ELISA based methods for the measurement of circulating high molecular forms of Acrp30

Transkript

1 UPTEC X ISSN APR 2006 SOFIE OLANDER ELISA based methods for the measurement of circulating high molecular forms of Acrp30 Master s degree project

2

3 Molecular Biotechnology Programme Uppsala University School of Engineering UPTEC X Date of issue Author Sofié Olander Title (English) ELISA based methods for the measurement of circulating high molecular forms of Acrp30 Title (Swedish) Abstract Adiponectin is an adipocyte-secreted hormone used as a marker for the metabolic syndrome. Adiponectin exists in several isoforms ranging from monomers up to large oligomers. Scientists have come to the conclusion that the ratio between the high molecular form of adiponectin and total adiponectin is the most significant marker. In this degree project four strategies for the measurement of high molecular weight forms of adiponectin were evaluated. One strategy was deemed successful, two needed further studies and the fourth was proven unsuccessful. Keywords Adiponectin, Acrp30, ELISA, T-Cadherin, Proteinase K, MAIIA Supervisors Hanna Johansson, Annika Andersson Mercodia AB Scientific reviewer Project name Leif Jansson Institutionen för medicinsk cellbiologi, Uppsala univeristet Sponsors Language ISSN English Supplementary bibliographical information Security Classification Pages Biology Education Centre Biomedical Center Husargatan 3 Uppsala Box 592 S Uppsala Tel +46 (0) Fax +46 (0)

4

5 ELISA based methods for the measurement of circulating high molecular forms of Acrp30 Sofié Olander Sammanfattning Adiponektin är ett hormon som utsöndras från fettceller i kroppen och är involverat i ämnesomsättningen. Proteinet existerar i flera former, från enstaka molekyler till komplex av tre (Low Molecular Weight - LMW) och även större komplex (High Molecular Weight - HMW). Friska individer har relativt höga nivåer av adiponectin i blodet, medan låga nivåer är kopplade till sjukdomar involverade i det metabola syndromet, vilka är fetma, diabetes typ 2 (även kallat åldersdiabetes) och hjärt- och kärlsjukdomar. Idag mäts den totala mängden adiponektin, alla olika komplex, som en markör för dessa sjukdomar. Studier har dock visat att förhållandet HMW/total adiponektin har en bättre korrelation till det metabola syndromet än den totala adiponektinkoncentrationen. Hög andel HMW visar på minskad risk för sjukdomar såsom diabetes typ 2, fetma och hjärt- och kärl sjukdomar. Mätning av HMW adiponektin är idag laborativt omfattande och mycket tidskrävande, de utgörs ofta av kromatografiska separationer och liknande tekniker. Därför finns det nu en stor efterfrågan på ett enkelt test för att mäta HMW adiponektin. I examensarbetet utvärderades fyra olika strategier för mätning av HMW adiponektin. Strategierna är baserade på antikroppar. Antikroppar är en del av kroppens egna immunförsvar, som känner igen icke kroppsegna ämnen genom att binda till dem. Två av strategierna är konstruerade med ELISA (Enzyme linked immuno sorbent assay) som plattform, en bygger på detektion med en HMW-specifik receptor och den sista på MAIIA II teknologin (Membrane assisted isoform immuno assay II). Examensarbetet visade att en av de fyra utvärderade strategierna, MAIIA II, har god potential. Tekniken prövades aldrig i projektet, men samtliga kriterier för den är uppfyllda. Övriga strategier kräver vidare studier med de olika komplexen var för sig. Examensarbete 20 p i Molekylär bioteknikprogrammet Uppsala universitet April 2006.

6

7 1. Introduction Metabolic syndrome Adiponectin Physiological and biochemical properties Activity Aim of study How to separate HMW from LMW Strategies used in this master s degree project Immunoassays Sandwich ELISA Competitive ELISA Membrane Assisted Isoform ImmunoAssay II (MAIIA II) Materials and methods Antibodies Antigens Buffers Receptor Enzyme Coating Labelling Biotin labelling Conjugation Separation and purification of recombinant and endogenous adiponectin Ammonium sulphate precipitation HiPrep Desalting Ion-exchange chromatography Gel-filtration Electrophoresis ELISA Competetive assay Sandwich assay Experiments Affinity Cross reactivity Purification of endogenous adiponectin Separation of recombinant isoforms of adiponectin High molecular weight tests - strategies Double monoclonal Receptor-based MAIIA II Enzyme-pretreatment Results Affinity

8 4.2 Cross reactivity C1q Mammalian Purification of endogenous adiponectin Ammonium sulphate precipitation Ion-exchange chromatography Gel filtration Separation of recombinant isoforms Ion-exchange chromatography Gel filtration High molecular weight assays - strategies Double monoclonal Receptor-based MAIIA II Enzyme pretreatment Discussion Affinity Cross reactivity Purification and separation of endogenous and recombinant adiponectin Double monoclonal assay Receptor-based assay MAIIA II Enzyme pretreatment Conclusions Acknowledgements References Appendix

9 1. Introduction This master s degree project has been carried out at Mercodia AB. The company was founded in 1991, is management owned and situated in Uppsala, Sweden. Mercodia AB develops, manufactures and markets high quality diagnostic immunoassay kits. The company specializes in ELISA assays for clinical as well as research applications, notably within cardiovascular disease, obesity and diabetes. These three diseases are parts of the metabolic syndrome. 1.1 Metabolic syndrome The so called metabolic syndrome is due to disturbed metabolism and consists of obesity, coronary disease and type 2 diabetes. The symptoms of this condition include: Hypertension Increased body weight/obesity Insulin resistance/glucose intolerance Dyslipidemia (High blood levels of LDL - Low density lipoprotein, low blood levels of HDL - High density lipoprotein, high blood levels of triglycerides) Prothrombotic state (High blood levels of fibrinogen or plasminogen) Proinflammatory state (High blood levels of C-reactive protein)[1] Thus, insulin, LDL, plasminogen, fibrinogen and C-reactive protein are examples of markers in blood used for diagnosis. The aim of this degree project was to evaluate the possibility to establish a diagnostic tool for another marker, adiponectin. 1.2 Adiponectin Also called: Acrp30 (30 kda adipocyte complement-related protein), GBP28 (Gelatin-binding protein), apm1 (Adipose most abundant gene transcript 1) [2] Physiological and biochemical properties Adiponectin is an adipocyte-secreted hormone, consisting of 244 amino acids with a molecular weight of approximately 30kDa (28-30kDa). It is one of the most abundant proteins in human blood, with an average concentration of 5-30µg/ml in healthy individuals [3]. The protein consists of four domains: one globular C-terminal, one collagen-like N-terminal, one signalling peptide and one hyper variable domain [4] (Figure 1). 4

