Editorial - Immunobiophysics: Advances and techniques.

被引量：- 发表：1970

Modified radioimmunoassay versus ELISA to quantify anti-acetylcholine receptor antibodies in a mouse model of myasthenia gravis.

In mouse models of myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies can be quantified to monitor disease progression and treatment response. In mice, enzyme-linked immunosorbent assay (ELISA) is the gold standard to quantify these antibodies. However, this method requires antigen purification, which is both time-consuming and expensive. In humans, radioimmunoassay (RIA)-which is more sensitive than ELISA-is commonly used to quantify AChR antibodies. At present, however, no commercial RIA kits are available to quantify these antibodies in mice. The aim of this study was to compare a modified commercial human RIA kit to two ELISA methods to detect AChR antibodies in an experimental autoimmune mouse model of MG (EAMG). C57BL/6 J mice were immunized with purified AChR from Tetronarce californica (T-AChR). Serum samples were analyzed by RIA and two ELISAs (T-AChR and purified mouse AChR peptide [m-AChR]). The modified RIA showed excellent sensitivity (84.1 %) and specificity (100 %) for the detection of AChR antibodies. RIA showed a good agreement with T-AChR ELISA (κ = 0.69) but only moderate agreement with m-AChR ELISA (κ = 0.49). These results demonstrate the feasibility of modifying a commercially-available RIA kit to quantify AChR antibodies in EAMG. The advantage of this technique is that it eliminates the need to develop the entire methodology in-house and reduces inter and intra-laboratory variability.

被引量：- 发表：1970

Optimizing the method for expressing human monoclonal antibodies from a single peripheral blood cell from vaccinated donors.

Human monoclonal antibodies are essential diagnostic and research tools and one of the most promising therapeutics. In the past years, single B cell technologies have evolved and over-come conventional methods' limitations, enabling the isolation of scarce B cell populations with desirable characteristics. In this study, we describe a simple and efficient method to isolate anti-gen-specific plasmablasts and memory B cells from hepatitis B virus vaccinated donors' peripheral blood and consequently amplification of immunoglobulin variable region genes. Amplified immunoglobulin variable region genes were cloned into expression vectors and transfected into a human cell line to produce human recombinant monoclonal antibodies. Major challenges in this protocol were isolation of antigen-specific B cells based on surface markers, recovering mRNA from a single cell for efficient amplification, and cloning the correct insert into a desired expression vector. The essential feature of our protocol was the separation of B cells from peripheral blood mononuclear cells before sorting. We identified antigen-specific binding B cells based on the expression of surface markers CD19, CD27, IgG, and binding to hepatitis B surface antigen. Efficient single-cell reverse transcription and polymerase chain reaction (RT-PCR) were achieved using a random primer mix and Kapa Hifi Hot Start Polymerase. Amplification efficiency differed among heavy and light chain variable regions (highest at heavy chain (68 %) and lowest at lambda light chain (22 %)). After co-transfection of HEK293T/17 with successfully cloned heavy and light chain vectors, 70 % of transfected cells produced recombinant monoclonal antibodies at a concentration ∼ 4 μg/ml and 7 % of them showed binding to HBsAg. Human monoclonal antibodies from peripheral blood have advantages over antibodies of mouse origin or phage display libraries, because of their high specificity and self-tolerance. Using the described protocol, we can generate fully human monoclonal antibodies to any other antigen for application in immunotherapy or basic research.

被引量：- 发表：1970

In vitro analysis of single chain variable fragment-based immunotoxins against Erythropoietin-producing hepatocellular A2 receptor overexpressed in breast cancer cells.

Breast cancer is one of the leading causes of cancer deaths worldwide. Thereafter, designing new treatments with higher specificity and efficacy is urgently required. In this regard, targeted immunotherapy using immunotoxins has shown great promise in treating cancer. To target a breast cancer cell, the authors used the antibody fragment against a receptor tyrosine kinase, EphA2, which is overexpressed in many cancers. This fragment was conjugated to a plant toxin, subunit A of ricin, in two different orientations from N to C-terminal (EphA2- C-Ricin and EphA2- N-Ricin). Then, these two immunotoxins were characterized using in vitro cell-based assays. Three different cell lines were treated, MDA-MB-231 (breast cancer) which has a high level of EphA2 expression, MCF-7 (breast cancer) which has a low level of EphA2 expression, and HEK293 (human embryonic kidney) which has a very low level of EphA2 expression. Moreover, binding ability, cytotoxicity, internalization, and apoptosis capacity of these two newly developed immunotoxins were investigated. The flow cytometry using Annexin V- Propidium iodide (PI) method indicated significant induction of apoptosis only in the MDA-MB-231 cells at different concentrations. It was also found that construct I, EphA2- C-Ricin immunotoxin, could bind, internalize, and induce apoptosis better than the EphA2- N-Ricin immunotoxin. In addition, the obtained data suggested that the N or C-terminal orientation conformation is of significant importance.

被引量：- 发表：1970

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by enzyme-linked immunosorbent assay.

Capsular polysaccharides of Streptococcus pneumoniae are used in pneumococcal polysaccharide and protein-conjugate vaccines. Cell-wall polysaccharide (C-Ps) is a critical impurity that must be kept at low levels in purified polysaccharide preparations. Hence, accurate and precise methods for determining C-Ps are needed. Currently available methods include nuclear magnetic resonance (NMR) spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both these methods suffer from their own limitations; therefore, we developed a simple and efficient enzyme-linked immunosorbent assay (ELISA) for accurate and precise quantification of C-Ps in samples of any serotype of pneumococcal capsular polysaccharide without interference. We quantified C-Ps in preparations of 14 serotype polysaccharides using newly developed ELISA method and compared the results with C-Ps values obtained using two previously reported methods, 1H NMR and HPAEC-PAD. The C-Ps value determined using 1H NMR for serotype 5 was 21.08%, whereas the values obtained using HPAEC-PAD and ELISA were 2.38% and 2.89% respectively, indicating some interference in 1H NMR method. The sensitivity of the ELISA method is higher because the sample is used directly unlike HPAEC-PAD method where sample is subjected to harsh treatment, such as acid digestion and quantify C-Ps based on peak area of ribitol or AAT. Furthermore, 1H NMR and HPAEC-PAD are expensive and laborious methods. Our work, underscores the simple and efficient ELISA that can be used for quantification of C-Ps in pneumococcal polysaccharide preparations.

被引量：- 发表：1970