Production and purification of rabbit igG antibodies against fasciola gigantica excretory- secretory antigen.pdf

NGUYEN TAT THANHNGUYEN TAT THANH UNIVERSITY True learning, true practice, true success, true future FACULTY OF BIOTECHNOLOGY GRADUATION THESIS PRODUCTION AND PURIFICATION OF Fasciola gi

NGUYEN TAT THANH

NGUYEN TAT THANH UNIVERSITY

True learning, true practice, true success, true future

FACULTY OF BIOTECHNOLOGY

GRADUATION THESIS

PRODUCTION AND PURIFICATION OF

Fasciola gigantica

EXCRETORY/SECRETORY ANTIGEN

Student’s name : Xuan-Thu ChauStudent ID : 1411530308Supervisor : Huu-Hung Nguyen, PhD

Ho Chi Minh City, 2019

NGUYEN TAT THANH

NGUYEN TAT THANH UNIVERSITY

True learning, true practice, true success, true future

FACULTY OF BIOTECHNOLOGY

GRADUATION THESIS

PRODUCTION AND PURIFICATION OF

Fasciola gigantica

EXCRETORY/SECRETORY ANTIGEN

: Xuan-Thu ChauStudent’s name

Ho Chi Minh City, 2019

NGUYEN TAT THANH UNIVERSITY

FACULTY OF BIOTECHNOLOGY

SOCIALIST REPUBLIC OF VIETNAM Independence - Liberty - Happiness

-oOo ASSIGNED TASK OF GRADUATION THESIS

1 Thesis’s title:

Production and purification of rabbit IgG antibodies against Fasciola

gigantica excretory/secretory antigen.

- Purification of polyclonal IgG antibodies from the immunized animals

- Assessment of bio-activity of purified IgG antibodies

4 Execution time: from 02/2019 to 08/2019

5 Supervisor: Huu-Hung Nguyen, PhD

Contents and requirements of this thesis were adopted by subject

HCM city, 2019

I also wish to express my deep gratitude to all members in laboratory MSc Thi- Phuong Nguyen, MSc Thi-Phuong-Thao Le, MSc Thi-Tuyet-Trinh Thai and Ms Ngoc-Kim-Thuy Dang who supported, encouraged, guided me through my study and their advice was invariably wise.

I am grateful to all friends, especially Thi-Truc-Phuong Nguyen, who have supported me with kindness, intelligence and always be beside me when I need

Finally, the heartiest thank to my family for their love, their resilience, their motivation and their belief in me throughout the period of my study

Xuan-Thu ChauFaculty of Biotechnology Nguyen Tat Thanh University

TABLE OF CONTENTS

ACKNOWLEDGEMENTS i

TABLE OF CONTENTS ii

ABSTRACT iv

LIST OF FIGURES V LIST OF TABLES vi

LIST OF ACRONYMS vii

INTRODUCTION ix

CHAPTER 1 LITERATURE REVIEW 1

1.1 Giant liver fluke Fasciola gigantica 1

1.1.1 Classification 1

1.1.2 Morphology 1

1.1.3 Life cycle 2

1.1.4 Fascioliasis epidemiology 3

1.1.5 Symptoms and signs 4

1.1.6 Diagnosis 4

1.2 IgG antibodies 5

1.2.1 Structure of IgG molecule and antigen-antibody interaction 5

1.2.2 Function of IgG molecule 7

1.3 Production and purification IgG antibodies 7

1.3.1 Adjuvant 7

1.3.2 IgG antibodies purification methods 8

1.4 Antibody applications 11

1.5 Quantity proteins using Quantity-one analysis software (BioRad) 12

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1.6 International and national research 12

1.6.1 International research 12

1.6.2 National research 14

CHAPTER 2 CONTENT AND METHODS 15

2.1 Place of administration 15

2.2 Contents 15

2.3 Methods 15

2.3.1 Rabbit immunization 15

2.3.2 Ouchterlony assay 16

2.3.3 Purification of IgG antibodies from immunized rabbit 16

2.3.4 SDS-PAGE 17

2.3.5 Indirect ELISA 18

CHAPTER 3 RESULTS AND DISCUSSION 20

3.1 Detection of specific IgG in rabbit after immunization with F gigantica E/S antigen 20

3.2 Antibodies titration 20

3.3 Immunoprecipitation of rabbit IgG by 45% saturated ammonium sulphate 21

3.4 Affinity chromatography using protein G column for IgG purification 22

3.5 Determination of purified IgG bio-activity by indirect ELISA 23

CONCLUSIONS AND RECOMMENDATIONS 25

REFERENCES 26

APPENDICES 29

Serodiagnosis of fasciolosis for specific IgG detection has a drawback that one can not discriminate a recent infection with that happened years ago or it is difficult to determine if the patients is recovering from infection Hence, the detection of stool parasite antigens, especially excretory/secretory (E/S) antigen is thought to be a better alternative for diagnosis of fasciolosis since it reflects the real parasite burden In this study, rabbit IgG antibodies specific for Fasciola gigantica’s excretory/secretory

