Isolation , Purification and Conjugation of polyclonal antibodies type Y from Immunized Hens ...

134 Pages·2012·2.37 MB·English

Republic of Iraq
Ministry of Higher Education & Scientific Research
University of Baghdad - College of Science
Biology Department

Isolation , Purification and Conjugation of
polyclonal antibodies type Y from
Immunized Hens against Hepatitis B
Surface Antigen

A Thesis
Submitted to the Council College of Science, University of
Baghdad in Partial Fulfillment of the Requirements for the
Degree of Master of Science in Biology/Microbiology

By
Najat Muayed Nafea
B.Sc. Biology ( Microbiology) / College of Science / University of Baghdad
(2008)

Supervised By
Dr. Raghad Al- Suhail Dr. Majeed Arsheed Sabbah

2012 1433
ﻢﻴﺣﺮﻟا ﻦﻤﺣﺮﻟا ﷲا ﻢﺴﺑ
ﻭَ ﻡْ ﻛُ َﻠﻣَ ﻋَ ُﷲ ﻯﺭَ ﻳَ ﺳَ ﻓَ ﺍﻭُﻠﻣَ ﻋْ ﺍ ﻝِ ُﻗ ﻭَ
ﻭَ ﻥَ ﻭﻧُ ﻣِ ﺅْ ﻣُ ﻟْ ﺍ ﻭَ ﻪُ ُﻟﻭﺳُ ﺭَ
ﺓِ ﺩَ ﺎﻬﺷﱠ ﻟﺍ ﻭَ ﺏِ ﻳْ ﻐَ ﻟْ ﺍ ﻡِﻟﺎﻋ ﻰﻟﺇ ﻥَ ﻭﺩﱡ ﺭَ ﺗُ ﺳَ
ِ ِ
ﻥَ ﻭُﻠﻣَ ﻌْ ﺗَ ﻡْ ﺗُ ﻧْ ﻛُ ﺎﻣﺑ ﻡْ ﻛُ ﺋُ ﺑﱢ ﻧَ ﻳُ ﻓَ

ﻢﻴﻈﻌﻟا ﻲﻠﻌﻟا ﷲا قﺪﺻ

Dedication
To who the Paradise is under her feet
to who I am debtor to her with all I reached. my love
My Mother.

To who supports me & provides me with the power. The Sun

of my sky
My Father.

To the lamp that lighting my way
My Brother (Ali).

To the angels of mercy, who support me in each step
My Sisters (Zainab, Huda & Luma).

To who did the impossible to make me success & happy
My aunt (Aom Ahmed).

