ACTAUNIVERSITATISUPSALIENSISUPPSALA2006

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 137

Application of proximity Ligationfor Detection of Proteins,Biomolecular Interactions, andSingle Copies of Pathogens

SIGRUN MARGRET GUSTAFSDOTTIR

ISSN 1651-6206ISBN 91-554-6531-5urn:nbn:se:uu:diva-6791

Publications

This thesis is based on the following papers, which are referred to in the text by their Roman numerals:

I Gullberg,M., Gustafsdottir,SM., Schallmeiner,E., Jarvius,J., Bjarnegård,M., Betsholtz,C., Landegren,U.& Fredriksson,S. (2004) Cytokine detection by antibody-based proimity ligation.Proc Natl Acad Sci USA 101(22) 8420-4

II Gustafsdottir,SM., Nordengrahn,A., Fredriksson,S., Wall-gren,P., Rivera,E., Schallmeiner,E., Merza,M., & Landegren,U. Detection of individual microbial pathogens by proximity liga-tion. Clinical Chemistry, in press.

III Gustafsdottir,SM., Rada-Iglesias,A., Schlingemann,J., Kamali-Moghaddam,M., Wadelius,C. & Landegren,U. Characterizing sequence specificity of DNA binding proteins by proximity liga-tion. Submitted.

IV Gustafsdottir,SM., Wennström,S., Fredriksson,S., Hägg,M., Wu,Y., Hamilton,A., Sebti,S., Landegren,U. In vitro analysis of inhibition of VEGF and VEGFR-2 by proximity ligation. Manu-script.

The first two authors of paper II contributed equally to the work.

Supervisor: Ulf Landegren Professor of Molecular Medicine Department of Genetics and Pathology Uppsala University Uppsala, Sweden

Faculty opponent: Per-Åke Nygren Professor of Molecular Biotechnology Department of Biotechnology Royal Institute of Technology (KTH) Stockholm, Sweden

Review board: Stefan Bertilsson Researcher in Limnology

Department of Ecology and Evolution Uppsala University Uppsala, Sweden

Niklas Dahl Professor of Clinical Genetics

Department of Genetics and Pathology Uppsala University Uppsala, Sweden

Mats Inganäs Director of ApplicationsGyros AB Uppsala, Sweden

Monica Nistér Professor of Pathology Department of Oncology-Pathology

Karolinska University Hospital Stockholm, Sweden

Fredrik Öberg Researcher in Hematology Department of Genetics and Pathology Uppsala University Uppsala, Sweden

Table of contents

Introduction.....................................................................................................7

Proteomics, before and now............................................................................8Early achievements, Swedish contributions ...............................................8Available assays for protein detection and quantification..........................9Immunoassays ..........................................................................................10

Homogenous immuoassays .................................................................10Heterogenous immunoassays...............................................................11Affinity reagents for protein analysis ..................................................12

Mass spectral analysis in combination with 2D gel electrophoresis ........15Protein arrays............................................................................................16Analysis of interactions between proteins and other biomolecules..........17

Protein-protein interactions .................................................................17Protein-DNA interactions ....................................................................18

Proteins in health and disease .......................................................................20Protein measurements in clinical practice ...........................................20Detection of pathogens in clinical samples..........................................21

Present investigations, a background ............................................................22

Present investigations....................................................................................24Paper I. Cytokine detection by antibody-based proximity ligation. .........25Perspectives on paper I.............................................................................26Paper II. Detection of individual microbial pathogens by proximity ligation......................................................................................................28Perspectives on Paper II ...........................................................................29Paper III. Characterizing the sequence specificity of DNA binding proteins using proximity ligation .............................................................31Perspectives on study III. .........................................................................32Study IV. In vitro analysis of inhibition of the interaction between VEGF and VEGFR-2 using proximity ligation. ..................................................33Perspectives on study IV ..........................................................................34

Future perspectives .......................................................................................36

Acknowledgements.......................................................................................39

References.....................................................................................................42

Abbreviations

2DGE Two-dimensional gel elecrophoresis AFM Atomic force microscopy ChIP Chromatin immunoprecipitation DNA Deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay ELISpot Enzyme-linked immunospot assay EMSA Electrophoretic mobility shift assay ESR Erythrocyte sedimentation rate HCG Human chorionic gonadotropin HNF4- Hepatocyte nuclear factor 4 alpha IL-2 Interleukin-2 IL-4 Interleukin-4 LOD Limit of detection MS Mass spectrometry ND50 50% Neutralization dose NMR Nuclear magnetic resonance PCR Polymerase chain reaction PDGF Platelet derived growth factor PLA Proximity ligation assay qPCR Quantitative PCR RCA Rolling circle amplification RIA Radioimmunoassay RNA Ribonucleic acid SCA Scintillation proximity assay SELEX Systematic evolution of ligands by exponential

enrichment SPR Surface plasmon resonance USF-1 Upstream stimulatory factor 1 VEGF Vascular endothelial growth factor VEGFR-1 VEGF receptor 1 VEGFR-2 VEGF receptor 2

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Introduction

The completion of the eagerly awaited human genome sequence [1, 2] has been described by some as the most important scientific achievement of all times. The information from the sequences alone is not very instructive however, and many “beyond-genome” projects are now dealing with func-tional annotations of the genome and its genes through e.g. analysis of the transcriptome [3-6], and the proteome [7-9].

The study of the protein complement of the genome, the proteome, repre-sents an enormous challenge. The proteome is far more dynamic and com-plex than the genome, protein levels change constantly over time and they are heavily affected by e.g. drugs and diseases. Proteins can differ in abun-dance, by up to ten orders of magnitude in human serum, and they are post-translationally modified in many different ways.

So far, most analyses of the proteome have been performed in a catalog-ing fashion, with large-scale identification of proteins, and recently with genome-wide gene expression analysis and protein localization [10] and global analysis of protein-protein interactions [11] in budding yeast.

New technologies and interdisciplinary strategies are needed to allow ef-ficient measurements of concentrations of proteins, secondary modifications, localizations in cells and tissues and interactions with proteins as well as other biomolecules both in health and disease.

Proximity ligation [12] is a newly developed technique that allows ex-tremely sensitive and specific detection of proteins by allowing the protein targets to be represented as DNA signatures that may subsequently be ampli-fied and detected by methods like PCR. Paper I in this thesis describes a way by which any matched pair of monoclonal antibodies or polyclonal antisera can be converted into proximity probes through attachment of oligonucleo-tide sequences, and the application of the proximity ligation technique for very sensitive measurements of cytokines in complex biological samples. Paper II illustrates an application of the technique for measurements of even single copies of microbial pathogens in common field samples using affinity probes raised against surface epitopes of the pathogens. Paper III reports the application of proximity ligation for analysis of transcription factors interact-ing with their cognate gene promotor elements, and paper IV, finally, de-scribes the analysis of inhibitors of ligand-receptor binding using proximity ligation.

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Proteomics, before and now

Early achievements, Swedish contributions Proteins have fascinated researchers since their discovery in the late eight-eenth century. This captivating group of molecules that constitutes more than half of the dry weight of cells, was named by the Swedish scientist Jöns Jacob Berzelius together with Dutch chemist Gerrit Mulder in 1838, adopt-ing the Greek word , meaning “primary”, or of “first rank” (pr -tos: first). Early protein scientists struggled with questions about the chemi-cal compositions of proteins, and their structure and function, as vividly described by the scientist Charles Tanford and Jacqueline Reynolds in their book “Nature’s Robots, a history of proteins” [13]. Since the early nine-teenth century, Swedish scientists have been in the protein research frontline [14], and many technologies that allow characterization, identification, puri-fication and detection of proteins, have been developed at Swedish laborato-ries.

Early achievements in isolation and characterization of proteins were made by chemistry Nobelist Theodor (The) Svedberg, and his students Arne Tiselius and Robin Fåhraeus in Uppsala. Svedberg and Fåhraeus isolated proteins by sedimentation using ultracentrifugation (an invention by Sved-berg), and described a method for determining molecular weights of proteins [15]. Svedberg and Tiselius investigated the possibilities of protein electro-phoresis, and Tiselius succeeded in separating human serum into four dis-tinct peaks, albumin, alpha-, beta, and gamma globulins, and he later demon-strated that antibodies belong to the gamma globulin fraction [16].

The careers of both Tiselius and Fåhraeus were very successful, Tiselius received a Nobel Prize in chemistry 1948 [17] for the separation of globulins or serum proteins and improvements in chromatography, and Fåhraeus be-came famous for the introduction of the erythrocyte sedimentation rate method, ESR [18], (later developed further by Alf Westergren [19]), a tech-nique still commonly used in clinical medicine.

