UNIVERSITI PUTRA MALAYSIA

MAPPING OF NUCLEOCAPSID PROTEIN (NP) EPITOPES AND NP-PHOSPHOPROTEIN (P) INTERACTIVE DOMAINS OF NEWCASTLE

DISEASE VIRUS WITH NP MONOCLONAL ANTIBODIES

RAHA AHMAD RAUS

IB 2006 8

MAPPING OF NUCLEOCAPSID PROTEIN (NP)

EPITOPES AND NP-PHOSPHOPROTEIN INTERACTIVE DOMAINS OF NEWCASTLE DISEASE VIRUS WITH NP MONOCLONAL

ANTIBODIES

RAHA AHMAD RAUS

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2006

MAPPING OF NUCLEOCAPSID PROTEIN (NP) EPITOPES AND NP-PHOSPHOPROTEIN (P) INTERACTIVE DOMAINS OF NEWCASTLE

DISEASE VIRUS WITH NP MONOCLONAL ANTIBODIES

By

RAHA AHMAD RAUS

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Doctor of Philosophy

October 2006

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Doctor of Philosophy

MAPPING OF NUCLEOCAPSID PROTEIN (NP) EPITOPES AND NP-

PHOSPHOPROTEIN INTERACTIVE DOMAINS OF NEWCASTLE DISEASE VIRUS WITH NP MONOCLONAL ANTIBODIES

By

RAHA AHMAD RAUS

October 2006

Chairman : Professor Datin Khatijah Mohd. Yusoff, PhD Institute : Bioscience The Newcastle disease virus (NDV) is an economically important poultry virus which

replicates in certain human cancer cells. This virus contains a negative single stranded

RNA genome which encodes for the nucleocapsid protein (NP), phosphorylated

nucleocapsid-associated protein, phosphoprotein (P); matrix protein (M); fusion

protein (F); haemaglutinin-neuraminidase protein (HN) and the RNA-directed RNA

polymerase, large protein (L).

The NP is the most abundant protein found in NDV. In this study, a panel of

monoclonal antibodies (mAbs) against NP was developed to study the NP-P

interactions in NDV. The spleen cells of Balb/C mice immunized with purified NP

obtained from the velogenic NDV strain AF2240 were fused with myeloma cells

(Sp2/0-Ag14 cell line). A panel of six mAbs were produced and characterized. Four

of the mAbs secreted immunoglobulins from class IgG2a with kappa light chains and

the remaining two were from class IgG1 with kappa light chains. Cross-reactivity test

against the NPs from other six NDV strains showed that mAbs, a2, a2s and b2 cross-

reacted with all NDV strains, while mAb b3 showed specificity towards the NP of

ii

strain AF2240, the strain that was used earlier to immunize the mice. The other two

mAbs, b4s and c1, demonstrated cross-reactivity amongst the various viral strains

with varying reactivities. These results indicate that certain epitopes recognized by the

mAbs were well conserved in all NDV strains whilst the other epitopes may have

undergone some structural changes.

The antigenic sites of NP bound by the mAbs were localized by Western blot

analysis. Four C- and N-terminally truncated NP mutants were purified from

Escherichia coli, blotted to the nitrocellulose membrane and probed with NP mAbs.

The results show that the antigenic sites bound by mAbs a2, a2s and b2 were located

within amino acids 441 to 489 of the C-terminal of NP. On the other hand, antigenic

sites that were recognized by the mAbs, b3 and b4s were located on the N-terminal

half of NP from 26 to 121 amino acid residues. MAb c1 bound to all C- and N-

truncated mutants indicating that the antigenic sites recognized by mAb c1 may be

located within amino acids 122 to 375.

