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 MAA Methacrylic acid MAC Membrane attack complex MASP1-3 MBL-associated serine protease 1-3 MASPs MBL-associated serine proteases MBL Mannose-binding lectin MS Multiple sclerosis NHS Normal human serum OPD O-phenylendiamine dihydrochloride RA Rheumatoid arthritis SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis SIRS Systemic inflammatory reaction syndrome sTCC Soluble terminal complement complex TCC Terminal complement complex TMB 3,3,5,5-tetramethylbenzidine WB Western blot

6

”The truth is, the science of Nature has been already too long made only a work of the brain and the fancy: It is now high time that it should return to the plainness and soundness of observations on material and obvious things [1]”

Robert Hooke (1665)

Robert Hooke (1665)

7 INTRODUCTION

Biomaterials

By one definition, a biomaterial is “a material that is intended to come in contact with blood or other tissues within the body” [2]. The increasing need for biomaterials in artificial aids and substitutes in modern medical therapeutics has provided a broad array of implantable and extracorporeal blood contact devices [1]. Commonly applied devices are stents, artificial organs, biosensors, catheters, heart valves [3], hemodialysers, prostheses, vascular grafts, oxygenators and miniature pumps [4], and it can be mentioned that more than 25 millions of patients in USA have some kind of artificial implant. Many more will however come in contact with extracorporeal and temporal biomaterial devices in their medicalization [2]. Materials commonly used as biomaterials in medical implants and extracorporeal devices are metals, ceramics, synthetic polymers [5], composites and glass [2].

As the need for and usage of biomaterials in medicine constantly increase, so do the requirements for increased biocompatibility and hemocompatibility (i.e. blood compatibility) [4]. The definition of biocompatibility are “the acceptance (or rejection) of an artificial material by the surrounding tissues and by the body as a whole” [6] and this implies that the biomaterial must not give rise to incompatibility reactions in the neighbouring surroundings nor systemic reactions which affect the entire host body. A biomaterial is thus required to function together with the local microenvironment supporting cell-biomaterial interactions [3], be non-inflammatic, be non-toxic as well as showing satisfactory tissue compatibility and hemocompatibility [6].

Blood-biomaterial interactions Initially in blood-biomaterial interactions (i.e. seconds to minutes after blood contact) the surface of an implanted biomaterial is enclosed with adsorbed host proteins. Proteins usually adsorbed to highest extent on the surfaces of polymeric biomaterials are albumin, fibrinogen, immunoglobulin G (IgG) and complement proteins [1]. The layer of spontaneously adsorbed proteins forms a common boundary between the blood/tissue and the biomaterial, and the composition of the adsorbed protein layer depends mainly on the physical-chemical properties of the biomaterial. The various compositions of adsorbed

8 proteins have shown to influence the immune system differently and it is suggested that the adsorbed protein layer is of major importance for the biocompatibility of biomaterials. Thus controlling the protein adsorption pattern by changing the composition of the biomaterial is one approach to enhance the biocompatibility and obtain a more host functional material [6]. In this degree project work, polymers of different monomers and crosslinkers were utilized to evaluate their complement activating properties (see Table I).

Polymers utilized in the degree project work Earlier work performed within the research group has led to the characterization of different novel polymers regarding their hemocompatibility. Among other things, complement activation potency, protein adsorption patterns and quantities of the adsorbed proteins have been evaluated. Two polymers (P1 and P2, see Figure 1) required further evaluation regarding their ability to induce antibody-independent complement classical pathway activation, as it has been shown that these polymers adsorb high amounts of complement component C1q when incubated in plasma (unpublished data). C1q was enriched on the surfaces of both polymers, but especially and to a higher extent on the surface of polymer P1. The polymers also showed low (P2) to intermediate (P1) adsorption of IgG.

Both polymers are in the size range of 25-63 µm, but they differ in composition and predicted properties (see Table I). Polymer P1 is predicted to be hydrophilic and negatively charged, while polymer P2 is predicted to be hydrophobic [2].

A B

20x 20x

Figure 1. Fluorescein isothiocyanate (FITC) labeled polymer P1 (A) and polymer P2 (B) visualized by fluorescence microscope in 20x magnification. (Image source: Kindly provided by Anna E. Engberg and Per Nilsson)

9 Table I. Composition and earlier results regarding the properties of utilized polymers P1 and P2 [2].

Polymer Monomer* Crosslinker* Functional Complement Adsorbed C1q*** Adsorbed IgG*** groups activation**

P1 MAA DAP -COOH +++ +++ +++ (JR0021)

P2 HEMA EGDMA -OH ++ +++ + (JR0062)

Methacrylic acid (MAA); N,N-diacryloylpiperazine (DAP); 2-hydroxyethyl methacrylate (HEMA); Ethylene glycol dimethacrylate (EGDMA).

*Crosslinker to monomer ratio 80:20. ** Activation levels indicated as follows: +++ > 150% increase in C3a generation compared to control; ++ 50- 150% increase in C3a generation compared to control. ***Adsorbation levels indicated as follows: +++ > 5-fold increased absorbance compared to polystyrene; ++ 2- 5-fold increased absorbance compared to polystyrene; + no increased absorbance compared to polystyrene.

The complement system

The immune system is responsible for host protection by identifying and neutralizing material perceived as being foreign or non-self, for example invading pathogenic microorganisms or altered host cells. The immune system can be divided into the innate immune system and the acquired immune system, where the innate immune system plays a crucial role as first-line defence against invading pathogens since prior exposure to the foreign material is not required. The complement system is mainly considered to be a part of the innate immune system [7] but also functions as an effector of aquired immunity [8].

Approximately 30 soluble and cell-bound proteins are associated with the complement system (for selected complement proteins important for this project, see Table II) [4] and about 5% of the total protein concentration in blood plasma is constituted by the soluble complement components [9]. Included are serine proteases of the chymotrypsin family, proteolytic enzymes containing the amino acid serine in their active site with the ability to activate proteins, including other enzymes, by enzymatic cleavage [10]. These serine proteases thus activate inactive proteases (i.e. zymogens) downstream the complement cascade and transfer the activating ability further down the continuing cascade [8].

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The main function of the complement system is to protect the host from invading pathogens (e.g. bacteria, viruses, fungi and parasites) [11] and to neutralize apoptotic and necrotic host cells. Neutralization of pathogens and altered host cells is achieved by opsonization with subsequent phagocytosis, lysis of foreign cells by the integration of the membrane attack complex (MAC) and generation of an inflammatory response after prosperous activation of one or several of the three complement pathways - the classical pathway, the lectin pathway and the alternative pathway (see Figure 2) [12].

Soluble and membrane-bound complement regulators cooperate to protect the host from damage at multiple levels in the complement cascade. Uncontrolled complement activation beyond normal limits can cause extensive damage to host cells and contribute to several inflammatory diseases and disorders such as rheumatoid arthritis (RA), multiple sclerosis (MS), Alzheimer’s disease, hyperacute graft rejection and systemic inflammatory reaction syndrome (SIRS) [13]. The tissue damaging effects of uncontrolled complement activation arise from the mediated immunological response induced by the anaphylatoxins C3a, C4a and C5a as well as host cell lysis by the membrane attack complex [4].

