H1N1 Influenza Virus (Swine Flu): A Comprehensive Insight into Escalating Catch-22 Scenarios

Introduction: Viruses have always been a major cause of various disastrous pandemics in mankind’s history. H1N1 became a threat when its original strain was first discovered back in the swine flu pandemic of 2009. It became highly catastrophic on a large scale because none of the therapeutic interventions and methodologies that were already present at the time were effective against the virus. Methods: A vast amount of literature and research is available regarding H1N1 influenza from different reputable sources online. The data was gathered following the contrasting and relative situations of 1918 as well as the 2009 pandemic in mind. The overall extracted material provides comprehensive insights into the ups and downs of H1N1 influenza from 1918 up to 2009. Results: H1N1 virus comprises of a huge potential to cause a pandemic of Influenza type A. The illness caused by the virus has a varying degree of severity depending on the immune function of the individual being under attack. The virus exploits droplet-based transmission mode for its spread from one host to another. The major center of escalation of the subtypes of virus mostly originates from different avian and swine species. Most notably subtypes H9N2 and H5N1 of influenza A, which aren’t easily transmissible among humans. Furthermore, the droplet-based transmission takes comparably less time to infect a population of thousands if not millions. This ultimately increases the overall death toll by several folds by initiating a constant wave of pro-inflammatory cytokine release among affected hosts. Conclusions: Since its discovery in 2009, researchers have developed antiviral drugs and vaccines to fight the virus, most of which have proven to be very successful in treating the interconnected complications. The present-day strategies are only efficacious until the current strains of influenza A do not produce resistance against these drugs. All the therapeutic techniques and methodologies that have been developed to confront the virus up until now have been described in this ample review