One of the mAb, a2s was further used as a tool in protein-protein interaction study

between assembled NP (NPNC) and P. The NPNC was purified from E. coli by

ammonium sulphate precipitation and sucrose garadient. In determining the

interaction regions of P that bind to NPNC, the mAb is used in immunoprecipitating

the radioactively labeled Ps-NPNC complex. The failure of certain P deleted mutants to

form complex with NPNC demonstrated that the regions of P which were deleted from

those mutants were responsible with the binding to NPNC. After 18 different N- and C-

terminally truncated P mutants were tested in the radioimmunoprecipitation assay, it

showed that the region of P that binds to NPNC is located within the internal region of

iii

C-terminal half of P, from amino acids 243 to279. In agreement with the

radioimmunoprecipitation results, protein binding assay, another assay that was

carried out to determine the P-NPNC interactive domain also showed that the

interactive domain was mapped to the internal region of the C-terminal half of P

(amino acids 224-279). A slightly bigger region of interaction domain was determined

by the latter assay compared to the former assay was due to its nature and higher

sensitivity of the assay. Nevertheless, both assays showed that the N-terminal half and

immediate C-terminal end of P is not involved in the binding of P to NPNC.

To further explore the interactions between P and NP, Far Western blotting was

carried out to determine the binding domain of P to NP monomer, NPO. The NPO was

obtained by fractionating the NPNC in SDS-PAGE. In this assay, the same deleted

mutants that are utilized in mapping P-NPNC interactive domain were also used. The

assay showed that amino acids 224-279 were indispensable for the P-NPO binding.

Interestingly, these were the same amino acids that were responsible for the P-NPNC

interaction. These results indicate that these amino acids were crucial for interaction

between P and NP and may play bigger roles in transcription and replication of viral

genome.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PEMETAAN EPITOP PROTEIN NUKLEOKAPSID (NP) DAN DOMAIN INTERAKTIF NP-FOSFOPROTEIN VIRUS PENYAKIT NEWCASTLE

MENGGUNAKAN ANTIBODI MONOKLONAL NP

Oleh

RAHA AHMAD RAUS

Oktober 2006

Pengerusi : Profesor Datin Khatijah Mohd. Yusoff, PhD Institut : Biosains

Virus penyakit Newcastle (NDV) adalah virus yang menyebabkan kerugian dalam

industri ternakan ayam dan juga boleh bereplikasi dalam sel kanser manusia tertentu.

Ia mengandungi genom bebenang tunggal (negatif) RNA yang mengkodkan protein

nukleokapsid (NP), protein fosforilasi yang bertuapan dengan nukleokapsid,

fosfoprotein (P), protein matriks (M), protein tuapan (F), protein hemaglutinin-

neuraminidas (HN) dan polimeras RNA, protein besar (L).

NP merupakan protein yang boleh didapati dengan banyak dalam NDV. Dalam kajian

ini, satu panel monoclonal antibodi (mAb) terhadap NP telah dihasilkan untuk

digunakan dalam kajian interaksi NP-P NDV. Sel limpa mencit Balb/C yang

diimunisasi dengan NP yang diperolehi daripada NDV veliogenik strain AF 2240

telah digabungkan dengan sel miloma (sel Sp2/0-Ag14). Satu panel yang terdiri

daripada enam mAb telah dihasilkan dan dicirikan. Empat daripada mAb tersebut

merembeskan immunoglobulin daripada kelas IgG2 dengan rantai ringan kappa dan

dua lagi daripada kelas IgG1 dengan rantai ringan kappa. Ujian reaksi silang terhadap

v

NP daripada enam strain NDV lain menunjukkan mAbs a2, a2s dan b2 bereaksi

silang dengan semua strain NDV, manakala mAb b3 menunjukkan spesifisiti terhadap

NP strain AF2240 iaitu strain sama yang digunakan untuk imunisasi mencit. Dua lagi

mAb iaitu b4s dan c1 menunjukkan reaksi silang secara variasi terhadap semua strain

tersebut. Keputusan ini menunjukkan terdapat epitop tertentu yang dikenalpasti oleh

mAb adalah terpelihara dalam semua strain NDV manakala epitop lain telah

mengalami perubahan struktur.