The classical pathway As the initial classical pathway component C1q associates with a Ca2+-dependent tetramer of the glycoproteins C1r and C1s (two of each), the C1 complex of the classical pathway is generated [14]. Binding of the C1 complex to different activating structures (details will be discussed further on) mediates classical pathway activation through the self- activation of C1r and subsequent cleavage of C1s. Once activated, serine protease C1s cleaves complement component C4 into the fragments C4a and C4b. The cleavage exposes a metastable thioester binding site on C4b, which allows C4b to attach to its target surface. The anaphylatoxin C4a is instead released into the fluid phase. Complement component C2 then binds to the surface-attached C4b and is also cleaved by C1s into fragments. The smaller fragment C2b is released, while the surface-attached complex of C4bC2a forms the classical C3 convertase. The C3 convertase cleaves the central complement component C3 into C3a and C3b [15]. C3a is an anaphylatoxin that stimulates inflammation while C3b, in addition to forming the classical C5 convertase in association with C4bC2a, also functions as an opsonin. By binding to target surfaces with its exposed reactive thioester, C3b promotes phagocytosis by inflammatory cells (e.g. macrophages and neutrophils) [8]. The formation of the classical

11 C5 convertase (C4bC2aC3b) leads to the enzymatic cleavage of complement component C5 and generation of the very potent anaphylatoxin C5a together with the first component of terminal pathway, C5b. All generated anaphylatoxins (i.e. C3a, C4a and C5a) can induce an inflammatory response and mediate chemotaxis, vasodilatation, cell activation and cell adhesion through binding to specific receptors on neutrophils, monocytes, macrophages, mast cells and smooth muscles cells [15].

The lectin pathway The lectin pathway is identical to the classical pathway with the exception of the initial activation step. Through the binding of C1q-like mannose-binding lectin (MBL) or ficolins to specific carbohydrates, such as N-acetylglucosamine [9] or lipopolysaccharides (LPS) [12] present on the surface of pathogens, MBL-associated serine proteases called MASPs becomes self-activated in conformity with C1r of the classical pathway. There are three kinds of MASPs associated with MBL and ficolins - MASP1, MASP2 and MASP3. Out of these only MASP2 is known to possess the ability to activate the lectin pathway through cleavage of C4 and C2 [9], even though MASP1 also is able to cleave C2. MASP2 thereby acts in a similar manner as C1s. The role of MASP1 and MASP3 is not yet established, but it is proposed that MASP1 contributes to the activation of MASP2 and has the ability to activate the alternative pathway through direct cleavage of C3 [16].

The alternative pathway Activation of the alternative pathway is mediated in an antibody-independent manner either through the spontaneous hydrolysis of C3 into C3(H2O) or by the attachment of precedently generated C3b to a foreign surface (e.g. pathogen or biomaterial) through the 2+ exposed reactive thioester. C3(H2O) binds to factor B in a Mg -dependent manner, activates the later and the complex C3(H2O)Bb is formed. Additional C3 molecules are cleaved by the complex, yielding fragments of C3a and C3b. The C3b fragment associates with Bb on a present target surface and the alternative C3 convertase is consequently assembled. As precedently generated C3b attaches to a surface through binding of its thioester to hydroxyl or amino groups on the surface, the binding of factor B and factor D to the C3b fragment is promoted. Factor D subsequently cleaves factor B into Ba and Bb and the formation of the alternative C3 convertase C3bBb is achieved. Properdin can help to stabilize the alternative C3 convertase and more C3b is thus generated, the alternative pathway thereby acts as an amplification loop. The alternative C3 convertase can bind additional C3b, which will lead to

12 the formation of the alternative C5 convertase (C3bBbC3b). The alternative C5 convertase, in conformity with the classical C5 convertase, cleaves C5 into C5a and C5b fragments [15].

The terminal pathway Generated C5b from the three complement pathways initiates the assembly of the terminal complement complex (TCC, sC5b-C9) directed against the cell membranes of target cells and pathogens [8]. In absence of a cell membrane, the sC5b-C9 remains in the fluid phase but when attached to a membrane, the complex is instead called the membrane attack complex (MAC) [11]. C5b complexed with complement component C6 can interact with lipid membranes and the additional binding of complement components C7 and C8 to the complex leads to further penetration of the target cell membrane [2]. As multiple complement components C9 bind to the C5b-C8 complex, a cylinder-like pore with a diameter of about 100 Å (1 Ångström = 10-10 m) is formed that causes cell damage through lysis by osmotic disruption [17-19].

Biomaterial-induced complement activation It is known that the adsorption of proteins on the surface of a biomaterial may be followed by conformational change of the protein and subsequent activation of the complement system. Two proteins known to undergo complement activating conformational changes are IgG [6] and complement component C3 [17]. The complement system can thus either directly be activated by the thioester-binding of C3b and C4b to free hydroxyl (-OH) and/or amino (-NH3) groups on the surface of a biomaterial or by the classical pathway through the binding of C1 complex to adsorbed immunoglobulin G (IgG) on the biomaterial surface [4].

13

Figure 2. A schematic overview of the different complement activation pathways. After activation, the classical and lectin pathways converge by formation of the classical C3 convertase, which associates with generated C3b and forms the classical C5 convertase. The alternative C3 and C5 convertase are generated through the assembly of fragments differing from the classical convertases, but the final objective of the three complement pathways is the initiation of the terminal pathway and assembly of the membrane attack complex (MAC). The terminal pathway is initiated by the cleavage of C5 into C5b and the anaphylatoxin C5a. C5b, C6-C8 and multiple molecules of C9 associate in the formation of the pore-like membrane attack complex that, when incorporated into cell membranes of target cells and pathogens, leads to the damaging of the cells and pathogens by lysis. (Image source: Adapted from Favoreel, H.W., et al., Virus complement evasion strategies.)

