Direct Detection of Antibody Concentration and Affinity in Human Serum Using Microscale Thermophoresis
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Direct Detection of Antibody Concentration and Affinity in HumanSerum Using Microscale ThermophoresisSvenja Lippok, Susanne A. I. Seidel, Stefan Duhr, Kerstin Uhland, Hans-Peter Holthoff, Dieter Jenne,
and Dieter Braun*,
Systems Biophysics, Center for Nanoscience, Physics Department, Ludwig Maximilians Universitat Munchen, Amalienstrasse 54,80799 Munchen, GermanyNanoTemper Technologies GmbH, Flossergasse 4, 81369 Munchen, GermanyCorimmun GmbH, Lochhamer Strasse 29, 82152 Martinsried, GermanyMax Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany
*S Supporting Information
ABSTRACT: The direct quantification of both the binding affinity andabsolute concentration of disease-related biomarkers in biological fluidsis particularly beneficial for differential diagnosis and therapymonitoring. Here, we extend microscale thermophoresis to targetimmunological questions. Optically generated thermal gradients wereused to deplete fluorescently marked antigens in 2- and 10-fold-dilutedhuman serum. We devised and validated an autocompetitive strategy toindependently fit the concentration and dissociation constant ofautoimmune antibodies against the cardiac 1-adrenergic receptor related to dilated cardiomyopathy. As an artificial antigen,the peptide COR1 was designed to mimic the second extracellular receptor loop. Thermophoresis resolved antibodyconcentrations from 2 to 200 nM and measured the dissociation constant as 75 nM. The approach quantifies antibody binding inits native serum environment within microliter volumes and without any surface attachments. The simplicity of the mix andprobe protocol minimizes systematic errors, making thermophoresis a promising detection method for personalized medicine.
Only a few physical forces are strong enough to inducetranslational motion of molecules in their nativeenvironment. Light fields or magnetic fields, for example, arenot sufficient. Electrophoresis is the dominant technique toseparate biomolecules on the basis of their size and structure.Unexpectedly, focused temperature fields can move biomole-cules, an effect termed thermophoresis. Temperature cannotdrive molecules over large length scales as electrophoresis does.Thermophoresis merely leads to a 20% concentration depletionover tens of micrometers when moderate temperaturedifferences on the 5 K scale are applied. However, this smalldepletion is easily detectable with fluorescence and providesdetailed information on the molecules interface.We devised a strategy to probe both the concentration and
affinity of biomolecules in bulk blood serum applyingmicroscale temperature gradients. In the recent past,thermophoresis has been used successfully to detect close toliterature binding constants for nucleotide aptamers as well asproteins binding to small molecules.13
Here, we established a combined dilution and autocompe-tition protocol to tackle immunological problems in bloodserum as the according native environment. Unlike methodsdepending on molecule immobilization to surfaces, microscalethermophoresis (MST) to measure binding affinity in bulk fluidrequires only a single unspecific fluorescent probe. Fluores-cence reports a change in thermophoretic depletion upon
binding, thus quantifying the latter within tens of seconds persample. We focused on the increasingly important group ofautoimmune diseases which are difficult to classify and treatusing standard immunological methods.Dilated cardiomyopathy (DCM) is a nonischemic heart
muscle disease characterized by dilation and impairedcontraction of the left or both ventricles. With a prevalenceof 300400 patients per million, DCM belongs to the maincauses of severe heart defects.4 Moreover, it is the prime reasonfor heart transplantations.5 Besides genetic, toxic, and infectiousfactors, autoimmune reactions are discussed as a putativetrigger for its appearance. In a notable number of DCMpatients, increased concentrations of autoantibodies againstseveral cardiac antigens, including membrane proteins such ascell surface adrenergic receptors, are found.68 Some of theseautoantibodies seem to injure the myocardium directly orindirectly.9 The 1-adrenergic receptor regulating heart activityrepresents a major autoimmune target in DCM (Figure 1a).Agonist-like antibodies found in DCM patients have beenshown to be associated with severe ventricular arryth-mias.7,1214 The prevalence of these antibodies varies from30% to 95% in DCM sera and from 0% to 13% in sera from
