Antigen-antibody affinity mapping: Characterization of monoclonal antibodies reactivity to viral proteins
Kashobwe, Lackson (2021)
Kashobwe, Lackson
2021
Bioteknologian ja biolääketieteen tekniikan maisteriohjelma - Master's Programme in Biotechnology and Biomedical Engineering
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2021-12-13
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-202111258650
https://urn.fi/URN:NBN:fi:tuni-202111258650
Tiivistelmä
Enteroviruses (EVs) belong to the Picornaviridae family, and their genomes are made up of single-stranded, positive-sense RNA. Infections from EVs can be asymptomatic or have a wide variety of symptoms, including paralysis, rash, poliomyelitis, hand-foot, mouth disease, encephalitis, and myocarditis. Also, human rhinoviruses (HRV) are widespread picornaviruses that cause the common cold and cost billions of dollars due to many missed work hours. Several monoclonal antibodies have been developed against EVs for the past decades. Some are specific to a single species or serotype, and others are group-specific. However, due to the vast array of enteroviruses circulating, better molecular tools would be needed to establish virus diagnostics. This thesis evaluated the diagnostic potential of five novel monoclonal antibodies against the eight viral proteins.
Eight recombinant Enterovirus structural proteins VP1s (CVA4, CVB1, PV1, EV-D68, RV-A89, RV-B14, RV-C3) were produced in E. coli and purified using glutathione and immobilized metal affinity chromatography. Also, two monoclonal antibodies (mAb) (4D12 and 9B9) were produced in rat hybridoma cells and purified in addition to three purified mAb (7C1, 12A4, 3A6) using affinity chromatography. The cross-reactivity of mAb to different VP1-proteins was mapped to generate the binding profile of each mAb using Western blot (WB), ELISA, and Biolayer interferometry (BLI).
It was discovered that the binding response of 3A6 mAb to eight VP1 in BLI and ELISA was superior to 9B9, 4D12, 7C1, 12A4, and Dako mAb. In ELISA and BLI measurements, 3A6mAb showed broad specificity and high affinity to CVB1 and E30. 12A4 and Dako mAb were found to react to CVA4, CVB1, PV1, and E30 in ELISA. Also, Dako mAb showed specificity and high affinity to CVB1 in BLI and Polio in ELISA. In contrast, 9B9, 4D12, and 7C1 had less affinity to the VP1 antigens in BLI, WB, and ELISA. Moreover, with further optimization, 3A6, 12A4, and Dako mAb can be used as a panel of antibodies to diagnose and distinguish EV infecting species based on the binding response kinetics and specificity.
Eight recombinant Enterovirus structural proteins VP1s (CVA4, CVB1, PV1, EV-D68, RV-A89, RV-B14, RV-C3) were produced in E. coli and purified using glutathione and immobilized metal affinity chromatography. Also, two monoclonal antibodies (mAb) (4D12 and 9B9) were produced in rat hybridoma cells and purified in addition to three purified mAb (7C1, 12A4, 3A6) using affinity chromatography. The cross-reactivity of mAb to different VP1-proteins was mapped to generate the binding profile of each mAb using Western blot (WB), ELISA, and Biolayer interferometry (BLI).
It was discovered that the binding response of 3A6 mAb to eight VP1 in BLI and ELISA was superior to 9B9, 4D12, 7C1, 12A4, and Dako mAb. In ELISA and BLI measurements, 3A6mAb showed broad specificity and high affinity to CVB1 and E30. 12A4 and Dako mAb were found to react to CVA4, CVB1, PV1, and E30 in ELISA. Also, Dako mAb showed specificity and high affinity to CVB1 in BLI and Polio in ELISA. In contrast, 9B9, 4D12, and 7C1 had less affinity to the VP1 antigens in BLI, WB, and ELISA. Moreover, with further optimization, 3A6, 12A4, and Dako mAb can be used as a panel of antibodies to diagnose and distinguish EV infecting species based on the binding response kinetics and specificity.