A Quantitative Method for Detecting Ara h 2 by Generation and Utilization of Monoclonal Antibodies

Peanut (Arachis hypogaea) is one of the most common food allergens that can induce fatal anaphylaxis, and Ara h 2 is one of the major allergen components involved in peanut allergy. The aim of this study was to develop a quantitative method for detecting peanut allergen using monoclonal antibodies against Ara h 2. The splenocytes of immunized mice were fused with myeloma cells (SP2/0), and stable mAb-producing clones were obtained by limiting dilution. mAbs against Ara h 2 were isolated from mouse ascites, and specificity was confirmed by immunoblotting. Five mAbs with high purity and specific reactivity were obtained, which were referred to as 1-2E10, 2-1D5, 3-1C5, 4-1C2, and 5-1G4, respectively. After screening different mAb combinations for development of a sandwich ELISA, we selected 5-1G4 as the capture antibody and 1-2E10 as the detection antibody for the measurement of Ara h 2 from which an optimal correlation between the Ara h 2 concentration and the OD value was obtained. This sandwich ELISA could specifically detect Ara h 2 in peanut extract at concentrations as low as 5 ng/mL and up to 10 μg/mL. These mAbs can, therefore, serve as quantitative diagnostic reagents for peanut and peanut product risk assessment.

Preparation and Biological Activity of the Monoclonal Antibody against the Second Extracellular Loop of the Angiotensin II Type 1 Receptor

The current study was to prepare a mouse-derived antibody against the angiotensin II type 1 receptor (AT1-mAb) based on monoclonal antibody technology, to provide a foundation for research on AT1-AA-positive diseases. Balb/C mice were actively immunized with the second extracellular loop of the angiotensin II type 1 receptor (AT1R-ECII). Then, mouse spleen lymphocytes were fused with myeloma cells and monoclonal hybridomas that secreted AT1-mAb were generated and cultured, after which those in logarithmic-phase were injected into the abdominal cavity of mice to retrieve the ascites. Highly purified AT1-mAb was isolated from mouse ascites after injection with 1 × 107hybridomas. A greater amount of AT1-mAb was purified from mouse ascites compared to the cell supernatant of hybridomas. AT1-mAb purified from mouse ascites constricted the thoracic aorta of mice and increased the beat frequency of neonatal rat myocardial cells via the AT1R, identical to the effects of AT1-AA extracted from patients’ sera. Murine blood pressure increased after intravenous injection of AT1-mAb via the tail vein. High purity and good biological activity of AT1-mAb can be obtained from mouse ascites after intraperitoneal injection of monoclonal hybridomas that secrete AT1-mAb. These data provide a simple tool for studying AT1-AA-positive diseases.

Gecko Crude Peptides Induce Apoptosis in Human Liver Carcinoma CellsIn Vitroand Exert Antitumor Activity in a Mouse Ascites H22 Xenograft Model

Aim. To investigate the anti-tumor effects and mechanisms of gecko crude peptides (GCPs)in vitroandin vivo.Methods. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was applied to measure the effects of GCPs on the HepG2 cell viability. Fluorescence morphology was used to identify apoptotic cells. A xenograft H22 liver cancer model was established in Kunming mice. The tumor-bearing mice were treated with daily intraperitoneal injections of normal saline (NS group) or GCPs (80, 40 or 20 mg/kg) for 10 days, or once per two days with 2 mg/kg doxorubicin (ADR group;n=10each). Serum tumor necrosis factor (TNF-α) and interleukin (IL)-6 were quantified using ELISA assay.Results. GCPs significantly inhibited the growth of HepG2 cells and induced typical apoptotic morphological features through increasing bcl-2/bax ratio in a dose- and time-dependent mannerin vitro. The tumor weights of the ADR group, GCPs (H) group, GCPs (M) group, GCPs (L) group were smaller compared to the NS group. While the white blood cell count, thymus index, spleen index were higher in the high dose GCPs group than the NS group (P<0.05), the VEGF expression in tumor tissue and serum TNF-αand IL-6 levels in the GCPs groups were lower than the NS group (P<0.05).