antigen were produced in rabbit IgG antibodies were then purified by affinity chromatography using protein G column and use tested for it activity by indirect ELISA The results show that 55 mg of IgG was obtained from 60 ml of rabbit serum and had purity of about 95% The purified IgG highly binds to excretory/secretory antigen in ELISA assay indicating for their use for development of a test kit for antigen detection in fasciolosis

IV

LIST OF FIGURES

Figure 1.1 The giant liver fluke Fasciola gigantica morphology 2

Figure 1.2 Fasciola gigantica eggs were observed under an optical microscope 2

Figure 1.3 Life cycle of Fasciola gigantica 3

Figure 1.4 IgG structure 6

Figure 2.1 Diagram summarizing the process of production and purification of rabbit IgG against F gigantica E/S antigens 18

Figure 3.1 The agglutination of F gigantica E/S antigen with antibodies produced in rabbit after immunization 19

Figure 3.2 Titration of F gigantica E/S antigen specific antibodies in rabbit sera after immunization 20

Figure 3.3 SDS-PAGE analysis of IgG after precipitation by 45% saturated ammonium sulphate 21

Figure 3.4 Capture of IgG by affinity chromatography using protein G column 22

Figure 3.5 Determination of purified IgG activity by ELISA The graph shows the OD value 23

vi

LIST OF ACRONYMS

ADCC: Antibody dependent cellular cytotoxicityAPC: Antigen presenting cell

CDC: Center of Disease Control and PreventiondH2O: Distilled water

ELISA: Enzyme-link Immunosorbent Assay

HLB: Hydrophile - lipophile balance

IgG: Immunoglobulin G

ISCOM: Immunostimulating complex

LAMP: Loop - mediated isothermal amplification

MWCO: Molecular weight cut - off

NK: Natural killer (natural killer cell)

PBS: Phosphate - buffered saline

PBS-T: Phosphate - buffered saline with tween 20

PCR: Polymerase chain reaction

SDS-PAGE: Sodium dodecyl sulphate - Polyacryamide gel ElectrophoresisTEMED: N, N, N’, N’ - Tetramethylendiamine

TMB: 3, 3’, 5, 5’ - Tetramethylbenzidine

TLR: Toll - like receptor

VL; Variable domain, light chain

viii

1 Rationale for this thesis

Human Fascioliasis (HF) is a foodbome neglected parasitic disease caused by

Fasciola hepatica and Fasciola gigantica Approximately 2.5 - 17 million people are

infected with HF and an increase of HF cases has been reported from many countries The highest prevalence of HF has been reported from highlands of South American (Bolivia, Peru), Nile Delta in Egypt, China, Spain and also Vietnam Vietnam is not only a country with a high incidence of disease due to low sanitation, a lack of control

in animal husbandry, but also has a tropical monsoon climate These are favorable conditions for the development of giant liver fluke, especially F gigantica, is thought

to thrive in Central and Central Highlands

In parallel with the emergence and strong development of parasitic infections

induced by F gigantica, the detection of diagnostic methods with high sensitivity,

accuracy and low cost is an important issue Diagnosis of HF is challenging Performances of parasitological approaches, based on the detection of parasite eggs in the stool, are not satisfactory Currently serological methods for the diagnosis of HF are mainly based on detection of mti-Fasciola IgG antibodies in patient sera Although, there have been some improvement in the development of immunological diagnostic tests for the diagnosis of HF, yet these tests suffer from insufficiency in sensitivity or/and specificity

Detection of antigens, rather than antibodies, seems to be a suitable approach in the diagnosis of HF Antigen can be detected in sera or stool of the fascioliasis patients Circulating antigen in serum disappears within a short time and most of the circulating antigens are in immune complex forms which are not freely available to be detected Therefore, antigenemia might not be an approriate method for the diagnosis of HF Detection of antigen in stool (coproantigens) seems to be a suitable alternative method for the diagnosis of HF

Several antigen detection assays with diverse sensitivity and specificity have been utilized for the diagnosis of HF, based on detection of Fasciola coproantigens

The principal approach for the detection of antigen in stool sample is a sandwich