10 QuickTime och en TIFF (okomprimerat)-dekomprimerare krävs för att kunna se bilden. Figure 1. Adiponectin monomer, modelled in imol. Adiponectin exists in different isoforms: monomers, trimers, hexamers, larger oligomers and isolated globular form (Figure 2, 3) [5]. Monomers associate to a trimer through the globular domain. Trimers associate to larger oligomers through the collagen like domain [6]. The protein has a ball and stick -like structure, where the C-terminal globular domain is the ball and the collagen-like domain is the stick (Figure 2)[5]. Figure 2. Freeze-etched rotary replicas of adiponectin. A: Trimer, B: Hexamer, C: Oligomer. Magnification is 70000x. Illustration used with permission from Harvey F. Lodish, Whitehead Institute for Biomedical Research [5] Activity Studies have shown that adiponectin concentration is reversely associated with type 2 diabetes, coronary disease and obesity, all together called the metabolic syndrome. Adiponectin decreases blood glucose and the free fatty acid serum concentration and increases insulin sensitivity [6]. Adiponectin has also been shown to have anti-inflammatory [3] and anti-apoptotic effects [7]. The different conformations of adiponectin have different activities. Some believe that isolated globular adiponectin (gad) exerts its major activity in skeletal muscle, where it stimulates the activation of AMPK (AMP-activated Protein Kinase). According to this view full length adiponectin also stimulates AMPK, but in the liver [8]. Others claim that both trimers and gad are active in the skeletal muscle, whereas larger oligomers are required in the liver (Figure 3) [9]. Activation of AMPK stimulates glucose uptake in the skeletal muscle and reduces gluconeogenesis in hepatocytes (liver cells). AMPK also leads to increased fatty acid 5

11 oxidation, through phosphorylation of coenzyme A carboxylase (ACC), an important regulator of fatty acid oxidation, and angiogenesis [8]. The anti-inflammatory effect of adiponectin is due to its ability to reduce secretion of TNF-α from monocytes, macrophages and foam-cells [3]. Figure 3. Structure and function of adiponectin. Illustration used with permission from Takashi Kadowaki, department of internal medicine, university of Tokyo [9]. Although the active form of adiponectin is not yet determined, most agree that it is the ratio HMW/total adiponectin which is important as a marker for the metabolic syndrome (oligomers larger than hexamers are considered HMW). Clinical trials have shown that the ratio is negatively correlated to insulin resistance and body weight, and positively correlated to HDL [10, 11]. The ratio and the total amount of adiponectin are also gender specific, since the levels of HMW and total adiponectin are lower in males [9]. 1.3 Aim of study Existing commercial adiponectin-elisas, measure total adiponectin. The producers do not mention if they measure the isoform mixture in blood or if they somehow pretreat the samples so that they contain only monomers/dimers/trimers/hexamers/oligomers, i.e. the total amount of adiponectin is measured. However as mentioned above, what one wants to measure is the ratio HMW/total adiponectin. Until recently, there have been no such HMW-tests available, the measurements have been done through separations, e.g. ultracentrifugation on a sucrose gradient or gelfiltration, and immunoblotting [10]. All these are very time consuming and labour intensive, alternative means for the measurement of HMW adiponectin is highly desirable. Therefore the aim of this master s degree project is to evaluate the possibilities to establish an ELISA assay for measurement of high molecular weight adiponectin. 6

12 1.3.1 How to separate HMW from LMW Theoretically there are multiple ways to separate the different adiponectin isoforms from one another. The separation can be performed either in the pretreatment steps or in the capture/detection step. To separate isomers there has to be differences at a molecular level, e.g. intra-molecular bonds, sensitivity to different chemicals/enzymes, pi, receptors, epitopes for antibodies, size etc. The different isoforms of adiponectin display some differences: Antibodies: Antibodies bind to different epitopes. Epitopes might be a part of a monomer, but it could also be the oligomer structure in itself. If the oligomer structure is the epitope the antibody should not recognize monomers. Another possibility is to use the same monoclonal antibody for both capturing and detection. If the three epitopes on the trimer also are positioned closely to the binding surface between the three monomers, binding of one antibody would block access to the other two epitopes and only one antibody would be able to bind to one trimer. Thus, a sandwich ELISA would only detect hexamers or larger oligomers. Receptors: A HMW adiponectin-specific receptor is known to exist [12]. pi: The isoforms of adiponectin have different pi [13]. Sensitivity to enzymes: Studies of adiponectin indicate that there are enzymes that digests monomers/dimers/trimers but not larger oligomers [14]. Intra-molecular bonds: Two of the three monomers in a trimer are linked by a disulfidebond. This sulfide bond is a requirement for the formation of hexamers and larger oligomers [5]. Size: Monomers have a molecular weight of 30kDa, trimers 90kDa etc. It has been shown that the molecular weight of the isomers appear differently in gels, for example hexamers appear to have a molecular weight of 230kDa and 12mers 440kDa [15] Strategies used in this master s degree project The indicated biochemical differences in adiponectin isoforms has led to the following strategies: Double monoclonal antibodies: The strategy makes use of two antibodies of the same clone. Hopefully the epitopes of the three monomers are situated near the binding surface in the trimer, so that only one antibody can adhere to one trimer, thus measuring hexamers and larger oligomers, but not trimers. Receptor based detection: The strategy makes use of a receptor specific for HMW adiponectin, T-Cadherin [12]. Membrane Assisted Isoform ImmunoAssay II (MAIIA II): The strategy makes use of the differences in pi [13, 16]. Pretreatment with enzyme: The strategy makes use of the different sensitivities of the isoforms to enzymedigestion [14]. 7