Tapak antigenik NP yang dikenalpasti oleh mAb telah diidentifikasi dengan analisis

blot Western. Empat mutan NP yang terpotong pada terminal-C and -N yang

diekstrak daripada Escherichia coli diblot kepada membran nitroselulosa dan diprob

dengan mAb NP. Keputusan menunjukkan tapak antigenik yang dikenalpasti oleh

mAb a2, a2s dan b2 terletak pada asid amino 441 hingga 489 terminal-C NP.

Manakala tapak antigenik yang dikenalpasti oleh mAb b3 dan b4s terletak pada

terminal-N NP dari asid amino 26 hingga 121. MAb c1 melekat pada semua mutan

NP yang terpotong pada terminal-C and –N dan ini menunjukkan tapak antigenik

yang dikenalpasti oleh mAb c1 terletak pada asid amino 122 hingga 375.

Satu daripada mAb iaitu a2s telah digunakan dalam kajian interaksi antara NP yang

berhimpun, NPNC dan P. NPNC diekstrak daripada E. coli dengan kaedah presipitasi

ammonium sulphate and gradien sukros. Dalam menentukan domain interaksi P yang

bergabung kepada NPNC, mAb tersebut digunakan untuk immunopresipitat pelbagai

kompleks mutan P-NPNC yang dilabel dengan radioaktif. Selepas 18 mutan P yang

terpotong pada terminal-C and –N diuji dengan asai radioimmunopresipitasi,

keputusan menunjukkan domain P yang bergabung dengan NPNC adalah terletak

vi

dalam bahagian dalaman terminal-C iaitu daripada asid amino 243 sehinnga 279. Asai

gabung protein juga meunjukkan domain P yang bergabung kepada NPNC adalah

terletak pada bahagian yang sama (asid amino 224-279). Domain interaksi yang

diidentifikasi oleh asai gabung protein adalah lebih besar daripada asai

radioimunopresipitasi adalah disebabkan bentuk tafsiran asai gabung protein yang

lebih sensitif berbanding asai imunopresipitasi. Walau bagaimanapun, kedua-dua asai

menunjukkan terminal-N dan hujung terminal-C P tidak terlibat dalam interaksi P

kepada NPNC.

Untuk mendalami kajian interaksi P dan NP, blot Western Far telah digunakan dalam

mengenalpasti domain P yang bergabung dengan monomer NP, NPO. NPO diperolehi

dengan fraksinasi NPNC di dalam SDS-PAGE. Dalam asai ini, mutan P yang sama

yang telah digunakan dalam kajian interaksi P-NPNC telah digunakan. Asai tersebut

menunjukkan asid amino 224 sehingga 279 adalah terlibat dalam interaksi P-NPO.

Yang menariknya, asid amino tersebut juga terlibat dalam interaksi P-NPNC. Ini

menunjukkan asid amino tersebut sangat penting dalam interaksi P-NP dan

kemungkinan memainkan peranan lebih besar dalam transkripsi dan replikasi genom

virus.

vii

ACKNOWLEDGEMENTS

Thanks to Allah, the God Almighty who has it made possible for me to finish my

PhD. course. It has not been easy for a mother of four children. And of course a debt

of gratitude is owed to the following people who have helped me throughout my PhD.

course. Only God knows how much I appreciate their contributions. Thank you very

much.

My main supervisor, Prof. Datin Dr. Khatijah Yusoff, for the help, advice and

guidance.

My co-supervisors, Prof Dr. Abdul Manaf Ali and Assoc. Prof. Dr. Tan Wen Siang,

for being understanding and be patient with me.

Dr. Majid Eshagi for technical advice and assistant.

My sponsors, International Islamic University of Islam, for financial support.

My dearest friends, Salina, Riha, Kah Fai and Sim, for being very encouraging and be

with me through good and bad times.