Najat
Acknowledgment
At the end of this work I thank God as always, who gave me the force & sailed my ship into the harbor of
success withstanding whatever meager obstacles came my way.
Mercy & peace are upon the prophet Mohammad & his relative & companions.
I would like to sincerely thank my respectful supervisor Dr. Raghad Al-Suhail for her scientific guidance,
support, encouragement, invaluable remarks. I learned from her how to be powerful student.
I express my gratitude to my respectful co-advisor Dr. Majeed A. Sabbah for his advising, fruitful scientific
instructions and encouragement.
Above all my deepest thank & respect to my country for granting me the research scholarship which
enables me to gain more knowledge & experience.
I express my gratitude to Dr. Hayfa H. Hassani & Dr. Hind Hussien who they supported me, encouraged me
& facilitated all of my official work.
I would like to take the opportunity to thank Dr. Allis Krekor, Dr. Ghazi Munim from Biotechnology Dept.
& Dr. Issam Al-Joumaili from Genetic Engineering & Biotechnology Institute for their scientific advising &
facilitating the use of some equipments.
The word cannot express my thankful and grateful for Dr. HamidReza Rahmani & Dr. Amir-hossein
Mahdavi from department of animal science/college of Agriculture/Isfahan University of Technology
(IUT),for their acceptance me as one of their students. Sincere thanks are expressed to Dr. Rahmani about his
unforgettable great attitudes with me, his support, his facilitating for all of my work during my staying there,
his scientific advising & for his kindly fatherhood treatment with me. Sincere thank are expressed to Dr.
Mahdavi who guided me through the tenure of my project in IUT. His profound knowledge on the subject &
his innovate ideas helped me to develop my project. He was integrally involved in the research.
Sincere thank are also expressed to Dr. S. Soleimanian-Zad from department of food science/ IUT for
giving me the permission to carry out some of my experiments in lab of food industry & for her scientific
advice.
Sincere appreciate & grateful goes to Mr. AhmedReza Mahmoudi from Avicenna Research Institute for his
arrangement for my work, for his fruitful instructions that helped me to carry out the purification & for a lot
of generous attitudes he had done for me. I would like to take the opportunity to thank his co-workers Mrs.
Roys Ghods, Mrs. Zahara Nochi, Miss. Sedegigheh Vafaei, Mr. Jafar Mahmoudian, Mrs. Leila Balaye Goli, ,
Mrs. Mahdokht Babaei, Miss. Maryam Darzi, Miss. Yasama Voshgani, Mrs. ElhamShaban, Mrs.
BanafshaTavangar, & Mrs. Mohtaram Vafakha who I got more knowledge & experience form them & who
they help me to pass my challenges.
Sincere thank goes to Mr. Molai & for my lab mates: Mr. Ali Ihssani, Mr. Ihssan Mogadas, Miss. Vajeha
Nikoofard, Miss. Mahsa Merbood, Miss. Shiva Olya, Miss. Azadah, Miss Zahra Vafaei, Mr. Mohammaedali
Abbasi and Mr. Jalal Baka for their assistance in handling with animal and be familiar in farm &department.
I would like to express my love & deep thank to the persons whom I will never meet their right by these
words my close friend in abroad Samaneh Tajik, Dr. Mahnaz Estaki, Sayeh Aghai, Zainab Rezaiefar, Zahra
Nochi, Yasama Voshgani, & others especially in dormitory 10 of IUT for their helping, supporting&
companionship which provided me with the comfort and encouragements.
I am indebted to Aom Ahmed & their sons, Aom Kawther, & Abo Kawther who they were as a family that
provided me with the warmth and support. I am sinking with their generous.
My humble thank to Mr. Muhanad Mustafa & Maan Zarzor from Syria for their great attitudes & helping
that they have done with me.
Special Thanks goes to Mr. Mosavi, Mr. Khoshoai, Miss. Satari, Mr. Karemi, Mr. Mohammadi, Mr. Maliki &
Mr. Norozi for their facilitating for my laboratory work.
My sincere thank to Mr. Mohammed Abdul-Rahamn with whom I could make this project successful. His
support has to be acknowledged. His suggestions have guided me towards the right path & his constant
interest on the progress & results of this project has made it possible for me to complete the project in the
stipulated time span.
I also express my special thanks to everyone work in laboratories of plant tissue/biotechnology research
centre / AL-Nahrain University especially Miss Ashoaq for their helping & supplying me with some materials
& tools.
I wish to thank Dr. Haidar Badri & his wife Dr. Hula Younis for their advising that helped me to keep my
life in proper perspective & balance.
I would like to thank Dr. Ayaid K. Zgair about his fruitful scientific advising.
I am grateful for Miss. Hind Adel & Mr. Muntadar who they help me to produce my thesis in this form.
My special thank to all of my close & kind friends who they always with me especially Zainab, Heba &
Noor.
I would like to express my deep appreciation to my family: my parents, My brother & my sisters, who
without their praying, love, concern & inspiration I would not accomplished my studies.
Finally, many individuals and their support have contributed to the successful completion of this project, I
would like to express my gratitude towards all these wonderful, outstanding and dedicated people who have
helped me in their own special ways.
Summary

Summary
Polyclonal antibodies presently available for diagnosis are of
mammalian source. These antibodies have some problems related with the
interference with the immunological assays which caused by human
complement system, rheumatoid factors, human anti-mouse antibodies
(HAMA) and human or bacterial Fc-receptors. The problems that related
with the mammalian antibodies enhanced the interest for developing suitable
alternative antibody that can overcome all of these drawbacks.
In this study, the production, purification and Horse Radish peroxidase
(HRP) conjugation of polyclonal IgY against Hepatitis B surface Antigen
(HBsAg) were carried out. Forty Single Comb White Leghorn (SCWL) hens
with the age of 36 weeks old were divided into control group and
experimental group of 20 hens each. Experimental group was immunized
intramuscularly with 20µg of hepatitis B vaccine in combination with
Freunds complete adjuvant for first immunization, with Freunds incomplete
adjuvant for first booster immunization and without adjuvant for second
booster immunization. Blood and eggs were collected weekly before and
during the ten weeks after the first immunization. The water soluble fraction
(WSF) containing IgY was extracted from egg yolk by water dilution
method. Sera and WSF were assayed for the presence of anti–HBsIgY by
ELISA. Anti–HBs IgY titer in sera and WSF was absent in control group
and at pre-immunization in experimental group. Whereas, it increased in
significantly (P<0.05) experimental group after the first immunization and
second booster immunization in sera and WSF, respectively. WSF with high
anti HBs IgY titer as detected by ELISA were used to isolate and purify IgY.
The IgY purification was based on ultra filtration and gel filtration–FPLC.
The purity of IgY was monitored by SDS–PAGE (under non–reducing
condition) and was confirmed by Western blot. A highly purified of 180
KDa with specific activity of 200 mIU/ml was obtained. One milligram of
the pure and active IgY was labeled with HRP. Sandwich ELISA was used
I
Summary

to determine the optimum titer of anti–HBs IgY–conjugate which was found
to be 1:20.
This study showed that laying hens can be used as an alternative source
for production of polyclonal antibodies against HBsAg. Moreover, these
antibodies can be labeled with HRP and used as secondary antibody in
ELISA for detection of HBs Ag in the sera of patients with HBV infections .
II
Contents

Contents
Subject Page number
Summary I
Contents III
List of contents
IV

List of tables X
List of figures XI
List of abbreviations XIII
Introduction 1

III