Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans

Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans

We aim to establish a sensitive and robust assay for estimation of systemic complement activation at the C3 component of complement in mouse and human plasma samples. In order to capture product activation iC3b and C3dg in the relevant specific and physiological manner we utilized construct that consists of sites iC3b / C3dg binds human complement receptor 2 (CR2) is attached to the Fc-portion of IgG mouse.

This construction C3dg and iC3b binding of both mice and humans. We construct CR2-IgG purified from mouse B cell line supernatant myeloma, J558L-CR2-IgG, with protein G affinity chromatography. CR2-IgG construct used to capture fragments of C3 in microtiter wells and anti-mouse or anti-human antibody-C3 is used to detect bound C3 fragments. Initially we tested the specificity of tests using purified fragment C3. Furthermore, using CR2-based test, we measured a signal of up to three-fold higher in activated rat serum compared to non-activated mouse serum, whereas serum activated from C3 knock-out mice did not give a signal.

We tested in vivo produced samples of the experimental mice; complement activation induced by injecting cobra venom factor or heat collected in C57bl6 mouse IgG, followed by the withdrawal of EDTA blood samples at the points of a different time and measurement iC3b / C3dg. We observed a clear time-dependent differences in the signal between samples with expected high and low complement activation. In addition, with the use of an assay to fragment C3 humans,

we observed that patients with systemic lupus erythematosus (SLE) (n = 144) had levels of iC3b / C3dg significantly higher compared with healthy individuals (n = 144) (p <0, 0001). We present two functional immunoassays, which is able to measure the systemic levels of C3 activation product iC3b and C3dg in mice and humans.

To our knowledge, this is the first test for the use of complement activation that are relevant capture physiological constructs such as CR2. This test will be a relevant tool when investigating a mouse model and human diseases involving the complement system.

Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans
Complement Receptor 2 Based Immunoassay Measuring Activation of the Complement System at C3-Level in Plasma Samples From Mice and Humans

Calcium phosphate nanoneedle gene delivery system based immunotherapy of cancer genetics.

Ovarian cancer has become one of the most common gynecologic cancer with a high mortality rate. However, conventional surgery together with combination chemotherapy is difficult to achieve ideal therapeutic effect. Although genetic immunotherapy is applied to an active immune response against the cancer, the lack of efficient in vivo gene delivery technique is still a constraint in clinical applications. To overcome these problems, minicircle DNA vector encoding a human anti-EpCAM / CD3 bispecific antibody (BsAbEPH) has been built.

In addition, various forms of calcium phosphate (Capo) biomaterials prepared. In particular, the capo-nanoneedle-mediated “perforated cell” transfection technology achieve high levels of gene expression in the peritoneal cavity. In intraperitoneal xenograft model with human ovarian cancer cell line SKOV3, Capo-DNA system nanoneedle / minicircle expressed BsAbEPH resulted in significantly retarded the growth of cancer and mouse extension of life span with limited toxicity.

And the system can be made as an off-the-shelf and products that are easy to use. Therefore, Capo-nanoneedle technology-based non-viral gene delivery will have great potential in clinical application.Correspondingly, human CFTR transgene expression was increased significantly by administration of cyclophosphamide compared with the group without treatment.

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These data indicate that sustained expression of the human CFTR transgene in the lungs of mice through a delivery vector can be achieved by repeated temporary immunosuppression.

Nathaniel

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