All the lab members of 143, 134 and Dept. of Microbiology and Biochemistry.

Especially my family (Mazlan, my husband; Haziq, Nabihah, A’qilah and Zahin, my

children) and my maid (Sumarsih), for their enormous support and patiently helping

me through the odd hours of my lab works.

To my parents, thanks for all your prayers.

viii

I certify that an Examination Committee met on 4th October 2005 to conduct the final examination of Raha Ahmad Raus on her Doctor of Philosophy thesis entitled “Mapping of Nucleocapsid Protein (NP) Epitopes and NP-Phosphoprotein (P) Interactive Domains of Newcastle Disease Virus with NP Monoclonal Antibodies” in accordance with Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degraee. Members of the Examination Committee are as follows: Raja Noor Zaliha Raja Abd. Rahman, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) Seow Heng Fong, PhD Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Raha Abdul Rahim, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Sharifah Syed Hassan, PhD Institut Penyelidikan Veterinar (External Examiner) _________________________

HASANAH MOHD GHAZALI, PhD Professor/ Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

ix

This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: KHATIJAH MOHD. YUSOFF, PhD Professor Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) ABDUL MANAF ALI, PhD Professor Faculty of Agriculture and Biotechnology Universiti Darul Iman (Member) TAN WEN SIANG, PhD Associate Professor Department of Microbiology Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) ___________________________ AINI IDERIS, PhD Professor/ Dean School of Graduate Studies Universiti Putra Malaysia Date: 16 JANUARY 2007

x

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions. ____________________ RAHA AHMAD RAUS Date: 11 NOVEMBER 2006

xi

TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL ix DECLARATION xi LIST OF TABLES xv LIST OF FIGURES xvi LIST OF ABBREVIATIONS xviii CHAPTER 1. INTRODUCTION 1 2. LITERATURE REVIEW 2.1 Antibodies 6 2.2 Production of Antibody 11

2.2.1 Monoclonal Antibodies (mAbs) Production by Hybridoma Technology 11

2.2.2 Production by Recombinant Antibody 16 2.3 Epitopes 21 2.4 Applications of mAb 23 2.5 Newcastle Disease Virus (NDV) 26 2.5.1 Nucleocapsid Protein (NP) 30 2.5.2 Phosphoprotein (P) 33 2.6 Protein-protein Interactions 37 3. METHODOLOGY 3.1 General Procedures 41 3.2 Escherichia coli, Virus and Vectors 41

3.3 Production of mAbs against NP 3.3.1 Cultivation of Myeloma cells 43 3.3.2 Immunisation 3.3.2.1 Preparation of Immunogen and

Immunisation 43 3.3.2.2 Titration of Antibody by Indirect

Enzyme-linked Immunosorbent Assay (ELISA) 44

3.3.3 Fusion 3.3.3.1 Preparation of Myeloma Cells 45 3.3.3.2 Preparation of Lymphocyte Cells 45 3.3.3.3 Fusion with Polyethylene Glycol (PEG) 46

3.3.4 Screening of Antibody-Producing Hybridomas 47 3.3.5 Limiting dilution of Hybridoma Cells 48

xii

3.3.6 Western Blotting 48 3.3.6.1 Sodium Dodecyl Sulphate

Polyacrylamide Gel Electrophoresis (SDS-PAGE) 49

3.3.6.2 Electroblotting 50 3.3.6.3 Immunodetection 51 3.3.7 Isotyping of mAbs 52 3.3.8 Cross-reactivity with NP from Local NDV

Isolates 52 3.3.9 The Bradford Assay 53

3.4 Localisation of Antigenic Sites of NP using NP mAbs by Radioimmunoprecipitation Method 53 3.4.1 Construction of Truncated NP Gene 3.4.1.1 Preparation of Competent Cells 54