Swedish protein pioneers have also been involved in the development of widely used techniques for protein purification, separation, and sequence determination. Descendants from the Svedberg laboratory have made a ma-jor contribution to the field of protein separation through methods for

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isoelectric focusing and partitioning (Svensson-Rilbe and PÅ Albertsson, both students of Tiselius), and purification of proteins. Famed biochemist Stellan Hjertén (still working in his laboratory at 78 years of age in 2006), got his PhD training under the supervision of Tiselius and has divided his career between chromatography and electrophoresis, making immense con-tributions in both fields. The development of methods based on hydroxyapa-tite for protein chromatography [20, 21], and suitable materials for separa-tion like crosslinked polyacrylamide [21, 22] and agarose [22] are a few of his achievements.

Jerker Porath, a member of the Tiselius lab, introduced cross-linked bead-shaped agarose for protein separation-the IMAC technique [23] that is widely used for purification of His-tagged recombinant proteins-and together with Per Flodin the cross-linked dextran [24]. Rolf Axén was an additional member of the Tiselius sphere who made discoveries leading to the birth of affinity-chromatography.

Examples of Swedish accomplishments in the field of immu-nodetection are the immunochemical double diffusion technique (Ö. Ouchterlony), and the rocket electrophoresis or immunoelectrophore-sis in antigen-coated gel, introduced by C. Laurell.

Yet another important technique in protein chemistry introduced by a Swedish scientist is the amino-acid sequencing method named after its inventor, Per Edman [25].

Available assays for protein detection and quantification Proteins are a very demanding class of biomolecules for analysis. There is little common chemical identity among the many different proteins, aside from the peptide backbone. Proteins are modified in many different ways, different splice variants frequently give rise to different proteins, and the differences in concentration range over at least ten orders of magnitude in human serum. The protein levels are not necessarily reflected in changes in mRNA levels [26] as not all transcribed mRNAs are translated, and protein turnover rates are not the same for different transcripts. In addition, many proteins are activated by post-translational modifications that cannot be evaluated by transcript analysis. Stathmin is an example of a disease marker, elevated in various cancers, that is phosphorylated in its active form and which must therefore be detected on the protein level.

The goals of research in proteomics (the term proteome first having been coined in 1996 [27]) is the analysis of concentrations of proteins, as well as their secondary modifications, localizations in cells and tissues, interactions with other biomolecules, and their structure and function. The number of technologies for protein detection and quantification, a field that will be in

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focus for this thesis, has expanded in the last decades from biochemical analyses of separate protein types to global protein measurements.

ImmunoassaysImmunoassays are by definition laboratory methods that use the specific binding of a protein antigen by an antibody/antibodies to identify and quan-tify substances of interest in samples of biological origin. Immunoassays are extensively used in the diagnostic field, and during the last decades there has been a tremendous increase in the number of available immunoassays, as well as their specificity, and sensitivity. Protocols have been established for production of monoclonal antibodies, in vitro maturation for creation of high-affinity binders etc. Radioisotopic labels were the first labels used in immunoassays, but due to environmental hazards these have gradually been replaced by fluorescence, chemiluminescence or enzyme turnover, and in recent years rolling circle amplification (RCA) or PCR, for increased assay sensitivity. Examples of immunoassays, both homogeneous and heterogene-ous, as well as different classes of binders will be described here below.

Homogenous immuoassays In homogeneous immunoassays, a signal, measurable from a label, is gener-ated by the interaction between an antibody and an antigen, or matter in competition for binding first to the measured substance. Classical examples of homogeneous immunoassays are the EMIT[28] and the CEDIA assays [29], followed by immunoprecipitation or immunoagglutination, FRET, fluorescence polarization, LOCI, and SPA.

Various agglutination assays are available, mainly based on latex mi-crobeads, for the direct or competition based detection of for example hor-mones and non-proteinaceous agents, like barbiturates, amphetamines and other drugs, in blood and urine. Agglutination assays are very simple to per-form, usually providing yes/no answers only, but they may also be used for example for determination of serum antibody titers.

Scintillation proximity assay (SPA) [30], is a commonly used homoge-neous-phase assay for protein measurements. SPA is based on the binding of a radioactive ligand to the surface of scintillant microbeads. Radiation by bound ligand leads to excitation of the scintillants, observable as emission of blue light that can be detected in scintillation counters. Beyond measure-ments of single proteins, the assay can be used for high-throughput screening of drug candidates where for example ligand-receptor interactions are the targets.

Luminescent oxygen channeling assay (LOCI) [31] is based on latex agglutination and channeling. Pairs of interacting ligands and receptors im-

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mobilized on polystyrene beads are formed at very low concentration, and detection is carried out by photochemically triggered chemiluminescence. Assay times are very short, and the method offers quantification over a 106-fold range of concentrations and a reported limit of detection of ~13.000 molecules of streptavidin, and ~250.000 molecules of thyrotropin in human serum [31].

Heterogenous immunoassays In heterogeneous assays, free analytes and antibody-antigen bound analytes need to be separated. An antibody is usually first immobilized on a solid surface with sequential additions of the other assay components, and exten-sive washes between each step.

The radioimmunoassay (RIA) was first described by Berson and Yalow [32] as a very sensitive assay for the detection of insulin in human plasma. Rosalyn Yalow indeed received the Nobel price in Physiology or Medicine in 1977 for the invention of the technology [17]. The assay is based on the displacement of radioactive by non-radioactive antigens bound by primary antibodies that are subsequently immunoprecipitated by a secondary anti-body. The radioactivity of the supernatant can then be related to the amount of the non-radioactive antigen being analyzed. It has been widely used in clinical laboratories and is very suitable for detection of small antigens and chemicals like hormones and certain abused drugs. Main drawbacks of the assay are the expense and other practical issues related to the use of radioac-tivity.

Various forms of the classical sandwich immunoassay have been the most widely used techniques for antigen or antibody detection in recent dec-ades, both for research and diagnostics [33]. Leif Wide with collaborators at the Uppsala University, did a follow-up to the work by Berson and Yalow, introducing a solid-phase competitive radioimmunoassay of proteins, for facilitated separation of free and antibody-bound antigens [34]. The non-competitive dual-specific sandwich test for allergy diagnostics was later introduced by L.Wide together with G. Johansson and H. Bennich [35] soon after the discovery of the immunoglobulin E. The sandwich assay requires simultaneous binding of the target to a solid-surface bound capture reagent and a labeled binding (detection) reagent, which ensures an increased speci-ficity of the assay in comparison with the competitive binding single-binder assay. The linear range of the dose-responsive curve is moreover increased in the sandwich assay [33]. The sensitivity of these assays is limited by the tendency to unspecific binding of the antibody reagents, which typically causes background signals that affect the limit of detection. The radioiso-topic labels have now largely been replaced by enzymatic labels (enzymatic linked immunosorbent assay, ELISA [36]) and fluorescence.

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The enzyme-linked immunospot assay ELISpot utilizes the ELISA tech-nique for quantitative detection of cells secreting specific antigens[37, 38]. The assay was initially developed for the detection of antigen-specific anti-body secreting B-cells [39, 40]. It is somewhat similar to the Jerne plaque assay invented by Nobelist Niels Jerne [41]. The ELISpot has proven to be a sensitive assay allowing analysis of individual cells, however limited by extreme intra-assay variation [42].

Immuno-PCR, developed by Charles Cantor and colleagues at Boston University [43], is an immunoassay that utilizes nucleic acid amplification instead of enzymatic turnover for signal enhancement, and thereby allowing very sensitive measurements of proteins with a reported detection limit of 580 molecules, as determined by agarose gel electrophoresis of the PCR products with ethidium bromide staining. The assay can be performed as a regular sandwich test or an indirect assay with the detection antibody or the secondary antibody labeled with one or more oligonucleotide sequences containing primer sequences for PCR amplification. The main limitation of the assay is related that of other sandwich assays; background signals due to unspecific binding of the antibodies, with the need for extensive washings and sufficient blockings [44].

An example of a newly developed immunoassay where PCR can be used for enhanced readout is the nanoparticle bio-bar-code based assay for pro-tein detection [45]. The format is a sandwich immuno-PCR assay, where the capture antibody is immobilized on a solid support (magnetic beads), and a secondary antibody is immobilized on nanometer size gold particles coated with numerous so-called DNA bio-bar-code molecules. After binding and extensive washes, the DNA strands can be released and detected by PCR or hybridization. A limit of detection of 30 attomolar human prostate specific antigen was demonstrated in goat serum. It is an obvious benefit to have multiple DNA labels per detection antibody for an increased signal in the PCR step, however, cross reactive binding and non-specific binding remain problems that can contribute to background signals.

