Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells

Hybridoma cells display an increase in antibody productivity following exposure to hypertonic conditions. However, the underlying mechanism is not well understood. In the present study, we hypothesize that the nuclear factor of activated T cells 5 （NFAT5）/tonicity enhancer binding protein （TonEBP） functions to increase the antibody productivity of hybridoma cells. NFAT5 is an osmosensitive mammalian transcription factor. However, its ubiquitous expression in various organs that are not bathed in hypertonic milieu suggests that NFAT5 may also regulate cell growth and function under isotonic conditions. In this study, we examined the expression of NFAT5 in hybridoma cells by Western blot analysis, and found that it increased significantly in hypertonic medium. To further define the function of NFAT5 in hybridoma cells, RNA interference technique was used to downregulate the expression of NFAT5 in SGB-8 cells （a hybridoma cell line）. In isotonic medium, antibody productivity ofhybridoma cells was reduced by downregulation of NFAT5 while cell proliferation was not influenced. The results presented here demonstrate that NFAT5 not only plays an important role in osmotic stress response pathway in hybridoma cells but also is essential for optimal antibody productivity.

Get effective polyclonal antisera in one month

According to the traditional immunization procedure, after the first injection of the sample A (emulsion of aimed antigen and Freund's complete adjuvant) to immunize rabbit, successive injections of the sample B (emulsion of aimed antigen and Freund's incomplete adjuvant) were followed every 2-4 weeks. In general,high titer of the corresponding polyclonal antisera will be observed after 4-5 injections of sample B in 3-4months. This report presents a simply modified procedure that was able to stimulate the antisera formation in one month and achieve enough avidity to satisfy either Western blot or immunohistochemistry analysis.It just applied an additional injection of the sample A to the rabbit at the 3rd day after the primary immunization injection. You could gain the high titer of the antisera right after the first sample B injection in one month. This method has produced the desired results in three different recombinant antigens with different molecular weight (5.9 KD-55 KD) expressed from prokaryotic or eukaryotic cells.

Humoral and cellular immunogenecity of DNA vaccine based on hepatitis B core gene in rhesus monkeys

INTRODUCTIONHepatitis B virus (HBV) is the most commonetiologic agent for infectious liver diseases. It isestimated that there are more than 250 millionchronic HBV carriersin the world today and thereis a significant association among persistentinfection, liver cirrhosis and hepatocellularcarcinoma[1-3].

Pathogenesis of coeliac disease:implications for treatment

INTRODUCTIONCoeliac disease(CD)is an enteropathy ,characterised by villous atrohy ,which occurs in genetically susceptible individuals .It affects mainly the proximal small intestine,and is caused by an intolerance to cereal storage proteins found in wheat ,barley and rye .

Development of protective antibodies to Streptococcus pneumoniae in healthy children

Aim: To study how immunity to Streptococcus pneumoniae normally develops in healthy children. Methods: Ninety two healthy children at 3 - 5, 7 - 9 and 13 - 15 years of age were recruited. No one of the children had previously been given pneumococcal vaccine. Serum was analysed for pooled antigens of the 23 most common pneumococcal polysaccharides with ELISA technique, and results are given in opitical density (OD). A three-level semi-quantitative system was used to assess degree of immunity to Streptococcus pneumoniae . Cut-off levels were OD ≤ 0.7 and OD ≥ 1.3, separating low, intermediate and high degree of immunity. Results: Median values for OD differed significantly between the groups, with OD 0.91, 1.18 and 1.10 respectively (p = 0.004). Levels were lower in the youngest age group, but from age 7-9 years, levels were similar trough out childhood. Twenty six percent of the children in the youngest age-group had a low degree of protection (OD ≤ 0.7) to Streptococcus pneumoniae . Such low levels were uncommon from 7 - 9 years of age, found in only 13% of the children. Conclusion: Protective antibodies to Streptococcus pneumoniae develops mainly during the preschool period. Thereafter, levels are stable throughout childhood up to the age of 15 years.