ELISA or indirect ELISA, using polyclonal or monoclonal or both antibodies Quite a few monoclonal or polyclonal antibodies have been produced against different antigens of F. gigantica and have been used in antigen capture ELISA for the

diagnosis of HF

Therefore, it is necessary to study the production of specific animal antibodies with high purity The purified antibody product not only helps diagnose and treat patients early, but also actively supply, which brings high economic value

The thesis “Production and purification of rabbit IgG antibodies against

Fasciola gigantica excretory/secretory antigen” was carried out to produce high

affinity and specific IgG antibody which can be use for development of parasitic diagnosis products

Chapter 1 Literature review

1.1 Giant liver fluke Fasciola gigantica

1.1.1 Classification

The giant liver fluke, Fasciola gigantica is important as a plant - borne trematode This parasite is the main cause of fascioliasis in ruminants and humans They live in the liver of cattle, buffaloes, sheep, goats, and swines, which have a significant impact on growth rate, development, and productivity of ruminants, and therefore, are considered economically significant ' In some occasions, fluke can infect human It has been reported that adult flukes have been recovered from the bile duct of people in Japan, Northern Iran, Thailand, and Vietnam 2 The classification

system of Fasciola gigantica is described below 3.

mm in diameter and lies in the anterior region and the ventral suker with 1.5 mm diameter, is located at the base of the cephalic cone The esophagus is well developed and divided into branched ceta extending to the posterior The testes are noticeably dendritic as well, situated anterior to the anterior testis, whereas the uterus is relatively

Chapter 1 Literature review

short and located anterior to the ovary The genital pore is located anterior to the ventral sucker Vitellaria are dendritic and scattered in both lateral fields of the body

Figure 1.1 The giant liver fluke Fasciola gigantica morphology 5.

F gigantica eggs vary from 156 to 197 pm in length and from 90 to 104 pm in

width The eggs are large, usually light golden brown in color and elliptical or oval in shape The operculum is small and inconspicuous, and the cell inside is unsegmented ?

Figure 1.2 Fasciola gigantica eggs were observed under an optical microscope 6

1.1.3 Life cycle

Fasciola parasites develop into adult flukes in the bile ducts of infected animals, which pass immature Fasciola eggs in their feces The next part of the life cycle occurs in freshwater In several weeks, the eggs hatch, producing a parasite form known as the miracidium, which then infects a snail host (Lytnnaea natalensis in

Africa and varieties of Lymnaea auricularia in Asia) Under optimal conditions, the

development process in the snail may be completed in 5 to 7 weeks, cercariae are then

2

Chapter 1 Literature review

shed in the water around the snail The cercariae lose their tails when they encyst as metacercariae (infective larvae) on water plants In contrast to cercariae, metacercariae have a hard outer cyst wall and can survive for prolonged periods in wet environments

5

Figure 1.3 Life cycle of Fasciola gigantica 7.

1.1.4 Fascioliasis epidemiology

Fascioliasis is a global disease In human, it has been reported from more than

75 countries worldwide Recognized areas of high transmission are the highlands of south America, the Nile valley, the Caspian sea basin, as well as east Asia and south - east Asia No countries can be considered free from the risk of fascioliasis8 WHO has sufficiented to justify the inclusion of fascioliasis in the list of important human parasitic diseases, with estimates of up to 17 million people or even higher depending upon hitherto unknown situations in many countries, mainly in Asia and Africa 9

In Viet Nam, fascioliasis is highly endemic, posing heavy burden in humans and ruminants, between 2000 - 4000 cases of fascioliasis each year occurring in all provinces, particularly in central provinces 10 In some studies, it was determined that the species causing fascioliasis in Vietnam belongs to Fasciola gigantica According

to local epidemiology in central provinces in Viet Nam, the prevalence of human

Chapter 1 Literature review

fascioliasis accounts for 5.4% The cases are mainly in women from 17 to 45 years old The number of women infected is three times higher than that of men 11

1.1.5 Symptoms and signs

After the larvae are ingested with contaminated food or water, a symptomless incubation period starts, lasting for a few days to a few months This is followed by an acute and a chronic clinical phase

• Acute phase: the acute phase, lasting 2-4 months, begins when the immature worms penetrate the intestinal wall and the peritoneum, the protective membrane surrounding the internal organs From here, they puncture the liver’s surface and eat their way through its tissues until they reach the bile ducts This invasion kills the liver cells and causes intense internal bleeding Typical symptoms include fever, nausea, liver swollen, skin rashes and extreme abdominal pain

