Anal. Chem. 2007, 79, 8601-8607
Particulate Platform for Bioluminescent Immunosensing Karin Fromell,† Greta Hulting,† Alexander Ilichev,†,‡ Anders Larsson,§ and Karin D. Caldwell*.†
Department of Physical and Analytical Chemistry, and Department of Medicinal Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden, and Semenov Institute of Chemical Physics RAS, Moscow, Russia
The present study examines pyruvate kinase-conjugated antibodies for potential use in ELISA applications. The conjugates had an acceptable stability, and the coupling inflicted only minor impairment on the kinase activity. To mimic the setup of an immunoassay under development, a test antigen (BSA) was attached to polystyrene nanoparticles. This arrangement was found to be suitable as solid support for presentation of antigens in sensitive bioluminescence assays. The nanoparticles were well characterized in terms of protein surface load and were used to establish the number of conjugate complexes needed to generate a detectable signal. Under the biochemical conditions employed here, the detection limit of the pyruvate kinase conjugate lies in the femtomole range. The clinical needs for ever more sensitive detection and quantification methodology has given rise to a large number of antibody-based analytical methods, where the antibody is carrying a label responsible for signal generation. When the signal is created by an appended enzyme with high turnover number, even short incubation times will lead to significant signal amplifications. While the choice of antibody dictates the selectivity and specificity of the detection technique, the sensitivity is determined by the signal generation mechanism together with the number of accessible binding sites of the analyte. When the ELISA is used in a biosensor analytical format, the antigen is generally captured by a solid-phase measuring surface and is subsequently exposed to the labeled antibody, which it binds if readily accessible. The tasks of determining antigen surface concentration and the limit of detection, i.e., the number of conjugate complexes required to yield a readable signal in the chosen sensor format, are nontrivial. In the present study, we have chosen to use nanoparticles with well-characterized protein surface coatings as reagents to establish the necessary sensitivity criteria. In addition to expanding available analytical area, these particles with their highly curved surfaces facilitate access and shorten the distance between reactants, in turn speeding up the reaction. Fluorescence-based detection is a frequently utilized technique due to its generally high S/N ratios and detection limits in the * To whom correspondence should be addressed. E-mail: Karin.Caldwell@ biosurf.uu.se. † Department of Physical and Analytical Chemistry, Uppsala University. ‡ Semenov Institute of Chemical Physics RAS. § Department of Medicinal Sciences, Clinical Chemistry, Uppsala University. 10.1021/ac0715118 CCC: $37.00 Published on Web 09/21/2007
© 2007 American Chemical Society
picomolar to-femtomolar range. Requirements for such performance are strong and well focused light sources for excitation paired with an effective capture of emitted light. This necessitates well-aligned and stably mounted optical components if one is to reach the high levels of sensitivity and the low coefficients of variation (CV) that are necessary for accurate diagnostic work. For measurement techniques that are to be used in mobile and miniaturized devices, these demands are at times prohibitive and other signal-producing principles are required. In recent years, analytical chemists have shown an increasing interest in detection techniques based on bioluminescence.1-4 Such techniques are by their very nature energy efficient, emitting photons in quantities directly proportional to the number of chemical bonds broken or generated in reactions that are catalyzed by highly specific enzymes. Because of the relative ease of performing efficient photon collection, the high quantum yields typically associated with such reactions and the low background signal stemming from the site-specific emission, bioluminescent reactions are good candidates for robust and sensitive signal generation. Firefly (Photinus pyralis) luciferase is an enzyme that converts the substrate luciferin into oxyluciferin under the emission of light in the 540-600-nm wavelength range. In addition to requiring the presence of oxygen and Mg 2+ ions, the reaction is energetically enabled by the presence of ATP, which binds to the luciferase and thereby activates the enzyme. The number of photons emitted in this oxidation reaction is a direct measure of the number of ATP molecules consumed in the process.5,6 The firefly luciferase molecule has two drawbacks in the ELISA context: first, it is structurally sensitive and loses its activity in most chemical conjugation efforts,7 and second, it has a relatively low turnover number.8 Therefore, strategies for analyte detection and quantification based on measurements of light emitted by the luciferase/ luciferin reaction either have involved other, more rapid types of luciferase, e.g., that from Cypridina noctiluca recently described,9 (1) Roda, A.; Pasini, P.; Mirasoli, M.; Michelini, E.; Guardigli, M. Trends Biotechnol. 2004, 6, 295-303. (2) Sato, A.; Klaunberg, B.; Tolwani, R. Comp. Med. 2006, 6, 631-634. (3) Issad, T.; Jockers, R. Methods Mol. Biol. 2006, 332, 195-209. (4) Doleman, L.; Davies, L.; Rowe, L.; Moschou, E. A.; Deo, S.; Daunert, S. Anal. Chem. 2007, 11, 4149-4153. (5) DeLuca, M.; McElroy, W. Biochemistry 1974, 5, 921-925. (6) Lundin, A.; Rickardsson, A.; Thore, A. Anal. Biochem. 1976, 75, 611-620. (7) Ugarova, N. N.; Enzyme Microb. Technol. 1982, 4, 224-228. (8) DeLuca, M. A; McElroy, W. D. Methods Enzymol. 1978, 57, 3-15. (9) Wu, C.; Kawasaki, K.; Ogawa, Y.; Yoshida, Y.; Ohgiya, S.; Ohmiya, Y. Anal. Chem. 2007, 79, 1634-1638.
