Dye-Protein Interactions: Protein staining and Dye-IgY, Dye-Dextran-IgY Complexes for Antigen Detection By Ikechukwu Anthony Achilonu B.Sc. (Hons.) (Abia) Nigeria Submitted in fulfillment of the Academic requirements for the degree of Master of Science In Biochemistry in the School of Molecular and Cellular Biosciences University of KwaZulu-Natal Pietermaritzburg January 2004. "Listen. to what is wise ana try to understand it. :res, beg for krtowCeefgei pleadfor insight. Look.-for it as hard as uouzaouidfor siluer or hidden. treasure. It is (joa who gives unsdomi from him comes k.-nowCeefge ana undetstandinq. 11 Proverb 21 2 - 4 ana6. This zoork is dedicated to myparentsl 5'Lntfiony ana'Iheresa 5'Lcfiiwnu. PREFACE The experimental work described in this dissertation was carried out in the Department of Biochemistry, University of KwaZulu-Natal Pietermaritzburg, from March 2002 to December 2003 under the supervision of Prof. J. P. D. Goldring. These studies represent original work by the author and have not been submitted in any form to any university. Where use has been made of the work of others, it has been duly acknowledged in the text. Ikechukwu Achilonu Prof. J. P. D. Goldring 16 January 2004. Supervisor 11 ABSTRACT In order to develop a cheaper alternative to the conventional enzyme-linked immunosorbent assay system, application of dye molecules as labels in immunoassay was investigated in this study. This chromogenic dye-antibody conjugate could be used in colourimetric immunodetection diagnostic assays that could be used in a rural African setting. The chemistry of the interaction between twenty-six dyes of anionic, cationic and ligand dye classes with IgY and other proteins were studied for protein detection and conjugation to antibodies. Out of the twenty-six dyes studied, Direct Red 81 proved to be a good protein stain on nitrocellulose and polyacrylamide gels with comparable sensitivity to Coomassie Blue R 250. Direct Red stained proteins faster « 5 min) than Coomassie Blue R 250 in polyacrylamide gels. Aurintricarboxylic Acid, Ethyl Red and Gallocyanine with carboxylic acid and/or hydroxyl functional groups were selected, activated with carbonyldiimidazole (CDI) to form amine reactive-imidazole intermediates and conjugated to anti-rabbit albumin IgY. Gallocyanine gave the best molar coupling ratio with IgY (76:1 dye:IgY). The dye-antibody conjugates were used to detect rabbit albumin on nitrocellulose. Aurintricarboxylic Acid-IgY and Gallocyanine-IgY detected 50 ng of rabbit albumin on nitrocellulose, which was 10 fold less sensitive than HRPO-IgY conjugate. Cross-linking of the antibodies by the dyes compromised the immunoreactivity of the Aurintricarboxylic Acid-IgY and Gallocyanine­ IgY conjugates. The immunoreactivity of Ethyl Red-IgY was not compromised. Anti-rabbit albumin IgY was conjugated to derivatized dextran as an alternative immunoassay reagent and used to detect rabbit albumin on nitrocellulose by staining the polysaccharide (dextran) in the immune complex with PAS reagent. IgY-dextran complex was able to detect 25 ng of rabbit albumin on nitrocellulose, but PAS staining resulted in high background staining of the nitrocellulose membrane. 111 Dextran-antibody conjugates may have better potential as immunodetecting reagent than dye-IgY conjugates, if a more sensitive and specific method of detecting the dextran in the Ag:Ab-dextran immune complex is developed. IV ACKNOWLEDGMENTS I wish to acknowledge Prof. Goldring for his effort in putting this work together. You never stopped encouraging me to see beyond this point. I thank you sincerely for your guidance. To other members of academic and non-academic staff, Prof. Dennison, Prof. Anderson, ---:; ------ Prof.~, Dr. Elliott, Dr. B ~~~~ , Mr. Yeg~~, Mrs. Jenine Jerry, Mr. Denzil Lakay, Mrs. Charmanine and Mrs. Agnes Zondi for been approachable in times of need. I feel honoured working with this wonderful team of researchers, academics and administrators. To my brothers, Chinonyerem, Chidozie and Chibunna, you guys have never been short of encouraging words to me. You have always wanted to follow your big brother's footsteps. Hope you guys will forge ahead toward earning a higher degree. To Mr. and Mrs. M. Moteane and family and Mr. and Mrs. M. C. Achilonu and Family, for all your support and encouragements that lead me so far in life. I thank you. To Mematsepang and Tsepang, it is through your effort that I have managed to get this far. Working hard to keep our school running in my absence and how you supported me always as a family. I am very grateful to God for having made it possible to know you. To Dr. Fidelis Esenjor, Mr. James Obuh and Mr. Celestine Amaechi, for all your encouragements and fatherly advice you gave me. To my