› manual › FMDV › FMDV 3Diff PAN Ag... · A V ûD ¨* < ¨â2019-04-26 · 74 2019 KSDV...

2019 KASV

2019 KASV

수의양돈정책포럼


V E T T O G E T H E R

일시 2019년 3월 26일 [화]장소 충남대학교 정심화국제문회회관 백마홀

2019.3.26 www.KASV.or.kr

www.KASV.or.kr

2019KASV


주최 주관 / 김현권 의원주최 주관 / 김현권 의원

2019 KSDV 수의양돈정책포럼62

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구제역바이러스 현장진단킷트 개발 및 사용례

-

정광면


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65구제역바이러스 현장진단킷트 개발 및 사용례

1

구제역바이러스 현장진단키트 개발 및 사용례

㈜메디안디노스틱

2019. 03. 26

2

1. 구제역바이러스 혈청형 구분 현장진단키트의 필요성

2. 구제역바이러스 현장진단키트의 개발 3. 구제역바이러스 현장진단키트의 민감도 / 특이도

4. 구제역바이러스 현장진단키트의 사용례

목차

3

1. 구제역바이러스 구 현장진단키트

4

Distribution of the seven endemic pools of Foot-and-Mouth Disease showing conjectured status of Foot-and-Mouth Disease in countries during 2015.



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5

Reference : OIE/FAO Foot-and-Mouth Disease Referene Laboratory Network Annual Report 2017


ELISA

PCR

PRRS 진단 및 현장진단 사(POCT)

이 개 사

monitoring

현장진단 사

(5~15min)

Culture

9 이 9 혈청형(7 ) 의 차


7 2016 1 1 ~ 2017 12 31 (2 )

2. 구제역바이러스 현장진단키트 개발- 역 구

구제역진단

Immunization of mouse

Cell fusion

Expansion and selection of cultures to be cloned

Isolation and expansion of clones

Antibody titration

Isolation of spleen cells Preparation of myeloma cells

Screening of hybridoma supernatants

Cloning

Production of ascitic fluids

Purification and Final testing of monoclonal antibodies

Monoclonal antibody production technology

2. 구제역바이러스 현장진단키트 개발 - FMDV 이 개발 scheme


www.KASV.or.kr


Colloidal gold Lateral Flow Immunochromatographic Assay

2. 구제역바이러스 현장진단키트 개발 – assay platform

10

FMDV O, A, Asia, Pan 100 mAb 제 mAb matching test Æ setotype(O,A,Asia1) specific & Pan

2. 구제역바이러스 현장진단키트 개발 – mAb 제 및 이 mAb 발

11 11 11

Nitrocellulose

membrane

Absorption pad

Gold

conjugate pad

Sample pad

Control line

Test line

Mouse anti-Malaria rAg specific mAb

Mouse anti-FMDV A specific mAb [6E82] (Capture)

C

Mouse anti-FMDV (all serotypes) specific mAb

[1E58] (Detector) conjugated gold nanoparticle

A

Positive at T line

Capture mAb

Detector mAb

FMDV binding by

sandwich method

FMDV O, A, Asia1 serotypes

Mouse anti-FMDV Asia1 specific mAb [5D39] (Capture)

Mouse anti-FMDV O specific mAb [4B14] (Capture) O

AS

Malaria rAg conjugated gold nanoparticle

Strip1 3Diff

2. 구제역바이러스 현장진단키트 개발 – 진단키트 구 및

12 12 12

Nitrocellulose membrane

Absorption pad

Gold conjugate pad

Sample pad

Control line

Test line

Mouse anti-Malaria rAg specific mAb

Mouse anti-FMDV (all serotypes) specific mAb [1E58] (Capture)

C

PAN

Positive at T line Capture mAb

Detector mAb

FMDV binding by sandwich method

FMDV all serotypes (O, A, Asia1, C, SAT1, SAT2, SAT3)

Malaria rAg conjugated gold nanoparticle

Strip2 PAN

Mouse anti-FMDV (all serotypes) specific mAb [1E58] (Detector) conjugated gold nanoparticle



