treatment training research development of chiken yolk immunoglobulin (igy) to some bacterial and...
TRANSCRIPT
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