Affinity reagents for protein analysis Good quality ligand binders, antibodies or other non-traditional binders, protein, peptide or nucleic acid-based, are the keys to successful immuno-chemical experiments. If the goals of proteomics research are to be fulfilled, specific ligands have to be raised against all members of proteomes. A joint European effort, “Proteome Binders” has now been established under the coordination of Prof. Michael J. Taussig, Babraham Institute, UK, for the collection of binders, antibodies, scaffolds or aptamers, against every single protein in the human proteome. These classes of binders will be briefly de-scribed here below.

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Affinity and specificity are two important features of ligand binders. The interaction between the ligand binders discussed here, and the antigen epi-topes are non-covalent and reversible. The affinity describes the strength of this interaction, by expressing the amount of binder-antigen complex that will be found at equilibrium. The specificity of ligand binders describes their ability to discriminate between the target protein and other related mole-cules. The binding of an antibody to an antigen, that was not used for stimu-lation of its production, is often referred to as cross-reactivity, a phenomenon which has been shown to be highly specific [46].

AntibodiesAntibodies, polyclonal or monoclonal, have been the most obvious choice

for use as affinity binders in recent decades. The hybridoma technique for the generation of monoclonal antibodies was described 1975 by Georges Köhler and César Milstein [47]; antibody-producing cells are immortalized through combination with tumor cells, which provides unlimited production of antibodies with the same specificity. The ingenious invention was one of the most important achievements in biomedicine in the 1970s, and led to the Nobel Prize in 1984 [17].

Antibodies are well designed by nature for protein recognition. They offer specific detection of proteins, long serum half-life, discrimination between protein antigen conformations, and detection of single amino acid substitu-tions. Antibodies have been used extensively in research and diagnostics, and several therapeutic antibodies have been evaluated in clinical trials. The advantages of antibodies are many and so are their disadvantages.

Examples of the disadvantages are the cumbersome production procedure, the limitation of target molecules e.g. due to toxicity, and the promiscuous nature of antibody binding.

Antibodies generated in vivo by immunization show a wide range of af-finities for their target molecules, from 10-4M-<10-10. The B-cell immune response occurs in two stages. The first stage generates low-affinity antibod-ies, mainly IgM isotypes, and in the second stage high-affinity antibody-generation is driven by the antigen stimulation. Second stage antibodies are mostly of IgG isotype.

Antibodies of higher affinities can be created by in vitro maturation by random mutagenesis of the heavy- and light chain variables with expression in phage-display vectors [48, 49], or in cell-free systems [50, 51].

SelexThe systematic evolution of ligands by exponential enrichment or SELEX

in short, was first described by Ellington and Szostak [52] and Tuerk and Gold [53]. The SELEX procedure is used to screen sequence-randomized nucleic acid libraries for high-affinity ligands (so-called aptamers) to mo-lecular targets. The selection is performed by the generation of a library,

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containing short (20-100 nucleotides) random sequences, DNA or RNA, flanked by common primer sequences. The library is subjected to the solid-phase bound target molecule in repeated cycles of selection by physical cap-ture and amplification, leaving out only the sequences with the highest affin-ity for the target after typically 8-15 rounds of selection. The aptamers can exhibit very high affinities [54], and combinatorial libraries, made of random oligonucleotide sequences contain more molecular diversity than similar protein libraries [55].

The advantages of nucleic aptamers for analytical purposes are several; the sequences are short and the synthesis is straightforward, aptamers may be raised against compounds that cannot be used for immunization due to e.g. toxicity, and they can be very stable in storage and may be subjected to rounds of denaturing and renaturing without any detectable loss in activity. The extension with nucleic acid oligomers for PCR or RCR applications is also straightforward.

Scaffold proteins for generation of bindersScaffold (framework) proteins, based on the immunoglobulin fold or of a

totally different structure, have been successfully used in the recent decade for the generation of high-affinity and specific ligand binders, while provid-ing advantages over in-vivo generated immunoglobulins. One example of the advantages of many scaffold-derived binders is the lack of disulfide bonds and free cysteines that allows cheap and rapid manufacture through bacterial expression. Different application formats have led to investigation of several different starting scaffold structures. Three examples are listed here:

Affibodies are derived from the Z-domain of the Staphylococcus aureusprotein A [56], and consist of three a-helices. They show reversible denatu-ration, and have been successfully utilized for a number of applications, both diagnostic and therapeutic. Lipocalins are compact one-domain proteins with a central ß-barrel structure. Engineered variants of lipocalin display high affinity and specificity for small molecules like fluorescein, and digoxi-genin. Lipocalins may also be divided into a ß-barrel-core and a highly vari-able region with ligand-binding properties similar to that of immunoglobu-lins [57]. Repeat proteins, like ankyrin-repeats have been used for genera-tion of high-affinity binders against a range of targets [58]. They are thermo-dynamically stable and display reversible folding behavior.

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Mass spectral analysis in combination with 2D gel electrophoresisMass-spectrometry (MS) is a very powerful and in fact indispensable tech-nique for de novo analysis of proteins and protein populations, analysis of primary sequences, post-translational modifications, and interactions of pro-teins from complex mixtures of samples [59].

It is based on the generation of mass spectra of peptides generated by fragmentation of the proteins to be analyzed. The mass spectra can then be matched against protein sequence databases for identification of the peptides and ultimately the parent proteins. Either electrospray ionization or matrix-assisted desorption/ionization can be used together with a total of four types of mass-analyzers that can be applied alone or in tandem for different pur-poses. The advantages and disadvantages of each of these are discussed by Aebersold and Mann [59]. Mass-spectrometry is clearly an unbeatable tool for fast identification of proteins, but there have been some technical issues related to the analysis: low peak resolution, that makes analysis of very complex mixtures of proteins difficult, and poor quantification (now solved by the isotope coded affinity tag (ICAT) approach [7]. The mass-spectrometry-based proteomics approach has been developing in recent years towards whole-protein analysis [60], tissue imaging [61] and analysis of high mass accuracy and resolution using mass tags [62].

Because of limitations of dynamic range, MS-analysis cannot be used without prior separation of the samples. A major separating technique is the two-dimensional gel electrophoresis (2-DGE) where proteins are separated on the basis of charge and mass [63]. Other techniques for protein separation are capillary electrophoresis and liquid chromatography. 2DGE is the most powerful technique available; 1000-5000 proteins can be separated in each gel. The main advantages of the technique are thus the separating power and the separation of post-translational modifications. The disadvantages are, however, that the first dimension separation is salt and contaminant sensi-tive, the method requires advances technical skills, different samples may need different protocols for analysis, and high and low molecular weight proteins as well as hydrophobic proteins are underrepresented. Protein arrays might become the new alternative for protein separation prior to the mass-spectrometry analysis, as it would be an easy way to purify proteins in a high-throughput fashion.

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Protein arraysThere are two types of protein arrays, analytical and functional arrays. The former types are used for detection and measurements of proteins in biologi-cal samples, while the latter group includes arrays with large sets of proteins immobilized for analysis of different biochemical activities. The protein arrays consist of immobilized antibodies, protein antigens or molecules that the proteins may interact with and can be applied for protein measurements, analysis of interactions between proteins, protein-lipids, protein-drugs, en-zymes-substrates, proteins-nucleic acids, receptor-ligands, and protein-carbohydrates. Current protein arrays, surface chemistries for protein immo-bilization on the solid surface, as well as different detection technologies are described by Zhu and Snyder [64]. Microarrays for DNA analysis have be-come a major success in the recent decade for analysis of genomic variations and expression profiling at the level of nucleic acids. For the past ten years or so, there has been a drive towards high-throughput measurements of ge-netic markers on a genome-wide scale, and microarrays serve the purpose by enabling parallel detection of hundreds of thousands of markers[65-67]. Ex-pectations of protein microarrays are high, the arrays could provide a power-ful tool for global analysis of proteins and their function, but so far success has been modest. The chemical nature of proteins is extremely diverse which makes it very difficult to provide truly universal protein microarrays. How-ever, considerable improvements of the protein arrays have been reported in recent years that allow immobilization of even entire proteomes on arrays [68]. New array formats have emerged recently, for example solution based arrays for increased flexibility [69], or silicon nanowire-based electronic arrays [70].