14 Table II. Selected proteins of the complement system [2, 7].

Protein Molecular weight Blood concentration Function (kDa) (µg/mL)

C1 750 100 The C1 complex initiates the classical pathway

C1q 410 75-150 Binds to antigen-antibody complexes, certain pathogens and structures

C1r 85 50 Activates C1s by proteolytic cleavage

C1s 85 50 The active serine protease cleaves C4 and C2

C2 102 20 C2a cleaves C3 and C5 as the active enzyme of the classical/ lectin pathway C3 and C5 convertases

C3 185 1000-2000 C3b functions as a component of the alternative pathway C3 and C5 convertases, classical/ lectin pathway C5 convertase as well as an opsonin

C3a is an anaphylatoxin that stim- ulates inflammation

C4 210 300-600 C4b functions as a component of the classical/ lectin pathway C3 and C5 convertases

C4a is an anaphylatoxin that stimulates inflammation

C5 190 80 C5b initiates formation of the membrane attack complex – MAC

C5a is an anaphylatoxin that stim- ulates inflammation

C6 110 45 Component of the MAC

C7 100 90 Component of the MAC

C8 155 60 Component of the MAC

C9 79 60 Component of the MAC

Factor B 93 200 Bb is the active enzyme of the alternative pathway C3 and C5 convertases

Factor D 25 1-2 Cleaves factor B associated with C3b

Properdin 56 per subunit 25 Stabilizes the alternative pathway C3 (up to four subunits) convertase

15 Complement component C1q Complement component C1q has a molecular weight of approximately 410 kDa [2] and is made up of 3 kinds of polypeptide chains (6 A-chains, 6 B-chains and 6 C-chains) with N- terminal collagen-like regions and C-terminal globular regions [14]. C1q, belonging to the family of collectins [19], is often referred to as a “bouquet of tulips” due to the six globular recognition domains linked together by collagen-like triple helical fibers forming a stalk (see Figure 3) [20]. C1q has several receptors that induce various immune effector functions, such as clearance of apoptotic cells, modulation of cellular cytotoxicity, increased surface expression of adhesion molecules and enhancement of phagocytosis as well as immunoglobulin secretion [14].

C1r C1s

C1q

Figure 3. The classical pathway initial C1 complex is comprised of three subunits - C1q, C1r and C1s. C1q is often referred to as a “bouquet of tulips” due to the globular heads and the collagen-like “stalk”. (Image source: Adapted from Favoreel, H.W., et al., Virus complement evasion strategies.)

Classical pathway complement activation is initiated by the binding of C1q to antigen- antibody complexes (IgG and IgM), surfaces of certain pathogens such as human immunodeficiency virus (HIV) [19], apoptotic cells [2], or structures such as lipopolysaccharides (LPS) and C-reactive protein (CRP) [21]. If more than one globular domain of C1q bind to a target, the C1 complex undergoes a conformational change and the associated zymogen C1r becomes self-activated. [19]. Since C1q binds only weakly to single immunoglobulin constant regions (i.e. Fc regions), multiple binding to Fc regions is required for complement activation. Multiple closely spaced Fc regions are found in antigen-antibody complexes enclosed with IgG and IgM and these are efficient activators of complement. Exactly where on the globular domains of C1q that IgG and IgM bind is not yet known, but it is proposed that C1q binds to the Cγ2 domain of IgG and Cμ3 domains of IgM present in the Fc regions [14].

16

The bovine complement system There are apparent differences between adult and fetal bovine complement systems regarding the quantity of complement components. The level of bovine complement components C1, C4, C5, C7 and C9 in adult bovine serum has shown to be high in comparison to complement components C2 and C8 in the same serum, as only low levels of these components have been detected. Interestingly, fetal bovine serum (FBS) only contains approximately 1-3% of C1 and C6 levels and 5-50% of C2, C4, C5, C7, C8 and C9 levels in comparison to adult bovine serum [22]. The level of C3 in FBS has shown to be extremely low, if detectable at all, but functional complement activity can still be present and must be evaluated before the possibility is dismissed [23].

Immunoglobulins

As mentioned earlier, the immune system can be divided into two parts - namely the innate and the aquired immune system. The aquired immune system, in contrast to the non- specific innate immune system, requires prior exposure to a foreign material to be able to combat it. Aquired immunity can be further divided into cell-mediated and humoral immunity, where the humoral immunity is mediated primarily by soluble proteins known as immunoglobulins.

General description Immunoglobulins (Igs) are a group of glycoproteins, also known as antibodies, made up of four polypeptide chains, two identical heavy chains (55-70 kDa) and two identical light chains (~24 kDa). The light chains are joined covalently to the heavy chains by a disulfide bond and the heavy chains are joined together by two disulfide bonds [7]. The different isotypes of heavy chains (α, δ, ε, γ and μ) determine the antibody class (IgA, IgD, IgE, IgG and IgM respectively) [18] and with the exception of heavy chains of IgM and IgE, all immunoglobulin heavy chains contain four constant regions and a hinge region (i.e. a proline- rich flexible region). The heavy chains of IgM and IgE lack a hinge region and thus only contain five constant regions. There are two types of light chains, κ and λ, and about 60% of human light chains are κ.

17 At their amino terminal end, both heavy chains and light chains comprise highly variable regions named fragment antigen-binding (Fab) regions. Within these variable regions, hypervariable complementarity-determining regions (CDRs) reside which compose the antigen-binding site and recognize specific antigen epitopes (see Figure 4). The constant regions of the carboxyl terminal ends of heavy and light chains do not contribute to antigen binding and recognition, but are responsible for immunobiological effector functions as these fragment crystallizable (Fc) regions interact with immunoglobulin receptors called Fc receptors found on many types of immune cells [24].

Figure 4. Schematic figure of the antibody antigen-binding site recognizing the unique region, the epitope, of a specific antigen. (Image source: Free image from Wikipedia, http://en.wikipedia.org/wiki/File:Antibody.svg)

Immunoglobulins are found in two forms, either membrane-bound on the surfaces of B cells or soluble secreted by plasma cell. Soluble immunoglobulins reside in the fluid fraction of the blood, the blood plasma, and in case of blood coagulation, the immunoglobulins are present in the residual blood serum [7]. About 80% of the total immunoglobulin content in blood serum is made up of IgG (see Table III) [24].

Table III. Properties of human IgG and IgM classes and subclasses [7, 18, 24].

Class/subclass Heavy chain MW Forms Serum concentration Classical pathway (kDa) (mg/mL) complement activation*

IgG1 γ1 146 Monomer 5-10 +

IgG2 γ2 146 Monomer 1.8-3.5 +/-

IgG3 γ3 170 Monomer 0.6-1.2 ++

IgG4 γ4 146 Monomer 0.3-0.6 -

IgM µ 900 Pentamer 0.5-2.0 ++

* Activation potency levels indicated as follows: ++ high; + moderate; +/- minimal; - none.

18 Immunoglobulins and complement activation As a part of the humoral immunity, soluble immunoglobulins circulate in the blood and functions as effectors since binding of an antigen triggers several mechanisms that causes elimination of the antigen [7]. Different immunoglobulin classes and subclasses have different functions in the immune system due to the differences in heavy chain constant regions. Most

IgG subclasses (not IgG4) and IgM can activate the complement system, while all other immunoglobulin classes lack this ability [10]. Activation of the classical complement pathway occurs when C1q binds to antibody-antigen complexes enclosed with these IgG and IgM [19]. IgG is not as efficient in activating the complement system as IgM, since the classical pathway activation requires binding of at least two Fc regions simultaneously and IgM consists of multiple Fc regions due to the pentameric structure [24].