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C

C O A Asia1

C

C

C

As A O

1) Sample loading to the rapid kit for FMDV O Ag detection

2) Primary antigen-antibody reaction in gold conjugate pad

3) Secondary antigen-antibody reaction on test lines in nitrocellulose membrane

4) Unbound molecules(antigen or antibody) are moved to absorption pad

O A Asia1

O A Asia1

O A Asia1


14 FMDV serotype A positive

FMDV serotype O positive FMDV serotype Asia1 positive

FMDV other serotype (SAT1, SAT2, SAT3, C) positive

Negative

2. 구제역바이러스 현장진단키트 개발 – 제 및

15

키트의 구성 성

사 사 요

2. 구제역바이러스 현장진단키트 개발 – 사

Yang-Ju Jincheon O/Hapcheon/KOR/2014

Gimje Boeun Jeongeup O1manisa O1/BFS/860/UK/67

Yunlin/Taiwan/97

ME-SA Euro-SA Cathay

Strip 3Diff 5.5 3.9 3.7 3.9 4.9 4.3 4.7 3.9 4.0

Strip PAN 4.5 3.9 3.7 3.9 4.9 4.3 3.7 3.9 4.0

5.5 3.9 4.7 3.9 >4.9 5.3 4.7 4.7 5.0

5.5 3.9 4.7 3.9 >4.9 5.3 4.7 4.7 5.0

MEDIAN

SVANOVA

PBM

Kit

Limit of Detection in FMDV O Strains (LogTCID50/ml)

SEA/Mya-98 ME-SA/ind-2001d

16

¾ Limit of Detection is Same or Better (10 times) than PBM(USA), SVANOVA

� Limit of Detection in FMDV O strain (9 different topotypes)

104.7 103.7 102.7 104.7 103.7 103.7 102.7 104.7 103.7 102.7

MEDIAN SVNOVA PBM

Hapcheon + + + w+ - -

+ + - - - -

3. 구제역바이러스 현장진단키트 / 이 – Analytical sensitivity


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Pocheon Yeoncheon Malaysia97 P1A-189 Iran05 A22 Iraq

FMDVA/SAU/2/2015

Asia/Iran-05 Asia/G-IV

Strip 3Diff 5.4 4.9 4.0 4.4 4.5 4.7

Strip PAN 5.4 4.9 4.0 4.4 4.5 3.7

>5.4 >4.9 5.0 4.4 4.5 4.7

5.4 4.9 5.0 4.4 5.5 4.7

MEDIAN

SVANOVA

PBM

Asia/Sea-97

Kit

Limit of Detection in FMDV A Strains (LogTCID50/ml)

17


� Limit of Detection in FMDV A strain (6 different topotypes)

105.0 104.0 103.0

MEDIAN SVNOVA PBM

Malaysia + + w+ w+ - -

+ + - - - -

103.0 103.0 104.0 104.0 105.0 105.0


MOG/05 CAM/9/80 Sharmir

G-V

Strip 3Diff 4.9 4.6 4.7

Strip PAN 4.9 4.6 4.7

4.9 5.6 >4.7

4.9 5.6 4.7

MEDIAN

SVANOVA

PBM

Limit of Detection in FMDV Asia1 Strains (LogTCID50/ml)

Kit

18


� Limit of Detection in FMDV Asia1 strain (3 different topotypes)

105.6 104.6 103.6

MEDIAN SVNOVA PBM

CAM/9/80 + + w+ w+ - -

+ + - - - -

103.6 103.6 104.6 104.6 105.6 105.6


SAT1 SAT2 SAT3 C

SAT1/BOT/1/68 SAT2/ZIM/5/81 SAT3/ZIM/4/81 C3/Resende/BRA/55

WZ(Ⅲ) WZ(Ⅱ) Euro-SA

Strip 3Diff - - - -

Strip PAN 5.6 4.9 4.6 3.7

5.6 >4.9 5.6 4.7

>5.6 >4.9 >5.6 4.7

SVANOVA

PBM

Limit of Detection in FMDV (LogTCID50/ml)

Kit

MEDIAN

19


� Limit of Detection in FMDV SAT1, SAT2, SAT3, C strain

105.7 104.7 103.7

MEDIAN SVNOVA PBM

C3 + w+ - -

+ + - - - -

103.7 103.7 104.7 104.7 105.7 105.7

- -


20

¾ No Cross-reaction with other viruses(5) including vesicular disease associated viruses

� Cross reactivity test (other vesicular disease associated viruses)

Vesicular stomatitis virus Negative Negative

Swine vesicular disease virus Negative Negative

Seneca valley virus Negative Negative

Porcine reproductive and respiratory syndrome virus Negative Negative

Classical swine fever virus Negative Negative

Virus Strip 3Diff Strip PAN

3. 구제역바이러스 현장진단키트 / 이 – Analytical specificity


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21

¾ No Cross-reaction with other viruses(5) including vesicular disease associated viruses

� Cross reactivity test (Serotypes)