Received: November 4, 2011Accepted: March 7, 2012Published: March 7, 2012
2012 American Chemical Society 3523 dx.doi.org/10.1021/ac202923j | Anal. Chem. 2012, 84, 35233530
unaffected controls.15,16 It is widely accepted that, in manypatients suffering from heart failure, a chronic adrenergicoverexcitation plays a harmful role.17 Permanent stimulation of1-adrenoceptors due to elevated levels of circulating catechol-amines in combination with autoantibodies could change theCa2+ homeostasis of cardiomyocytes, resulting in metabolic andelectrophysiological disturbances which are responsible fortachyarrhythmia and sudden death.18 It was also shown thatDCM autoantibodies against 1-adrenergic receptors increasethe activity of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA).19 Therefore, the detectionof 1-adrenergic receptor autoantibodies in human serum as anindicator for dilated cardiomyopathy and the development ofspecific antigens are important steps toward reliable diagnosticsand treatment of the disease. The autoimmune antibodiestarget, the second extracellular receptor loop,10,11 is mimickedby the candidate peptide drug COR1 (Figure 1b). Thus, COR1competes with the binding of autoimmune antibodies to thereceptor. In animal models, COR1 was shown to prevent heartfailure and reverse existing cardiac insufficiency (manuscript inpreparation).Although quantitative autoantibody analysis is crucial to
characterize and discriminate different autoimmune diseasetypes and optimize their treatment, it still remains challenging.Enzyme-linked immunosorbent assays (ELISAs) are widelyused to detect and quantify disease-related biomarkers, butdisplay several disadvantages. Surface immobilization, initialstrong dilution as well as tedious washing steps, enzymaticsignal amplification, and normalization to a standard curveoften cannot be avoided, resulting in unpredictable influenceson the results. The optimization of surfaces and linkers hasimproved the binding protocols of ELISA; however, it is time-consuming and often depends on the studied binding reaction.Linking small peptides to surfaces in a way that all bindingepitopes are presented in the same manner as in solutionremains a pertinent obstacle.20,21 The surface density of thecaptured proteins is hard to predict.22,23 Additionally, thenonspecific binding of molecules of interest and plasma/serumposes a challenge. Thus, absolute biomarker quantification viaELISA remains difficult. Despite these limitations and pitfalls,the popularity and application depth of ELISA are enormous.
Only a few solution-phase alternatives to ELISA are beingutilized. As radioimmunoassays (RIAs) fulfilled these principlesvery closely, they were popular until the 1970s. However, theneed for radioactive labeling and purification of the analyteprobe complexes from the free radioactive probe by asecondary reagent is a significant disadvantage compared tothe later developed ELISA.A number of recent approaches are based on fluorescence
resonance energy transfer (FRET) between two labeled ligandsto detect binding in biological fluids or inside cells.24 Forexample, homogeneous time-resolved fluorescence (HTRF)25
detects binding by proximity in bulk fluids, while in the bead-based alphascreen26 singlet oxygen is used as a chemicalproximity signal.In contrast to the above methods, microscale thermophoresis
is much simpler to set up, as only one binder is labeled and themeasurement follows a straightforward mix and read protocol.It is sample-efficient and immobilization-free and allowsmeasurements in bulk fluids. As shown here, it can be usedfor simultaneous and quantitative analysis of antibodyconcentration and affinity, making it a promising alternativeto the commonly used techniques.