Protein G Protein G is an immunoglobulin-binding protein derived from the cell walls of certain 8 - Streptococcus with the capacity of binding the Fc region of IgG with high affinity (Ka~10 M 1) [24]. Protein G binds all human IgG subclasses and in affinity chromatography, protein G is widely used for purification of IgG by attachment of the recombinant proteins to inert supports like sepharose or agarose [25].

Blood

The blood cells (i.e. erythrocytes, leukocytes and thrombocytes) comprise 40-45% of total human blood volume of 3 to 6 liters and the remaining liquid fraction is called blood plasma. Blood plasma consists of 90% water and the remaining percentage is made up of soluble small molecules and macromolecules, including proteins, glucose, fatty acids and ions. The concentration of proteins in human plasma is about 60-80 g/L [2], out of which approximately 90% is comprised of the ten most abundant proteins [26]. Important plasma proteins among others are albumin, transport proteins, proteins of the complement and coagulation systems and immunoglobulins.

If the blood is allowed to coagulate and the clot is removed by centrifugation, blood serum is obtained. The difference between blood serum and plasma is thus that serum contains all proteins as the plasma, except for the coagulation proteins [2]. For functional

19 studies of the complement system in vitro, serum is preferable to plasma since it is a simplified system, however it should be noted that plasma provides more relevant physiological in vivo indications [27] due to interactions between the complement system, the coagulation system and the different blood cells [4].

AIM

The aim of this degree project work was to prepare and evaluate IgG and IgM free sera with functional complement activity for complement studies regarding antibody-independent and biomaterial-induced classical pathway activation.

20 MATERIALS AND METHODS

Figure 5. Flow chart summarizing approaches and methods evaluated for antibody-independent and biomaterial-induced classical pathway activation studies.

Fetal bovine serum

Detection of bovine Igs FBS is naturally deficient of immunoglobulins. Presence of immunoglobulins, if any, is solely a result of infection, as the immunoglobulins are not transferred across the placenta [28]. To verify the deficiency, the detection of bovine Igs was performed in fetal, newborn and adult bovine serum (newborn and adult bovine serum served as positive control samples) from GIBCO®. The sera were diluted in two-fold dilutions (1:1-1:16,384) with PBS (phosphate buffered saline containing 10 mM phosphate buffer and 0.15 M NaCl). Of each serum dilution, 100 µl were transferred to an unwashed polystyrene microtiter ELISA plate (Nunc Maxisorp Immunoplates, Copenhagen, Denmark) and PBS served as blank. The plate was incubated shaking for 60 minutes at room temperature (RT). After washing three times with washing buffer (PBS containing 0.05% Tween 20), bound bovine immunoglobulins were

21 detected using 100 µl of a polyclonal rabbit anti-bovine-immunoglobulins-HRP (Dako, Glostrup, Denmark) diluted 1:500. After incubation for 60 minutes at RT (shaking) and washing, staining followed by 100 µl of o-phenylendiamine dihydrochloride (OPD, 1 mg/mL, Sigma, St. Louis, MO, USA) in citrate buffer (0.35 mM containing 70 mM Na-phosphate, pH

5.0) with H2O2 (1 µl/mL buffer). The reaction was stopped with 100 µl 1 M sulfuric acid

(H2SO4) and the absorbance was measured with an ELISA reader (SpectraCount, Packard, Canberra Company, Australia) at 490 nm.

Biotinylation of anti-human-C3c

To remove preservatives (Na-azide, NaN3) from anti-human-C3c (Dako, Glostrup, Denmark) before biotinylation, a protein desalting spin column (Thermo Scientific, Rockford, IL, USA) was used according to manufacturer’s instructions. The column was initially equilibrated three times with coupling buffer containing 0.1 M NaHCO3 (pH 8.3) and 0.5 M NaCl. After washing, 100 µl of anti-human-C3c was added to the column and the column was centrifuged at 1500g for 2 minutes. To the antibody, 900 µl of coupling buffer and 100 µl of biotin amidohexanoic acid N-hydroxysuccinimide ester (Sigma, St. Louis, MO, USA) dissolved in dimethyl sulfoxide (8 mg/mL, DMSO) was added and the biotinylation reaction was allowed to proceed for 30 minutes in RT. The mixture was dialyzed at 4ºC over night against 250 mL of PBS to remove excess biotin reagent. NaN3 was added to a final concentration of 0.05%.

Detection of complement activation by measuring C3c As FBS has shown to comprise low levels of complement components [22], the complement activity was evaluated by detection of the C3 fragment C3c [29]. Several attempts to detect C3c and achieve complement activation in FBS were done by different experimental methods and set-ups. Dilution of samples was performed with PBS and biotinylated anti-human-C3c was diluted with washing buffer (PBS containing 0.05% Tween 20). Parameters varied in the different experimental set-ups were dilution of biotinylated anti- human-C3c, dilution of fetal, newborn and adult bovine serum and addition of bovine IgG to physiological concentration (~13.5 mg/mL) for enhancement of complement activation.

The same method procedure was used for all experimental set-ups as follows. After serum preparations, samples of 100 µl were incubated in an unwashed polystyrene microtiter ELISA plate for 60 minutes at 37ºC. The plate was then washed three times with washing

22 buffer. C3c depositions in the wells were detected with 100 µl of biotinylated anti-human- C3c, incubation 60 minutes shaking at RT, followed by washing. The wells were incubated for 15 minutes with 100 µl HRP-conjugated streptavidin (Amersham, Little Chalfort, UK) diluted 1:500 with working buffer containing 1% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) and 10 mM EDTA (ethylenediaminetetraacetic acid). After washing and staining with 100 µl 3,3,5,5-tetramethylbenzidine (TMB, 6 mg/mL, Serva, Heidelberg,

Germany) in Na-acetate buffer (0.11 M, pH 5.5) containing H2O2 (1µl/mL buffer), the absorbance was measured with an ELISA reader at 450 nm.

Biomaterial-induced complement activation study Polymers P1 and P2 were incubated with undiluted FBS or normal human serum (NHS, 2.5 mg/mL) in heparinized 2 mL Eppendorf tubes for 60 minutes at 37ºC with continuous rotation (20 rpm). As controls, serum samples incubated in the same manner but with no added polymers were used. After incubation, the polymer particles were centrifuged down and the supernatants was transferred to Eppendorf tubes containing EDTA (complexes metal ions necessary for the complement system and thereby inhibits further activation) to a final concentration of 10 mM and put on ice. To the unwashed polymer particles, 400 µl 2% SDS in PBS were added to elute adsorbed proteins and the tubes were incubated for 20 minutes at 37ºC with rotation. After centrifugation the eluates were transferred to Eppendorf tubes containing EDTA (to a final concentration of 10 mM). All samples were stored at –80ºC until sandwich-ELISA measurements of C3a and sTCC.

