Optimal Design of Microarray Immunoassays to Compensate for Kinetic Limitations
2006; Elsevier BV; Volume: 5; Issue: 9 Linguagem: Inglês
10.1074/mcp.t500035-mcp200
ISSN1535-9484
AutoresWlad Kusnezow, Yana V. Syagailo, Sven Rüffer, Nina Baudenstiel, Christoph Gauer, Jörg D. Hoheisel, David Wild, Igor Goychuk,
Tópico(s)Nanofabrication and Lithography Techniques
ResumoIn this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the am and low fm ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios. In this report we examine the limitations of existing microarray immunoassays and investigate how best to optimize them using theoretical and experimental approaches. Derived from DNA technology, microarray immunoassays present a major technological challenge with much greater physicochemical complexity. A key physicochemical limitation of the current generation of microarray immunoassays is a strong dependence of antibody microspot kinetics on the mass flux to the spot as was reported by us previously. In this report we analyze, theoretically and experimentally, the effects of microarray design parameters (incubation vessel geometry, incubation time, stirring, spot size, antibody-binding site density, etc.) on microspot reaction kinetics and sensitivity. Using a two-compartment model, the quantitative descriptors of the microspot reaction were determined for different incubation and microarray design conditions. This analysis revealed profound mass transport limitations in the observed kinetics, which may be slowed down as much as hundreds of times compared with the solution kinetics. The data obtained were considered with relevance to microspot assay diffusional and adsorptive processes, enabling us to validate some of the underlying principles of the antibody microspot reaction mechanism and provide guidelines for optimal microspot immunoassay design. For an assay optimized to maximize the reaction velocity on a spot, we demonstrate sensitivities in the am and low fm ranges for a system containing a representative sample of antigen-antibody pairs. In addition, a separate panel of low abundance cytokines in blood plasma was detected with remarkably high signal-to-noise ratios. Microarray immunoassays are gaining in importance as an analytical platform for the high throughput detection of proteins in biological fluids (1Kusnezow W. Hoheisel J.D. Antibody microarrays: promises and problems.BioTechniques (suppl.). 2002; : 14-23Crossref PubMed Scopus (142) Google Scholar, 2Haab B.B. Antibody arrays in cancer research.Mol. Cell. 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Schneider E.M. Hug H. Advances in recombinant antibody microarrays.Clin. Chim. Acta. 2004; 343: 17-35Crossref PubMed Scopus (104) Google Scholar), and diverse assay formats (2Haab B.B. Antibody arrays in cancer research.Mol. Cell. Proteomics. 2005; 4: 377-383Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 5Kusnezow W. Pulli T. Witt O. Hoheisel J.D. Solid support for protein microarrays and related devices.in: Schena M. Protein Microarrays. Jones and Bartlett Publishers, Sudbury, MA2004: 247-284Google Scholar, 7Angenendt P. Progress in protein and antibody microarray technology.Drug Discov. Today. 2005; 10: 503-511Crossref PubMed Scopus (268) Google Scholar). These can be roughly grouped into antibody, antigen, and reverse phase microarrays as well as multispotting techniques. Nevertheless the applicability of current antibody microarrays for profiling complex biological specimens, which was one of the main tasks that originally motivated the overall development of this technology, is still restricted at the moment. If simple detection strategies are used, such as protein labeling with dyes or haptens, the best detection thresholds achieved tend to be in the nm to mid-pm range (2Haab B.B. Antibody arrays in cancer research.Mol. Cell. Proteomics. 2005; 4: 377-383Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 3Nielsen U.B. Geierstanger B.H. Multiplexed sandwich assays in microarray format.J. Immunol. Methods. 2004; 290: 107-120Crossref PubMed Scopus (174) Google Scholar, 8MacBeath G. Protein microarrays and proteomics.Nat. Genet. 2002; 32: 526-532Crossref PubMed Scopus (745) Google Scholar). Powerful signal-generating systems such as rolling circle amplification (9Schweitzer B. Roberts S. Grimwade B. Shao W. Wang M. Fu Q. Shu Q. Laroche I. Zhou Z. Tchernev V.T. Christiansen J. Velleca M. Kingsmore S.F. Multiplexed protein profiling on microarrays by rolling-circle amplification.Nat. Biotechnol. 