13 1.4 Immunoassays Immunoassays are diagnostic tools based on immunoreactions between antigens and antibodies. The most common immunoassay is ELISA (enzyme linked immuno-sorbent assay). This technique is based on detection of antigens in a sample through enzyme-coupled antibodies or antigens. Detection using enzyme-coupled antibodies is based on spectrophotometric analysis. A spectrophotometer sends a light beam through the sample and light is absorbed by the molecules in the sample, thus decreasing the radiation energy [17]. Through measurement of incoming (P 0 ) and outgoing (P) light, the absorbance (A) can be calculated, A= log(p 0 /P) [18]. The absorbance is proportional to the concentration of the absorbing molecule in the sample. For immunoassays the absorbing molecule is most often the digested substrate of the enzyme coupled to the detection antibody. A commonly used enzyme is horse radish peroxidase (HRP). The reaction between the enzyme and its substrate is brought to a colorimetric end through addition of an acidic stop-solution, after which the plate is analyzed for absorbance at 450nm [17] Sandwich ELISA In a sandwich ELISA a specific antibody, raised against the antigen of interest, is immobilized on a surface, for instance a microtiter plate. The antigen is captured by this specific antibody and then detected by an enzyme-coupled specific antibody (this antibody being raised against a different epitope on the antigen). Detection is possible through addition of the enzyme s substrate. Thus, the higher the signal, the higher the antigen concentration (Figure 4) [17]. X mol/l 100*X mol/l Figure 4. Sandwich ELISA. Calibration curve and construction. Illustration used with permission from Mercodia AB. 8

14 1.4.2 Competitive ELISA In this approach a non-specific antibody, is immobilized onto the surface. This antibody recognizes antibodies produced in a certain species, that is the species used to produce the antigen-specific-antibody. A specific antibody is added to the plate together with the sample and a labelled antigen, called a tracer. The combined addition of sample and tracer creates a competition for the specific antibody, hence the name competitive ELISA. The signal will be reversely correlated to the concentration (Figure 5) [17]. X mol/l 100*X mol/l Figure 5. Competetive ELISA. Calibration curve and construction. Illustration used with permission from Mercodia AB Membrane Assisted Isoform ImmunoAssay II (MAIIA II) MAIIA II is a relatively new immunoassay technique, developed in collaboration between Uppsala University and Pharmacia Diagnostics (Phadia). This method separates different isoforms based on their pi, affinity or their hydrophobic properties. Detection is made through a carbon-black labelled antibody. In this project the protein, adiponectin, was to be separated based on its pi. The assay involves four steps (Figure 6): 1. Sample application. The sample is applied on a nitro-cellulose membrane suitable for ion-exchange chromatography. 2. Separation. The proteins are allowed to migrate on the membrane. The flow is maintained by capillary forces from a buffer reservoir and an absorbent sink. The isomers are retained in different zones of the membrane depending on their pi. 3. Elution. The flow is changed to run perpendicular to the separation. The eluted proteins are captured, in a capture-zone, by immobilized antibodies specific for the protein of interest. 4. Detection. Detection of the protein is achieved by adding carbon-black labelled specific antibodies, which are allowed to flow through the capturing-zone [16]. The carbon-black is then detected with a flat-bed scanner, measuring 24 pixels/mm with 4096 grey-scale levels. The detection limit for a 10-minute-test (standard) is 0,02µg/L. The end-result of MAIIA II is an isoform-profile, showing concentration versus charge (pi) [16]. 9

15 Conc. analyte Charge Figure 6. Procedure of MAIIA II technology. Illustration Used with permission from Maria Lönnberg, center for surface biotechnology, Uppsala university. 2. Materials and methods 2.1 Antibodies A search for antibodies targeting adiponectin, resulted in ten antibodies specific for human adiponectin. Five of these (A-E), from three different suppliers, were evaluated for affinity, specificity and kinetics towards the antigen. Four of the antibodies (A-D) were from mouse and one from rat (E). Antibody A and B are both directed towards the globular domain of human adiponectin, B shows no cross reactivity to rmadiponectin (recombinant mouse adiponectin) or rradiponectin (recombinant rat adiponectin)*. Antibody C is mouse-specific with a minor cross reaction to human adiponectin*, D is directed towards the collagendomain in the human adiponectin*, E is specific for the globular domain in mouse adiponectin but shows a 100% cross reactivity to rhadiponectin (recombinant human adiponectin)*. *According to the manufacturer 2.2 Antigens When evaluating the antibodies a recombinant human antigen (1), derived from mouse myeloma cells, was used. In stability experiments this antigen was found to be unstable. This finding led to an evaluation of five different antigens (1-5) from four suppliers. All of the antigens were recombinant, one expressed in mouse myeloma (1), one in E.coli (2) and three in HEK (Human embryonic kidney) cells (3-5). Native electrophoresis of the five antigens revealed their isoform profiles (Figure 7). 10

16 Figure 7. Isoform profiles of antigen1-5. Lane 1-2: antigen 1, 3-4: antigen 2, 5-6 antigen 3, 7-8: antigen 4, 9-10: antigen 5, 11: protein ladder. Antigen 1 consists of one, high molecular, isoform. Antigen 3-5 consists of 5 different isoforms. Antigen 2 not detected. Scanning and printing of the gel have added false bands. 2.3 Buffers In the early experiments and evaluations a standard sample buffer (Mercodia AB) was prepared. This buffer was used to dilute samples, calibrators and conjugates. During the project, the buffer was modified and optimized for the proteins to be diluted. 2.4 Receptor One of the four approaches to measure HMW adiponectin requires a receptor targeting HMW. This receptor, T-Cadherin, was labelled with biotin and used as a detector, instead of a conjugate antibody. 2.5 Enzyme The only existing human HMW adiponectin ELISA [14] includes a pretreatment step using Proteinase-K. According to the manufacturer, the enzyme selectively digests all forms of adiponectin except HMW. After treatment with the enzyme, the remaining HMW adiponectin oligomers are detected using a sandwich assay. For the enzyme pretreatment approach, proteinase K was required. The activity of proteinase K is expressed as: 1U digests 1µmol tyrosin per minute [19]. 2.6 Coating The plates used for coating are Maxisorp nunc-plates (Göteborgs termometer fabrik, GTF). Binding of antibodies to the plate is achieved through several interactions: The plates are γ-radiated, which breaks the polymers in the plastic, making the surface negatively and positively charged. The negative and positive residues on the antibodies will bind to this surface. Hydrophobic interactions between the antibodies and the plate. Weaker interactions mediated by e.g. van der Waahl forces also occurs [17]. 11