Detection of complement activation by measuring C3a with sandwich-ELISA An estimation of complement activation can be obtained by measuring the generation of anaphylatoxin C3a [11]. A polystyrene microtiter ELISA plate was initially coated at 4ºC over night with a monoclonal anti-human-C3a 4SD17.3 (Clinical Immunology, Uppsala, Sweden). The plate was then incubated shaking with 200 µl working buffer for 30 minutes at RT to block remaining protein-binding sites. Supernatants and eluates from the polymer activation experiment were individually diluted in working buffer and 50 µl of diluted samples, zymosan-activated serum (served as standard in two-fold dilution, ng/mL) and C3a control diluted 1:1,000 was put on the plate. After incubation with shaking for 60 minutes in room temperature shaking, the plate was washed with washing buffer and 50 µl of the biotinylated detection antibody anti-human-C3a (Clinical Immunology, Uppsala, Sweden) diluted 1:300 in working buffer was added to the plate. The plate was once again incubated for 60 minutes in

23 RT shaking and subsequently washed three times. The wells were incubated for 15 minutes with 50 µl streptavidin-HRP diluted 1:500 with working buffer. After washing and staining with TMB (50 µl) as described earlier, the absorbance was measured with an ELISA reader at 450 nm. Evaluation of obtained results was performed using the software DeltaSoft (BioMetallics Inc., Princeton, NJ, USA).

Detection of complement activation by measuring sC5b-C9 with sandwich-ELISA Detection of soluble C5b-C9 (TCC) is an important tool to measure C5 activation and thus complement activation [11]. A polystyrene microtiter ELISA plate was coated at 4ºC over night with monoclonal anti-human-C9 aE11 (Diatec Monoclonals AS, Oslo, Norway) as the capturing antibody. After blocking of remaining protein-binding sites with 300 µl working buffer, 100 µl of diluted serum and eluates, zymosan-activated serum (served as standard in two-fold dilution, AU/mL) and sC5b-C9 control diluted 1:25 in working buffer were added to the plate and incubated for 60 minutes at RT shaking. Subsequent incubation for 60 minutes at RT shaking with 100 µl polyclonal rabbit anti-human-C5 (Dako, Glostrup, Denmark) followed by anti-rabbit-immunoglobulin-HRP (Dako, Glostrup, Denmark), both diluted 1:500 with working buffer, resulted in detection of sC5b-C9 by TMB staining (100 µl) as described earlier and subsequent absorbance measurement at 450 nm. Evaluation of obtained results was performed using the software DeltaSoft (BioMetallics Inc., Princeton, NJ, USA).

Human serum

IgG-depletion using a HiTrapTM Protein G column Since no established protocol for IgG-depletion of NHS without interference of complement activity existed, a new method had to be developed. Initially the IgG-depletion was performed in absence of NaCl (sodium chloride) but since this also lead to the depletion of C1q, the protocol below was established. C1q has affinity for Sepharose and the presence of NaCl is crucial to avoid C1q binding to Sepharose.

The HiTrapTM Protein G HP 1 mL column (GE Healthcare, Uppsala, Sweden) with a binding capacity of more than 25 mg human IgG was initially equilibrated with 10 column volumes of binding buffer (20 mM Na-phosphate buffer containing 10 mM EDTA and 0.5 M NaCl, pH 7.0) at 1 mL/minute. The NHS, also containing 10 mM EDTA and 0.5 M NaCl,

24 was applied onto the column (3 mL) and the first mL of throughput was discarded. The following 1.5 mL of NHS was the desired IgG-depleted fraction and was kept on ice until dialyzation against 2 L PBS over night and storage at –80ºC. After washing with 5 mL of binding buffer, bound IgG was eluted using 10 column volumes of elution buffer (0.1 M glycine-HCl, pH 2.7). Finally the column was washed with 10 mL of binding buffer and 5 mL of 20% ethanol for storage in at 4ºC. The IgG-depletion was evaluated by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) with separating gel 12% under reduced conditions followed by silver staining according to manufacture’s instructions (Bio- Rad, Hercules, CA, USA). Precision Plus ProteinTM Standards (Bio-Rad, Hercules, CA, USA) served as molecular weight marker.

Detection of IgG Initially a polystyrene microtiter ELISA plate was coated over night at 4ºC with 150 µl anti-human-IgG (Dako, Glostrup, Denmark) diluted 1:200 in PBS. After washing three times with washing buffer, blocking of remaining protein-binding sites using 300 µl working buffer. Two-fold diluted IgG-depleted human serum (1:1-1:8,192) and NHS (1:100-1:819,200) in working buffer was added to the plate of 100 µl and incubated for 1 h in RT. Working buffer served as blank. Bound IgG was detected by incubation for 1 h in RT with 100 µl of a mixture of biotinylated and non-biotinylated anti-human-IgG diluted 1:800 in working buffer. HRP- conjugated streptavidin diluted 1:500 followed washing and was incubated for 15 minutes at RT shaking. After washing, staining followed with 100 µl of OPD as described earlier and the absorbance was measured with an ELISA reader at 490 nm.

IgM-depletion using a HiTrapTM IgM column

Ammonium sulphate ((NH4)2SO4) was, according to manufacturer’s instructions, supposed to be used in the IgM-depletion using a HiTrapTM IgM column. After several trial and errors to achieve IgM-depletion as well as maintaining functional complement activation, the protocol below for IgM-depletion of NHS with best results was established. No regard of C1q-depletion was taken (compensating C1q-addition afterwards if necessary was possible), since NaCl in early experimental set-ups was shown not to possess the ability to promote

IgM-depletion as (NH4)2SO4 did and thus interfered with the depletion.

The HiTrapTM IgM Purification HP 1 mL column (GE Healthcare, Uppsala, Sweden) with a binding capacity of 5 mg IgM was equilibrated with 5 column volumes of each of the

25 following buffers - binding buffer (20 mM Na-phosphate containing 10 mM EDTA and 0.8 M

(NH4)2SO4, pH 7.5), elution buffer (20mM Na-phosphate containing 10 mM EDTA, pH 7.5) and regeneration buffer (20 mM Na- phosphate with 30% isopropanol and 10 mM EDTA, pH 7.5). Following equilibration with 5 mL binding buffer, 3 mL of NHS containing 0.8 M

(NH4)2SO4 and 10 mM EDTA was applied onto the column. The first mL was discarded and the following 2 mL were collected and put on ice until storage at –80ºC. After column washing with 5 mL binding buffer, the bound IgM was eluted by applying 10 column volumes of elution buffer. Regeneration was obtained with 7 mL of regeneration buffer and the column was re-equilibrated with 5 mL of binding buffer. The column was stored in 20% ethanol at 4ºC. The IgM-depletion was evaluated by SDS-PAGE with subsequent WB (western blot) analysis using a biotinylated anti-human-IgM (Dako, Glostrup, Denmark) diluted 1:400 in working buffer and DAB (3,3-diaminobenzidine) staining. Precision Plus ProteinTM Standards served as molecular weight marker.