2002; 20: 359-365Crossref PubMed Scopus (495) Google Scholar) or detection by resonance light-scattering colloidal gold particles (10Saviranta P. Okon R. Brinker A. Warashina M. Eppinger J. Geierstanger B.H. Evaluating sandwich immunoassays in microarray format in terms of the ambient analyte regime.Clin. Chem. 2004; 50: 1907-1920Crossref PubMed Scopus (77) Google Scholar) need to be applied to improve the limit of detection (LOD) 1The abbreviations used are: LOD, limit of detection; GPTS, (3-glycidoxypropyl)trimethoxysilane; IFNG, interferon-γ; IL, interleukin; KLH, keyhole limpet hemocyanin; TCM, two-compartment model; TG, thyroglobulin; anti-IFNG, monoclonal anti-human interferon-γ antibody; anti-KLH, affinity-isolated anti-hemocyanin antibody; anti-TG, monoclonal anti-thyroglobulin antibody; NHS, N-hydroxysuccinimide; PEG, polyethylene glycol. 1The abbreviations used are: LOD, limit of detection; GPTS, (3-glycidoxypropyl)trimethoxysilane; IFNG, interferon-γ; IL, interleukin; KLH, keyhole limpet hemocyanin; TCM, two-compartment model; TG, thyroglobulin; anti-IFNG, monoclonal anti-human interferon-γ antibody; anti-KLH, affinity-isolated anti-hemocyanin antibody; anti-TG, monoclonal anti-thyroglobulin antibody; NHS, N-hydroxysuccinimide; PEG, polyethylene glycol. and to attain the low fm range analyzing complex biological samples. Successful commercial examples include the Clontech antibody microarray system containing ∼380 well characterized antibodies (11Sukhanov S. Delafontaine P. Protein chip-based microarray profiling of oxidized low density lipoprotein-treated cells.Proteomics. 2005; 5: 1274-1280Crossref PubMed Scopus (23) Google Scholar) and Zeptosens, protein microarrays enabling detection to 10 pg/ml analyte in serum (12Pawlak M. Schick E. Bopp M.A. Schneider M.J. Oroszlan P. Ehrat M. Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis.Proteomics. 2002; 2: 383-393Crossref PubMed Scopus (220) Google Scholar).However, this situation is inconsistent with the performance predicted by ambient analyte theory (13Ekins R.P. Ligand assays: from electrophoresis to miniaturized microarrays.Clin. Chem. 1998; 44: 2015-2030Crossref PubMed Scopus (269) Google Scholar, 14Ekins R. Chu F. Multianalyte microspot immunoassay. The microanalytical 'compact disk' of the future.Ann. Biol. Clin. 1992; 50: 337-353PubMed Google Scholar). The ready availability of standard scanner resolutions of 0.1 and less Cy molecules/μm2, antibodies with a picomolar affinity (10−9–10−11 m), and typical density of binding sites in the range of 105/μm2 should ideally allow achievement of attomolar sensitivity even with simple detection approaches. Nevertheless to the best of our knowledge such a high sensitivity has not been demonstrated thus far without using expensive amplification techniques.In addition to some known reasons such as low stability of antibody molecules, strong background signal due to inevitable protein adsorption on all kinds of surfaces, or even insufficient sensitivity of the detection approach itself, we have demonstrated recently a strong domination of the mass transport constraints in the reaction kinetics on examples of a typical antibody microspot assay (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar, 16Kusnezow W. Syagailo Y.V. Goychuk I. Hoheisel J.D. Wild D.G. Antibody microarrays: the crucial impact of mass transport on assay kinetics and sensitivity.Expert Rev. Mol. Diagn. 2006; 6: 111-124Crossref PubMed Scopus (58) Google Scholar, 17Klenin K.V. Kusnezow W. Langowski J. Kinetics of protein binding in solid-phase immunoassays: theory.J. Chem. Phys. 2005; 122: 214715Crossref PubMed Scopus (21) Google Scholar). Due to a small binding area, the microspot kinetics depends intrinsically on the analyte concentration. Therefore, even if ideal mass transport-independent incubation conditions could be achieved, the reaction on a microspot may still require many hours, or even tens of hours, to reach the thermodynamic equilibrium at relatively low analyte concentrations (L0), i.e. at L0 ≪ Kd where Kd is the binding affinity constant in m. In contrast to this, the reaction may be accomplished relatively fast at L0 ≫ Kd (few minutes or seconds). To enable analysis of the mass transport dependence of binding reactions on microspots, a mathematical tool was developed in our previous study (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar). This tool is based on the so-called two-compartment model (TCM), which is widely used for interaction studies using the Biacore instruments (18Goldstein B. Coombs D. He X. Pineda A.R. Wofsy C. The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore.J. Mol. Recognit. 