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Analysis of interactions between proteins and other biomolecules

Protein-protein interactions A majority of all proteins function in cooperation with other proteins and other biomolecules. Standard techniques for detection of protein-protein interactions include amongst others the extensively used yeast two-hybrid technique [71] that is based on reconstitution of functional transcriptional activator in yeast and allows detection of novel interactions as well as analy-sis of known interactions. Chemical crosslinking is one of the oldest tech-niques available for studying of protein-protein interactions. The technique can be used for a wide range of applications, from screening of interacting partners to the retrieval of structural information for interacting proteins [72]. It is also used for the linking of interacting proteins for various im-munoprecipitation and other pull-down assays. Coimmunoprecipitationis another popular technique for the analysis of known interactions as well as for identification of partners that interact with a known protein [73].

Examples of biophysical techniques for the identification and characteri-zation of protein-protein interactions are mass spectrometry, atomic force microscopy (AFM), and surface plasmon resonance (SPR).

Under the appropriate conditions the interacting proteins, assemblies of multiprotein structures, as well as macromolecular complexes remain intact and can thus be analyzed via their mass spectra [74, 75]. AFM [76] has been successfully adapted for example ligand-receptor interactions [77]. SPR can be used for quantitative studies of protein-protein interactions, as well as the interactions of proteins with other molecules, e.g. nucleic acids and lipids [78-80].

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Protein-DNA interactions Many different proteins interact with nucleic acids, RNA or DNA. Replica-tion, recombination, transcription, and restriction are controlled by sequence specific protein-DNA interactions.

A considerable number of methods are available for investigations of DNA-protein interactions in vitro or in vivo. Some address single pairs of interacting biomolecules at the time, while others elucidate protein-DNA interactions in a global fashion. Examples of common methods for low-throughput analysis of sequence specificity of DNA binding proteins are gel retardation techniques, nitrocellulose binding assays [81], Southwestern blotting [82], and reporter constructs in yeast [83].

EMSA, or electrophoretic mobility shift assay, is the technique that dominates in vitro analysis of interactions of binding of DNA binding pro-teins to its target nucleic acids. Gel retardation has been utilized for that pur-pose since the late 1960s, but the method commonly used today was intro-duced by Fried and Crothers in 1981 [84]. The method is based on the dif-ference in the electrophoretic mobility between a slowly migrating DNA-protein complex and fast migrating non-bound DNA strands in non-denaturing acrylamide or agarose gels. The supershift assay was a later im-provement of the technique [85] which allows antibody-based identification of a DNA-binding protein in a specific band in a gel retardation assay. In spite of its tremendous popularity, the method suffers from drawbacks such as the requirement for large amounts of input material, and for radioactive labeling of the nucleic acids. It is moreover not suitable for analysis of tran-sient interactions.

Fluorescent polarization or anisotropy measurements allow characteri-zation and equilibrium determinations of both protein-protein and protein-DNA interactions. The measurements, based on rotational motion of fluores-cently labeled macromolecules in solution, are particularly useful for low affinity interactions not detectable by electrophoretic methods [86, 87]. As mentioned for the analysis of interactions between proteins, electrospray ionization mass-spectrometry can be used for detection of non-covalent interactions involving proteins with other molecules, like DNA. Electros-pray-ionization-MS provides identity and stoichiometry of the interacting molecules, not easily obtained by other techniques [88, 89]. The drawbacks are mainly the high sample consumption and the need for purification of samples before analysis.

High-throughput analysis of DNA-protein interactions has been made possible in the recent years by utilization of DNA microarray technology. Chromatin immunoprecipitation with DNA microarray readout, ChIP-on-chip analysis, is a powerful technique that allows genome-wide binding patterns of transcription factors in vivo [90]. In this technique, protein com-plexes are crosslinked to chromatin in the cell nucleus. The chromatin is then

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fragmented and immunoprecipitation with an antibody raised against a spe-cific transcription factor is used for isolation of the transcription factor of interest together with bound DNA elements. The cross-link is subsequently reversed and the DNA strands are identified on DNA microarrays. The fragmentation of the chromatin generates DNA strands that can be several hundred base pairs long. The resolution of the analysis is for that reason low, and the sequence specificity of the binding has to be verified by reference methods like EMSA at all times.

The ChIP-on-chip method is not the sole technique that utilizes DNA mi-croarrays for high-throughput analysis of protein-DNA interactions. The George Church laboratory at Harvard Medical School has used double stranded DNA microarrays for investigation of protein binding patterns. Affymetrix single stranded DNA arrays were converted enzymatically into double stranded arrays that were subsequently used for e.g. demonstration of sequence specific cleavage of restriction enzymes [91].

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Proteins in health and disease

Protein measurements in clinical practice Measurements of proteins are among the most commonly ordered clinical lab tests. Examples of tests that indicate general health and nutritional status include measurements of total protein concentration in blood serum or urine samples, or the erythrocyte sedimentation rate (ESR). As previously men-tioned the method was invented by R. Fåhraeus, and the most acknowledged ESR-protocol was introduced by A. Westergren in 1921. It is one of the old-est laboratory tests still in use, being both simple and cheap. The test reflects changes in the concentration of fibrinogens in the blood, which is an unspe-cific indicator of inflammation and tissue injury. An example of a more di-rected protein-assay that has been used historically for monitoring active infections and inflammation is the C-reactive protein assay. The C-reactive protein (CRP) is an acute-phase protein, produced in the liver in response to elevated concentration of interleukin-6, and discovered in the 1930s by Tillet and Francis [92]. The CRP test was originally a simple precipitin test but it can now be performed with specific affinity probes. Standardized immuno-assays allow analysis of a wide variety of protein disease markers. Autoanti-bodies directed against a range of autoantigens are among the diagnostic criteria for autoimmune diseases [93], and glycated hemoglobins are used to monitor progression of diabetes [94]. Pregnancy tests are usually performed by measurements of the hormone human chorionic gonadotropin (hCG) which is produced early in pregnancy. The HCG agglutination test was in-troduced by the young physician Leif Wide in 1960 [95]. It was the first simple test for pregnancy, and the first immunoassay diagnostic kit available on the market.

There has been enormous progress in the field of laboratory diagnostics during the recent fifty years or so. Major scientific inventions like immuno-assays, PCR, mass-spectrometry etc together with rapid development of computer technologies have revolutionized biomedical research, and offered new opportunities for clinical applications as reviewed by Clinical Chemis-try editor Robert Rej [96]. High-throughput technologies make monitoring of global gene expression through mRNA measurements, as well as changes in large sets of protein concentration (mainly cytokines) possible. These techniques have mostly been confined to research laboratories, and they have

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unfortunately not yet made their way into the routine clinical laboratory test-ing.

Detection of pathogens in clinical samplesPCR [97] has revolutionized the detection of pathogens of microbial origin as well as research on infectious diseases by offering fast and specific detec-tion of pathogens with single-copy sensitivity. There are circumstances, however, where the analysis of the surface antigens or the humoral antibody response to a pathogen might be a more valuable diagnostic tool than the detection of the nucleic acids of the pathogen. Examples of these are chronic infectious diseases, strain variability of the pathogen which complicates oli-gonucleotide design for nucleic acid detection, and pathogens that lack nu-cleic acid components (prions). The use of cell tissue culturing is a common means of isolating and identifying microorganisms, but the methodology can only be applied on organisms where in vitro cell culture is possible, the technique may be unsafe due to the handling of potential harmful organisms or impractical. Various immunoassays are widely used for detection of pathogens from clinical specimens today. Most of them offer specific analy-sis but with drastically lower sensitivities compared to PCR. There are many immunologic diagnostic kits available that enable rapid detection of different microbes in only a few minutes. Some of the immunoassay formats are listed below together with examples of pathogens that are assayed with these methods. Direct agglutination is used for detection of typhoid antibodies (known as the Widal test) while hemagglutination is used for detection of certain viral diseases such as influenza and measles. The classical sandwich ELISA is used for detection of a wide range of antigens, like Helicobacterpylori, syphilis and cholera. The indirect ELISA is used for detection of patient humoral immunoresponse, e.g. antibodies against the human immu-nodeficiency virus. Direct immunofluorescence is used for detection of surface antigens of for example Salmonella typhi, Neisseria meningitidis,and rabies. Indirect immunofluorescence, on the other hand, is used for antibody detection for diagnosis of e.g. syphilis [98]. The methods men-tioned above are suitable for detection of a single pathogen species at the time only, the assay procedures are in some cases quite complicated, and the researchers are often left with subjective analysis and interpretation of the results. Ideally, for the detection of an unknown pathogen, analysis should allow simultaneous detection of nucleic acids and protein antigens, prefera-bly the analysis should be carried out in a multiplexed fashion to enable si-multaneous searches for several causative agents at the same time, the results should be easy to interpret, and the assays should be suitable for use in doc-tors offices or in the field without requirements for advanced technical train-ing.