• Chronic phase: The chronic phase begins when the worms reach the bile ducts where they mature and start producing eggs These eggs are released into the bile and reach the intestine, where they are evacuated in faeces, thereby completing the transmission cycle Symptoms include intermittent pain, jaundice and anaemia Pancreatitis, gallstones and bacterial infections may also occur Patients with chronic infections experience hardening of the liver (fibrosis) as a result of the long - term inflammation

1.1.6 Diagnosis

1.1.6.1 Coprological examination

Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis l2 This method is usually done by finding F gigantica eggs in stool (fecal) specimens examined under a microscope More than one specimen may need to be examined to find the parasite Sometimes eggs are found by examining duodenal contents or bile Infected people do not start passing eggs until they have been infected for several months People do not pass eggs during the acute phase of the infection when juvenile worms migrate through the intestinal wall to the peritoneal cavity (at 1 week), penetrate the liver parenchyma (at 5 to 7 weeks), and pass into the biliary tract where they ultimately

4

Chapter 1 Literature review

reach maturity (at 2 months and more) 13 Once the worms have matured, diagnosis still remains difficult, since eggs are frequently excreted at irregular intervals l4

1.1.6.2 Immunological techniques

Immunological techniques to detect worm - specific antibodies in serum samples or worm - specific antigens in serum or stool samples, they are usually more sensitive than the commonly used coprological examination Detection of antibodies in serum by ELISA with excretory/secretory (E/S) antigens is a frequently used diagnostic tool, is considered a sensitive and reliable means of diagnosing acute infections and can be used as an adjunct to fecal analysis for the diagnosis of latent and chronic infections 15 Specific antibodies to F gigantica may be detectable within

2 to 4 weeks after infection, which is 5 to 7 weeks before eggs appear in stool l6

Serum samples and stool samples were also studied by sandwich ELISA and indirect ELISA to detect circulating antigens and parasite coproantigens, respectively Circulating antigens were not detected in patients with patent infections However, coproantigens were clearly detected in all patients with patent infections Both the circulating antigen assay and the coproantigen detection assay have an important advantage over antibody detection for diagnosis, in that the presence of antigens implies active infection Thus, the circulating antigen and coproantigen assays could

be a simple, fast, and accurate diagnosis tool for human fascioliasis

1.1.6.3 Molecular techniques

Aside from conventional coprological and serological diagnostic methods, there are also several molecular methods available such as polymerase chain reaction (PCR) and loop - mediated isothermal amplification (LAMP) based on the detection of liver fluke DNA in faeces l7

1.2 IgG antibodies

1.2.1 Structure of IgG molecule and antigen-antibody interaction

IgG, the most abundant class in serum, constitutes about 80% of the total serum immunoglobulin IgG molecule has a common structure of four peptide chains This structure consists of two y (gamma) heavy (H) chains, and two K (kappa) or two À (lambda) light (L) chains Each light chain is bound to a heavy chain by a disulfide

Chapter 1 Literature review

bound, and by such noncovalent interactions as salt linkages, hydrogen bonds, and hydrophobic bonds, to form a heterodimer (H-L) 18

Antigen - antibody interaction is referred to by many terms involving many different actors Some samples include: 19

Table 1.1 Summary of some of the terms used to describe antigen - antibody interaction.19

Antigen Antibody

An antibody is a protein molecule (immunoglobulin) that has one or more combining sites called paratopes An antigen is a general term for a molecule that may trigger antibody generation, with potentially many different features surface Antigenic determinants are those surfaces features of the antigen that are complementary to an antibodies combining site 19

The capability of an antigen to trigger an immune response, the reactivity, and likelihood that it will bind to an antibody is called the antigenicity or immunogenicity

of the antigen The ability or likelihood of an antibody or B-cell receptor to bind with

an antigen is referred to as the antibody s or receptors specificity 19

These general terms are defined by, and abstract the chemical properties of the antigenic determinant and binding site respectively The specificity of an antibody may be relevant to other antibodies and may be relative to antigenic determinants Equally, the antigens immunogenicity may be relative to other antigens, other antigenic determinants, or to antibodies l9

6

Chapter 1 Literature review

LEGEND Fab Fragment, antigen-binding

CH VL VH

Fragment, crystallizable Constant domain, Light Chain Constant domain, Heavy Chain Variable domain, Light Chain

Variable domain, Heavy Chain Hypervariable Region Variable Region

Constant Region

Figure 1.4 IgG structure20

1.2.2 Function of IgG molecule

IgG antibodies are the only antibodies that are passed from mother to fetus through placenta IgG antibodies play an important role in protecting the body against from pathogens IgG antibodies have several effector functions including21:

