respectively. In vitro, anti-N'-fla-b but not anti-N'-fla-Sm IgG specifically recognized recombinant and Pa endogenous flagellin type b proteins, total bacterial lysates of Pa type b, and inhibited Pa01 invasion into A549 cells. In vivo, administration of anti-N'-fla-b afforded a remarkable improvement in survival in lethal peritonitis (90% vs. 12% in control; p<0.001) and burn infection (83% vs. 8-17% in control groups; p<0.005) Pa models. These findings would suggest that the N'-terminal domain of Pa flagellin harbors critically important bioactive domains and that an antibody-targeted, neutralization approach directed at this region could provide a novel therapeutic strategy to combat Pa infection.

Hepatitis C virus infection in dialysis and chronic liver patients: Viraemia dependent anti-E2-antibody response.

Cryptic hepatitis C virus (HCV) infection relates to patients infected chronically with HCV that are seronegative but have HCV-RNA. These patients are not identified by the standard serological tests for HCV, which are based on detection of antibodies to core, NS3 and NS5 antigens. They will, therefore, be wrongly diagnosed as non-infected, and are considered as a potential risk for others. Cryptic HCV infection in dialysis units occurs frequently and, due to medical procedures, is a major factor for contracting the virus when unrecognised. This study was conducted in order to assess the humoral immune responses to E2-antigen in sera of patients infected chronically with HCV. Recombinant E2 protein in enzyme linked immunosorbent assay (ELISA) and Western blot (WB) were used to test the occurrence of anti-E2 antibodies in the sera of patients from the liver clinic and of dialysis patients. The presence of E2 antibodies was found to be correlated with the presence of HCV-RNA and with viral load. Antibodies to the E2 protein could be detected in as many as 30% of the sera from dialysis patients with cryptic HCV infection (HCV-RNA only). The results suggest that detection of anti-E2 antibodies may enhance significantly HCV serological standard testing; especially among patients on dialysis, and that antibodies to envelope E2 protein appear to depend on and correlate with the presence of HCV particles.

The Trimera mouse: a system for generating human monoclonal antibodies and modeling human diseases.

A Trimera mouse is constructed through a three-step process. Firstly, a normal mouse host is rendered immuno-incompetent by a lethal split-dose total body irradiation. Secondly, the myeloid and erythroid lineages are reconstituted by transplantation of bone marrow cells from a genetically immune-deficient mouse donor. Thirdly. the resulting preconditioned mouse is transplanted with human cells or tissues that can be maintained in the foreign, yet supporting, environment for a considerable period of time. Immunization of Trimera mice, engrafted with human immune cells, induces a strong human immune response, thereby enabling generation of human therapeutic monoclonal antibodies (mAbs) via hybridoma technology. Transplantation of infected human tissue into the preconditioned mice results in the creation of Trimera mouse models for human diseases.