>Magister Scientiae - MSc / Human Coronaviruses (HCoVs) are found within the family Coronaviridae (genus, Coronavirus) and are enveloped, single-stranded, positive-sense RNA viruses. Infections of humans by coronaviruses are not normally associated with severe diseases. However, the identification of the coronavirus responsible for the outbreak of severe acute respiratory syndrome (SARS-CoV) showed that highly pathogenic coronaviruses can enter the human population. The SARS-CoV epidemic resulted in 8 422 cases with 916 deaths globally (case fatality rate: 10.9%). In 2004 a group 1 Coronavirus, designated Human Coronavirus NL63 (HCoV-NL63), was isolated from a 7 month old Dutch child suffering from bronchiolitis. In addition, HCoV-NL63 causes disease in children (detected in approximately 10% of respiratory tract infections), the elderly and the immunocompromised. This study was designed to express the full length nucleocapsid (N) proteins of HCoV-NL63 and SARS-CoV for antibody production in an animal model. The NL63-N/pFN2A and SARSN/ pFN2A plasmid constructs were used for this study. The presence of the insert on the Flexi ® vector was confirmed by restriction endonuclease digest and sequence verification. The sequenced chromatographs obtained from Inqaba Biotec were consistent with sequences from the NCBI Gen_Bank. Proteins were expressed in a KRX Escherichia coli bacterial system and analysed using 15% SDS-PAGE and Western Blotting. Thereafter, GST-tagged proteins were purified ith an affinity column purification system. Purified fusion proteins were subsequently cleaved with Pro-TEV Plus protease, separated on 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. The viral fusion proteins were subsequently used to immunize Balbc mice in order to produce polyclonal antibodies. A direct ELISA was used to analyze and validate the production of polyclonal antibodies by the individual mice. This is a preliminary study for development of diagnostic tools for the detection of HCoV-NL63 from patient samples collected in the Western Cape. / South Africa

Antibodies are Y-shaped proteins. In the human body, antibodies areproduced by plasma cells, mainly T and B cells which are included inthe adaptive immune system. The production of antibodies is stimulatedby antigens. The binding between an antigen-specific antibody and itsantigen can be like the interconnect between a lock and a key.Therefore, antibodies are widely used as diagnostic tools for avariety of diseases but most importantly cancer. Some rapid diagnostictests are completely dependent on the specificity and reactivity ofantibodies such as UBC® Rapid produced by IDL Biotech AB. Therefore,the quality of these antibodies is important. This master thesis at IDL Biotech aimed to validate six anticytokeratinantibodies that are currently used in several rapid cancerdiagnostic tests produced by IDL. Antibody validation is a processwhere specificity, selectivity and reproductivity of an antibody isdemonstrated through specific laboratory investigations. During thisthesis, two laboratory methods were used to validate antibodies,namely, isoelectric focusing electrophoresis and the Attana QuartzCrystal Microbalance based biosensor. Isoelectric focusing electrophoresis (IEF) is a method that determinesproteins pI-values which can then reveal information about posttranslationalmodifications and protein sustainability during storage.IEF revealed changes in pI-values in two antibodies: AB2 and AB4. Attana biosensor analysis on AB1-5 showed that all antibodies havehigh specificity, reactivity and relatively high affinity to theircytokeratin targets. It also revealed that 4 antibodies (AB1 and AB3-5) have lower cross-reactivity with other cytokeratins than theirtarget cytokeratins compared to AB2. Keywords: Antibody validation, Isoelectric focusing, QCM, Attanabiosensor, biosensors, rapid diagnostics, epithelial carcinomas.