17 Coating is performed in two steps: A solution containing the antibodies to be coated is dispensed into the wells, and allowed to incubate over night. During this step the antibodies react to and bind to the surface of the plate. After incubation with the antibody solution, the plate is washed and blocking buffer is added. This buffer contains protein binding to free surfaces on the plate, thus blocking areas where no antibody is bound and stabilizing the bound antibodies. The plates are incubated over night, after which they are aspirated, dryed and packed. Approximately 30% of the coated antibody is active and available for reaction in the well [17]. Three of the adiponectin specific antibodies (A,B,E) were coated (1µg/well, ph 7), for use in sandwich assay. A rabbit anti-mouse antibody was coated (1,2µg/well, ph 9), for use in the competitive assay. 2.7 Labelling Detection in an ELISA-test is carried out through enzyme labelling. This is performed either trough direct linking of the antigen/antibody to an enzyme (conjugation) or trough a bridge e.g. biotin-strepavidin. The enzyme used is horse radish peroxidase (HRP). In direct conjugation HRP is linked to the antibody/antigen while when using a bridge, the enzyme is linked to streptavidin and the antibody/antigen is linked to biotin (biotinylation) [17]. Biotin (vitamin H) and streptavidin (albumen) are two naturally occurring molecules, with an affinity for one another stronger than the affinity between antibody and antigen [20] Biotin labelling For initial studies in the competitive assay, two of the antigens, 1 and 5, were labelled with biotin (to produce a tracer), and then detected with HRP-conjugated streptavidin. The reason for biotinylation is that biotin is small (454 Da) compared to HRP (44kDa), and therefore less likely to interfere with the association between antibody and antigen than HRP. Also, conjugation requires a larger amount of protein for labelling than biotinylation [17]. Biotinylation was also performed on the HMW-specific receptor, T-Cadherin. The biotinylation was carried out in a 1:20 molar excess of biotin and incubated in room temperature for one hour Conjugation Biotinylation is a good initial step, but direct conjugation is more robust and is preferred in the final product. Direct conjugation excludes one step in the assay procedure, streptavidin- HRP addition, which simplifies the test. Moreover the streptavidin has a tendency to unspecific binding to molecules/surfaces, thus giving a high background signal [17]. Conjugation of antibodies was carried out at a molar concentration of 1:1, ph 9,2, for two hours at room temperature, followed by reduction with sodium borohydride for one hour. During conjugation the antibody and the enzyme are linked together by a Schiff s base 12

18 reaction. In this reaction enzyme carbohydrate chains are oxidized into aldehydes which are then linked to ε-amino groups on the antibody. The reduction ensures that the conjugation reaction stops by reducing the aldehydes on the enzyme [21]. Three of the antibodies (A, B, E) were conjugated. The conjugations were analyzed by an enzyme study and an immunoreactivity study. 2.8 Separation and purification of recombinant and endogenous adiponectin Ammonium sulphate precipitation Ammonium sulphate precipitation was used as a first step in purification of endogenous adiponectin. In this method proteins are precipitated at different salt-concentrations. The precipitation-process is due to the aggregation of hydrophobic surfaces in the proteins. The elevated salt-concentration increases hydrophobic interactions through weakening of the liquid-layer surrounding the proteins [18] HiPrep Desalting The next step in the protein purification process is ion-exchange chromatography. Since this method is based on the capturing of charged residues and then elution by a saltgradient, the protein first needs to be desalted. The desalting of precipitated sera was performed on a HiPrep 26/10 Desalting column (Amersham bioscience AB), using a triethanolamin buffer, ph~7, Ion-exchange chromatography Ion-exchange chromatography was performed both on sera, for endogenous purification, and on recombinant adiponectin for separation of different isoforms. This method separates particles according to their pi, since charged particles are retained in the column and eluted with a saltgradient. For both the separation and the purification, a MonoQ-column (HR 5/5 respectively 10/100 GL, Amersham bioscience AB) was used and the proteins were eluted with triethanolamine buffer, ph ~7,7, with a continuous gradient of NaCl, 0-100% Gel-filtration For the finishing step in the purification of endogenous adiponectin, gel filtration chromatography was used. The method was also used to separate isoforms of recombinant adiponectin. This method separates particles according to size, since smaller particles are retained longer than larger ones. The gel filtration for endogenous purification was performed on a HiLoad 16/60 Superdex 200 prepgrade column (Amersham bioscience AB), but for the separation of recombinant isoforms a Superdex /300 GL column was used. For both purposes a PBS buffer was used as eluent. 13

19 2.9 Electrophoresis Native electrophoresis was used as a control method during the purification, separation and sample-pretreatment studies. This method separates particles according to size, since small particles moves faster through the gel. The electrophoresis was performed under native conditions, using a Tris-HCl ready-gel and a tris-glycin running buffer. The gels were run in a miniprotean-3-cell system, at a constant voltage of 200V for 35 minutes (Bio-Rad Laboratories, Inc.) ELISA Competetive assay For competitive studies the following conditions were used: Coated rabbit anti-mouse-antibody: 1,0µg/well. Calibrators: 0,05, 0,1, 0,2, 0,4, 0,8, 1,6, 3,2, 6,4µg/ml. Serum samples to be tested were diluted 1/30. Tracer: 0,2µg/ml. Anti-adiponectin antibody: 0,5µg/ml. Streptavidin: 1,76µg/ml, 1 part streptavidin-hrp + 19 parts streptavidin. TMB, HRP-substrate. Stop-solution, an acidic solution used to reach the calorimetric endpoint. The tests were performed as followed: 50µl/well calibrator/sample, tracer and anti-adiponectin antibody are added to the plate, incubation 2h, room temperature (RT) on a plate shake. The plate is washed six times with 700µl wash buffer (Mercodia AB). 150µl/well streptavidin is added to the plate, incubation 2h, in room temperature on a plate shake. The plate is washed six times with 700µl wash buffer (Mercodia AB). 200µl/well TMB is added to the plate, incubation 15 minutes, in RT. 50µl/well stop-solution is added, the plate is shaken on a plate shake for a few seconds to ensure that the reaction is stopped, and the plate is analyzed in an ELISA plate reader at 450nm (Sunrise, TECAN) Sandwich assay For studies with the sandwich assay, the following conditions were used: Coated anti-adiponectin antibody: 1,0µg/well. Calibrators: 3, 10, 30, 100, 300ng/ml. Serum-samples to be tested are diluted 1/100. Conjugated anti-adiponectin antibody: ~0,8µg/ml. TMB, HRP-substrate. Stop-solution. 14

