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Trial report of Anigen

Bovine Brucella Diagnostics

ANIGEN B. Brucella Antibody

Rapid Test Kit & ELISA Kit

Introduction

Principle : Indirect Sandwich Method – (LPS capture)-(blood,milk)-(Protein G detector) – LPS extracted from cell

Purpose : Brucella abortus antibody titration in bovine whole blood, plasma, serum and milk

[Rapid Test Kit] [ELISA Kit]

Anigen B. brucella Ab Diagnostics

Principle of B. Brucella Rapid Test Kit

Rose Bengal & Milk Ring Test Anigen Rapid Test Anigen’s

Advantage

Principle -Serum : Rose Bengal

-Milk : Milk Ring Test

Chromatographic Immunoassay

Specimen -Rose Bengal : Serum

-Milk Ring Test: Milk

Whole blood, serum, plasma, Milk

available

All specimen

are available

Method

- Rose Bengal

1) Preparation of serum

2)1 drop of Rose bengal reagent +

1 drop of serum : Mixing

3) Interpret the result within 4 min.

- Milk Ring Test(MRT)

1) Adding of 1ml of milk into a tube

2)Adding of 30ul of MRT reagent

3) Waiting for 1 hour at 37℃

4) Reading the result

1) Adding 1 drop of specimen

2) Adding one drop of assay

diluent

3) Reading result at 20 min.

Easy Test

procedure

Reading

Method

- Rose Bengal : Agglutination?

- MRT : Changing of blue color of Milk

Positive : 2 line

Negative : 1 line

Simple

Required

testing time

Min.one day 20 Min. Rapid Testing

1st Screening Method [Rose Bengal Test, Milk Ring Test vs. Rapid Kit]

SVANOVIR : Sweden Anigen : Korea

Principle ELISA ELISA

Specimen Serum & Milk Serum & Milk

Method Similar Similar

Reading Method Complicated result calculation

OD of neg. standard PP= ------------------------------------- X 100 OD of pos. standard

Serum cut off : PP>25

Milk cut off : PP>10

Simple result calculation

Required testing time 2 hours 10 min. 2 hours

2nd Confirmatory Method [Reference ELISA kit vs Anigen ELISA Kit]

Clinical Field Trial - Anigen B. Brucella Ab Rapid Test -

Korea Kyonggi-Do Province Livestock and Veterinary Service

KLVS

Performed by

Sensitivity and Specificity - of serum specimen -

Anigen

Rapid

+ 145

- 182

SVANOVIR

ELISA

+ -

139 6

3 179

142 185

Anigen

Rapid

+ 118

- 35

Rose

Bengal

+ -

118 0

35 0

153 0

Sensitivity : 97.9%

Specificity : 96.8%

- Rose Bengal Test

: Current 1st screening assay

- Specimens : serums from KLVS

- SVANOVIR ELISA Kit

: Current confirmatory assay

-Specimens : serums from KLVS

Sensitivity : 77.1%

Specificity : 100.0%

Sensitivity : 87.1%

Specificity : 98.6%

Anigen

Rapid

+ 30

- 36

SVANOVIR ELISA

+ -

30 0

1 35

31 35

Anigen

Rapid

+ 30

- 36

Milk Ring Test

+ -

26 4

1 35

27 39

Sensitivity : 96.7%

Specificity : 97.2%

- Milk Ring Test : Current 1st screening assay

- Milk specimens : from random 66 cows KLVS

- SVANOVIR ELISA : Current confirmatory assay

- Milk specimens : from random 66 cows KLVS

Sensitivity : 96.2%

Specificity : 89.7%

Sensitivity : 96.5%

Specificity : 93.4%

Sensitivity and Specificity - of Milk specimen -

A. Specimens: Serum and Milk specimens from same cow of randomly selected 66 cows of KLVS

B. Assay Method :Anigen B. Brucella Ab Rapid Kit

Serum Total

+ -

Milk

+ 30 0 30

- 1* 35 36

Total 31 35 66

* Serum test showed weak positive

The serum and milk shows similar result between serum and milk:

Agreement rate: 98.5%

Agreement rate - Serum and Milk specimen -

Detection Limit Study - serum and milk specimens -

No. of serum specimens No. of milk specimens

Titer(2n) Titer

Result of Detection Limit Study

A. Serum : Anigen rapid test is more 256 ~ 1,024 times sensitive than Rose Bengal

B. Milk: Anigen rapid test is more 2 ~ 4 times sensitive than Milk Ring Test

Study principle : Comparison of detection limit of diluted serum and milk specimens

