Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

This protocol describes the development of a colloidal gold immunochromatographic test strip based on the sandwich format that can be used to ...

National Chiayi University

The ability to acquire fully human monoclonal antibodies (mAbs) with pre-defined specificities is critical to the development of molecular tags for the analysis of receptor function in addition to promising immunotherapeutics. Yet most of the arriving affinity maturated and complete human immunoglobulin G (IgG) molecules, which are actually derived from single human B cells, have not widely been used to study the conserved self antigens (Ags) such as CD152 (cytotoxic T lymphocyte antigen-4, CTLA-4) because proper hosts are lacking.

Site-directed in vitro immunization leads to a complete human monoclonal IgG4 lambda that binds specifically to the CDR2 region of CTLA-4 (CD152) without interfering the engagement of natural ligands.

CD152 or cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential receptor involved in the negative regulation of T cell activation. Because of its profound inhibitory role, CD152 has been considered a sound susceptible candidate in autoimmunity and a persuasive target for cancer immunotherapy for over a decade. However, the precise roles played by this molecule continue to emerge. In particular, recent evidence suggests that CD152 is also important in the homeostasis and function of a population of suppressive cells, termed regulatory T cells (Treg). In this review, we discuss the recent progress and main features of monoclonal antibodies (mAbs) targeting CD152 and examine how each mAb prepared to a distinct epitope may impact differently upon CD152 modulation depending on its demonstrated regulatory role acting as an agonist, antagonist, or inverse agonist.

Immune intervention with monoclonal antibodies targeting CD152 (CTLA-4) for autoimmune and malignant diseases.

The visualization of fluorescently labeled protein gels is required for the analysis of electrophoretic results or manually picking protein bands or spots from gels. To accomplish this task, an UV light table is generally utilized, but may be hazardous to operators. The blue light transilluminator is another apparatus that may be used for this purpose, but is sometimes insufficient for revealing weak fluorescent signals. In this study, we invented a new setup utilizing a backlit blue light plate for illuminating fluorescently stained protein gels. This method employs Snell's law and allows for the direct visualization of fluorescent signals in protein gels without the use of a filter or filter glasses. This safe, convenient, economic, and effective setup was found to be an ideal alternative for illuminating fluorescent protein gels in proteomic experiments.

Direct visualization of fluorescent signals in protein gels using a backlit blue light plate.

The inhibition of dihydrofolate reductase (DHFR) by antifolates is a common practice both in cell culture and in chemotherapy. Surprisingly, antifolate resistance was also observed in cultured murine myeloma cells (SP2/0) in the presence of human plasma (HP); thus, we used a proteomic approach to identify novel plasma biomarker(s) for this condition. In contrast to the in vitro antifolate response, metabolic enzymes and translation machinery proteins were found to be up-regulated in the presence of HP. The antifolate resistance inherent in HP may be explained by a simultaneous promotion of cell proliferation and the maintenance of DNA integrity. Furthermore, the factor(s) was found to be extrinsic, heat stable and very small in size. Adenine, a supplemented additive in erythrocyte preservation, was subsequently identified as the contributing factor and exogenous addition in cultures reversed the cytotoxicity induced by antifolates. Importantly, adenine-containing blood components, which may provide enhanced survival to otherwise sensitive antifolate-targeted cells, showed a dose-dependent adverse effect in transfusion recipients receiving antifolate (methotrexate) medications. These findings not only highlight a previously unnoticed role of adenine, but also emphasize a novel mechanistic link between transfusion and subsequently reduced survival in patients taking methotrexate.

A proteomics-based translational approach reveals an antifolate resistance inherent in human plasma derived from blood donation.

Prion diseases such as Bovine Spongiform Encephalopathy (BSE) and new variant Creutzfeldt-Jakob disease (nvCJD) have caused a major safety concern in cell cultures using fetal calf serum (FCS). In this study, we found that screened and tested human plasma (HP) obtained from blood centers may be an ideal alternate nutrient substitute to FCS for culturing hybridoma. In addition to the inherent safety, a ten-fold increase in the fusion efficiency has been observed if the HP was used as the nutrient supplement instead of FCS. Subsequently, a broader antibody repertoire may be recovered. The HP supplement was found to promote the growth of hybridoma cells but no impact on antibody secretion. Interestingly, this effect of enrichment was only observed for HP, but not plasma from other animals. Unidentified murine hybridoma cloning factors other than IL-6 may specifically reside in human blood.

Increasing hybridoma viability and antibody repertoire after the cell fusion by the use of human plasma as an alternative supplement.