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Present investigations, a background

Proximity ligation, PLA, is a novel protein detection method that was first described by Fredriksson et al. in 2002 [12]. Detection of proteins by PLA depends upon the simultaneous binding of a pair of probes, in a manner similar to PCR and sandwich immunoassays. The free ends of the probes come into close proximity after pairwise binding to the target. The free ends are then lined up by a so-called splint oligonucleotide that hybridizes to and enables joining of the probes by DNA ligase (Figure 1). The newly formed DNA sequence can then be detected and quantified by real-time quantitative PCR (qPCR) [99].

Figure 1. The PLA depends upon binding of a pair of affinity probes, here antibod-ies (black and red). These affinity probes are extended by single stranded oligonu-cleotide probes (black wavy lines), that form a new DNA sequence upon ligation. The newly formed sequence serves as a sort of a barcode that reflects the presence of the specific target protein and that can be detected and quantified by PCR.

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Proximity probes The proximity probes consist of protein binding moieties conjugated to

ligatable single stranded DNA-oligonucleotides, each having either a free 5’ or 3’ end. The protein binding units reported in the paper by Fredriksson et al. were single-stranded DNA aptamers selected from combinatorial libraries through the SELEX procedure. Aptamers are ideally suited for the proximity ligation assay; the extension by the ligatable single-stranded oligonucleo-tides is straightforward, and the SELEX in vitro selection and amplification yields binders with high affinity. Unfortunately, the number of available aptamers suitable for proximity ligation is very limited. The procedure of generating pairs or triplets of aptamers appropriate for proximity ligation via modified SELEX procedure is currently under investigation in our lab (M. Kamali-Moghaddam & O. Ericsson, unpublished results). Protocols for the conversion of pairs of monoclonal antibodies or polyclonal antisera into proximity probes have recently been established (Paper I). The ligatable oligonucleotides can either be covalently linked to the antibodies via chemi-cal crosslinkers or via biotin-streptavidin linkage. The antibody-based probes used in the papers II-IV were solely created by streptavidin-biotin linkage. Examples of covalently conjugated antibody-based probes are shown in pa-per I.

Assay formats The assay can be performed in a homogenous or heterogeneous fashion,

as described previously [12, 100]. The homogenous assay requires no washes or phase separations, and it offers fast analysis with minimal hands-on time and only two additions of reagents. The heterogeneous format can provide measurements with lower limits of detection and larger dynamic range than the homogeneous assay, and it allows removal of agents that in-hibit the reaction. The heterogeneous assay format was mainly used in paper II.

Biological samplesWe have mainly evaluated PLA for the analysis of cytokine and growth

factors in different types of biological samples. The platelet derived growth factor (PDGF) has been assayed in cell culture media, spinal fluid and serum samples as described by Fredriksson et al. PDGF levels have also been monitored in human keratinocytes [101], kidney glomeruli cells [102], and recently in neural stem cells (A. Erlandsson et al. submitted), and embryonal tissue (M. Enarsson et al unpublished results). Papers I-IV describe the PLA analysis in various other complex biological samples. Minimal changes have been made to the standard assay protocol to adapt the assay to the analysis of each of the biological samples. All of these changes are described in papers I-IV.

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Present investigations

Figure 2. The areas of the four studies I-IV presented in this thesis

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Paper I. Cytokine detection by antibody-based proximity ligation. The first paper on proximity ligation [12] described the very sensitive and specific protein detection provided by the proximity ligation mechanism. The proximity probes that were used for the protein detection were DNA aptamers, generated by the SELEX procedure.

This study describes approaches to the generalization of the method, by introducing protocols for the conversion of any polyclonal or matched monoclonal antibody pairs into proximity probes. The conjugation of the single-stranded proximity probes was performed either by covalent crosslinking or via biotin-streptavidin interaction. The covalent conjugation of monoclonal antibodies by succinimidyl-4-(p-maleimidophenyl)-butyrate crosslinker and detection of human insulin with the covalently conjugated proximity probes is described in the study. The conjugation of proximity probes to antibodies via biotin-streptavidin was performed by conjugation of thiol-modified proximity probes to maleimide-derivatized streptavidin, fol-lowed by incubation with a biotinylated antibody.

Affinity purified and biotinylated antibodies raised against interleukin 2 (IL-2), interleukin 4 (IL-4), vascular endothelial growth factor (VEGF), and PDGF were split in two aliquots each, and conjugated by biotin-streptavidin interaction to generate proximity probes having either free 5’ or 3’ ends. The limit of detection was at the low femtomolar level for all assays. The per-formance of the assays was furthermore investigated in biological samples. The limits of detection for IL-2 and IL-4 were not affected by the presence of 100% FCS in the analysis. VEGF analysis on cell supernatants from em-bryonic kidney cells induced by hypoxia together with normoxic controls correlated well with results generated by a commercial ELISA.

Glomeruli tissue samples from conditional PDGF knockout mice and wild type mice were analyzed for PDGF using proximity probes based on poly-clonal antibodies raised against PDGF, and the results correlated well with previously reported results [102] where DNA-aptamers were used for detec-tion of PDGF in the same samples. Many biological samples are available in minute amounts only, and some of the glomeruli samples could not be ana-lyzed by conventional ELISAs due to limiting sample amounts and insuffi-cient sensitivity of the analysis technique.

We evaluated the relation between proximity probe concentration, protein binder affinity, and the signal to noise ratio. The results were calculated us-ing experimentally determined background and a formula describing the amount of complexes formed between target proteins and a pair of the prox-imity probes, given the dissociation constant for the affinity probes. The calculations identified the optimal reagent concentrations to achieve the highest signal/noise ratios. Irrespective of the affinities of the ligand binders,

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low concentrations (5 pM) of both proximity probes give rise to the highest signal/noise ratios.

Perspectives on paper IAntibodies remain the primary choice for affinity probes in various methods for protein-detection. Antibodies are well designed by nature for specific detection of proteins, and the development of protocols for conversion of antibodies into proximity probes greatly increases the flexibility of the prox-imity ligation assay.

Monoclonal antibodies are in many ways more suitable than polyclonal antibodies for protein detection, due e.g. to the homogeneity of the reagents leading to low intra- and inter-lot variability, and highly reproducible ex-perimental results. Nonetheless, polyclonal antibodies are quite suitable as a source for affinity probes to be used in proximity ligation. Individual poly-clonal antisera typically recognize mixtures of epitopes on the target protein which is advantageous for the proximity ligation mechanism that requires binding of a pair of probes to the same target molecule. Also, the polyclonal antibody preparations are typically more tolerant to slight denaturation of the target proteins than are monoclonal antibodies.

As described earlier in this thesis, the affinity of the protein binding moie-ties is a very important factor. As presented in paper I, a ten-fold increase in sensitivity for each of the proximity probes reduces the limit of detection 100-fold. If the dissociation constants are known for the affinity probes, the limit of detection can be calculated. However, this is true for covalently con-jugated antibody probes only. Proximity probes generated by biotin-streptavidin linkage may form aggregated structures that affect the affinity and the sensitivity of the detection.

The quality of the antibody conjugation is extremely important for the ex-perimental results, free DNA strands and antibodies in the proximity probe preparation affect both the signal and the background of the analysis as demonstrated in the paper.

Streptavidin-biotin conjugated proximity probes are convenient for many detection purposes. Many antibody manufacturers offer biotinlyated antibod-ies, the coupling to streptavidin-DNA conjugate is straightforward, and the antibody conjugates can be stored at 4ºC for over a year without a decrease in performance. For some purposes, covalent crosslinking might be a better choice for generating proximity probes. The antibody-streptavidin-complexes can be quite bulky, and affinity probes relying on biotin-streptavidin binding are probably not a good choice for many cell-based applications.

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Figure 3.The limit of detection (LOD) for proximity ligation in comparison to three other technologies. The LOD expressed as concentration of target protein is shown on the X-axis, and the Y-axis illustrates the amount of target detectable. From a review article: Proximity ligation assays for sensitive and specific protein analyses [100]. Illustrated by Simon Fredriksson.

The limit of detection is very low for the four cytokines and growth factors analyzed in this study. The LOD for analysis of all of these proteins by PLA in comparison with three other methods, Western blotting (R&D Systems), commercial individual ELISAs (R&D Systems), and a multiplexed sandwich immunoassay with rolling-circle amplification of signals in an array format [103] are shown in figure 3.