20 The tests were performed as followed: 25µl/well calibrator/sample is added to the plate. 100µl/well conjugated anti-adiponectin antibody, is added to the plate, incubation 2h, RT on a plate shake. The plate is washed six times with 700µl wash buffer (Mercodia AB). 200µl/well TMB is added to the plate, incubation 15 minutes, RT. 50µl/well stop-solution is added, the plate is shaken on a plate shake for a few seconds to ensure that the reaction is stopped, and the plate is analyzed in an ELISA-platereader at 450nm (Sunrise, TECAN). 3. Experiments 3.1 Affinity When developing an ELISA prototype, the affinity between different antibodies and the antigen of interest has to be studied. The antibodies were first tested for affinity to the antigen by titration of tracer, and the achieved signals were used in deciding which antibodies to use. The study involved antibody A, B, C and D, at a concentration of 1µg/ml. The tracer, made from antigen 1, was detected with streptavidin-hrp as described for the competitive assay. Antibody E could not be tested, since the coated antibodies for the competitive assay are rabbit-anti-mouse and antibody E is derived from rat. Antibodies achieving good signals in the competitive assay were then evaluated in couples, in a sandwich assay. In this experiment three antibodies were coated, conjugated (A, B and E) and tested against each other. The calibrators used were made from antigen 1. The sandwich assay reveals if the antibodies function as pairs. That antibodies do not function as a pair can have two explanations: The epitopes for the two antibodies might be situated too close, and thus both antibodies cannot bind simultaneously (steric hindrance). Alternatively, the first antibody to bind the antigen, might induce a conformational change of the antigen, so that the second antibody cannot bind [17]. 3.2 Cross reactivity When working with antibodies, there is always a risk for cross reaction between the antibody and proteins similar to the antigen of interest. To find these proteins a homology search, BLAST, was performed. The search resulted in one closely structure and sequence related serum-protein, namely complement factor C1q. A competitive test for C1q was constructed, using antibodies A and B. Tracer and calibrators were made from antigen 1. Cross reaction can also occur between the antibody and an antigen from a different species, and can be either positive or negative. If there is high cross reactivity between two species the antibodies can be used to establish assays for both species. The disadvantage is that the assay cannot differentiate the two species-specific proteins in analogy testing, e.g. when proteins from one species is tested in another [17]. Species tested for cross reactivity in this study is dog, cow, mouse, rat and pig. The species cross reactivity tests were performed in the competitive format, using sera from the different species and antibodies A and B. The tracer and calibrators were made from antigen 1. 15

21 3.3 Purification of endogenous adiponectin The strategy for the purification of endogenous adiponectin from serum includes four steps: Ammonium sulphate precipitation High Prep desalting Ion-exchange Chromatography Gel Filtration Chromatography The first precipitation experiment was performed to achieve the specific salt concentrations needed for the salting-out of adiponectin. Sera, buffered with Tris-HCl ph8, was treated with salt at concentrations ranging from 10%-90% saturation in parallel. The precipitates and supernatants were tested for immunoreactivity, using a competitive assay (tracer 0,1µg/ml, tracer and calibrators were made from antigen 5). From these studies, a suitable salting-out concentration was established, 20% respectively 40%, which was used in the following studies. Serum, from which the protein was to be purified, was first buffered with a Tris-HCl buffer ph 8, and then precipitated with an ammonium sulphate concentration of 20%, to salt-out unwanted proteins. The pellet was saved and the supernatant was then precipitated at a concentration of 40%, to precipitate adiponectin. Both pellets were redissolved in PBS, after which a fraction of the dissolved pellets and the supernatant from the precipitation with 40% ammonium sulphate were desalted, using a pierce protein desalting spin column. The desalted protein solutions were then analyzed with a competitive assay (tracer and calibrators made from antigen 1), to ensure that adiponectin was located in the redissolved pellet from the precipitation at 40%. The dissolved pellet from precipitation with 40% ammonium sulphate was then applied to a High Prep desalting column. Fractions containing protein were pooled and applied to an ionexchange column, and fractionated. The fractions were analyzed, using a competitive assay (tracer and calibrators made from antigen 5 respectively 4). Fractions, representing each peak in the immunoassay, were then pooled. Each pooled peak from the ion-exchange chromatography was applied separately to a gel filtration column. Fractions were collected and analyzed, using a competitive assay (tracer and calibrators were made from antigen 5 and 4). Fractions containing adiponectin were pooled for later use. 3.4 Separation of recombinant isoforms of adiponectin The recombinant adiponectin was delivered as a mixture of isoforms. For analysis of HMWadiponectin, the isoforms had to be separated. The separation was done through gel filtration or ion-exchange chromatography. Antigen 3, 30µg, was applied to a gel filtration column. Fractions were collected and analyzed, using a competitive assay (tracer 0,1µg/ml, tracer and calibrators were made from antigen 1). Fractions containing adiponectin were pooled for later use. 16