1999; 12: 293-299Crossref PubMed Scopus (92) Google Scholar, 19Schuck P. Minton A.P. Analysis of mass transport-limited binding kinetics in evanescent wave biosensors.Anal. Biochem. 1996; 240: 262-272Crossref PubMed Scopus (190) Google Scholar, 20Schuck P. Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport.Biophys. J. 1996; 70: 1230-1249Abstract Full Text PDF PubMed Scopus (206) Google Scholar). TCM dissects the mass transport-dependent binding into two steps: (i) transport of the analyte from the bulk compartment to the reaction area (reaction compartment) and (ii) the subsequent binding process. Using this model, the overall reaction rate in the antibody microspots was found to be strongly impaired by the mass transport constraints, being capable of prolonging the times required for a solution reaction by many orders of magnitude. As a consequence of this, the saturation of signal intensity on a highly affine antibody spot can be achieved only after unrealistically long incubation of tens, hundreds, or even thousands of hours (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar, 16Kusnezow W. Syagailo Y.V. Goychuk I. Hoheisel J.D. Wild D.G. Antibody microarrays: the crucial impact of mass transport on assay kinetics and sensitivity.Expert Rev. Mol. Diagn. 2006; 6: 111-124Crossref PubMed Scopus (58) Google Scholar).In contrast to other kinetically relevant effects, e.g. related to antibody immobilization (partial denaturation, heterogeneous affinity, steric hindrances for binding, etc.), the strong mass transport limitation of the reaction kinetics seems to be the primary physicochemical shortcoming of the current microarray technology. Therefore, the kinetically relevant optimization of microarray parameters aimed to reduce mass transport limitations has to be one of the first and most important considerations in protein microspot assay design. We carried out a kinetic analysis experimentally and using a modified TCM theory to assess some basic design characteristics of a microarray immunoassay such as incubation geometry, spot size, stirring, and binding site density on a spot. Additionally we investigated possible reaction mechanisms underlying the signal development on the antibody microspot and tried to understand the physicochemical nature of several experimental microarray characteristics. Finally by optimizing a conventional assay format with a simple signal generation and detection approach, we achieved am and low to mid-fm sensitivities for several labeled antigens. Moreover we were able to detect a group of low abundance cytokines in the blood plasma with very high signal-to-noise ratios.EXPERIMENTAL PROCEDURESMaterials—All chemicals and solvents were purchased from Fluka (Taufkirchen, Germany), Sigma, or SDS (Peypin, France) unless stated otherwise. Untreated slides were purchased from Menzel-Gläser (Braunschweig, Germany). Milk powder, (3-glycidoxypropyl)trimethoxysilane (GPTS), recombinant human interferon-γ (IFNG), monoclonal anti-human interferon-γ antibody (anti-IFNG), keyhole limpet hemocyanin (KLH), and affinity-isolated anti-hemocyanin antibody (anti-KLH) were obtained from Sigma. Thyroglobulin (TG) and monoclonal anti-thyroglobulin antibody (anti-TG) were obtained from HyTest Ltd. (Turku, Finland). For profiling experiments, anti-IL1A, anti-IL1B, anti-IL2, anti-IL4, anti-IL6, anti-IL8, anti-IL10, anti-IL12B, IL15, anti-interferon-α, anti-IFNG, anti-tumor necrosis factor α, anti-transforming growth factor β1, anti-granulocyte-macrophage colony-stimulating factor 2, anti-vascular endothelial growth factor, and anti-fibroblast growth factor 2 were obtained from Acris Antibodies (Hiddenhausen, Germany).Fabrication of Antibody Arrays—Homemade epoxysilanized slides were manufactured according to the following protocol. Untreated slides were washed with ethanol, then etched overnight by immersion in 10% NaOH, cleaned by sonicating for 15 min, rinsed four times in water, washed twice in ethanol, and derivatized in a 100% GPTS solution at room temperature for 3 h. After silanization, GPTS-treated slides were washed thoroughly with dichloroethane and dried with N2. The reason for usage of the homemade slides was their lower variability in comparison with the different batches of commercial epoxysilane-coated slides. PBS buffer supplemented with 0.5% trehalose was used as a spotting buffer (21Kusnezow W. Jacob A. Walijew A. Diehl F. Hoheisel J.D. Antibody microarrays: an evaluation of production parameters.Proteomics. 