Coupling of anti-human-IgM to CNBr-activated Sepharose™ 4B Preservatives were removed from 500 µl anti-human-IgM (5.6 mg/mL) by dialyzation against coupling buffer. Of the CNBr-activated Sepharose™ 4B (Pharmacia Biotech, Uppsala, Sweden), 2.0 g were transferred to a 50 mL Falcon tube and activated by 1 mM HCl two times with subsequent centrifugation at 1000 rpm for 5 minutes. The dialyzed antibody was added to 12 mL coupling buffer and UV280 nm was measured with NanoDrop for later control of successful coupling. After washing of the Sepharose with coupling buffer and centrifugation at 1000 rpm for 5 minutes, the antibody-coupling buffer mixture was added to the Sepharose and the ligand was allowed to bind for 30 minutes at RT. Centrifugation at

1000 rpm for 10 minutes was followed by UV280 nm which showed successful coupling and the Falcon tube was filled with blocking buffer (0.2 M glycine, pH 8.0) for 2 h at RT. The anti- human-IgM-coupled Sepharose was washed with washing buffer (0.1 M Na-acetate containing 0.5 M NaCl, pH 4.0) and coupling buffer alternately (three times with each buffer) with centrifugation at 1000 rpm for 10 minutes in between and finally three times with PBS.

The anti-human-IgM-coupled Sepharose was stored in PBS containing 0.05% NaN3.

IgM-depletion using anti-human-IgM coupled to CNBr-activated Sepharose™ 4B In a new attempt to deplete NHS from IgM, a protocol for IgM-depletion using anti- human-IgM to CNBr-activated Sepharose™ 4B was developed and evaluated. As mentioned earlier in connection with the HiTrapTM IgM column, NaCl has potency to interfere with IgM-

26 depletion. This was also the case in early experimental set-ups of this method and therefore no regard of simultaneous C1q-depletion in the protocol was taken as NaCl was excluded.

Washing of 1 mL anti-human-IgM coupled Sepharose with PBS was followed by the application of 2 mL EDTA-treated NHS and subsequent incubation (shaking) for 30 minutes at RT. After centrifugation at 1000 rpm for 5 minutes, the serum was retained on ice until storage at –80ºC. The Sepharose was regenerated with 8 mL of 0.1 M Tris buffer containing 0.5 M NaCl (pH 8.5) and 0.1 M Na-acetate buffer containing 0.5 M NaCl (pH 4.0) alternately three times with centrifugation at 1000 rpm for 5 minutes in between. The anti-human-IgM- coupled Sepharose was finally washed with PBS three times and stored in 0.05% NaN3 in PBS. The IgM-depletion was evaluated by SDS-PAGE with subsequent WB analysis using a biotinylated anti-human-IgM diluted 1:400 in working buffer and DAB staining. Precision Plus ProteinTM Standards served as molecular weight marker.

Biomaterial-induced complement activation study Different serum variations were prepared – IgG-depleted serum containing C1q, IgG- depleted serum containing IgG and IgG-depleted serum containing both C1q and IgG. IgG- depleted C1q (1.18 µg/µl) and IgG (165 µg/µl, Beriglobin, CLS Behring, King of Prussia, PA) were added to the sera in physiological concentrations of 75 µg/mL and 13.5 µg/µl respectively. Finally all sera were diluted 1:8 in VBS++ (veronal buffered saline containing 0.15 mM Ca2+ and 0.5 mM Mg2+, pH 7.4) to eliminate contribution of the alternative pathway to complement activation. NHS, also diluted 1:8, served as comparison control and IgG- depleted serum containing C1q and IgG served as control sample (also incubated with continuous rotation but without added polymers). Start values of IgG-depleted serum (start value 1) as well as IgG-depleted serum containing both C1q and IgG (start value 2) were also obtained by EDTA-treatment of the sera.

The same procedure for biomaterial-induced complement activation as described earlier was used with some exceptions. Polymers P1 and P2 were incubated with 6.7 mg polymer/mL of each serum instead of 2.5 mg polymer/mL as described before and samples of both polymers were also pre-incubated with 100 µl of C1q in 1% HSA in PBS for 30 minutes at 37ºC before addition of IgG-depleted serum diluted 1:8.

27 Detection of complement activation by measuring C3a with sandwich-ELISA The same protocol as earlier described was used for measuring C3a with sandwich- ELISA. For dilution of supernatants and eluates from the biomaterial-induced complement activation study, see Appendix.

Statistics Significant outliers were detected by Grubb’s test (α = 0.05, GraphPad Quickcalcs at http://www.graphpad.com/quickcalcs/, GraphPad Software Inc., La Jolla, CA, USA).

28 RESULTS

Fetal bovine serum

Detection of bovine Igs HRP-conjugated polyclonal anti-bovine-immunoglobulins followed by OPD-staining was used to detected bovine immunoglobulins in FBS (newborn and adult bovine serum served as positive control samples). The result (see Figure 6) showed no detectable immunoglobulins in the screened FBS and this indicates that the serum consequently was deficient of immunoglobulins.

2 1,8 1,6 1,4 Fetal bovine serum 1,2 1 Newborn bovine serum 0,8 0,6 Adult bovine serum 0,4 0,2 Absorbance490 nm 0 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1,024 1:2,048 1:4,096 1:8,192 1:16,384 Serum dilution

Figure 6. Detection of bovine immunoglobulins in fetal, newborn and adult bovine serum at various dilutions using polyclonal anti-bovine-immunoglobulins-HRP followed by OPD-staining. The result showed that the screened FBS contained no immunoglobulins in contrast to newborn and adult bovine serum.

Detection of complement activation by measuring activation markers C3c, C3a and C5b-C9 Several attempts to detect and achieve complement activation in FBS were done by different experimental methods and set-ups. Despite various measurements of complement activation markers (i.e. the generation of C3 fragment C3c, C3a and C5b-C9), no complement activation could be detected in the screened FBS (data not shown).

29 Human serum

IgG-depletion using a HiTrapTM Protein G column The IgG-depletion of NHS was performed by the employment of a HiTrapTM Protein G column with affinity for all human subclasses of IgG. After SDS-PAGE with subsequent silver staining, the resulting gel showed a prosperous IgG-depletion, as IgG heavy chain and light chain bands (at approximately 50 kDa and 25 kDa, respectively) were less intense as compared to NHS (see Figure 7). The stained gel further indicated that the depleted serum comprised approximately the same protein profile as NHS and no noticeable unspecific protein depletion could be seen.

Figure 7. Silver stained gel visualizing normal (A-C) and IgG-depleted human serum (D-F) applied to the gel in three different protein concentrations (from left to right for each serum; 10 µg/mL, 5 µg/mL and 2,5 µg/mL). Prosperous IgG-depletion could be seen as the IgG-depleted serum showed less intense immunoglobulin heavy and light chain bands compared to NHS.

Detection of IgG To determine the extent to which IgG was depleted, the detection of IgG was done by sandwich-ELISA measurements followed by OPD-staining. The result indicated close to complete IgG-depletion as the IgG-depleted serum had an Ig concentration equivalent to NHS diluted 1:102,400 (data not shown). This provided an approximately IgG concentration of 0.13 ng/mL in comparison to the IgG concentration in NHS of approximately 13.5 mg/mL.