EXPERIMENTAL SECTIONSetup. A fluorescence microscope (AxioScope Vario, Carl
Zeiss GmbH, Germany) was modified by a 1480 nm infraredheating laser (Fibotec Fiberoptics, Germany) generating localtemperature gradients in solution (Figure 2a and SupportingInformation).1,2 Most of the laser power is absorbed by theprobe volume. Back-reflected light is absorbed by an IR filterplaced above the fluorescence emission filter, and stray light isblocked by housing the capillaries. The maximal temperatureincrease was measured to be 8 K in the beam center usingBCECF (2-7-bis(carboxyethyl)-5(6)-carboxyfluorescein) andfinite element calculations (Figure 2b).27,28 With the initialtemperature jump set at time t = 0 s, the cold fluorescencesignal FI was averaged from 6 to 2 s, while the warmfluorescence signal FII was between 1.2 and 3.7 s (Figure 2c).Error bars of fluorescence F are the result of at least fourmeasurements.
Peptides. The dominant epitope in the second extracellulardomain of the 1-adrenergic receptor is the sequenceARRCYND forming a disulfide bridge with the cysteine ofthe first extracellular loop.11,34 COR1, an 18-mer cyclic peptidewith the sequence ADEARRCYNDPKCSDFVQ and the N-and C-termini linked via a peptide bond, represents this secondextracellular loop. COR1 was labeled with a red dye, D2(Figure 1c, Cisbio Bioassays, France).
Sample Preparation. Measurements were conducted inphosphate-buffered saline (PBS) with 0.1% Tween 20 andhuman serum. Serum from unaffected donors was collected inS-monovettes (Sarstedt, Germany), incubated at room temper-ature (30 min), and centrifuged (10 min, 12000g). Thesupernatant was aliquoted and stored at 20 C.
Measurement Protocols. For measurements with varyingantibody concentration, titration series of antibodies in PBSwere prepared using Quali-PCR-Tubes (Kisker, Germany). Aserial dilution was set up, starting with a concentration of 2.6M polyclonal antibody (pAB), with a 2-fold dilution down to2.6 nM. Separately, a 40 nM COR1-D2 stock was prepared inPBS with 0.2% Tween 20 or blood serum. The monoclonalantibody concentration ranged from 4000 to 1.6 nM with 200nM COR1-D2. The antibody and COR1 dilution series were
Figure 1. Target pathway. (a) Activation of the 1-adrenergic receptorvia a -adrenoceptoradenylyl cyclase protein kinase A cascade. ThecAMP-dependent PKA phosphorylates Ca2+ channels, and theresulting calcium influx increases the contractility of the myocard.(b) The extracellular loop of the 1-adrenergic receptor is mimickedby the peptide sequence COR1. (c) The labeled COR1 variant is usedto detect binding with thermophoresis.
Analytical Chemistry Article
dx.doi.org/10.1021/ac202923j | Anal. Chem. 2012, 84, 352335303524
mixed 1:1 to obtain final measurement samples. For theautocompetition assay, a serial dilution of unlabeled COR1from 4000 to 42 nM was set up in PBS. Separately, a stocksolution containing 40 nM labeled COR1-D2 was mixed withtwice the final pAB concentration in human serum at a 1:1ratio, resulting in a total COR1 concentration (labeled andunlabeled) from 2000 to 21 nM. For the measurements in 10%human serum, the stock solutions of COR1-D2 and pAB wereset up in 20% human serum and 80% PBS. After 2 h ofincubation at room temperature, about 5 L of each sample wasfilled into capillaries, which were then closed with sealing wax(NanoTemper, Germany).
RESULTS AND DISCUSSIONThermophoresis. The movement of molecules in a
temperature gradient2729 is modeled phenomenologically bythe drift velocity v = DT,i grad T with temperature gradientgrad T. The proportionality constant DT,i is termed thethermal diffusion coefficient or thermophoretic mobility.The index i distinguishes between the bound and unboundstates of the molecule. The drift is counterbalanced by massdiffusion:
= j c D T D cgrad gradi i i i iT, (1)
with diffusion coefficient Di and molecule concentration ci.Integration with a temperature-independent DT,i and Di yields asteady-state concentration of cT,i = ci exp(ST,iT) at theposition where the temperature is increased by T. The Soretcoefficient ST,i is defined by the ratio ST,i = DT,i/Di. A number ofways to measure thermophoresis have been devised in thepast.3032 Typically, the depleted concentration is lower, cT,i