12

4

13

5

13

5

17

7

15

7

0

2

4

6

8

10

12

14

16

18

1 2 3 4 5

래피드 진단시약 Rose BengalAnigen Rapid

5120

1280

160 80 16040

5120

1280

0

1000

2000

3000

4000

5000

6000

1 2 3 4

래피드 진단시약 MRTAnigen Rapid

OIE Evaluation

Result

Clinical Field Trial - Anigen B. Brucella Ab ELISA Kit -

Korea Kyonggi-Do Province Livestock and Veterinary Service

KLVS

Performed by

Anigen

ELISA

+ 130

- 141

SVANOVIR

ELISA

+ -

127 3

0 141

127 144

Sensitivity : 100%

Specificity : 97.9%

( SVANOVIR ELISA : current confirmatory assay)

Sensitivity and Specificity - serum specimens -

A. Specimens: Total 271 of positive and negative cow serums from KNVRQS

B. Assay Method : Comparison of Anigen ELISA and SVANOVIR ELISA Kit(Sweden)

( KNVRQS: Korea National Veterinary Research and Quarantine Service)

Anigen

ELISA

+ 69

- 116

SVANOVIR

ELISA

+ -

68 1

0 116

68 117 185

Sensitivity and Specificity - of Milk specimen -

A. Specimens: Total 185 of positive and negative cow milk from KLVS

B. Assay Method : Comparison of Anigen ELISA and SVANOVIR ELISA Kit(Sweden)

Sensitivity : 100%

Specificity : 99.1%

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1 2 3 4 5 6 7 8 9

Anigen SVANOVA

0.000

0.500

1.000

1.500

2.000

2.500

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Anigen SVANOVA

Dilution (2n) Dilution (2n)

Comparison of detection limit in serum Comparison of detection limit in milk

OD OD

Detection Limit Study - of serum and milk specimens -

Study principle :

Comparison of detection limit of diluted positive serum confirmed by Rose Bengal

and milk confirmed by Milk Ring Test

Anigen B. Bruella Ab ELISA kit showed the detection limit:

- Anigen ELISA Kit is 2,048 times sensitive than Rose Bengal Test in serum

- Anigen ELISA Kit is 128 times sensitive than Milk Ring Test in milk

It showed that Anigen ELISA showed similar detection limit with SVANOVIR ELISA Kit in serum and milk

Anigen

Comparison

Anigen Rapid Kit Anigen ELISA Kit

Serum Milk Serum Milk

Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity

Rose Bengal

/Serum 77.1% - - - - - - -

MRT/Milk - - 96.2% 89.7% - - - -

SVANOVA

B.Brucella ELISA 97.9% 96.8% 96.7% 97.2% 100% 97.9% 100% 99.1%

- Anigen B.Brucella Ab Rapid test kit has better sensitivity and specificity than Rose

Bengal test kit and Milk Ring test.

- Anigen B.Brucella Ab ELISA test kit has better sensitivity and specificity than a

commercially available SVANOVIR ELISA

- Using Anigen B.Brucella Ab Rapid test kit is very simple, quick and it does not

need any special equipments. Therefore, Anigen B.Brucella Ab Rapid test kit will be

suitable to be adopted in clinical laboratory of basic hospital for screening of

Brucella abortous.

Summary of Accuracy

A. Specimen : 65 serums of Rose Bengal test positive

B. Comparative assay: Tube agglutination reaction

C. Positive Result : Anigen Rapid Test-91%, Tube Agglu.-88%

Assay Tube agglutination

Total Positive Negative

Anigen B. brucella

Ab Rapid Test

Positive 55 4 59

Negative 2* 4 6

Total 57 8 65

Anigen B.brucella Ab Rapid Kit (91%) is more sensitive than the Tube

agglutination reaction method (88%)

Additional Validation 1

Bovine

Brucellosis

Conventional Test Method

Rose Bengal Test

Milk Ring Test

PPD Skin Test

(purified protein derivative)

Result in within 10 Min.

Weak points :

- Laboratory Testing

- 1 day required

- RB : Serum only

- MRT : Milk only

Bovine TB

Weak points :

- 3 days required

- Low specificity

Immediate Testing at Farm

Testing in whole blood, serum, Milk.

Anigen Rapid Test Kit

(Bovine Brucella Ab, Bovine TB Ab)

Immediate Preventive Action

Isolation, Disinfections,

Protective slaughter

Reducing the damage

Clean

Application of Rapid Kit at Cattle Farm

Thank you for your attention