3.4.1.2 Polymerase Chain Reaction (PCR) 54 3.4.1.3 Extraction of Plasmid 58

3.4.1.4 Restriction Enzyme Digestion 59 3.4.1.5 Isolation of DNA Fragments from

Agarose Gels 59 3.4.1.6 Ligation and Transformation 60

3.4.1.7 Screening for the Transformants 61 3.4.2 In Vitro Transcription and Translation 61 3.4.3 Radioimmunoprecipitation 62

3.5 Localisation of Antigenic Sites of NP using NP mAbs by Western Blotting Method 63

3.6 NP-P Interaction Assay 3.6.1 Construction of Truncated P Genes 65 3.6.2 NPNC-P Interaction by Radioimmunoprecipitation 68 3.6.3 NPNC-P Interaction by Protein Binding Assay 69 3.6.4 NPO-P Interaction by Far Western 69 4. RESULTS 4.1 MAbs against NP 71 4.1.1 Hybridomas 71 4.1.2 Specificity of Hybridomas Producing Antibodies 72 4.2 Characterization of mAbs against NP 72

4.2.1 Isotyping of mAbs 75 4.2.2 Specificity of mAbs with Local NDV Isolates 75

4.3 Localisation of NP Antigenic Sites Using In Vitro Translated NP Mutants 77

4.3.1 Amplification of Full Length and Truncated NP Genes 77

4.3.2 Cloning of Full Length and Truncated NP Genes into pCITE-2a 80 4.3.3 In Vitro Transcription and Translation of Full

Length and Truncated NPs 84 4.3.4 Radioimmunoprecipitation 84

4.4 Localisation of NP Antigenic Sites Using Purified NP Mutants 87

xiii

4.5 NPNC -P Interaction Analysis 94 4.5.1 Cloning of Truncated P Genes into pCITE-2a 94 4.5.2 In Vitro Transcription and Translation of

Truncated Ps 101 4.5.3 Immunoprecipitation Analysis 104

4.5.4 Protein Binding Assay Analysis 109 4.6 NP0-P Interaction by Far Western 115 5. DISCUSSION 5.1 Production of mAbs against NP 119

5.2 Characterization of mAbs against NP 122 5.3 NP Antigenic Sites 126

5.4 NPNC -P Interaction 131 5.5 NP0-P Interaction 139 6. CONCLUSION 144 REFERENCES 147 BIODATA OF THE AUTHOR 167

xiv

LIST OF TABLES Table Page 2.1 Properties of the immunoglobulins 9

3.1 E. coli and vectors 42 3.2 Local NDV isolates 42 3.3 Primers used in generating NP deletion mutants 57 3.4 Primers used in generating P deletion mutants 67

4.1 Biological properties of mAbs against NP of NDV strain AF2240 73

4.2 Specificity of mAbs with the some local NDV isolates using

indirect ELISA 76 4.3 Summary of radioimmunoprecipitation shown in Figures 4.19

and 4.20 110 4.4 Summary of Far Western shown in Figure 4.22 118 5.1 Domains of P that bind to NPNC and NPO 134

xv

LIST OF FIGURES Figure Page 2.1 Schematic diagram of antibody structure 7 2.2 The general procedure of monoclonal antibody production by

hybridoma technology 12 2.3 Schematic representation of small antibodies molecules 19 2.4 Schematic diagram of Newcastle disease virus 29 2.5 Structure of the C-terminal half of phosphoprotein 36 3.1 NP gene of NDV AF2240 (Kho et al., 2001a) 56 3.2 P gene of NDV AF2240 (Kho et al., 2002) 66 4.1 Specificity of the mAbs against NP 74 4.2(a) Agarose gel electrophoresis of amplified 5’-end deleted NP

fragments full length 78 4.2(b) Agarose gel electrophoresis of amplified 3’-end deleted NP

fragments full length 79 4.3 Schematic representation of the deletion mutants of NP 81 4.4 Antigenic peptides software analysis of NP (according to the