22 Antigen 3, 10µg, was applied to an ion-exchange column. Fractions were collected and analyzed, using a sandwich assay. Antigen 4, 40µg, was applied to an ion-exchange column. Fractions were analyzed, using a competitive assay (Streptavidin-HRP 1µg/ml, 1 part streptavidin-hrp, 9 parts streptavidin) (tracer and calibrators were made from antigen 5 respectively 4). The positive fractions were pooled, concentrated with Vivaspin (ultrafiltration device used to concentrate biological samples, Vivascience Ltd) and run through electrophoresis to analyze the separation. 3.5 High molecular weight tests - strategies Double monoclonal The double monoclonal assay was constructed with the same monoclonal antibody used for capturing and detection. A sandwich assay using antibody A/B for capturing and detection, was constructed in parallel (A: Conjugate ~0,8µg/ml: 1 part conjugate, 2 parts unconjugated antibody; calibrator made from antigen 3) (B: Conjugate ~0,8µg/ml: 1 part conjugate, 1,5 parts unconjugated antibody; calibrator made from antigen 3). A sandwich assay using antibody A/B for capturing and antibody B/A for detection, using the same conditions, was constructed as a control Receptor-based According to literature, there is a receptor for high-molecular adiponectin, T-Cadherin. This receptor provide a good basis for an HMW assay [12]. The assay was constructed with a monoclonal antibody immobilized on the plate, antibody A, and the biotinylated receptor as the detector (calibrator was made from antigen 4). The receptor was added at molar concentration of 6:1 (hexamers) to the recombinant sample (5,5µg/ml receptor: 6,4µg/ml rhadiponectin). Detection was done through addition of streptavidin-hrp (1µg/ml, 1 part streptavidin-hrp + 9 parts streptavidin) MAIIA II The MAIIA II technology is based on the possibility to separate isomers based on their pi and the availability of antibodies directed to the protein of interest [16]. For this master s degree project MAIIA II was not tested, but the requirements for the use of this technology was. To test the possibility to separate isomers based on their pi, ion-exchange chromatography was performed on both endogenous and recombinant adiponectin (see chapter ). Conjugation of antibody A with HRP was performed, to investigate if the antibodies could be labelled (see chapter 3.7.2) Enzyme-pretreatment Today there is one commercially available adiponectin assay, where the producer claims to measure HMW adiponectin. This assay makes use of an enzyme pretreatment with proteinase K, where the enzyme cleaves only low molecular structures. For this project, this strategy for the measurement of HMW adiponectin was reproduced and evaluated [14]. 17

23 Pretreatment with proteinase K was performed at 37 C for one hour. The samples were then evaluated using a sandwich assay and electrophoresis. For samples evaluated in electrophoresis the enzyme was added in 150:1 (enzyme U:mol tyrosin in adiponectin). For samples evaluated in ELISA, the enzyme was added at a concentration of 30U/ml, at a volume series ranging from 2,5µl to 20µl (molar concentrations :1 (monomers)), to 5µl sample (calibrators were made from antigen 4). 4. Results 4.1 Affinity When tested in the competitive format, antibodies A and B had a good affinity for the tracer, while antibodies C and D did not yield any signal (Figure 8). 4 3,5 3 2,5 2 A B C D 1,5 1 0, Concentration ng/ml Figure 8. Affinity assay, competitive format. Tracer in concentrations ranging from 12,5-400ng/ml was tested against the four antibodies. Antibody A and B showed good reactivity, antibody C and D did not bind to the tracer. The affinity studies, in a sandwich-format, revealed that only two of the antibodies worked as a pair, namely A and B. Both of these antibodies functioned well as conjugates and when immobilized on the plate. Combinations between antibody A/B and E did not produce any signal (Figure 9). 18

24 4,5 4 3,5 3 2,5 2 E/A B/A A/E B/E A/B E/B 1,5 1 0, Concentration ng/ml Figure 9. Affinity assay in sandwich format, simultaneous incubation. Two of the antibodies functions as a pair: A and B. 4.2 Cross reactivity C1q Homology searches resulted in one closely sequence- and structure-related serum protein, complement factor C1q (Figure 10). sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN sp Q15848 ADIPO_HUMAN sp Q9BXJ2 C1QT7_HUMAN -MLLLGAVLLLLALPGHDQETTTQGP GVLLPLPKGACTGWMAG MFVLLYVTSFAICASGQPRGNQLKGENYSPRYICSIPGLPGPPGPPGANG ::**.. : :..*: :. :* : **.. * IPGHPGHNGAPGRDGRDGTPGEKGEKGDP GLIGPKGDIGET SPGPHGRIGLPGRDGRDGRKGEKGEKGTAGLRGKTGPLGLAGEKGDQGET ** *: * ******** *******. ** * *** *** GVPGAEGPRG FPGIQGRKGEPGE GAYVYRSAF GKKGPIGPEGEKGEVGPIGPPGPKGDRGEQGDPGLPGVCRCGSIVLKSAF * *. **.* ** :* :** *: *: * :*** SVGLETYVTIPNMPIRFTKIFYNQQNHYDGSTGKFHCNIPGLYYFAYHIT SVGITTSYPEERLPIIFNKVLFNEGEHYNPATGKFICAFPGIYYFSYDIT ***: *..:** *.*:::*: :**: :**** * :**:***:*.** VYMKDVKVSLFKKDKAMLFTYDQYQENNVDQASGSVLLHLEVGDQVWLQV LANKHLAIGLVHNGQYRIKTFDAN-TGNHDVASGSTVIYLQPEDEVWLEI : *.: :.*.::.: : *:*.* * ****.:::*: *:***:: YGEGERNGLYADNDNDSTFTGFLLYHDTN FFTDQNGLFSDPGWADSLFSGFLLYVDTDYLDSISEDDEL :.:.. :. ** *:***** **: Figure 10. Sequence alignment, Complement factor C1q and hadiponectin. * indicates conserved residues, : indicates conserved substitutions,. indicates semi-conservative substitutions. Data from CLUSTAL W (1.82) multiple sequence alignment. C1q exist in human blood in concentrations ranging from 75 to 150µg/ml [22], in the cross reactivity test, concentrations from 3,44 to 220µg/ml were tested (Figure 11-12). 19

25 2,5 2 1,5 Calibrators C1q 1 0,5 0 0, Concentration µg/ml Figure 11. Cross reactivity, complement factor C1q, antibody A. 3 2,5 2 1,5 Calibrators C1q 1 0,5 0 0, Concentration µg/ml Figure 12. Cross reactivity, complement factor C1q, antibody B. The result indicated a cross reaction of 0,2% for antibody A and 1,2% for antibody B (Table 1). Table 1. Cross reactivity, complement factor C1q tested against antibody A and B. Cross reaction calculated through: Measured concentration Actual concentration 100 Antibody A Antibody B Actual Measured concentration concentration Mean cross (µg/ml) (µg/ml) Cross reactivity reactivity 220 0,438 0,20% 55 0,142 0,26% 0,20% 220 2,166 0,98% 55 0,738 1,34% 1,20% 20