The low limit of detection and sample consumption (1 µL) allows analy-sis of biological samples where the amount available is very small, and not analyzable by other common immunoassays. Minimal changes have been made to the standard assay protocol for detection of biological materials. Many samples of biological origin may be difficult to analyze by homogene-ous PLA, however, due to the complexity and agents inhibiting the reaction. There are several known immunoassay-interferents present in human body fluid samples, antigen-mimicking molecules like analyte fragments or differ-ent isoforms, or protein-binding agents like autoantibodies, heterophilic an-tibodies, and carrier proteins [104]. Heterogeneous PLA might provide a

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convenient way of removing inhibitory agents by washes and concentration of the target protein on a solid surface.

Paper II. Detection of individual microbial pathogens by proximity ligation In this paper we describe how PLA can be utilized for specific detection of individual molecules of virus and bacteria.

Protein-based pathogen detection methods can provide valuable informa-tion about ongoing infections, but the methods available, mainly cultivation, isolation, agglutination tests and ELISAs, are much less sensitive than nu-cleic acid based methods, for example PCR. We describe how monoclonal antibodies raised against surface antigens of a parvovirus and an intracellular bacterium, previously tested and validated in capture ELISAs, can be used for highly sensitive detection of these pathogens (figure 4).

The assay can be performed in a homogenous format with only two addi-tions of reagents, or in a heterogeneous format. In the heterogeneous assay, the pathogen is captured via a biotinylated antibody to a streptavidin coated surface of a 96-well microplate. The heterogeneous format allows removal of inhibitory agents through washing and achieves increased assay sensitiv-ity, partly due also to removal of unbound reagents that cause a background signal in the homogeneous format. We compared the assay performance using only one monoclonal antibody with assays where two or even three monoclonals were used. The assays with one and two monoclonals were performed in a homogeneous format, and the one with three monoclonals was used a heterogeneous assay where one of the antibodies was used to capture the agents on a solid support. The assay specificity increases when more probes are used in the analysis and the limit of detection is reduced due to decreased background as a result. A heterogeneous assay with three monoclonal antibodies provides a highly sensitive and specific assay for pathogen detection in complex samples, with the possibility to remove in-hibitory agents by washings. On the other hand the homogenous assay offers rapid analysis with only two reagent additions and no time-consuming blocking or washing steps. By diluting solutions of known titers of the virus and the bacterium, the limit of detection (LOD) could be investigated. The LOD of PLA was compared to that of the ELISAs and real-time PCR analy-sis on the genomes of the pathogens. The PLA was shown to be far more sensitive than the ELISA analysis and equally sensitive as real-time PCR of the nucleic acid sequences of the infectious organisms. We further analyzed sets of positive and negative field samples for each pathogen, and compared the results with previous findings.

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Perspectives on Paper II There is a need for reliable detection methods for pathogens both in research and in diagnostics, and protein-based microbe detection methods are impor-tant complements to nucleic-acid based detection technologies. By choosing good sets of affinity binders for analysis by PLA in a homogeneous or het-erogeneous format, even single copies of microbes can be detected in com-plex biological samples like tissue or fecal samples.

PLA should also afford advantages for serotyping microorganisms, by identification of the different strains of the pathogen using sets of affinity binders that target the strains specifically. This approach has been investigated through detection of different strains of foot and mouth disease virus (A. Nordengrahn unpublished results).

Figure 4. Pathogen detection by proximity ligation. Repeated protein determinants present on the surface of the pathogen to be analyzed (green) are bound by several pairs of proximity probes, with either free 5’ or 3’ ends (black or grey ribbons). The assay enables detection of individual pathogens.

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Specific and sensitive multiplexed pathogen detection assays are desperately needed both in human and veterinary medicine as well as for biowarfare detection. Clinical symptoms are seldom specific for single pathogens, so multiplexed detection can provide quick diagnosis in situations where sev-eral pathogens can cause the symptoms, e.g. viruses causing acute respira-tory tract infections, which is a significant cause of mortality. If the proximity ligation mechanism could be successfully developed for multiplexed detection it would most certainly possess major advantages over other techniques for pathogen detection in diagnostics and for monitoring of biowarfare agents, given its unique specificity and sensitivity on the level of protein detection.

Another important issue, especially in the field of veterinary medicine, is assay cost. National or international eradication programs are frequently executed in order to eliminate infectious agents in livestock. The programs exist until the disease-causing organism is eradicated, and the country is declared free of the disease. When the eradication programs are in progress, large numbers of animals are screened for the pathogen, and positive animals are slaughtered. Mass animal testing, a heavy economic burden on farmers, demands cheap assays, and today ELISA-based screening can be performed for 1-2€/sample in eradication programs. The costs for ELISA-based diag-nostics for other applications may be up to 10€/sample (M. Merza, senior vice president Svanova Biotech AB, personal communication).

The assay cost also plays a role in general veterinary medicine as it is not economically favorable to spend more on an animal than the meat price paid by the slaughterer. This reasoning usually doesn’t apply on companion ani-mals as their owners are willing to pay more for the welfare of the animals.

By using alternative means for detecting the ligation products in the PLA procedure, assay costs could certainly be reduced. Multiplexed proximity ligation assays with a signal amplification mechanism other than PCR could therefore become a promising alternative for monitoring diseases in live-stock. The high sensitivity of the assay could moreover enable sample pool-ing and thereby save analysis costs.

In pathogen detection the most costly events are false positives when en-tire cattle stocks are needlessly slaughtered. Lowering the risk of generating false positive results would dramatically reduce the costs associated with slaughtering of livestock.

In future visions, the incorrect evacuation of entire cities from biowarfare threats would be vastly expensive. Assay specificity, in terms of lower num-bers of false positives, could be improved by simultaneously detecting the virus/bacteria particle surface proteins with PLA and its nucleic acid by standard qPCR. Only samples scoring positive in both assays would be con-sidered truly positive. Perhaps could both assays even be run in multiplex in the same reaction tube.

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Paper III. Characterizing the sequence specificity of DNA binding proteins using proximity ligation Chromatin immunoprecipitation [105, 106] together with DNA microarray readout (ChIP-on-chip) [90] is a powerful method for in vivo analysis of interactions of transcription factors with their cognate promotor elements on a genome-wide scale. The resolution of such analysis is fairly poor however; sonication is utilized for fragmentation of the genomic DNA which gener-ates fragments that may be up to several hundred base pairs in length. Com-puter programs are used for retrieving the putative binding sequence within each DNA fragment, and the sequence binding specificity of the protein is then tested in vitro, mainly by electrophoretic mobility shift assay (EMSA) [84]. EMSA suffers from the drawbacks that reagent and sample consump-tion is high, and the assay procedure is complicated and time-consuming. It may be practical when analyzing only a few sequences at the time, but high-throughput analysis of hundreds or even thousands of sequences is not pos-sible.

Figure 5. Analysis of protein-DNA interactions via proximity ligation. A DNA binding protein (green) binds a partially double-stranded target DNA oligomer hav-ing a single-stranded extension with a free 3’ end (grey ribbon). A proximity probe, consisting of an antibody against the DNA binding protein conjugated to an oli-gonucleotide with a free 5’ end (black ribbon) can also bind the DNA-binding pro-tein. Upon simultaneous binding, the free DNA ends come into proximity and can be ligated together by a DNA ligase. The newly formed DNA sequence can then be amplified and detected by PCR.

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We have established a protocol for analysis of interactions of DNA binding proteins with promotor elements. The protein-binding sequence to be ana-lyzed consists of double-stranded oligomers of 20-25 basepairs, that are ex-tended by a single-stranded proximity probe sequence with a free 3’ end. The other proximity probe consists of an antibody raised against the DNA-binding protein, conjugated to a single-stranded proximity probe sequence with a free 5’ end, coupled via streptavidin-biotin linkage. The 3’ free prox-imity probe is incubated with a nuclear lysate containing the DNA-binding protein. The 5’ free proximity probe, consisting of the antibody-conjugate, is then added to the incubation. Binding of the DNA-binding protein to the proximity probe with a free 3’end, and subsequent binding of the antibody-based proximity probe having a free 5’ end to the DNA-binding protein, leads to close proximity of the free ends of the probes. These can be ligated in a joint ligation and PCR step, and the newly formed DNA strand can be detected and quantified by real-time PCR (figure 5). In this study we ana-lyzed DNA-binding interactions of three proteins, p53, HNF4- , and USF-1. P53 was used as a model system, as the protein and the promotor sequences that the protein binds to are well characterized. The interactions were ana-lyzed in nuclear lysates and the lysates were serially diluted and analyzed in order to investigate the limit of detection. All experiments included negative controls, consensus sequences with mutated binding sites, promotor se-quences specifically bound by other proteins, and short probes consisting only of the double stranded probe part, used to outcompete the positive sig-nal.

