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Preparation and Evaluation of Immunoglobulin Free Sera for Biomaterial-Induced Complement Activation Studies

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Preparation and Evaluation of Immunoglobulin Free Sera for Biomaterial-Induced Complement Activation Studies School of Natural Sciences Degree project work Preparation and Evaluation of Immunoglobulin Free Sera for Biomaterial-Induced Complement Activation Studies Nadia Vickius Subject: Biomedical Science Level: Advanced level Nr: 2010:BV2 Preparation and Evaluation of Immunoglobulin Free Sera for Biomaterial-Induced Complement Activation Studies Nadia Vickius Degree Project Work, Biomedicine 30 ECTS Master of Science Supervisor: Prof. Kristina Nilsson Ekdahl School of Natural Sciences Linnaeus University SE-391 82 KALMAR SWEDEN Examiner: Prof. Bengt Persson School of Natural Sciences Linnaeus University SE-391 82 KALMAR SWEDEN The Degree Project Work is included in the Study programme Biomedical Chemistry 240 ECTS Abstract As the need for and usage of biomaterials in medicine constantly increase, so do the requirements for increased biocompatibility and hemocompatibility. Initially in blood- biomaterial interactions, the surface of an implanted biomaterial is enclosed with adsorbed host proteins and the composition of the adsorbed protein layer depends mainly on the physical-chemical properties of the biomaterial. It is known that the adsorption of proteins on the biomaterial surface may be followed by conformational changes of the adsorbed proteins and subsequent activation of the complement system. For example, binding of complement component C1q to IgG and IgM associated with biomaterial surfaces mediates complement classical pathway activation. The aim of this degree project work was to prepare and evaluate IgG and IgM free sera with functional complement activity for complement activation studies. Further complement studies necessitated IgG and IgM free sera, since two novel polymers with different compositions needed evaluation regarding their ability to induce antibody-independent complement classical pathway activation. Initially, immunoglobulin deficient fetal bovine serum was evaluated regarding complement activity, but no detectable complement activation was present. Different methods for depleting human serum of IgG and IgM were instead utilized and evaluated. From the results, it can be concluded that a close to complete IgG-depletion of human serum is achievable with serum maintaining low but functional complement activity. None of the applied methods for IgM-depletion were however successful and necessitate further optimization and evaluation. 2 POPULÄRVETENSKAPLIG SAMMANFATTNING Biomaterial är en viss typ av material som är speciellt ämnade att komma i kontakt med blod eller vävnader inom kroppen. Denna kontakt kan ske i samband med behandling av olika sjukdomstillstånd, då biomaterial i form av slangar, katetrar, dialysmembran, proteser osv. används i försök att mildra symptom eller bota sjukdomen i fråga. Det första som sker när ett biomaterial kommer i kontakt med blod är att det täcks med kroppens egna blodproteiner. Sammansättningen på proteinlagret avgörs av biomaterialets egenskaper och lagret har avgörande betydelse för hur biomaterialet uppfattas av kroppen. Trots att biomaterial är avsedda att hjälpa patienter kan kroppen reagera mot det kroppsfrämmande materialet och ge upphov till inflammationsreaktioner, vilka i värsta fall kan leda till svåra biverkningar. Därför är det ytterst viktigt att det finns kunskap om hur kroppen uppfattar och reagerar på olika biomaterial samt hur biomaterial kan förändras för att ge upphov till så få biverkningar som möjligt. Komplementsystemet utgör en del av det medfödda immunförsvaret och består av flera komponenter som cirkulerar i blodet. Huvudfunktionen är att skydda kroppen från allt kroppsfrämmande, exempelvis bakterier, virus och material, och förändrade egna celler genom neutralisering samt upphovet av ett inflammatoriskt svar. Ofrivillig aktivering av komplementsystemet kan ske när ett biomaterial kommer i kontakt med blod och detta är ett signifikant problem som måste bemästras genom att producera material som är mer förenliga med kroppen. Genom att förändra komponentsammansättningen hos ett biomaterial kan kroppsförenligheten öka och det är ytterst viktigt att nya biomaterial testas för hur de aktiverar komplementsystemet. Syftet med detta examensarbete var att ta fram och utvärdera antikroppsfria serum, där komplementsystemet var intakt med bibehållen aktivitet. Detta var nödvändigt för att senare kunna genomföra komplementstudier, där fokus låg på huruvida två olika nya biomaterial med olika komponentsammansättningar orsakade aktiveringen av komplementets klassiska aktiveringsväg utan närvaro av antikropparna IgG samt IgM. Initialt utvärderades serum från ofödda kalvar, naturligt fritt från antikroppar, men det visade sig att komplementsystemets aktivitet var otillräcklig. Senare gjordes försök att avlägsna IgG samt IgM från humant serum och resultaten visar att IgG kan avlägsnas med fungerande komplementaktivitet kvarvarande i serumet, medan använda metoder för att avlägsna IgM inte var framgångsrika. 