Phage libraries are a versatile source of recombinant antibody fragments directed against a wide variety of antigens. Recombinant antibodies have the advantage that they can be engineered to improve their binding or other characteristics. A chicken single chain variable fragment (scFv) phage library was panned against the 16 kDa antigen of Mycobacterium tuberculosis. Three phage displayed antibodies were obtained which bound specifically to the antigen. In soluble scFv format, however, they produced low ELISA signals. For this reason they were able to be used as models for antibody engineering. Three mutant sub-libraries were created by random mutagenesis. High stringency panning of the mutant sub-libraries against the target antigen yielded stronger binders which produced ELISA signals of up to eleven times higher than the parent scFvs. An increase in affinity was confirmed by surface plasmon resonance. One mutant scFv with a single amino acid exchange also showed an increase in the yield of scFvs it produced. Upon shortening the linker sequence between the heavy and light chains, size exclusion chromatography showed that multimerisation had occurred. Dimers, trimers and tetramers were formed thus increasing the avidity of the scFvs. Tetramers derived from the unmutated scFv showed the greatest improvement in ELISA binding. To improve expression and purification, an alternate bacterial expression vector with a histidine tag was investigated. For this series of experiments the coding region for a chicken scFv directed against VP7 of bluetongue virus was transferred from the pHEN1 display vector to the pSANG 14-3F vector, which fuses the scFv gene to a bacterial alkaline phosphatase gene. A bi-functional chicken scFv-alkaline phosphatase fusion protein that exhibits both alkaline phosphatase activity and specific antigen binding was expressed in Escherichia coli and purified in a single step via metal affinity chromatography. Furthermore the scFv-AP fusion protein was directly detected in ELISA without the use of secondary detection reagents. This study confirms that the strategies used can efficiently enhance the characteristics of chicken scFvs. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

The cytochromes P450 (P450s) are important enzymes involved in metabolic pathways, which use exogenous and endogenous substances as their substrate for various enzymatic reactions. These enzymes can also use precarcinogens as their substrate and activate them into carcinogens, which leads to a cancer development. If the P450s are induced, the cancer risk increases. Some chemopreventive compounds may induce the P450s and thus be harmful to the human body. Therefore it is necessary to pay enough attention to a study of the mechanism of action of P450s and the influence of the chemopreventive compounds on the activity of cytochromes P450. mRNA expression of most of the P450s isoforms is detected in a number of healthy (nontransformed) tissues, viz. liver, brain, heart, colon, kidney or placenta. Nevertheless there are a few P450s isoforms which mRNAs are expressed at relatively low levels in the nontransformed tissues, whereas the expression in the transformed tissues is significantly higher. One of these P450s is CYP2W1, which can be used as a prognostic marker for colorectal cancer - therefore it is useful to be able to detect a presence of this enzyme in various tissues. A detection of P450s can be accomplished by using a method Western blot. In this method, the immunodetection is achieved by using...

Currently, an increasing attention is being paid to phytochemicals as one of the most widely used chemopreventive compounds, generally considered as health-promoting and safe. Flavonoids representing a large group of phytochemicals are present in many dietary supplements formulated from natural sources. The consumption of these concentrated phytochemicals has dramatically increased in the recent decade. It appears, however, that the ingestion of flavonoids might be associated with some adverse effects. Some flavonoids are known modulators of enzymes involved in phase I and phase II metabolism of xenobiotics biotransformation, thus their induction may result in an increase of carcinogen activation. In this study, the effects of selected flavonoid compounds -naphthoflavone, - naphthoflavone, myricetin, and dihydromyricetin, and carcinogens (BaP, PhIP) on phase II metabolism enzymes, sulfotransferases (SULT), have been investigated. To determine the induction of SULT, antibodies for their immunodetection have been developed. Peptide antigens derived from sequences of selected rat sulfotransferases rSULT1A1, 1B1, 1C1, 1C2, 1C1/2, 1E1, and 2A1, were used as KLH conjugates for hen immunization to obtain yolk anti-peptide antibody (IgY). Fractions of IgY were isolated from eggs yolks by simple...

Pseudomonas Aeruginosa is a common gram-negative bacterium present in the environment. It causes severe infections in immunosuppressed patients. Cystic fibrosis patients are especially at risk of being infected with Pseudomonas Aeruginosa. Ongoing studies are preformed to find alternative therapies to antibiotics, due to increased resistance. One new treatment is intake of specific IgY antibodies against Pseudomonas Aeruginosa as an oral therapy. The problem today is that IgY solutions must be kept frozen until consumed.  In this study we examined the possibility to freeze-dry specific IgY antibodies without losing any activity or specificity of the antibodies. This would be more convenient of patients, as well as it makes transportation and storage easier.  The methods used were ELISA for control of activity, western blot analysis and SDS-PAGE gel for control of specificity. Three different batches of the IgY anti-Pseudomonas Aeruginosa solution were tested. The results showed that no loss in activity occurred that would affect clinical outcome or change of specificity in the antibodies after freeze-drying appears. This indicates that it is possible to replace the liquid antibody to a freeze-dried powder.

Human respiratory system is in constant contact with heterogeneous environmental agents, which can possibly become initiators of inflammatory responses. Mechanical barriers and balance between the innate and adaptive immunity provide protection from external agents without initiation of any inflammatory response. Under certain circumstances, overreaction of the immune system might occur and leads to the production of various inflammatory cytokines and mediators such as TNF-α and GM-CSF factors or interleukins IL-1ß and IL-6. Respiratory system of the human suffering from cystic fibrosis is unable to defend against bacterial infections effectively. Chicken egg immunoglobulins (IgY) could eventually become a suitable tool for the protection from opportunistic pathogens, because they, when bound to antigen, don't activate the complement cascade. One method of determining the immunogenicity of the studied agent is the exposure of experimental animal and further measurement of inflammatory cytokine concentration. In this thesis, the potential inflammatory effect of IgY, IgY-derived Fab fragments and ovalbumin was determined after an exposure of Wistar rats by long-term inhalation. Correct implementation of inhalation experiment was confirmed via production of specific antibodies against the inhaled...