TREATMENT

TRAINING

RESEARCH

DEVELOPMENT OF CHIKEN YOLK IMMUNOGLOBULIN (IGY) TOSOME BACTERIAL AND BIOTOXIN AGENTS

Colonel A/Prof. Le Van Dong et al.,Vietnam Military Medical University

INTRODUCTION

Big size animals require large amount of antigens;

Blood collection and Ab purification is complicated process;

Blood can not be harvested very often, that limits the yield of Ab obtain from each immunized animal.

Antiserum production

Chicken immune system

Chicken antibodies: IgM, IgA and IgY. IgY can be transferred from hen’s blood to egg and

accumulated in the yolk (egg yolk immunoglobulin: IgY)

Transfer of IgY from hen to chick

IgY from hen blood → egg yolk → chick.

IgG from mother blood → placenta and umbilical cord → fetus.

Antigen

Advantages of IgY

IgY applications

Diagnosis: Immunological tests. Treatment: Passive immunotherapy.

Specific IgYs have been made and used for diagnosis and treatment on animals

Diarrhea caused by viruses and enterobacteria; Respiratory diseases: influenza virures A H5N1

and H1N1; Hand foot mouth disease; Candida albicans; Snake venoms, toxins and other bio-toxins.

Clinical application of IgY

Prevention of P. aeruginosa infection in patient having cystic fibrosis.

Prevention of decay with mouth washing solution contain IgY to Staphyloccocus mutans.

Prevention of gastric ulcer with yogurt contained IgY against urease from H. pylori.

IgY production is simple method that can provide mass quantity of antibody with low cost, suitable for developing country.

TREATMENT

TRAINING

RESEARCH

MATERIALS1. Pseudomonas aeruginosa 2. Vibrio cholerae3. Cholerae toxin4. Urease from Helicobacter pylori

Reagents

Freund’s complete adjuvant. Freund’s in-complete adjuvant. Peroxidase conjugated anti-IgY antibody. Bacterial culture media. Reagents for IgY extraction and ELISA test.

Egg laying hens

Imported hen Domestic hen

Antigen preparation and immunization

Bacterial/toxin antigens

Egg laying hens

Chicken eggs

Egg yolk

Purified IgY

Protein

Antigen preparation and immunization

Detection of specific Ab in hen blood

Detection of specific Ab in egg yolk

Extraction of egg yolk protein

Purification of IgYIgY-based biological products

RESULTS

Seroconversion of P. aeruginose specific IgY in hen blood before and after immunization

IgY specific to P. aeruginose

0

10

20

30

40

50

60

70

1 2 3 4 5

Hiệ

u g

iá k

hán

g t

hể

(x10

00)

Lần gây miễn dịch

Lô 1

Lô 2

Lô 3

An

tibo

dy

tite

rs (

x10

00

)

Immunization times

Gr 1

Gr 2

Gr 3

IgY specific to P. aeruginose in egg yolk

Reactivity of P. aeruginose specific IgY in egg yolks after 5th immunization

0

0.2

0.4

0.6

0.8

1

1.2

1/500 1/1k 1/2k 1/4k 1/8k 1/16k 1/32k 1/64k

Độ pha loãng kháng thể IgY

OD

(4

50

nm

)

Chứng âm

Lô 1

Lô 2

Lô 3

Egg yolk dilution

Neg. Ctr.

Gr 1

Gr 2

Gr 3

Reactivity of IgY specific to P. aeruginose against each bacterial strain

Reactivity of IgY specific to P. aeruginose extratced from egg yolk against each bacterial strain

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1/500 1/1k 1/2k 1/4k 1/8k 1/16k 1/32k 1/64k

Độ pha loãng kháng thể

OD

(45

0 n

m)

1100P2

79P16

IM27P11

9HP13

79P16

7P8

36P8

Chứng âm

Egg yolk dilution

Neg. Ctr

IgY extraction and purificationIon-exchange chromatography spectrum of the extraction product.

SDS-PAGE of extraction and purification products.

Reactivity of specific IgY in each chromatography factions as tested with ELISA

P. aeruginose agglutination with IgY in liquid medium

Strain No. 6P11 M27P11 79P16 9HP13 1100P2 7P8 36P8

Agglutination reactivity

(+++) (++) (++) (++) (+) (+) (+)

P. aeruginose inhibition with IgY in solid medium

A) Strain 27P11 (27 mm) B) Strain 79P16 (28 mm)

C) Strain 9HP13 (26 mm) D) Negative control, Strain 27P11 (10 mm)

Treatment of heat experimental burn wounds infected with P. aeruginose

Quantities of P. aeruginose at the wound surface before and after treatment

Kinetic of bacterial quantities at wound surfaces during the treatment

Day 2 Day 3 Day 6 Day 9 Day 12

Den

sit

y o

f b

acre

ria/

cm2

(Lg

)

0.9% NaCl

Conclusion

We have successfully applied IgY antibody technology in production of Ab antidotes to some bacteria and bio-toxins at laboratory scale.

The obtained IgYs show specific reactions to immunized antigens as tested in vitro and in vivo.

TREATMENT

TRAINING

RESEARCH

Thank you

AcknowledgmentsDr. Nguyễn Đặng DũngDr. Tim SunnaryDr. Đỗ Hoàng LongA/Prof. Lê Thu Hồng

Dr. Đỗ Minh TrungDr. Đỗ Khắc ĐạiDr. Hoàng Trung KiênDr. Nguyễn Ngọc Tuấn