감염초기 진단을 위한 고양이와 개의 IgM에 특이적인 단일클론항체 개발 및 적용

다국어 초록 (Multilingual Abstract)

Many households around the world including those in Korea and the United States, are living with companion animals, and the ratio of such households is constantly increasing. Among these companion animals, cats and dogs account for the highest percentage of household animals. With the increase of the number of households living with these companion animals, including cats and dogs, the risk of infection with the diseases is also growing. In particular, among cat- or dog-related diseases, there are zoonotic diseases that cause infection in humans. Therefore, it is essential to prevent spread of these diseases by rapidly diagnosing them. IgM is the first antibody that is generated during infection; if IgM is detected in a rapid diagnostic test (RDT) kit, it can faster and more accurately confirm infection and time of the infection. In this study, 6C3E11 and 4E5D7 were developed as monoclonal antibodies (mAbs) specific for cat IgM and dog IgM, respectively, for purpose of diagnosing various diseases related to cats and dogs at the early stage of infection. When comparing these antibodies with commercially available antibodies (GM-20A, GM-40A), the diagnostic efficiency of 6C3E11 was proven to be better than GM-20A, and 4E5D7 showed efficiency similar to that of GM-40A. The newly developed cat IgM or dog IgM specific mAbs can be used to rapidly diagnose various cat- or dog-related diseases at the early stage of infection.

국문 초록 (Abstract)

한국과 미국을 포함한 전 세계 많은 가정에서 반려동물과 함께 생활하고 있으며 그 비율 또한 증가하고 있는 추세이다. 이러한 반려동물 중 고양이와 개가 가장 높은 비중을 차지하고 있다. 고양이와 개를 포함한 반려동물과 함께 생활하는 가구가 점차 증가함에 따라 그에 대한 관련 질병의 감염 위험성 역시 높아지고 있다. 특히 고양이 또는 개 관련 질병 중에는 사람에게도 교차 감염을 유발하는 인수공통질병이 존재한다. 따라서 고양이와 개를 포함한 반려동물의 질병 감염여부를 빠르게 진단하여 질병의 확산을 막는 것이 무엇보다 중요하다. IgM은 질병 감염 시 가장 먼저 생성되는 항체로써 이를 진단한다면 질병의 감염 여부 및 감염 시기를 보다 신속하고 정확하게 확인할 수 있다. 본 연구에서는 고양이 및 개와 관련된 다양한 질병을 감염 초기에 진단하기 위한 목적으로, 고양이 IgM과 개 IgM에 특이적인 단일클론항체로써 6C3E11과 4E5D7을 각각 개발하였다. 이들 항체를 이용하여 시중에 판매 중인 항체와 진단효율을 비교한 결과, 6C3E11의 경우, 시중에 판매중인 항체보다 우수한 감도를 보였고 4E5D7의 경우, 비슷한 감도를 나타냈다. 새롭게 개발한 고양이 IgM 또는 개 IgM 특이 단일클론항체를 이용하여 신속진단키트제작 시, 인수공통감염질병을 포함한 다양한 고양이 또는 개 관련 질병들을 감염초기에 신속하게 진단할 수 있을 것이다.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• 1. Companion animals in Korea and America, especially Cat and Dog 1
• 2. Several diseases related to Cat and Dog 4
• 3. Various diagnostic methods for diagnosing diseases 6
• 4. Rapid diagnostic test (RDT) kit 7
• Ⅱ. MATERIALS AND METHODS 10
• 1. Network analysis of relation with human in Cat or Dog related diseases 10
• 2. Development of anti-Cat IgM and anti-Dog IgM mAbs from mouse 11
• 2.1 ELISA coating 11
• 2.2 Immunization 11
• 2.3 SP2/0 Myeloma cell cultivation 12
• 2.4 Preparation of immunized splenocytes 12
• 2.5 Cell fusion 12
• 2.6 Screening of hybridomas 13
• 2.6.1 Hybridomas producing anti-Cat IgM antibodies 14
• 2.6.2 Hybridomas producing anti-Dog IgM antibodies 14
• 2.7 Establishment of monoclonal hybridmas: Sub-cloning 14
• 2.8 Cell stock 14
• 2.9 Ascites production for mAbs purification 15
• 2.10 mAbs purification 15
• 2.11 Characterization analysis of purified mAbs 16
• 2.11.1 Antibody isotype analysis 16
• 2.11.2 Determination of antibody Kd value 17
• 2.12 Gold nanoparticle synthesis and antibody conjugation 18
• 2.13 Rapid diagnostic test kit production and configuration 18
• 2.14 Dot assay 20
• 2.15 Validation of cross reactivity in RDT strip 20
• 2.16 Clinical evaluation using IgM positive specimens 20
• 2.17 Validation of the candidate by a comparative evaluation 24
• 2.18 Serial Dilution-Lateral Flow Assay (SD-LFA) 24
• Ⅲ. RESULTS 25
• 1. Network analysis of relation with human in Cat or Dog related diseases 25
• 2. Immunization and monoclonal hybridomas production 25
• 3. Anti-Cat IgM mAbs 28
• 3.1 Screening of hybridomas 28
• 3.2 Validation of cross reactivity 28
• 3.3 Sub-cloning for obtain monoclonal hybridoma 28
• 3.4 Cross reactivity validation of monoclonal hybridomas 34
• 3.5 Ascites extraction and mAbs purification 34
• 3.6 Properties of purified mAbs specific for cat IgM 34
• 3.7 Dot assay using gold conjugated mAbs 39
• 3.8 Validation of cross reactivity in RDT strip 39
• 3.9 Clinical evaluation using cat IgM positive specimens 42
• 3.10 Validation of the candidate using comparative evaluation 42
• 3.11 Serial Dilution-Lateral Flow Assay (SD-LFA) 42
• 4. Anti-Dog IgM mAbs 48
• 4.1 Screening of hybridomas 48
• 4.2 Validation of cross reactivity 48
• 4.3 Sub-cloning for obtain monoclonal hybridoma 48
• 4.4 Cross reactivity validation of monoclonal hybridomas 54
• 4.5 Ascites extraction and mAbs purification 54
• 4.6 Properties of purified mAbs specific for dog IgM 54
• 4.7 Dot assay using gold conjugated mAbs 59
• 4.8 Validation of cross reactivity in RDT strip 59
• 4.9 Clinical evaluation using dog IgM positive specimens 62
• 4.10 Validation of the candidate using comparative evaluation 62
• 4.11 Serial Dilution-Lateral Flow Assay (SD-LFA) 62
• Ⅳ. DISCUSSION 68
• Ⅴ. REFERENCES 70
• ABSTRACT (in KOREAN) 76
• ACKNOWLEDGEMENT (in KOREAN) 77