Rekombinant Antikor Fragmentlerinin Üretimi ve Genetik Modifikasyonuyla Sitokin Ölçümüne Yönelik İmmünoassay Geliştirilmesi

Abstract

Cytokines, which play an important role in the regulation of immune responses and inflammation, are used as biomarkers in the diagnosis of many diseases. During this immune response to pathogens or diseases, cytokine concentrations change rapidly and can lead to a severe condition such as sepsis if not controlled. Therefore, the measurement of cytokine levels is of great importance in early diagnosis and treatment, and sensitive, rapid, and economical tests need to be developed. The most sensitive and specific methods for measuring components in biological samples are immunosensors using antibodies as receptor molecules. Monoclonal antibodies (mAbs), which are used as receptor molecules in biosensors due to their high affinity and specificity, have an important place in diagnosis and treatment; however, their production costs are high. Small, non-glycosylated antibody fragments can be produced in high yield in prokaryotes, reducing the time and cost required. In this context, scFvs, single-chain variable fragments, are small (25 kDa) proteins with similar specificity and affinity to mAbs. Since scFvs can be modified more easily than IgGs due to their size, they can be immobilized on sensor surfaces using different methods. The ability of scFvs to bind to the surface with higher density and orientation increases the number of functional antigen binding sites, and thus the sensitivity of biosensors can be increased. The aim of this thesis study is the recombinant production of various anti-cytokine scFvs in Escherichia coli (E. coli) bacteria, and the development of highly sensitive immunoassays. The main goal was to utilize the advantages of bacterial production (low cost, ease of process control) to produce an easy, rapid, sensitive, and low-cost immunoassay. Anti-TNFα, anti-IL-6, anti-IL1β, and anti-IFNγ scFvs produced in a limited number of studies had problems regarding yield, recovery, stability, binding activity, and solubility. In order to improve solubility, stability, binding activity, and production yields, the use of E. coli SHuffle® T7 Express strain was the first target for the production of scFvs in soluble form thanks to its less oxidizing cytoplasmic environment; cloning steps were successfully completed in line with the designs of the gene cassette. As a result of optimizations at the gene and process level, low protein solubility and low purification yields, which are common in bacterial recombinant protein production, were obtained; to solve the problem, active protein was obtained from inclusion bodies with sarcosyl. This method has the advantage of obtaining protein in higher yields without the use of denaturing agents and laborious experiments such as refolding. The yield of protein production from inclusion bodies was 8-10 times higher than that from the soluble fraction, and scFvs were obtained with high yield and purity compared to the literature (>95% purity, 23 mg/mL). In addition to anti-cytokine scFvs, this technique can also be used for other recombinant proteins to be produced in E. coli. Following expression, scFvs purified by immobilized metal affinity chromatography (IMAC) were characterized by SDS-PAGE, Western blot, and ELISA, and then initially labeled with AuNp and lateral flow immunosensors were produced. Due to non-specific binding at the test site, ELISA-based assays were preferred. As a result, mAb fragments (scFv) were used in the ELISA method often employed in commercial mAb-based kits for cytokine measurement. Stable tests with a limit of detection (LoD) of 49 pg/mL and an affinity of Kd=0.77 μM were produced and contributed to the literature for a biotechnological product that can play a role in the diagnosis of many diseases.