• Neutralization of microbes and toxins

• Opsonization of antigens for phagocytosis by macrophages and neutrophils

• Activation of the classical pathway of complement

• Antibody - dependent cellular cytotoxicity (ADCC) mediated by NK cells

• Neonatal immunity: transfer of maternal antibody across placenta and gut

• Feedback inhibition of B cell activation

1.3 Production and purification IgG antibodies

1.3.1 Adjuvant

Adjuvant are compounds that may enhance the magnitude, breadth, and longevity of specific immune responses to antigens, as well as direct the quality of immune response, but have minimal toxicity or lasting immune effects on their own

22

The adjuvant property of a molecule increases with the length of the sugar side chain and the HLB value have high hydrophile - lipophile balance (HLB) value However, adjuvants are conventionally classified into the following categories:

Chapter 1 Literature review

mineral compounds, bacterial products, oil - based emulsions, the immunostimulating complexs (ISCOMs) and liposomes23 Based on their mechanism of action, adjuvants have been categorized into two broad groups: the particulate vaccine - delivery systems that target antigens to antigen presenting cells (APCs) and the immunostimulatory adjuvants that directly activate such cells through specific receptors as Toll - like receptors (TLRs) resulting in inflammatory responses that amplify the innate immune response The ultimate aim is to activate the innate system

to respond more rapidly to infection and for the adaptive immune response to become more specific 24

Freund’s Adjuvant is one of the most commonly used adjuvants in research It

is used as a water - in - oil emulsion It is prepared from non - metabolizable oils (paraffin oil and mannide monooleate) If it also contains killed Mycobacterium tuberculosis and known as Complete Freund’s Adjuvant (CFA) Without the bacteria

it is known as Incomplete Freund’s Adjuvant (IFA) It is generally assumed that incomplete (IFA) and complete Freund’s Adjuvant (CFA) act by prolonging the lifetime of injected autoantigen, by stimulating its effective delivery to the immune system and by providing a complex set of signals to the innate compartment of the immune system, resulting in altered leukocyte proliferation and differentiation 25 The main disadvantage of Freund’s Adjuvant is that it can cause granulomas, inflammation

at the inoculation site and lesions The mycobacteria in CFA attracts marcophages and other cells to the injection site which enhances the immune response For this reason, the CFA is used for the initial injections To minimize side effects, IFA is used for the boots

1.3.2 IgG antibodies purification methods

IgG is a divalent antibody and the most prevalent antibody in serum accounting for about 10% - 20% of plasma protein In peripheral blood, the IgG antibody population has an average half - life of 23 days and is the only isotype that crosses the placenta 26 IgG antibodies play an important role in treating infectious diseases In order to use IgG antibodies in some techniques such as ELISA, Immuno - Western Blotting, IgG must be purified To purify IgG, some of following methods can be used

8

Chapter 1 Literature review

- Precipitation of IgG from serum by saturated Ammonium Sulphate (NH4)2SO4

- Purification of IgG by affinity chromatography using protein A/G sepharose

- Purification of IgG using gel filtration chromatography

- Purification of IgG using ion exchange chromatography

1.3.2.1 Precipitation of IgG antibodies by Ammonium Sulphate

Precipitation using ammonium sulphate is one of the oldest method and widely applicable approach for partial purification of antibodies In solution, a large number

of water molecules are bound to the sulphate ion reducing the amount of water available to interact with the protein molecules At a particular concentration of (NH4)2SO4, an insufficient quantity unbound water will remain to keep a given protein species in solution, resulting in precipitation A saturated solution of ammonium sulphate is added to serum to precipitate the antibody Ammonium sulphate is useful for both polyclonal and monoclonal IgG isolation 27

IgG can be precipitated at 40% - 50% ammonium sulphate saturation or solid salt ammonium sulphate depending on species and subclass of antibodies In order to precipitate rabbit IgG antibodies, 45% ammonium sulphate saturation are commonly used

1.3.2.2 Affinity chromatography using protein A/G sepharose

The ability of proteins to bind specifically to other molecules is the basis of affinity chromatography In this technique, ligand molecules that bind to the protein of interest are covalently attached to the beads used to form the column Ligands can be enzyme substrates or other small molecules that bind to specific proteins In a widely used form of this technique, antibody - affinity chromatography, the attached ligand is

an antibody specific for the desired protein An affinity column will retain only those proteins that bind the ligand attached to the beads, the remaining proteins, regardless

of their charges or masses, will pass through the column without binding it However,

if a retained protein interacts with other molecules, forming a complex, then the entire complex is retained on the column The protein bound to the affinity column are then eluted by adding an excess of ligand or by changing the salt concentration or pH 28