프로콜라겐 III형 N-말단 펩타이드의 신속한 분석을 위한 형광 센서 “퀜치바디” 개발

학위논문(석사) -- 서울대학교대학원 : 공과대학 협동과정 바이오엔지니어링전공, 2021.8. 신승협.Procollagen type III N-terminal peptide (PIIINP) is a major biomarker of growth hormone which is on the prohibited substances list by the World Anti-Doping Agency (WADA). The recently developed and conducted PIIINP analysis methods are radio immunoassay (RIA) or fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA), and these have issues with radiation safety, time-consuming, and expensive equipment. Therefore, the development of an analysis method that can overcome these shortcomings is required. To this end, we tried to develop a high-throughput doping analysis method using a fluorescence-based antibody sensor called “Quenchbody. The quenchbody consists of a single chain variable fragment (scFv) and a fluorophore which emits fluorescence depending on the presence or absence of an antigen The sequence of anti-PIIINP scFv was obtained from a hybridoma cell and cloned scFv was expressed in E. coli. Inclusion body refolding was performed to obtain more scFv, and fluorescence was conjugated to native and refolded anti-PIIINP scFv that were confirmed to have antigen binding affinity against PIIINP. Finally, dose-dependent fluorescence signal were confirmed with a fluorescence spectrophotometer. The best limit of detection (LOD) and limit of quantitation (LOQ) were calculated as 1.64 nM and 3.89 nM for TAMRA-labeled quenchbody with native anti-PIIINP scFv, according to the five-point logistic curve regression. Furthermore, with 2 nM of quenchbody, the analysis could be performed within 30 minutes from the experimental preparations to validations. Thus, we confirmed the high-throughput and high sensitivity capabilities of quenchbody required for a new doping analysis method.프로콜라겐 III형 N-말단 펩타이드는 성장 호르몬의 주요한 바이오 마커로서 성장호르몬은 세계반도핑기구의 금지약물목록에 올라와 있다. 이에 따라, 근래에 개발된 방사면역측정법이나 형광기반의 샌드위치 효소결합 면역흡착검사와 같은 방법으로 프로콜라겐 III N-말단 펩타이드를 검출하고 있으나, 이들 방법은 방사능 안전성, 긴 분석 소요 시간, 값비싼 장비의 사용과 같은 문제가 있다. 그러므로 기존 분석법의 문제를 해결할 수 있는 새로운 방법의 개발이 요구되고 있는 상황이다. 따라서 본 연구에서는 형광기반의 항체 센서인 퀜치바디를 만들어 고속 도핑 분석법을 개발하고자 하였다. 퀜치바디는 단일 쇄 가변 단편 형태로 만들어진 항체와, 항원의 유무에 따라 형광을 방출하는 형광 분자로 이루어진다. 항프로콜라겐 III형 N-말단 펩다이드의 단일 쇄 가변 단편의 서열은 하이브리도마 세포로부터 얻어졌으며 재조합된 단백질은 대장균에서 발현되었다. 많은 양의 단일 쇄 가변 단편을 얻기 위해 봉입체 재접힘이 시도되었으며, 항원 결합성이 확인된 천연 및 재접힘 단일 쇄 가변 단편에 형광을 부착하였다. 마지막으로 형광광도계를 이용하여 퀜치바디의 항원 농도에 따른 형광 세기의 증가를 확인하였다. 그 결과로 5개 변수 로시스틱 곡선 회귀 분석에 따라 TAMRA-라벨된 천연 단일 쇄 가변 단편을 사용한 퀜치바디가 각각 1.64 nM과 3.89 nM의 검출한계와 정량한계를 가져 가장 우수한 것으로 나타났다. 또한, 이는 2 nM의 퀜치바디를 이용해 실험 준비부터 결과 검증까지 30분 이내에 분석을 진행할 수 있었다. 따라서 우리는 새로운 도핑 분석법에 필요한 신속성과 고감도성을 퀜치바디에서 확인할 수 있었다.Abstract 1 Contents 3 List of figures 5 List of tables 7 1. Introduction 8 2. Materials and methods 12 2.1. Chemicals and materials 12 2.2. Strains and vectors 13 2.3. DNA construction 16 2.3.1. ELISA of anti-PIIINP IgG from hybridoma 35J22 16 2.3.2. cDNA extraction of anti-PIIINP IgG 16 2.3.3. Anti-PIIINP IgG4 expression in HEK293-F 18 2.3.4. ELSIA of anti-PIIINP IgG4 from HEK293-F 19 2.3.5. Anti-PIIINP scFv construction 19 2.4. Production of anti-PIIINP scFv 20 2.4.1. Expression optimization 20 2.4.2. MBP fusion anti-PIIINP scFv expression 21 2.4.3. Molecular chaperone co-expression of anti-PIIINP scFv 21 2.4.4. Native anti-PIIINP scFv purification 22 2.4.5. MBP cleavage and purification 22 2.5. Refolding of anti-PIIINP scFv 23 2.5.1. Inclusion body isolation 23 2.5.2. Optimization of disulfide bond formation 24 2.5.3. Optimization of refolding additives 25 2.5.4. Refolding scale-up: step-wise dialysis refolding 26 2.6. ELISA of native and refolded anti-PIIINP scFv 27 2.7. Fluorescence labeling and purification 28 2.8. ELISA of quenchbody 29 2.9. Fluorescence measurement 29 3. Results 31 3.1. ELISA of anti-PIIINP IgG from hybridoma 35J22 31 3.2. ELISA of anti-PIIINP IgG4 from HEK293-F 34 3.3. Production of anti-PIIINP scFv 36 3.3.1. Native anti-PIIINP scFv 36 3.3.2. MBP fusion anti-PIIINP scFv 39 3.3.3. Molecular chaperone co-expression 41 3.4. Refolding of anti-PIIINP scFv 43 3.4.1. Inclusion body isolation 43 3.4.2. Optimization of disulfide bond formation 43 3.4.3. Optimization of refolding additives 44 3.4.4. Refolding scale-up: step-wise dialysis refolding 45 3.5. ELISA of native and refolded anti-PIIINP scFv 49 3.6. Fluorescence labeling and purification 51 3.7. ELISA of quenchbody 56 3.8. Dose–dependent fluorescent response 58 4. Discussion and conclusion 63 5. References 66석