method of Kolaskar and Tongaonkar, 1990) 82 4.5 Construction of the pCITE-2a containing the PCR products 83 4.6 BamHI-XhoI restriction analysis of the pCITE-2a harboring

full length and truncated NP genes 85 4.7 In vitro translated full length and truncated NPs 86 4.8 Schematic representation of the deletion mutants of NP 88 4.9 PCR of full length and truncated NP genes from recombinant

clones constructed from the previous study 89 4.10 SDS-PAGE (12 %) of purified full length NP and truncated NP

mutants 90 4.11 Western blot analyses of the purified truncated NP 91

xvi

4.12 Western blot analyses of the truncated NP to determine NP antigenic sites 93

4.13 Schematic representation of the deletion mutants of P 95 4.14 PCR amplification of truncated P gene 96 4.15 BamHI-SacI restriction digestion of the pCITE-2a harboring

5’-end truncated P genes 98 4.16 BamHI-SacI restriction digestion of the pCITE-2a harboring

3’-end truncated P genes and full length P gene 100 4.17 In vitro translated N-terminal truncated P 102 4.18 In vitro translated full length and C-terminal truncated P 103 4.19 Radioimmunoprecipitation of in vitro translated products of

N-terminal deletion P mutants 105 4.20 Radioimmunoprecipitation of in vitro translated products of

C-terminal deletion P mutants 108 4.21 Protein binding assay of truncated P mutants (radioactively

labelled P mutants) 111 4.22 Far Western of N- and C-terminal truncated P mutants 116 5.1 SDS-PAGE (12 %) of allantoic fluid of NDV isolates 124 5.2 Hydrophobic sequence software analysis of NP (according to

the method of Kyte and Doolittle, 1982) 128 5.3 Schematic representation of NP antigenic sites of several

negative stranded RNA viruses 130

5.4 Schematic representation of P domains of several negative stranded RNA viruses that binds to NPNC. 136

5.5 Schematic representation of P domains of several negative

stranded RNA viruses that binds to NPO. 142

xvii

LIST OF ABBREVIATIONS

ABTS 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic)

ATP adenosine triphosphate

BCIP bromochloroindoyl phosphate

bp base pair

BSA bovine serum albumin

C constant domain

oC degree Celcius

cDNA complementary deoxyribonucleic acid

CDR complementarity-determining region

CE conformational epitope

CITE cap-independent translation enhancer

cpm count per minute

DEA diethanolamine

dH2O distilled water

DMEM Dulbeccos’s modified Eagle’s medium

DMSO dimethylsulfoxide

DNA deoxyribonucleic acid

dNTP deoxyribonucleoside triphosphate

DTT 1,4-dithiothreitol

EDTA ethylenediaminetetraacetic acid

ELISA enzyme-linked immunosorbent assay

F fusion protein

Fab fragment antigen binding

xviii

FACS fluorescence-activated cell sorter

Fc crstallizable fragment

FBS fetal bovine serum

Fv variable fragment

H heavy chain

h hour

HAMA human anti-mouse antibodies

HAT hypoxanthine, aminopterin and thymidine media

HGPRT hypoxanthine guanine phosphoribosyl transferase

HN haemaglutinin-neuraminidae protein

hPIV3 human parainfluenza virus type 3

HT hypoxanthine and thymidine media

hRSV human respiratory syncytial virus

Ig immunoglobulin

IPTG isopropyl-β-D-thiogalactopyranoside

kDa kiloDalton

L large protein (polymerase)