2003; 3: 254-264Crossref PubMed Scopus (266) Google Scholar). The antibodies were spotted using an SDDC-2 Micro-Arrayer from Engineering Services Inc. (Toronto, Canada) and SMP15, SMP10, SMP3, and SMP2 pins (TeleChem). The slides were spotted with antibody concentrations of 2 mg/ml in spotting solution if not stated otherwise. It was initially found that different pins produce spots with similar antibody density at this antibody concentration (variation less then 15%; data not shown). The slides for all kinetics experiments were produced so that only one spot could centrally be positioned in every incubation chamber. After spotting the slides were incubated at 4 °C overnight and subsequently blocked for 3 h at room temperature in PBST (PBS with 0.005% Tween 20) supplemented with 4% milk powder. The slides for profiling experiments were spotted with 2 mg/ml antibody in spotting solution. So-called FullArea chips were made by pipetting 30 μl of 150 μg/ml anti-IFNG in spotting buffer on the bottom of a Flexiperm well (Sigma). The Flexiperms were of 3.3-mm well radius and 10-mm height and fixed on the slide surface using double adhesive tape. FullArea chips, where the bottom of the incubation chamber is completely coated by antibodies, were designed to be comparable to the classical ELISA incubation geometry. Incubation and blocking was done as described above.Antigen Labeling and Incubation—Antigen solution of 1 mg/ml was labeled with the monofunctional NHS ester of Cy3 dye (Amersham Biosciences) as recommended by the manufacturer. Unreacted dye was blocked from further reaction by adding hydroxylamine to a final concentration of 1 m and separated from the labeled proteins by PD-10 columns (Sephadex™ G-25, Amersham Biosciences).The slides before incubation were first rinsed for a few minutes in PBS and dried by centrifugation to remove an aqueous film on the slide surface that may potentially prolong and modify the reaction kinetics due to the additional diffusion time through this film. Incubation of the microarrays with antigens always occurred in Flexiperms and with the incubation volume of 100 μl (PBS, 4% skim milk, and 0.01% sodium azide) unless stated otherwise. For stirring we usually used the SlideBooster (Advalytix, Brunnthal, Germany), which allows for the simultaneous incubation of up to 12 slides with high agitation efficiency so that e.g. up to 48 reactions in Flexiperm format could be processed in parallel. The surface acoustic wave mixing technology of the instrument has no dead volume and allows for the agitation of various reaction geometries (cover glass, microtiter plate well, or open drop) making it well suited for our investigations (22Toegl A. Kirchner R. Gauer C. Wixforth A. Enhancing results of microarray hybridizations through microagitation.J. Biomol. Tech. 2003; 14: 197-204PubMed Google Scholar). The SlideBooster agitation parameters were optimized as indicated previously (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar). To test the suitability of the incubation geometry, the following incubation chambers were also used: "slidebox" (chamber size, 15 × 26 × 80 mm), EasySeal with dimensions 16 × 28 × 0.5 mm (ThermoHybaid Ltd., Ashford, UK), and Flexiperm as mentioned above. In contrast to EasySeals and Flexiperms, slideboxes were incubated on a shaker. To avoid potential photobleaching effects especially at long incubation times, all incubation chambers were isolated from light. Incubations were performed under stirring and non-stirring conditions. After the incubation, the slides were rinsed several times with PBST.For profiling experiments, 1 mg of serum was labeled with 100 μg of biotin-PEG4-NHS (Quanta-Biodesign), and the unreacted reagent was separated from the proteins using Microcon centrifugal