26 In a sandwich, constructed with antibody A and B, the cross reaction would be ~0,002%. Even though the concentration of C1q in human blood is ~70-150µg/ml [22], that is ~2-30 times higher than adiponectin, C1q will still be out of the measurement interval and will therefore not interfere with the assay Mammalian Mammalian cross reactivity test shows that antibody A reacts to serum from most mammals, in particular pig, mouse and rat. This antibody could be used to construct a general mammalian test. Antibody B reacts strongly with mouse and possibly cow, but not with any other species. Antibody B is more species-specific and could be used for analogy testing etc. (Figure 13) A B Dog A Dog B Cow A Cow B Rat A Rat B Mouse A Mouse B Pig A Pig B Human Blank Sample Figure 13. Cross reactivity, mammals, antibody A and B. The highest calibrator is 3,2µg/ml, therefore concentration readings above this level are not reliable, these samples have a concentration >3,2µg/ml. Antibody A reacts to all samples, antibody B only to mouse. 4.3 Purification of endogenous adiponectin Ammonium sulphate precipitation Immunoreactivity studies of the pellets and supernatants from the first precipitation experiment showed that adiponectin was mainly found in pellets from precipitation with 20-40% ammonium sulphate (Figure 14). 21

27 2,5 2 1,5 1 Pellet Supernatant 0,5 0 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% -0,5 % saturated ammonium-sulphate Figure 14. Ammonium sulphate precipitation of sera, salt concentrations ranging from 10-90%. Adiponectin is precipitated at salt concentrations >20%, a large quantity of adiponectin is salted out between 20 and 40%. As a result a procedure for salting-out adiponectin was established: Step one: Precipitate unwanted proteins at an ammonium sulphate concentration of 20%. Step two: Precipitate adiponectin at an ammonium sulphate concentration of 40%. Precipitation of serum, for the purification of endogenous adiponectin, was done according to the procedure mentioned above. Analysis of the precipitation fractions show that a large amount of adiponectin is present in the pellet from precipitation with 40% ammonium sulphate, the first pellet and the supernatant only contains small amounts of adiponectin (Figure 15) Serum Pellet 20% Pellet 40% Supernatant 40% Sample Figure 15. Ammonium sulphate precipitation of sera, salt concentrations are 20 respectively 40%. A large amount of adiponectin is present in the pellet from the precipitation at 40%, trace amounts of adiponectin are present in the other pellet and in the supernatant. 22

28 4.3.2 Ion-exchange chromatography The protein was desalted and applied to an ion-exchange column, the achieved fractions were tested for immunoreactivity. Fraction B15-D14 were immunoreactive (Figure 16), four pools were made from the fractions. A:B15-C5, B:C6-C15, C:D1-D6, D:D7-D14, these were applied separately to a gel filtration column. After the ion-exchange chromatography, 73% of the activity remained Amount of protein Immuno-reactivity (ng/ml) A6 A10 A14 B3 B7 B11 B15 C4 C8 C12 D1 D5 D9 Fraction D13 E2 E6 E10 E14 F3 F7 F11 F15 Figure 16. Separation of ammonium sulphate precipitated serum, ion-exchange chromatography. The immunoreacivity studies indicated presence of adiponectin in fraction B15-D14. Four pools were made: B15-C5, C6- C15, D1-D6 and D7-D Gel filtration The four pools prepared from ion-exchange chromatography (pool A, B, C and D) were applied separately to a gel filtration column. The chromatograms show different profiles for the separate pools (Figure 17) poola poolb poolc poold Fraction Figure 17. Purification of endogenous adiponectin, gelfiltration. Four pools prepared from fractions from ionexchange chromatography separation were run separately. 23

29 Immunoreactivity studies of the pools show that pool A has a low reactivity, B and D moderate and C a high reactivity (Figure 18). Pool C and D show reactivity in the same fractions, indicating that the reactive proteins in the pools are of the same size. After the gelfiltration 58% of the activity remained. The total activity loss for the purification is 42% PoolA PoolB PoolC PoolD Fraction Figure 18. Purification of endogenous adiponectin, gelfiltration. Four pools prepared from fractions from ionexchange chromatography separation were run separately. The four pools were tested for immunoreactivity, poola gave low signals, B and D moderate signals, while poolc had a high reactivity. 4.4 Separation of recombinant isoforms Ion-exchange chromatography A recombinant adiponectin sample, antigen 3, consisting of a mixture of isoforms, was applied to the column. The achieved chromatogram displays 5 peaks, the last one being the largest. In immunoreactivity studies only one peak was detectable, the last one in the chromatogram (Figure 19). This peak is thought to be the largest isoform present in the recombinant sample. 24

30 2,5 2 1,5 1 Immunoreactivity (450nm) Amount of protein (mau*10) 0, ,5-1 Fraction Figure 19. Separation of recombinant isoforms of adiponectin, ion-exchange chromatography. Chromatogram and immunoreactivity study of separation of antigen 3. The separation yielded 5 peaks, one peak was immunoreactive. Repeating the ion-exchange chromatography yielded similar results. Antigen 4 was used, an antigen with approximately the same isoform profile. 5 peaks were detected, around fraction 10, 30, 60, 70 and 80. The first two peaks had a low protein concentration (Figure 20). 8,000 7,000 6,000 5,000 4,000 3,000 2,000 1,000 0, Fraction Figure 20. Separation of recombinant isoforms of adiponectin, ion-exchange chromatography. Chromatogram from separation of different isoforms from antigen 4. Five peaks are detected, the first two peaks have a low protein concentration. The fractions were pooled, 4-14; 23-34; 52-60; 66-68; 79-85, tested for immunoreactivity (Figure 21) and electrophoresis was performed (Figure 22). The immunoreactivity studies provided low signals for all pools; pool gave a small increase, though still very low. The electrophoresis showed no protein in pool 1-4 whereas pool 5 displayed six bands. 25