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or have involved the enzyme in soluble form, as in the DNA sequencing technique referred to as pyrosequencing, where the required ATP is generated in the actual sequencing reaction.10 In the present study, we set out to explore a sandwich ELISA strategy in which the “second antibody” is conjugated not to the luciferase but to the enzyme pyruvate kinase that produces the necessary ATP by phosphorylation of ADP according to the following stoichiometric relationship: pyruvate kinase
ADP + phospho(enol)pyruvate 98 ATP + pyruvate >luciferase ATP + luciferin + O2 98 AMP + oxyluciferin + CO2 + pyrophosphate + light
By comparison with the luciferase, the kinase is rather easily manipulated with conventional conjugation chemistry without significant loss of activity. Its turnover number, (240 s-1) according to Reynard et al.,11 is slightly under three times that of the firefly luciferase (96 s-1),8 and its stability upon storage is good, both factors that speak in favor of examining its potential use in second antibody conjugates for ELISA applications. In order to evaluate the signal strength provided by a pyruvate kinase-conjugated second antibody, we have attached a test antigen (bovine serum albumin, BSA) to polystyrene (PS) nanoparticles. Suspensions of such coated particles have been allowed to bind the kinaseconjugated antibody, and the complex has been evaluated for information about detection limits of the system. The present work is part of a feasibility study aiming at developing a robust pointof-care device for sensitive diagnostics. EXPERIMENTAL SECTION Materials. Polystyrene particles with a nominal diameter of 240 nm (10% solids) were produced by Bangs Laboratories, Inc. (Fishers, IN). Pluronic F108 modified with a pyridyl disulfide group (F108-PDS, Cellink) was kindly donated by Allvivo Inc. (Lake Forest, CA). BSA, pyruvate kinase (PK) from rabbit muscle, (EC 2.7.1.40), and phospho(enol)pyruvate (PEP) were purchased from Sigma. Adenosine diphosphate (ADP) was from Fluka, and D-luciferin and luciferase (EC 1.13.12.7) were from BioThema AB (Handen, Sweden). The antiBSA IgY antibodies were supplied by the Department of Medical Sciences, University Hospital, Uppsala, Sweden. For all bioluminescence measurements, a “reaction cocktail” was prepared by mixing 1 µL of luciferin (10 mg/mL), 4 µL of luciferase (0.5 mg/mL), and 10 µL of ADP (5.2 µM) per sample. The ADP was 99% pure (HPLC) according to the supplier; still, the 1% ATP impurity was enough to give a strong initial light intensity, especially in a sensitive system. Therefore, the reaction cocktail was always incubated at least 20 min before measurement to ensure that most of the ATP was used up by the luciferinluciferase reactants. The bioluminescence measurements were performed in 50 mM Gly-Gly buffer containing 1 mM TRIS, 5 mM MgSO4, and 5 mM KCl (pH 7.6); this solution is referred to as “buffer A” below. A CCD camera was used to follow light (10) Ahmadian, A.; Ehn, M.; Hober, S. Clin. Chim. Acta 2006, 1-2, 83-94. (11) Reynard, A. M.; Hass, L. F.; Jacobsen, D. D.; Boyer, P. D. J. Biol. Chem. 1961, 8, 2277-2283.