units (Millipore, Schwalbach, Germany). After 14 h of incubation with 300 μl/well, the slides were washed two times for 5 min each with PBST and incubated for 30 min with 100 nm ExtrAvidin (Sigma) labeled with Dy647-NHS (Dyomics) in the SlideBooster. Finally the slides were washed five times (5 min each) with PBST.Scanning and Data Analysis—Fluorescence signals were recorded using a ScanArray5000 unit (Instrument Co.) and analyzed with the GenePix software package (Axon Instruments). The results were stored and managed in an appropriate Microsoft Access database. All data points in this work represent an average of three to six individual measurements obtained.The development of the microspot intensities over time was analyzed using the analytical solution of two-compartment theory (18Goldstein B. Coombs D. He X. Pineda A.R. Wofsy C. The influence of transport on the kinetics of binding to surface receptors: application to cells and BIAcore.J. Mol. Recognit. 1999; 12: 293-299Crossref PubMed Scopus (92) Google Scholar, 19Schuck P. Minton A.P. Analysis of mass transport-limited binding kinetics in evanescent wave biosensors.Anal. Biochem. 1996; 240: 262-272Crossref PubMed Scopus (190) Google Scholar, 20Schuck P. Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport.Biophys. J. 1996; 70: 1230-1249Abstract Full Text PDF PubMed Scopus (206) Google Scholar) as described previously (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar), S(t)=S∞(1−W(c exp(−Γt))/W(c))(Eq. 1) where S∞ = SmaxL0/(L0 + Kd), S and Smax are the maximum steady state, and current and maximally attainable signal intensities, respectively, all three parameters expressed in signal units (SU); Kd = k−/k+ where k+ and k− are the association and dissociation rate constants (m−1 s−1 and s−1, respectively); L0 is the initial analyte concentration (m); and t is the time in seconds. W(x) in Equation 1 is the Lambert special function defined as the solution of the equation W(x)exp(W(x)) = x (23Corless R.M. Gonnet G.H. Hare D.E.G. Jeffrey D.J. Knuth D.E. On the Lambert W function.Adv. Comput. Math. 1996; 5: 329-359Crossref Google Scholar), c = aexp(a) where a = k+L0Smax/(k−Smax + Km(Kd + L0)) is a dimensionless parameter that measures the deviation of the kinetics in Equation 1 from an ideal single exponential kinetics, and Γ = (k− + k+L0)/(1 + k−Smax/Km(Kd + L0)) is the rate of approaching the steady state where Km is the phenomenological mass transport constant (SU × m−1 s−1). Alternatively the experimental data can be fitted to the following equation. Γt=a S(t)S∞−ln (1−S(t)S∞)(Eq. 2) Equation 1 can be obtained by inversion of Equation 2 and vice versa. To describe the initial development of signal intensity (S(t) ≪ S∞), one can derive from Equation 2 a simple linear expression, S(t)≈ S∞Γ1+a t=Smaxv0t(Eq. 3) where v0 is the initial binding reaction velocity which is given by 1/v0 = 1/videal + 1/vm where videal = k+L0 is the ideal initial binding velocity and vm = KmL0/Smax is the mass transport contribution. In the case of mass transport-limited binding (vm ≪ videal), the initial binding velocity equals approximately the mass transport velocity, v0 ≈ vm.Under non-stirring conditions, the corresponding binding velocity and mass transport rate can be found from the following approximation. Let us assume a fully absorbing disc (videal → ∞) of radius R homogenously covered by antibodies with the density of binding sites ρ. All of the remaining surface is assumed to be totally reflecting. In the stationary Smoluchowski limit, the rate of absorption by such a disc is known to be KS = 4DRL0 where D is the bulk diffusion coefficient of the analyte molecules (in cm2/s). The number of antibodies with antigen bound increases with time as N(t) = KSt, and the signal increases accordingly as S(t) = SmaxN(t)/ Nmax where Nmax = πR2ρ is the total number of antibodies available. On the other hand, in the considered limit we have S(t) = Smaxvmt with vm = KmL0/Smax. From this it follows that KmL0πR2ρ = SmaxKS and thus km=4DSmax/(πρR).