best friend and soul mate, Martha Marenga, for all the good times we had and will always have. Thanks for all your help in organizing my data and providing me with the computer that helped to facilitate the production of this dissertation. You are an angel. To my close friends and colleagues Habtom Habte, Pamela Mkhize, Dele Ebeye, Tammy Hiltunen, MaryAnn Chetty, Tim Smallie, Jacqui Foster, Laura Huson and Fabian 'Djemba Djemba' Fon and for your moral courage and lovely working environment you guys v provided. Biochemistry can be a pain. But with you guys, Biochemistry was nothing but fun. To my 'Bollywood' friends, Alicia Naidoo, Ektha Papli, Kovashni Pillay, Thilognee Subramani, Eulashni Chuntharpursat and Ureshnie Govender for making feel like an Indian. It is wonderful knowing you guys. I cherish all the good times we had. To all the staff and students of SBS Maseru Lesotho for all your support. The journey began from SBS Maseru. Thank you for providing me with the path to academic achievement. To the memory of my grandparents: Pa Achilonu Eke, Ma Catherine Agbaraji and Mr. Emma Agbaraji. Wherever you oaks are, I know you are proud of your Grandson. Till we meet again. VI CONTENTS PREFACE 1 ABSTRACT ii ACKNOWLEDGEMENTS iii CONTENTS vi LIST OF FIGURES xiii LIST OF TABLES xvi ABBREVIATIONS xvii CHAPTER 1: LITERATURE REVIEW 1 1.1 Introduction to dyes and dye applications 1 1.2 The basic concept of colours 4 1.3 Overview of the classification of dyes 6 1.3.1 Anionic (acid) dyes 7 1.3.2 Cationic (basic) dyes 7 1.3.3 Reactive dyes 8 1.3.4 Direct dyes 9 1.3.5 Disperse dyes 9 1.3.6 Ligand (chelating) dyes 10 1.4 Classification and chemistry of dyes used in this study 10 1.4.1 Acridine Orange [C.1. 46005] 11 1.4.2 Alcian Blue 8GX 11 1.4.3 Aurintricarboxylic Acid 12 1.4.4 Azure A [C.I. 52005] 13 1.4.5 Azure B [C.1. 52010] 14 1.4.6 Basic Fuchsin [C.1. 42500] 14 1.4.7 Bromocresol Purple 15 1.4.8 Cibacron Blue 3GA 16 1.4.9 Coomassie Blue R 250 [C.1. 42660] 17 1.4.10 Direct Red 75 [C.1. 25380] 17 vu 1.4.11 Direct Red 81 [Cl. 28160] 18 1.4.12 Eriochrome Black T 19 1.4.13 Ethyl Red 19 1.4.14 Evans Blue [Cl. 23860] 20 1.4.15 Gallocyanine 20 1.4.16 Hematoxylin [Cl. 75290] 21 1.4.17 Malachite Green HCI [Cl. 42000] 22 1.4.18 Methyl Green 23 1.4.19 Methyl Orange [Cl. 13025] 23 1.4.20 Napthol Blue Black [C.I. 20470] 24 1.4.21 Ponceau S [C.I. 27190] 25 1.4.22 Primuline 25 1.4.23 Reactive Black 5 26 1.4.24 Reactive Red 120 27 1.4.25 Trypan Blue [Cl. 23850] 27 1.5 Introduction to principles and mechanisms of dye binding (dyeing) 28 1.5.1 Dye binding by covalent forces 29 1.5.2 Dye binding by ionic (coulomb) forces 29 1.5.3 Dye binding by hydrogen bonding 30 1.5.4 Dye binding by non-polar and hydrophobic interactions 30 1.5.5 Dye binding by van der Waal forces 31 1.5.6 The relationship between dye structure and dye-substrate interactions 32 1.5.7 Physicochemical properties of macromolecules in relationship to dye binding 33 1.5.7.1.1 Chemistry of dye binding to proteins 34 1.5.7.1.2 Chemistry of dye binding to nucleic acids 36 1.5.7.1.3 Chemistry of dye binding to carbohydrates 37 1.5.8 Effects of solvents and solvent properties on dye binding 38 1.6 Introduction to direct colourimetric immunodetection 39 1.6.1 FITC-labelled antibody detection system 39 viii 1.6.2 Antibody-labelled colloidal dye detecting system .40 1.6.3 Dye-encapsulated latex bead detecting system .41 1.6.4 Colloidal-immunogold detecting system .41 1.7 Chicken egg yolk immunoglobulins (IgY) as an antibody model .42 1.7.1 Basic structure of IgY 43 1.7.2 Immunochemistry of IgY 47 1.7.3 Utilizing evolutionary differences of chicken IgY .47 1.8 Chemistries of bioconjugation and modification of antibody molecules .48 1.9 Aim and objectives of the study 54 CHAPTER 2: MATERIALS AND METHODS 56 2.1 Introduction 56 2.2 Materials 56 2.3 Ethics 58 2.4 Isolation and purification of chicken IgY from chicken egg yolks 58 2.4.1 Reagents 59 2.4.2 Procedure 59 2.5 Affinity purification of anti-rabbit albumin IgY 60 2.5.1 Reagents 62 2.5.2 Preparation of rabbit albumin affinity column using AminoLink® coupling gel 63 2.5.3 Method for immunoaffinity purification of chicken anti-rabbit albumin IgY 64 2.5.4 Storage of affinity-purified polyclonal chicken anti-rabbit albumin IgY 64 2.6 Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 64 2.6.1 Reagents 66 2.6.2 Preparation of polyacrylamide gel and evaluation of IgY purification on reducing Laemmli SDS-PAGE 67 IX 2.7 Secondary structure and sequence analysis of chicken IgY A-chain and u-chain regions 69 2.8 Enzyme-linked immunosorbent assay (ELISA) 69 2.8.1 Reagents 71 2.8.2 Method 71 2.9 Detection of proteins blotted on nitrocellulose membrane using range of dyes 72 2.9.1 Preparation of dye staining reagents 72 2.9.2 Method