LB Luria-Bertani

LE linear epitope

LPMV La Piedad Michoacan Mexico virus

M matrix protein

mA milliampere

mAb monoclonal antibody

MCS multiple cloning site

mg milligram

xix

min minute

ml milliliter

mM millimolar

mRNA messenger ribonucleic acid

µg microgram

µl microliter

µm micrometer

µM micromolar

NBT nitro-blue tetrazolium chloride

NDV Newcastle disease virus

nm nanometer

NP nucleocapsid protein

NPO nucleocapsid monomer

NPNC assembled nucleocapsid

NP-40 nonidet P-40

OD optical density

P phosphoprotein

PBS phosphate-buffered saline

PBS-T phosphate-buffered saline containing Tween 20

PCR polymerase chain reaction

PEG polyethylene glycol

pNPP p-nitropheyl phosphate disodium

PVDF polyvinylidene difluoride

RNA ribonucleic acid

RSV respiratory syncytial virus

xx

xxi

RT room temperature

S second

SB standard binding buffer

scFv single-chain variable fragment

SDS sodium dodecyl sulphate

SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis

TAE tris-acetate EDTA buffer

TBS tris buffered saline

TBS-T tris buffered saline containing Tween 20

V variable domain

VH variable domain of heavy chain

VL variable domain of light chain

VSV vesicular stomatitis virus

v/v volume for volume

w/v weight for volume

CHAPTER 1

INTRODUCTION

One of the immune responses to eliminate pathogens and other foreign molecules

from our body is by secreting antibodies that act specifically against them. Antibodies

are produced in large quantities by plasma cells which are developed from precursor

B cells. However, Kohler and Milstein (1975) successfully produced the antibodies in

vitro through permanent tissue culture cell lines. These antibodies are produced by

hybridoma cells which are generated from a fusion of immunized B-cells and

myeloma tumor cells. These antibodies are considered monoclonal (mAb) as they are

secreted from a single cell or clone. Therefore these antibodies are specific against a

particular epitope of an antigen. Although initially, these mAbs which were produced

had no practical application, further investigations by other scientists have resulted in

many beneficial discoveries. At present, the production of mAbs has resulted in the

generation of an industry with an economic impact measured in billions of dollars.

MAbs with specificity to tumour-associated antigens have been applied in various

diagnostic assays in vitro and as well as in vivo by using gamma-emitting radio-

labelled mAbs to identify the location of tumor in patient. It has also been used to

treat cancerous and non-cancerous diseases and act as a carrier to various toxin,

cytostatic drug and radionuclides to treat diseases (Govindan et al., 2005). Poisoning

with digoxin and other potent, low formula mass poisons, such as colchicine and

tricyclic antidepressants could be successfully treated using specific mAbs fragment,

Fab (Flanagan and Jones, 2004). Identification of bacteria, virus and protozoa in

clinical samples are easily conducted by using mAbs that are specific against each

microbe. For certain bacterial and viral infections, the use of neutralizing mAbs

confers impressive protection against the pathogen (Mc Cann et al., 2005).

Other than its obvious contribution in clinical medicine, the use of mAbs has a strong

impact towards the knowledge of biological sciences. For example, in neuroscience

research, mAbs provide purified membrane associated proteins such as pumps,

channels, receptors and cell-adhesion molecules so that their structures could be

studied at high resolution. MAbs to cytoskeletal proteins, organelles, and protein

kinases have revealed that specific molecules are concentrated in anatomically distinct

regions of the cell and protein kinase has been shown to be a major postsynaptic

constituent in many synapses. The antibodies are also used to identify functionally

related subpopulations of neurons and describing neural cell lineages (Valentino et

al., 1985).

In the present study, mAbs are produced using the hybridoma technology. These were

then characterized and, used to map the NP antigenic sites and to study protein-

protein interactions in Newcastle disease virus (NDV). This particular virus infects

many avian species. In many countries worldwide, NDV infections result in great

economic loses amounting billions of dollars as infections of chicken with velogenic

strain of NDV causes up to 100% mortality. NDV contains a negative single stranded

RNA genome which encodes at least six structural proteins, the nucleocapsid (NP),

phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) and

large polymerase (L) proteins. Among these proteins, the NP, P and L proteins are

involved in viral transcription and replication. The interaction between P and NP is

2