(Eq. 4) Furthermore the overall reaction becomes obviously mass transport-limited when the experimentally estimated Damkoehler number, Da−exp= videalvm=k+Smaxkm,(Eq. 5) is large, Da-exp ≫ 1. For this particular model with Km in Equation 4, Da−theo= k+πRρ4D(Eq. 6) where Da-theo is the theoretically determined Damkoehler number. It grows linearly both with the spot radius R and with the antibody density ρ, implying that smaller spots with lower antibody density are preferable to minimize mass transport limitations under the model assumptions.When the stirring is applied, the situation becomes more complicated. In this case, the mathematical response of the rate of mass transport or the Damkoehler number to stirring intensity, diffusion coefficient, spot radius, and density of binding sites is currently not known. Nevertheless the two-compartment model remains a powerful and insightful tool for kinetic analysis if the rate constant of mass transport is experimentally determined. On the phenomenological grounds, an effective diffusion coefficient can be introduced for all the remaining parameters. The effective diffusion coefficient might then be determined using Equation 4 used merely for its definition. However, to have an experimental definition of the effective diffusion coefficient that does not depend on the two-compartment modeling, we determined it experimentally from the FullArea chip measurements (from non-stationary initial binding kinetics) as described below.To analyze the data obtained from FullArea chips, an equation from the reaction-diffusion theory by Stenberg and Nygren (24Nygren H. Stenberg M. Immunochemistry at interfaces.Immunology. 1989; 66: 321-327PubMed Google Scholar, 25Stenberg M. Nygren H. A diffusion limited reaction theory for a solid-phase immunoassay.J. Theor. Biol. 1985; 113: 589-597Crossref PubMed Scopus (32) Google Scholar, 26Stenberg M. Nygren H. Kinetics of antigen-antibody reactions at solid-liquid interfaces.J. Immunol. Methods. 1988; 113: 3-15Crossref PubMed Scopus (182) Google Scholar) was used. S(t)= 2L0Smaxρ Dt/π(Eq. 7) Previously determined affinity parameters for anti-IFNG (15Kusnezow W. Syagailo Y.V. Ruffer S. Klenin K. Sebald W. Hoheisel J.D. Gauer C. Goychuk I. Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface.Proteomics. 2006; 6: 794-803Crossref PubMed Scopus (93) Google Scholar) (k+ = 527,000 m−1 s−1, k− = 0.000322 s−1, and Kd = 611.01 pm as measured by Biacore) as well as binding site density (ρ = 10−11 mol/cm2) were used for this mathematical analysis. Affinity of anti-TG was also measured using Biacore (k+ = 45,520 m−1 s−1, k− = 0.000998 s−1, and Kd = 21.93 nm). We observed that in the presence of stirring the initial FullArea chip kinetics was described by a square root dependence (as in Equation 7). For this reason, the experimental definition of the effective diffusion coefficient in the presence of stirring was obtained by applying Equation 7 for the corresponding data analysis.RESULTSImpact of Different Incubation Geometries on Signal Development—To find the optimal assay geometry, three different designs were tested under stirring and non-stirring conditions. The geometry slidebox (15 × 26 × 80 mm) may be considered as an unlimited incubation chamber (Fig. 1i). "Flexiperm" has classical well dimensions with a radius of 3.3 mm and height of 10 mm. "EasySeal" is a typical flat incubation geometry for microarrays with dimensions 16 × 28 × 1 mm. To determine the most effective stirring method, the slideboxes were incubated on a shaker at 150 rpm. This condition was found to be the most intense, leading to the highest attainable signals in this system.Development of signal intensities was observed on anti-IFNG spots printed with SMP3 pins (spot radius, about 90 μm) with a 200 pm concentration of IFNG. Note this analyte concentration is below the Kd value of the IFNG antigen-antibody pair (about 611 pm), and therefore the progression curves were close to the maximal duration. The well chamber was the best performing system and required stirring for more than 20 h to reach maximum signal intensity (Fig. 1ii, A). In comparison with this, the incubation using flat EasySeals resulted in up to a 6-fold decrease in signal intensity at the initial time points, whereas only 30–40% of the maximum could be attained after 2 days using this format (Fig. 1ii, C). Under non-stirring condition, it was impossible to reach the saturation of signal intensity for any of the geometries tested. Also application of the standard glass coverslip usually used for microarray incubations revealed a strong decrease in signal velocity even in comparison with the E
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