Vesicular stomatitis virus Negative Negative

Swine vesicular disease virus Negative Negative

Seneca valley virus Negative Negative

Porcine reproductive and respiratory syndrome virus Negative Negative

Classical swine fever virus Negative Negative

Virus Strip 3Diff Strip PAN


22

¾ No Cross reaction between different serotypes

� Culture supernatants containing different type of FMDV (A, Asia, C, SAT1, 2, 3)

Strip PAN

O A Asia 1 PAN

O(9) Positive Negative Negative Positive

A(6) Negative Positive Negative Positive

Asia 1(3) Negative Negative Positive Positive

SAT 1(1) Negative Negative Negative Positive



C(1) Negative Negative Negative Positive

Type(No. of strains)

Results

Strip 3Diff


23

¾ Higher Sensitivity than PBM, SVANOVA in every serotypes

� Clinical Sensitivity

1) Test samples used in this test were already determined as positive by PCR or ELISA 2) No serotype-cross reaction between test line

MEDIAN2) PBM SVANOVA

Saliva 70% (13/18) 0% (0/13) 67% (6/9)Vesicular fluid 100% (3/3) 100% (1/1) NT

Tissue 94% (44/47) 86% (25/29) NTTotal 88% (60/68) 60% (26/43) NT

AVirus-Spiked Saliva

(=Total)100% (70/70) 10% (5/25) 40% (8/20)

Tissue 100% (1/1) 100% (1/1) NTVirus-Spiked Saliva 100% (60/60) 52% (31/60) 50% (6/12)

Total 100% (61/61) 52% (32/61) 50% (6/12)

SAT1 100% (20/20) NT 0% (0/4)

SAT2 100% (20/20) NT 0% (0/4)

SAT3 100% (20/20) NT 0% (0/4)

C 100% (20/20) 75% (15/20) 100% (4/4)

Sensitivity1)

(No. of Positive/No. of Samples)

O

Serotype Type of Samples

Asia1

Virus-Spiked Saliva(=Total)

3. 구제역바이러스 현장진단키트 / 이 – Clinical sensitivity

24

¾ FMDV negative Saliva in Korea (RT-PCR)

9 Specificity in bovine: 100% (n=92/92)

9 Specificity in swine: 99.5% (n= 398/400)

� Total 99.6% specificity (n= 490/492)

� Clinical Specificity

3. 구제역바이러스 현장진단키트 / 이 – Clinical specificity


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25

구

17/07/07 10-2017-0086716 구제역바이러스 혈청형 O 용 단 이의 용도 민 ( 역 )

스

17/10/13 10-2017-0133574 구제역 바이러스 혈청형 A 용 단 이의 용도 민 ( 역 )스

17/11/21 10-2017-0155390 구제역 바이러스 혈청형 Asia1 용 단 이의 용도 민 ( 역 )

스

26

및

27

2017 Global FMD Research Alliance, p63, 2017

발

28

Date Country Estimated lesion age (days)

Lesion site

Sample type

Svanova Median (pan)

Median (serotype)

Lab result

Note

21/11/2017 Kenya 1-2 Oral Epithelium - - - SAT2 Svanova buffer used in median kits

21/11/2017 Kenya 1-2 Oral Epithelium + (weak) - - SAT2 Svanova buffer used in median kits

28/11/2017 Kenya 7-10 Foot Epithelium - + (weak) - SAT2 Svanova buffer used in median kits

28/11/2017 Kenya 4-5 Oral Epithelium - - - SAT2 Svanova buffer used in median kits

27/02/2018 Nepal 2-3 Oral Epithelium - + + (O) O

13/03/2018 Nepal 1-2 Foot Epithelium Not recorded

+ + (O) O


+ + (O) O

20/03/2018 Nepal 2 Foot Epithelium Not recorded

- - O

20/03/2018 Nepal 2 Foot Epithelium Not recorded

+ + (O) O

20/03/2018 Nepal 2 Oral Epithelium Not recorded

+ + (O) O


+ + (O) O

20/03/2018 Nepal 2 Oral Epithelium Not + Not recorded O

FAO (2017. 11. 21 ~ 2018. 03. 20)

4. 구제역바이러스 현장진단키트 사용례


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29

FAO (2017. 11. 27 ~ 2018. 12. 04) 4. 구제역바이러스 현장진단키트 사용례

30

FAO (2017. 11. 27 ~ 2018. 12. 04) 4. 구제역바이러스 현장진단키트 사용례

31

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Mongolia - SCVL (2018. 03. 30) 4. 구제역바이러스 현장진단키트 사용례

type A (epitelium) A/Asia/Sea-97-2016

type Asia1 (epithelium)-2005

State Central Veterinary Laboratory(SCVL) in Mongolia

32

사 .

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