3 TABLE OF CONTENTS TABLE OF CONTENTS ABBREVIATIONS ______________________________________________________ 6 INTRODUCTION _______________________________________________________ 8 Biomaterials _______________________________________________________________ 8 Blood-biomaterial interactions _____________________________________________ 8 Polymers utilized in the degree project work __________________________________ 9 The complement system ____________________________________________________ 10 The classical pathway ___________________________________________________ 11 The lectin pathway______________________________________________________ 12 The alternative pathway _________________________________________________ 12 The terminal pathway ___________________________________________________ 13 Biomaterial-induced complement activation__________________________________ 13 Complement component C1q______________________________________________ 16 The bovine complement system ____________________________________________ 17 Immunoglobulins__________________________________________________________ 17 General description_____________________________________________________ 17 Immunoglobulins and complement activation_________________________________ 19 Protein G _____________________________________________________________ 19 Blood____________________________________________________________________ 19 AIM ____________________________________________________________________ 20 MATERIALS AND METHODS ________________________________________ 21 Fetal bovine serum ________________________________________________________ 21 Detection of bovine Igs __________________________________________________ 21 Biotinylation of anti-human-C3c___________________________________________ 22 Detection of complement activation by measuring C3c _________________________ 22 Biomaterial-induced complement activation study_____________________________ 23 Detection of complement activation by measuring C3a with sandwich-ELISA _______ 23 Detection of complement activation by measuring sC5b-C9 with sandwich-ELISA ___ 24 Human serum ____________________________________________________________ 24 IgG-depletion using a HiTrapTM Protein G column ____________________________ 24 Detection of IgG _______________________________________________________ 25 IgM-depletion using a HiTrapTM IgM column ________________________________ 25 Coupling of anti-human-IgM to CNBr-activated Sepharose™ 4B_________________ 26 IgM-depletion using anti-human-IgM coupled to CNBr-activated Sepharose™ 4B ___ 26 Biomaterial-induced complement activation study_____________________________ 27 Detection of complement activation by measuring C3a with sandwich-ELISA _______ 28 Statistics _____________________________________________________________ 28 RESULTS ______________________________________________________________ 29 Fetal bovine serum ________________________________________________________ 29 Detection of bovine Igs __________________________________________________ 29 Detection of complement activation by measuring activation markers C3c, C3a and C5b- C9 __________________________________________________________________ 29 Human serum ____________________________________________________________ 30 IgG-depletion using a HiTrapTM Protein G column ____________________________ 30 Detection of IgG _______________________________________________________ 30 4 IgM-depletion using a HiTrapTM IgM column and anti-human-IgM coupled to CNBr- activated Sepharose™ 4B ________________________________________________ 31 Biomaterial-induced complement activation study_____________________________ 31 DISCUSSION __________________________________________________________ 33 ACKNOWLEDGEMENT_______________________________________________ 36 REFERENCES _________________________________________________________ 37 5 ABBREVIATIONS APW Alternative pathway of complement activation CDRs Complementarity-determining regions CPW Classical pathway of complement activation CRP C-reactive protein DAB 3,3-diaminobenzidine DAP N,N-diacryloylpiperazine DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraacetic acid EGDMA Ethylene glycol dimethacrylate ELISA Enzyme-linked immunosorbent assay FBS Fetal bovine serum Fc region Fragment crystallizable region FITC Fluorescein isothiocyanate HEMA 2-hydroxyethyl methacrylate HIV Human immunodeficiency virus HRP Horseradish peroxidase IgA Immunoglobulin A IgD Immunoglobulin D IgE Immunoglobulin E IgG Immunoglobulin G IgM Immunoglobulin M Igs Immunoglobulins LPS Lipopolysaccharides LPW Lectin pathway of complement activation
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