30 IgM-depletion using a HiTrapTM IgM column and anti-human-IgM coupled to CNBr- activated Sepharose™ 4B The IgM content of the assumed IgM-depleted sera was evaluated by WB analysis followed by DAB staining. The result clearly showed (see Figure 8) that none of the applied methods successfully depleted NHS of IgM, although the HiTrapTM IgM column showed some IgM-depletion as IgM was detected in the eluate. Additional purifications by anti- human-IgM coupled to CNBr-activated Sepharose did not result in extended IgM-depletion.

Figure 8. WB analysis followed by DAB staining visualizing HiTrapTM IgM column eluate (A), HiTrapTM IgM column throughput serum (B), fractions of serum after one, two and three times of IgM-depletion using anti- human-IgM coupled to CNBr-activated Sepharose™ 4B (C-E) and IgM-containing IgG-depleted serum (F) for comparison. The resulting blot clearly showed unsuccessful IgM-depletion by both applied methods, as IgM still was present in the serum and the IgM content had not decreased significantly compared to the non-IgM-depleted serum (F).

Biomaterial-induced complement activation study Similar complement activation levels in the different sera subjected to polymers P1 and P2 (see Figure 9) were indicated. Potential elevation of complement activation in the IgG- depleted serum after preincubation with C1q could be seen, as well as a possible activation enhancing effect by IgG-addition. P1 appeared to induce activation to a higher extent than P2 in NHS, the sera containing both C1q and IgG as well as in the IgG-depleted serum after preincubation of polymer with C1q. P1 also potentially showed to adsorbed more C3a to the surface regardless of sera in opposite to P2, which appeared to have a higher concentration of

31 C3a in the supernatants than in the eluates. Control sample indicated elevated activation in comparison to start value.

P1

1400 1200 1000 800 Eluate

600 Supernatant

[C3a] (ng/mL) [C3a] 400 200 0 NHS +C1q +IgG +C1q Pre-C1q Start Start Control +IgG value 1 value 2 sample

P2

1200

1000

800 Eluate 600 Supernatant 400

(ng/mL) [C3a]

200

0 NHS +C1q +IgG +C1q Pre-C1q Start Start Control +IgG value 1 value 2 sample

Figure 9. Generation of complement activation marker C3a in various sera subjected to polymer P1 and P2 for 60 minutes at 37ºC. Data are presented as concentration of C3a (ng/mL) in supernatants and adsorbed to the polymers after incubation. Start values of IgG-depleted serum (start value 1) and IgG-depleted serum containing both C1q and IgG (start value 2) as well as control sample are also shown for comparison

32 DISCUSSION

To develop and engineer biocompatible materials and devices, it is necessary to have full knowledge of the different fundamental mechanisms underlying the biological responses to implanted biomaterials and extracorporeal devices. Complement activation after biomaterial-blood contact is a significant problem to overcome in the enhancement of biomaterial hemocompatibility and it is crucial to test novel biomaterials for their potency to activate the complement system. The focus in this degree project work was on preparing and evaluating immunoglobulin-free sera for further antibody-independent and biomaterial- induced complement activation studies.

Immunoglobulin deficient FBS was evaluated regarding complement activity but no complement activation could be detected in the screened serum despite various measurements of complement activation markers. This was not surprising as earlier publications had indicated low levels of complement components in FBS, but the possibility of functional complement activity could not be dismissed until the serum was evaluated thoroughly. The second approach to obtain an immunoglobulin free serum was to deplete NHS of IgG and IgM by different depletion methods. The depletion of C1q in addition to IgG and IgM was inevitable without the presence of NaCl and in various experimental set-ups, different concentrations of NaCl in samples and buffers were evaluated to see whether C1q depletion could be avoided and immunoglobulin depletion promoted simultaneously. One probable reason for simultaneous C1q depletion could be attraction between the large positive protein and negatively charged Sepharose. It is also possible that C1q is depleted bound to captured IgG and IgM, as C1q can be isolated using affinity chromatography with IgG and IgM [30]. Using the HiTrapTM Protein G column, IgG was successfully depleted despite NaCl present and C1q remained in the serum to indefinite extent. Increased concentration of NaCl did not result in lesser C1q depletion and the lowest concentration was further utilized to minimize serum protein distress. The presence of NaCl was, however, shown to prohibit IgM- depletion and neither of the applied methods for IgM-depletion was thus successful when NaCl was present. Initially it was evaluated if NaCl could substitute ammonium sulphate (in description from manufacturer) in promoting IgM binding when using a HiTrapTM IgM column, as ammonium sulphate usually is used to precipitate proteins [18] and precipitation of plasma was seen. NaCl did not however contribute significantly to binding and no regard of C1q-depletion was taken as NaCl was excluded. Serum instead of plasma was used and no

33 precipitation using ammonium sulphate could be seen, conceivably due to lack of coagulation proteins such as fibrinogen (physiological plasma concentration of 1.5-4.5 g/L [31]). The possibility of precipitation of complement proteins was considered but as the method did not show prosperous results, further evaluation was not performed regarding complement functionality. According to manufacturer of CNBr-activated Sepharose 4B, NaCl could be used to elute bound ligands. This offered no ground for high expectations and as predicted IgM-depletion was prevented in presence of NaCl. Although NaCl was excluded and several depletion processes was performed sequentially, the method still was not successful in depleting NHS of IgM. One possible reason for this might be that too low concentration of capturing antibody was coupled to the Sepharose. The coupling procedure was however effectively achieved and could not significantly contribute to the method failure. Another issue using this method was unavoidable dilution of the serum, as the serum was diluted for every time the depletion procedure was repeated. Because of intended dilution to eliminate the effect of the alternative pathway on complement activation, indeterminate dilution was undesirable and this is something that necessitates further optimization and evaluation to avoid using this method.