31 0,16 0,14 0,12 0,1 0,08 Immuno-reactivity 0,06 0,04 0, Fractions Figure 21. Separation of recombinant isoforms of adiponectin, ion-exchange chromatography. Immunoreactivity from separation of different isoforms, antigen 4. Weak signal is detected from pool 4, fraction 66-68, a weak to no signal is seen in other pools Figure 22. Separation of recombinant isoforms of adiponectin, ion-exchange chromatography. Lane 1. Protein ladder; lane 2: proteinase K; lane 3: pool 1; lane 4; pool 2; lane 5: pool 3; lane 6: pool 4; lane 7: pool 5. Scanning and printing of the gel has added false bands Gel filtration Gel filtration of recombinant antigen 3, revealed 4-5 peaks (Figure 23). Peak number 1, B1- B13, was the largest peak and the only one being immunoreactive. 26

32 0,9 0,8 0,7 0,6 0,5 0,4 Immuno-reactivity (µg/ml] Amount of protein (mau) 0,3 0,2 0,1 B1 B3 B5 B7 B9 0 B11 B13 B15 C2 C4 C6 C8 Fraction C10 C12 C14 D1 D3 D5 D7 D9 Figure 23. Separation of recombinant isoforms of adiponectin, gel filtration chromatography. Immunoreactivity and chromatogram from separation of different isoforms, antigen 3. Four peaks were detected during the separation, one of these was immunoreactive, namely peak number one. 4.5 High molecular weight assays - strategies Double monoclonal The signal decreases significantly when the same antibody was used for capturing and detection (Figure 24). This is the case for both antibody A and B. 2,5 2 1,5 1 Immobilized antibody:conjugate B:A A:A A:B B:B 0, Concentration (ng/ml) Figure 24. Calibrator curves from sandwich assay using antibodies A and B. Serum with a high concentration of adiponectin yields a significantly higher signal when measured in the double monoclonal assay. Serum with a low concentration of adiponectin does not show any significant differences using the standard sandwich assay or the double monoclonal assay (Figure 25). 27

33 Adiponectin concentration (ng/ml) Immobilized antibody:conjugate B:A A:A A:B B:B 50 0 Serum low Serum Figure 25. Adiponectin concentrations in serum. Concentrations measured using calibrator curves in figure Receptor-based Final pools, A, B, C and D, from the endogenous purification, were tested against T- Cadherin, together with a recombinant sample. Pool D yielded a signal higher than the blank, the signal from the other samples were lower than the blank (Figure 26). 0,25 0,2 0,15 0,1 0,05 0 Blank PoolA PoolB PoolC PoolD rhadiponectin 6,4µg/ml Sample Figure 26. Detection with T-Cadherin. Final pools from endogenous purification were tested against T- Cadherin. Only pool D yielded a higher signal than the blank MAIIA II Ion-exchange chromatography, during purification of endogenous adiponectin, was successful in separating the different isoforms of adiponectin (Figure 16). Conjugation of antibody A and HRP yielded immuno reactive conjugates with low background signals (Figure 27). 28

34 4,5 4 3,5 3 2,5 2 Immunoreactivity, OD (450nm) Amount of protein (mau/20) Enzymatic activity, OD (450nm) 1,5 1 0, Fraction Figure 27. Conjugation of antibody A and HRP. The graph displays the chromatogram from the separation procedure and the results from immunoreactivity and enzymatic activity tests Enzyme pretreatment Pretreatment of serum and recombinant adiponectin with Proteinase K shows a decreasing signal when analyzed in sandwich assay. The signal decreases at a steady pace when titrating with higher amounts of enzyme (Figure 28). 1,8 1,6 1,4 1,2 OD (450nm) 1 0,8 OD 0,6 0,4 0, ,5µl 5µl 7,5µl 10µl 12,5µl 15µl 20µl 0 2,5µl 5µl 7,5µl 10µl 12,5µl 15µl 20µl Serum Calibrator Figure 28. Serum and recombinant adiponectin treated with Proteinase K before immunoreactivity studies. The signal decreases as more enzyme is added. Electrophoresis studies on Proteinase K pretreated recombinant adiponectin, antigen 3, reveals that the enzyme degrades all of the isoforms in the sample (Figure 29). 29

35 Figure 29. Electrophoresis on Proteinase K pretreated adiponectin, antigen 3. Lane A: Protein ladder, lane B: untreated antigen 3, lane C: Proteinase K pretreated antigen 3, lane. Lane three displays low molecular proteins in a smear, no high-molecular species detected. Scanning and printing of the gel has added false bands. 5. Discussion 5.1 Affinity The affinity studies revealed that only two of the antibodies reacted with the antigen, A and B. These two were then used to construct the sandwich assay, they were also used separately for the competitive assay. Antibody C and D did not bind to the antigen. Antibody E was tested in pairs with antibody A and B, which did not give any signal. The reason for the non-reactivity displayed by the three antibodies could be that: C is directed towards mouse adiponectin and only cross-reacts to a lesser extent with human adiponectin. D is directed towards the collagen-domain. Since trimers adhere to each other through the collagen domain and form a bouquet, the collagen domains will eventually be surrounded by globular domains, making the collagen domains unaccessible to antibodies. The antigen used for the affinity studies was antigen 1, from electrophoresis studies (Figure 7), antigen 1 was determined to contain only oligomers. Antibody E should have reacted with the antigen, it is directed towards globular domain of mouse adiponectin, but has shown 100% cross reactivity to rhadiponectin. In the affinity experiment the antibody showed no affinity for the antigen. The reason for this could be: o The animal, that the antibody was derived from, might have been immunized with an antigen slightly different from the one used in the affinity test. o The affinity for the antigen could be to low for an ELISA, but high enough for other uses, for example immuno-blotting. o It is possible that antibody E and A/B has epitopes too close to each other on the antigen, so that they will not work in a pair. This is however unlikely, since A and B can work as a pair. The construction using antibody B immobilized on the plate and antibody A as a conjugate gave a higher signal than A immobilized and B as a conjugate. Since the competitive assay did not display that difference (result not shown), it was probably due to better conjugation of antibody A. The conjugates were later balanced with non-conjugated antibody to achieve the same signals. 30