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evolution from reactions taking place in microtiter wells of 200µL volume, while a Berthold FB12 luminometer was used to follow reactions in 5-mL ATP-free round cuvettes of polypropylene from Sarstedt (Nu¨mbrecht, Germany). Methods. Validation of Light Emission by the Chosen Reagents. The ability to relate a given quantity of ATP to a recorded amount of emitted light in the presence of a given reaction mixture was determined using a CCD camera, which had been previously calibrated with a photodiode to ensure proportionality between diode emitted light and camera current. This camera was mounted in a light-tight box and positioned a fixed distance from a platform with a 96-well microtiter plate (NUNC) made of white opaque plastic for minimal cross talk between wells. The output from the camera was fed to a PC, which imaged the entire plate at 5-s intervals. Light emissions from the wells of interest were first measured prior to the addition of ATP (or PEP, as appropriate) and subsequently during an actual reaction. The readings were then corrected by subtraction of the background. To each 200-µL well was added a mixture composed of 0.75 mM luciferin and 1.2 µM luciferase in buffer A. The oxidation of luciferin started immediately upon addition of ATP, and the reaction proceeded with the emission of light until all ATP had been consumed. In the case of an initial ATP concentration of 0.5 µM, the emission reached its maximum value after 80 s and had decayed to zero after 3 × 104 s. At this point, a new aliquot of ATP was added and the emission curve was faithfully reproduced. The integrated photon output curve was therefore considered to be proportional to the amount of ATP in the well. Preparation of (Anti-BSA)IgY antibodies. Laying White Leghorn hens were immunized intramuscularly in the breast muscle with 100 µg of BSA emulsified with an equal volume of Freund’s adjuvant (Difco Laboratories, Detroit, MI). After the initial immunization, the animals received three booster injections within 2-week intervals and thereafter every 2-3 months to ensure that the titer remained at a high level. Freund’s complete adjuvant was used for the primary immunization, and Freund’s incomplete adjuvant was used for all other immunizations. Eggs were collected continuously after the initial immunization. The antibodies were purified from the egg yolk by the PEG method of Akita and Nakai,12 and subsequently affinity purified on a BSA column. Bound antibodies were eluted with 0.1 M glycine, pH 2.25, and collected IgY fractions were pooled and dialyzed against 0.02 M Na2HPO4, 0.15 M NaCl, 0.02% NaN3, pH 7.2. Preparation of Enzyme-Antibody Conjugate. To conjugate the PK enzyme with the IgY antibody, pyridyl disulfide groups were first introduced to the enzyme to allow disulfide linkage to a free thiol on the antibody. This was done by the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP).13 Aliquots of 10 µL of 3 mM SPDP were added to 0.5 mL of PK (1.1 mg/mL), and the resultant mixture was incubated for 20 min at room temperature. Excess reagent was removed by gelfiltration using a NAP 5 column (GE Healthcare). Since the antibodies have no accessible thiol groups for conjugation, the same thing must be repeated for the IgY antibody, followed by reductive cleavage to obtain a free SH group for conjugation. An additional 10-µL aliquot of 30 mM SPDP was added to 0.4 mL of (12) Akita, E. M.; Nakai, S. J. Immunol. Methods 1993, 160, 207-214. (13) Carlsson, J.; Drevin, H.; Axe´n, R. Biochem. J. 1978, 173, 723-727.
IgY (1.18 mg/mL), and the resultant mixture was incubated for 30 min at room temperature, followed by gel filtration using a NAP 5 column. The antibody conjugate was then reduced with DTT (25 µL 100 mM at room temperature for 20 min) After desalting, using a NAP 10 column, the product was immediately transferred to the previously SPDP-modified pyruvate kinase. The conjugation was allowed to proceed for 1 h at room temperature after which the product was stored at +4 °C for periods exceeding 1 week. The concentration of pyridine-2-thione released by cleavage with DTT can be determined by measuring the absorbance at 343 nm, using 8080 M -1 cm -1 as the extinction coefficient. This concentration is equivalent to the concentration of pyridyl disulfide groups in the protein. The degree of substitution was estimated to be 1.2 per IgY molecule. The effect of the SPDP-reagent on the PK activity was explored by adding 10-µL aliquots of 1, 3, 10, or 30 mM SPDP reagent to one and the same amount of PK (0.25 mL, 1.1 mg/mL). The samples were then incubated for 30 min and subsequently desalted using a NAP 5 column. To measure the activity, an aliquot of 27.5 ng of each SPDP-modified PK preparation was added to 235 µL of buffer A and 15 µL of reaction cocktail; see above. The background was