Intracerebral hemorrhage (ICH) is associated with high mortality and disability, and hyperglycemia worsens the clinical and neurological outcomes of patients with ICH. In this study, we utilized proteomic approaches to investigate the role of hyperglycemia in ICH. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ) in adult Sprague-Dawley male rats; ICH was induced by stereotaxic infusion of collagenase/heparin into the right striatum. It was observed that the size of induced hemorrhage was significantly larger in the hyperglycemic group (n=6 in each group). On the first day after ICH, an apparent decrease in the bilateral grasp was also observed for the lesioned hyperglycemic rats compared with normoglycemic ones. When employing 2-DE and MS to examine the proteomes of perihematomal and control regions in individual hyperglycemic and normoglycemic rats, eight differentially expressed protein targets were identified. Most noteworthy, in response to ICH significant increase of albumin was ubiquitously observed in the brains of normoglycemic rats but not in the brains of hyperglycemic rats. Coincidentally, more significant neuronal apoptosis were found in the perihematomal regions of hyperglycemic rats. These observations described suggest the protection role of albumin in acute stage of ICH, which may be dependent on different blood sugar levels.

Investigation of the effect of hyperglycemia on intracerebral hemorrhage by proteomic approaches.

Growth inhibition and apoptotic/necrotic phenotype was observed in nanogold particle (AuNP)-treated human chronic myelogenous leukemia cells. To elucidate the underlying cellular mechanisms, proteomic techniques including two-dimensional electrophoresis/mass spectrometry and protein microarrays were utilized to study the differentially expressed proteome and phosphoproteome, respectively. Systems biology analysis of the proteomic data revealed that unfolded protein-associated endoplasmic reticulum (ER) stress response was the predominant event. Concomitant with transcriptomic analysis using mRNA expression, microarrays show ER stress response in the AuNP-treated cells. The ER stress protein markers' expression assay unveiled AuNPs as an efficient cellular ER stress elicitor. Upon ER stress, cellular responses, including reactive oxygen species increase, mitochondrial cytochrome c release, and mitochondria damage, chronologically occurred in the AuNP-treated cells. Conclusively, this study demonstrates that AuNPs cause cell death through induction of unmanageable ER stress.

Identification of the nanogold particle-induced endoplasmic reticulum stress by omic techniques and systems biology analysis.

The consumption of cetacean meat is geographically common and often of undetermined sustainability. Besides, it can expose humans to contaminants and zoonotic pathogens. The illegality of possessing cetacean meat was likely under-reported in some countries due to lack of attention paid by the officials although DNA analysis of market products helped to show such practices. We developed two monoclonal antibodies against synthetic peptides of myoglobin (Mb) for constructing a rapid immune colloidal gold strip. Only cetacean Mb is capable of binding to both antibodies and presents positive signal while the Mb from other animals can bind only 1 of the antibodies and presents negative result. The strip for cetacean meat would be an applicable and cost-effective test for field inspectors and even the general public. It contributes to increase the reporting capacity and coverage of illegal cetacean meat possession, which has implications for global cetacean conservation and public health.

Rapid immune colloidal gold strip for cetacean meat restraining illegal trade and consumption: implications for conservation and public health.

Intracerebral hemorrhage (ICH) is associated with high mortality and neurological deficits, and concurrent hyperglycemia usually worsens clinical outcomes. Aquaporin-4 (AQP-4) is important in cerebral water movement. Our aim was to investigate the role of AQP-4 in hyperglycemic ICH.

Hyperglycemia exacerbates intracerebral hemorrhage via the downregulation of aquaporin-4: temporal assessment with magnetic resonance imaging.

Interferon-beta (IFN-β) treatment may not be effective in neuromyelitis optica (NMO). Whether the poor response to IFN-β is related to long spinal cord lesions (LSCL) or the NMO disease entity itself is unclear. We evaluated the spinal cord involvement of patients with multiple sclerosis (MS) and NMO, as well as the response after receiving IFN-β. Forty-nine MS and 21 NMO patients treated with IFN-β for at least 2 years from 2002-2008 were enrolled in this study and the treatment response was analyzed 2 years post-treatment. In the study, spinal cord lesions were present in 57.1% (28/49) of the MS patients, of which 16.3% (8/49) presented spinal cord lesions longer than 3 vertebral segments (LSCL). Responses to IFN-β treatment were seen in 69.3% (34/49) of all the MS cases, of which the appropriate response rates were 76.1% (16/21) in MS patients without spinal cord lesions and 37.5% (3/8) in patients with LSCL. Only 14.2% (3/21) of NMO patients responded to IFN-β treatment. In conclusion, spinal cord lesion is common in MS patients in Taiwan. Both NMO and MS patients with LSCL had a poor response to IFN-β treatment. NMO patients had a worse response to IFN-β treatment than MS patients with LSCL, which shows that the crucial structural defect is something other than LSCL such as the elevated serum IL17 level in NMO compared to MS.

Li-Te Chin

Department of Microbiology and Immunology/Graduate Institute of Biomedical and Biopharmaceutical Sciences

National Chiayi University

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

Li-Te Chin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology and Immunology/Graduate Institute of Biomedical and Biopharmaceutical Sciences

National Chiayi University

Development of a Colloidal Gold-based Immunochromatographic Test Strip for Detection of Cetacean Myoglobin

Department of Microbiology and Immunology/Graduate Institute of Biomedical and Biopharmaceutical Sciences