Since none of the applied methods successfully depleted NHS of IgM, the antibody- independent and biomaterial-induced classical pathway activation could not be thoroughly studied. Some potential results were however obtained using the IgG-depleted serum and will be discussed below. However, it is kittle and difficult to draw any concrete conclusions as the study was performed to few times to provide accurate and reliable results. The generation of complement activation marker C3a in the biomaterial-induced complement activation study overall showed similar complement activation levels in the different sera subjected to both polymers. Both polymers showed adsorption of C3a on the surface to a higher extent than earlier findings on the polymers from the research group (unpublished data). This could conceivably be explained by the fact that the sera were diluted before polymer incubation and the amount of generated C3a was less than in undiluted serum and thus adsorbed on the surface of the polymers without saturation. As the sera was diluted 1:8 before incubation, the alternative pathway was also inactivated and no amplification loop present to enhance complement activation. The amplification loop of the alternative pathway has shown to contribute as much as 80% to complement activation initiated by the classical pathway [4]. The elevated complement activation in IgG-depleted serum after preincubation of polymers with C1q could indicate antibody-independent complement activation, however it

34 must be noted that IgM still was present in the serum and could induce activation as well. IgG appeared to enhance complement activations as predicted. The complement activation in NHS appears to be approximately 100-fold higher than in the serum containing both C1q and IgG when the two sera in fact ought to have similar contents and activity. Possible explanations for the decreased activation could be distressed proteins after several and various depletion methods or limited functional activity in added C1q. Enhancement of complement activation will further on be evaluated with the addition of known complement activators. When comparing control sample and start value 2 (containing the same serum), the control sample showed elevated activation probably due to heat and rotation during incubation. Some small differences between the polymers were however revealed, as complement activation in the prepared sera varied slightly when studied in detail. In general, hydrophilic and carboxylate-containing polymers such as P1 adsorb less protein [6]. Interestingly, P1 adsorbed more C3a on the surface regardless of sera in opposite to P2, which had a higher concentration of C3a in supernatants than in eluates. One possible reason for the larger amount of adsorbed C3a to P1 could be attraction between the nitrogen-containing and thus negatively charged crosslinker DAP and the positively charged C3a. P1 also appeared to induce complement activation to higher extent than P2 in some sera and this is in conformity with earlier finding, as P1 has shown to be a potent activator of complement system probably due to the large amounts of adsorbed C1q and IgG (unpublished data). However, it must be emphasized that the focus of this degree project work was to prepare and evaluate sera for further antibody-independent and biomaterial-induced complement studies and it is kittle to draw any conclusions from obtained results before further repeated experiments have been performed.

35 CONCLUSIONS

- FBS is deficient of immunoglobulins. - No detectable complement activity is present in FBS. - A close to complete IgG-depletion of NHS is achievable. - IgG-depleted NHS has low but functional complement activity. - None of the applied methods for IgM-depletion successfully depleted NHS of IgM.

To be able to thoroughly study the antibody-independent and biomaterial-induced classical pathway activation, further evaluation regarding the immunoglobulin free serum is necessary. Two further aspects to evaluate would be the possibility to accomplish a complete IgM-depletion by optimization of existing methods as well as the possibility to enhance the complement activation in the immunoglobulin free serum with known complement activators.

ACKNOWLEDGEMENT

First and foremost, I would gratefully like to acknowledge Prof. Kristina Nilsson Ekdahl for the opportunity to perform this degree project work and for the chance to enter the thrilling world of complement activation. Further, Anna E. Engberg (Ph.D.) is acknowledged for her never-ending enthusiasm and goofiness, as well as support regarding everything from experimental set-ups to encouraging peptalks. Two members of the research group possessing enormous amounts of knowledge, Per H. Nilsson and Kerstin Sandholm (both MSc.), are also acknowledged for their helpful guidance and inspiration during this degree project work. My former academy classmate Gustaf Olsson (MSc.) is acknowledged for his motivating chitchats, computational aid and for always being close to hand (nearby laboratory facilities is great).

Last but not least, I would like to thank my family, my boyfriend Nicklas and my best friend Jessica for supporting me during the time this degree project work was carried out. I am so grateful that I have got you and that you always believe in me.

36 REFERENCES

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37 16. Takahashi, M., et al., Mannose-binding lectin (MBL)-associated serine protease (MASP)-1 contributes to activation of the lectin complement pathway. J Immunol, 2008. 180(9): p. 6132-8. 17. Gros, P., F.J. Milder, and B.J. Janssen, Complement driven by conformational changes. Nat Rev Immunol, 2008. 8(1): p. 48-58. 18. Wilson, K., Walker, J., Principles and Techniques of Biochemistry and Molecular Biology. 6 ed. 2005: Cambridge University Press. 19. Favoreel, H.W., et al., Virus complement evasion strategies. J Gen Virol, 2003. 84(Pt 1): p. 1-15. 20. Gaboriaud, C., et al., Structure and activation of the C1 complex of complement: unraveling the puzzle. Trends Immunol, 2004. 25(7): p. 368-73. 21. Skoglund, C., et al., C1q induces a rapid up-regulation of P-selectin and modulates collagen- and collagen-related peptide-triggered activation in human platelets. Immunobiology. 22. Triglia, R.P. and W.D. Linscott, Titers of nine complement components, conglutinin and C3b-inactivator in adult and fetal bovine sera. Mol Immunol, 1980. 17(6): p. 741- 8. 23. Linscott, W.D. and R.P. Triglia, The bovine complement system. Adv Exp Med Biol, 1981. 137: p. 413-30. 24. Kindt, T.J., Goldsby, R.A., Osborne, B.A., Kuby, J., Kuby Immunology. 6 ed. 2007: W.H. Freeman and Company. 25. GE Healthcare, Instructions for usage of HiTrap Protein G HP. 2006. 26. Pernemalm, M., R. Lewensohn, and J. Lehtio, Affinity prefractionation for MS-based plasma proteomics. Proteomics, 2009. 9(6): p. 1420-7. 27. Lachmann, P.J., Preparing serum for functional complement assays. J Immunol Methods. 352(1-2): p. 195-7. 28. Gould, E.A., et al., Detection of antibody to bovine syncytial virus and respiratory syncytial virus in bovine fetal serum. J Clin Microbiol, 1978. 8(2): p. 233-7. 29. Janssen, B.J., et al., Structures of complement component C3 provide insights into the function and evolution of immunity. Nature, 2005. 437(7058): p. 505-11. 30. Gadjeva, M.G., et al., Interaction of human C1q with IgG and IgM: revisited. Biochemistry, 2008. 47(49): p. 13093-102. 31. Boyd, J.H., et al., Fibrinogen decreases cardiomyocyte contractility through an ICAM-1-dependent mechanism. Crit Care, 2008. 12(1): p. R2.

38 APPENDIX

Table IV. Supernatant and eluate dilutions for biomaterial-induced complement activation study. P1 Supernatant Eluate NHS 1:100, 1:250, 1:500, 1:1000 1:250, 1:500, 1:1000 + C1q 1:100, 1:250, 1:500, 1:1000 1:250, 1:500 + IgG 1:100, 1:250, 1:500, 1:1000 1:250, 1:500 + C1q + IgG 1:100, 1:250, 1:500, 1:1000 1:250, 1:500 Pre-C1q 1:100, 1:250, 1:500, 1:1000 1:250, 1:500

P2 Supernatant Eluate NHS 1:100, 1:250, 1:500, 1:1000 1:250, 1:500 + C1q 1:100, 1:250, 1:500, 1:1000 1:250 + IgG 1:100, 1:250, 1:500, 1:1000 1:250 + C1q + IgG 1:100, 1:250, 1:500, 1:1000 1:250 Pre-C1q 1:250, 1:500, 1:1000 1:250

Start value 1 1:250, 1:500 Start value 2 1:250, 1:500 Control sample 1:250, 1:500

39