The consensus binding sites of HNF4- and USF-1 on a genome wide scale have recently been analyzed by ChIP-on chip [107]. The results gener-ated by PLA were found to correlate well with those obtained by EMSA for analysis of binding specificities of HNF4- and USF-1 using three (HNF4-

) and five (USF-1) putative binding sequences. We next chose fifty sequences, randomly selected from the list of HNF4-

positives as analyzed by ChIP-on-chip, designed proximity probes that included the sequences, analyzed them by PLA, and compared the results with the computer prediction of the data. The PLA results correlated well with the computer predictions, but the results did not, however, correlate with the chip signal intensities. This could be explained by the fact that the DNA fragments from the chip-on-chip experiments consist of genomic DNA, and the lengths are very different from those in the in vitro-basedproximity ligation assay.

Perspectives on study III.We have shown that PLA can be used for analysis of interactions between transcription binding factors and their cognate promotor sequence elements.

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The method can be useful for in vitro verification of results generated invivo, such as from ChIP analysis with microarray readout. The method might also be useful for the evaluation of effects of polymorphisms in known tran-scription factor binding sites. PLA has many obvious advantages over exist-ing methods for in vitro analysis of protein-DNA interactions. PLA is a non-radioactive assay, the reagent consumption is very low, and the very high sensitivity enables the analysis of as few as one to ten cells. The assay pro-cedure takes less than four hours, including the PCR and ligation step which takes a couple of hours. The analysis is performed in homogenous format; the hands-on time is very limited with only three additions of reagents, which makes it suitable for automation.

The analysis of hundred and fever sequences is possible in the assay for-mat described above. A multiplexed setup would be needed for truly high-throughput analysis, with each DNA molecule with single stranded 3’ end sequence coded with a unique ID-tag. PCR amplification would then be car-ried out with common primer pairs and the products sorted on DNA tag mi-croarrays. The number of transcription factors per analysis could also be increased by the introduction of a protein-specific tag sequence in the prox-imity probe consisting of the antibody conjugate.

So far we have focused on analysis of transcription factors with synthetic DNA probes, in a fashion somewhat similar to EMSA. We are planning to develop the assay further to analyze binding of transcription factors to ge-nomic DNA on a global scale; with sequence information retrieval through high-throughput sequencing (J. Schlingemann unpublished work).

Study IV. In vitro analysis of inhibition of the interaction between VEGF and VEGFR-2 using proximity ligation.Receptor-ligand interactions constitute approximately 60% of the estimated proteinaceous drug targets, and investigations of these interactions are of a obvious importance for the development of compounds that target the bind-ing. Angiogenesis, and especially VEGF signaling through its receptors, is an attractive target in cancer treatment, and there are currently clinical trials ongoing for a number of inhibitors of VEGF signaling applied in several different cancers.

An example of a therapeutic drug directed against VEGF is the famed drug Avastin™ (Bevacizumab). It is a monoclonal antibody that inhibits VEGF, and that has been shown to improve progression-free survival when used in combination with chemotherapy in the treatment of various cancers, compared to chemotherapy alone [108, 109].

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The drug candidates are not necessarily of antibody origin only, modified nucleic acid aptamers selected against VEGF by the SELEX procedure have been shown to bind to the protein with high affinity and effectively inhibit the binding of VEGF to its receptors [110]. The creation of low molecular weight drug candidates that mimic regions of the contact surfaces between the interacting proteins is also a subject of intense investigation [111].

In this study we analyzed the interaction of VEGF-A with two of its re-ceptors, VEGFR-1(Flt-1) and VEGFR-2 (KDR) by PLA. The VEGFR-2 is the main inducer of proliferation, survival and vascular permeability [112]. Accordingly targeting of the interaction of VEGF with VEGFR-2 is of great importance [113-115]. We analyzed the inhibitory effects of a number of agents that target VEGF-A or VEGFR-2 and thereby block the receptor-ligand binding. We studied a couple of antibodies, a DNA/RNA aptamer, a low molecular weight compound, and a recombinant growth factor. The specificity was tested by the use of inhibitors raised against related proteins, serving as negative controls.

The results were compared with those obtained by immunoblotting of immunoprecipitated phosphorylated anti-VEGFR-2 antibody in porcine aor-tic endothelial cells expressing VEGFR-2. We calculated the approximate 50% neutralization dose (ND50) for each of the inhibitors by PLA and by immunoblotting of the phosphorylated VEGFR-2.

The analysis of the inhibitors by PLA generate dose-response data com-parable to those obtained by immunoblotting of the phosphorylated VEGFR-2, although the actual ND50 values differs somewhat, presumably because of the lower concentrations of VEGF used in PLA.

Perspectives on study IV The development of a new drug is a costly business; typically it may take 15 years and cost on average $880 million, as reported by Boston Consulting Group 2001 [116]. The sizes of drug-candidate libraries generated by com-binatorial chemistry demand development of techniques suitable for auto-mated high-throughput screening. There has been a drive towards miniatur-ized assays, with slimmed reagent consumption and assay costs. According to Perkin Elmers CSO Neil Cook who highlights the importance of highly informative screening methodologies [116], high-throughput analysis must be complemented. Many tools can be used in the procedure of reducing the complexity of drug candidate libraries to a handful of compounds that can be evaluated carefully in vitro and subsequently in vivo. Mass spectrometry as well as NMR analyses together with in silico screening helps bring out in-formation of the characteristics of target binding sites [117-119], one exam-ple being ligand docking to cell surface receptors.

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The most promising compounds could then be tested, preferably in a ho-mogenous in vitro setup. The candidates that pass the test can be evaluated in cell-based assays and by more extensive and costly in vivo studies.

Rapid, inexpensive, and highly informative homogenous in vitro assaysare thus important steps in the development of protein-targeted drugs. Prox-imity ligation is an assay that allows medium-throughput analysis of com-pounds with dose response curves, possibilities to calculate ND50 values as well as the evaluation of binding kinetics of the drug compounds.

The throughput of the assay is currently limited by the PCR step. With the generation of dose responsive curves for inhibitors of ligand-receptor bind-ing with duplicate measurements, ~30 inhibitors using 96 well plates or ~120 inhibitors using a 384 well plates could be analyzed / person*instrument* 8 hour working day. The candidates scoring high in the in vitro assay could subsequently be analyzed in situ, using the same affinity binders. The prox-imity ligation in situ assay (P-LISA) (Söderberg O, Gullberg M, Jarvius, M et al submitted) is a promising alternative for in situ analysis of drug effetcs at the cellular level, or even localization of the drug and its effects directly in tissue samples from treated subjects.

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Future perspectives

Recent progress in the fields of molecular biology, genomics, proteomics, and computer technologies make it possible to address previously unmet needs in biomedicine, and international research bodies promote research through global collaboration paving the way for new discoveries. One exam-ple of an organized effort in the field of proteomics is the human proteome organization initiative, HUPO (www.hupo.org). HUPO unites leading labo-ratories in life sciences, bioinformatics, mass spectrometry, systems biology, pathology, and medicine, in large-scale proteomics projects aimed at unrav-eling fundamental biological processes and studies of human diseases through technologies in proteomics. The Swedish Human Proteome Re-source Center (HPR) has launched a program through collaboration between scientists at the Royal Institute of Technology in Stockholm and the Uppsala University that aims at high-throughput generation of mono-specific anti-bodies against all human proteins with tissue profiling. The project is de-scribed in more detail at the homepage www.proteinatlas.org.

The methods available for protein detection are not sufficiently stream-lined for the necessary automation that could allow truly large-scale protein analysis, so development of new technologies is ultimately the key to pro-gress in proteomics.

Proximity ligation is a promising method, with a wide variety of possible applications. I have described in this thesis how pairs of monoclonal anti-bodies or polyclonal antibodies can be converted into proximity probes, and how microbial pathogens can be detected by proximity ligation in complex field samples. Furthermore how proximity ligation can be used for the detec-tion of transcription factors with gene promotor elements, and the inhibition of receptor-ligand interactions.

The success in the field of pathogen detection as well as the analysis of other protein disease markers depends on the multiplexing capacity of the assay, and the PLA is currently being adapted for multiplexed cytokine analysis in biological samples (S. Fredriksson, unpublished results). A few technical issues need to be solved, good protocols for conjugation of affinity probes to the proximity probes are crucial, different chemical properties of the target proteins may demand different assay conditions, and the proximity probe sequence design must be carefully undertaken.

