Isolation, purification and conjugation of polyclonal antibodies type y from immunized hens against hepatitis B surface antigen

Other Title(s)

عزل و تنقية و ربط أضداد عديدة النسيلة نوع Y من الدجاج الممنع ضد المستضدات السطحية لفايروس التهاب الكبد الوبائي نوع B

Dissertant

Nafi, Najah Muayyad

Thesis advisor

Sabbah, Majid Arshid
al-Suhayl, Raghad Ghalib

Comitee Members

Jasim, Hamid M.
al-Azzawi, Raghd H. M.
Melconian, Alice K.

University

University of Baghdad

Faculty

College of Science

Department

Department of Biology

University Country

Iraq

Degree

Master

Degree Date

2012

English Abstract

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 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.

Main Subjects

Biology

Topics

• Diagnosis
• Hepatitis B

No. of Pages

107

Table of Contents

• APA
• MLA
• AMA

Language

English

Data Type

Arab Theses

Record ID

BIM-603211