The analysis of some biological samples may benefit from solid-phase as-say formats. Solid-phase setups offer improved specificity and sensitivity of

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the analysis as well as the removal of inhibitory agents. The dynamic range of the assay increases as the probe concentration can be increased without adverse effects on the background. The “hook effect”, typical for homoge-neous immunoassays, the effect of the saturation of proximity probes that limits proximity probe-target complex formation, can moreover be avoided in a solid-phase setup. The assay format poses some technical challenges, careful blocking of the solid support is necessary in order to minimize unde-sired adsorption of the antibody reagents to the surface. Furthermore the immobilized affinity probe may be distanced from the solid surface with the aid of a spacer, which can lead to increased flexibility and improved antigen capture efficiency. The multiplexed proximity ligation approach could also benefit from the solid-surface assay format, due to larger dynamic range and removal of the characteristic immunoassay “hook effect” that would allow measurements of samples without extensive dilutions.

The proximity ligation assay is dependent upon highly specific and high-affinity binders for the creation of proximity probes. The sensitivity of the assay can be calculated from the dissociation constant for the affinity binder as described in the thesis, which provides a tool for prediction of the assay performance and for the choice of suitable reagents for analysis.

One might ask when extremely sensitive measurements of protein targets are important. Leigh Anderson and Norman Anderson discuss the clinical importance of measurements of low, medium, and high-abundance proteins in their article “The Human Plasma Proteome, history, character and diag-nostic properties” [120]. Some cytokines, as well as other mediators of sig-naling pathways, bind with very high affinities to their cognate receptors, so their active physiological concentrations have so far been estimated to be very low. The local concentrations could however be much greater than ex-pected. The cytokine IL-2, both produced and used by T-cells in a process referred to as autocrine secretion, has been estimated to reach a local concen-tration of 1-100 mM which is much higher than the 10 pM dissociation con-stant for the binding to the Il-2 receptor [121]. It may on the other hand be very important to be able to analyze small amounts of foreign proteins, of microbiological pathogens and parasites entering the circulation, one exam-ple being the infectious form of prion proteins.

A triple binder proximity ligation assay has recently been developed (E. Schallmeiner unpublished results) that allows extremely sensitive measure-ments of low-abundant proteins in body fluids. Due to its requirement for simultaneous binding of three different probes to distinct epitopes on the target molecule, it can also be used for analysis of protein complexes.

Proteomics analysis is not limited to detection and quantification of pro-teins only. Genomics and medicine professor Stanley Fields gives his defini-tion of the proteomics term in the article “proteomics in genomeland” [122]: “Proteomics includes not only the identification and quantification of pro-teins, but also the determination of their localization, modifications, interac-

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tions, activities, and, ultimately, their function." The proximity ligation has already proven useful for in situ analysis and localization of endogenous proteins and protein-protein interactions in individual cells and tissue sam-ples. (P-LISA; O. Soderberg, M. Gullberg, and M. Jarvius submitted). P-LISA has also provided means to analyze modifications of proteins in indi-vidual cells (M. Jarvius, unpublished). The proximity ligation mechanism has thus shown to be valuable for analysis of protein concentrations, interac-tions, localizations, and modifications, and I believe that it might also be-come a useful tool for the remaining fields of protein analysis.

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Acknowledgements

I’m very grateful that I got the opportunity to do my PhD at the De-partment of Genetics and Pathology under the supervision of Ulf Landegren. It has been immensely fun to experience the traditions of the student life here in Uppsala and the social activities at the student nations. The laboratory is however the place where I’ve spent most of my waking hours during the last few years, and the work has been a sheer pleasure thanks to my colleagues. They all deserve a big thank you.

I would like to express my gratitude to:

My supervisor Ulf Landegren, for all the encouragement, your con-fidence in my work, and your infectious interest in science. Thanks for providing such a creative and fun working environment!

Mats Nilsson, associate professor, for interesting discussions on sci-ence and non-science in front of the white board in the corridor. And for occasionally lending me your lovely pets.

The Landegren/Nilsson group, Jonas Jarvius for help with computa-tional and electrical problems. Masood for your excellent cloning as-sistance, and for spreading joy in the lab. Ola Söderberg, former fish-industry worker in Iceland, for help with cloning and antibody-stuff. Edith, for help with various proximity ligation problems and for fun times at conferences, especially the Florence conference. Malin for sharing your terribly good taste of sweets (liquorice-chocolate fusions) with your husband, and your great BBQ parties. Jenny for introducing me to the blob-business, and for good company together with Chatarina at the Santorini conference (with the mental hats) last sum-mer. Henrik and Jonas Melin, that sometimes lock themselves inside a room with a big and very expensive mill (fräs), and Olle for discus-sions on the splint effect, and great parties. Johan Stenberg, for ex-plaining the importance of titles and the horrors of “särskrivningar”.

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Mathias, for your optimistic view on most things. Lena, for keeping order on everything in the lab and for excellent collaboration on vari-ous projects. Carolina R. for the organization of the EMBO stuff. Our “new” people: Tim, Magnus, Sara, Kalle, Anders, Carolina W., Mia, Jörg, and Katerina. I’m looking forward to get to know you all better. Thanks Jörg for a great collaboration on the protein-DNA project.Lore, your presence at the groupmeetings is very rewarding.Thanks to the former members of the groups, DOA, Anette, Maritha, Anders I., Marek, Andrey, Simon, Johan Banér, Fredrik and Mats Gullberg. Simon, my co-supervisor, for introducing me to the wonders of proximity ligation and to the Janne Vängman movie genre, and for your hilarious after parties. Johan Banér, for all the jokes, and for keeping track on the departure to Farmen, 11:30 sharp every day. Fredrik Dahl, king of Escobar, for uniting the whole HUGO confer-ence (even the British ladies) in Montpellier in a conga dance. Mats Gullberg for always keeping track on the literature.

The Olink people, for interesting discussions and collaborations on various projects.

My hardworking students, especially Nora, Rebecka, Henrik,Jonas H., Lucas, and Andreas.

My collaborators, Ann Nordengrahn and Malik Merza, for excellent collaboration on the pathogen detection. Ann for teaching me a lot about viruses and ELISAs and the trip to Pirbright last year. Alvaro Rada-Iglesias and Claes Wadelius for the collaboration on protein-DNA interaction, and Stefan Wennström and Maria Hägg for the work on the VEGFR-2 inhibition. Ola Rollman and Sofi Forsberg, Mathias Bjarnegård and Christer Betsholtz, Karin Forsberg-Nilson, Mia Enar-son, Nils Heldin, Kristoffer Rubin, and Dominique Vanhence.

The Ann-Christine Syvänen-group, especially Chrisse, Lovisa, Katarina, Karin, Lotta, Annika, Lily, Johanna, Andreas, Ulrika, and Per, for the great joint group-meetings, fun parties, and good company at conferences.

The Department of Biochemistry and Molecular Biology, Univer-sity of Iceland, Jón Jóhannes Jónsson (J3), Jónína, Gummi, Gunni, Helga, Hans Þormar, Hans Tómas, Eiríkur, Barki, Alex, Lina, and Aðalheiður; for introducing me to laboratory work and to the wonders

41

of molecular biology. The pubs with the dancing on the tables, the horrid-email-club, and the regular visits to the University Hospital cantina when lasagna was served.

Uppsala friends, The UGSBR class 2000-2001, especially Frida (part of the Simons Slaves-team), Larry, and Adam (from a later UGSBR class) for being such good friends. Good luck with your theses! Caro-line and Per, for your marvelous dinner parties and Comitia for the very secret and very fun activities. The people from the chamber mu-sic course for the great autumn term 2001. Arna og Karvel, and re-cently ASK, for all the fun times, for the trips to Sälen, Öland, and the many Settlers tournaments.

Ingileif, Inger Rós, Hrönn, Hrafnhildur, Rannveig, Steinunn, Lóa, Margrét Vala & Magga, My darling friends that I have spent many happy hours together with during the last 14 years. Thanks for the parties, the camping tours, and the trips abroad during my stay here in Sweden. And for the very entertaining visits to the cottage on the is-land of Mön, the summers of 2003 and 2005

The Svanholms, for introducing me to the Snerike-nation and the Snerike-choir, and for always making me and Snævar feel welcome at Pilvägen 25.

My family in Iceland, for the constant support and all the encourage-ment and the parcels that I’ve regularly received with Icelandic news-papers and candy.

Snævar, for agreeing on moving to Uppsala together with me, we’ve had great fun here during the last five years. Thank you for patiently describing what to do when the computer turns moody in the middle of the thesis writing, and thanks for taking care of all the cooking dur-ing the last few months. Fdm.

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