measured before addition of 10 µL of PEP (0.89 mg/mL). Unless otherwise noted, the luminescence was measured using a FB12 luminometer (Berthold Detection Systems). To determine the degree of substitution, the absorbance at 343 nm was measured before and after addition of 25 µL of DTT (100 mM). Preparation of Particles. Pluronic F108-PDS is an end groupactivated triblock copolymer with two relatively hydrophilic and mobile poly(ethylene oxide) (PEO) blocks and a centrally located hydrophobic poly(propylene oxide) block responsible for the adsorption. The PEO side chains have been activated by the introduction of a pyridyl disulfide group (PDS), to which thiolcontaining molecules can be covalently attached. The Pluronic F108-PDS surfactant was adsorbed to 240-nm PS particles by incubating a particle suspension containing 2% 240-nm PS particles in Milli-Q water with 10 mg/mL F108-PDS overnight. After coating, the particles were separated from excess surfactant by centrifugation using a table-top centrifuge (14 000 rpm, 20 min). The supernatant was removed, and the pellet was resuspended in PBS buffer. This procedure was repeated three times. Particle Fixation of Antigen (BSA). BSA molecules were prepared for attachment to the polymeric surfactant by reaction with the SPDP reagent described above. This was done by adding 20 µL of 30 mM SPDP to 1 mL (2 mg/mL) of BSA. After 30-min incubation, excess reagents were removed by desalting with a NAP 10 column. The introduced 2-pyridyl disulfide was then cleaved with DTT to give the thiolated BSA. Excess DTT and the produced pyridine-2-thione were removed by gel filtration with a NAP 10 column. The thiolated product was rapidly transferred to the suspension of F108-PDS-coated particles and incubated for 1 h at room temperature. Unbound BSA was removed by washing the particles three times using a suspension/centrifugation (14 000 rpm, 20 min) procedure. After each cycle, the supernatant was removed and the pellet was resuspended in PBS. Characterization of the Particles Using SdFFF. The theoretical basis for deriving sample size/mass from SdFFF retention
data has been described in detail elsewhere.14-16 The retention ratio, R, i.e., the ratio of the column void volume, V0, to the observed retention volume Vr, is experimentally determined under a given applied field and is expressed by
R ) V0/Vr ) 6λ[coth (1/2λ) - 2λ]
(1)
This retention ratio is the basis for determining the λ-value from which the mass of the particles can be calculated according to
λ ) kT/[ma(1 - Fc/Fa)Gw]
(2)
where k is the Boltzmann constant, T the temperature, ma the mass of the particle, Fc the density of the carrier liquid, Fa the density of the particle, w the channel thickness, and G the applied gravitational field. When the mass of the bare particle is known, the mass of the adsorbed or attached layer can be determined as
λ ) kT/[(ma(1- Fc/Fa)) + (mb(1- Fc/Fb))Gw]
(3)
where mb is the mass of the adsorbed/attached material and Fb is its density. The density of the bare particles F108-PDS and BSA has been determined to 1.053, 1.186, and 1.35 g/cm3, respectively, using a PAAR density meter (model DMA60+DMA602). The SdFFF system used in this work is a prototype of the commercially available SdFFF system from Postnova Analytics (Landsberg, Germany). Descriptions of the instrumentation and operation have been reported before.16,17 The sizing of the core particles were performed in 0.1% FL 70, while the coated particles were sized in 2.5 mM NH4HCO3 + 0.1% F108. The spin rate was kept at 1200 rpm, the relaxation time was set to 17 min, and the flow was maintained at 2.1 mL/min throughout each experiment. Testing of Ability To Bind Antigen. The PK-IgY (antiBSA) conjugates were added in six different amounts, ranging from 0 to 700 nmol, to Eppendorff tubes containing 30 µL of PS-F108BSA particles ( ∼7.5 × 1010 PS particles) and PBS to obtain a total volume of 850 µL. After 2 h of incubation on a shaker at room temperature, the particles were washed three times as above and resuspended in 50 µL of PBS. The conjugate activity was measured by mixing 50 µL of each particle suspension with 185 µL of buffer A and 15 µL of reaction cocktail. After background measurement, 10 µL (4.3 mM) of PEP was added and the resulting luminescence recorded each minute during a 5-min measuring time. Determination of Limit of Detection. The BSA particles to which 700 nmol of conjugate was added appeared to have reached saturation, since further additions resulted in no further uptake. These conjugate-containing particles were resuspended in 100 µL of PBS after final washing. From the particle suspension, aliquots (14) Giddings, J. C. Science 1993, 260, 1456-1465. (15) Caldwell, K. D.; Li, J-T.; Li,J-M.; Dalgeish, D. G. J. Chromatogr. 1992, 604, 63-71. (16) Moon, M. H. In Field-Flow Fractionation Handbook; Schimpf, M. E., Caldwell, K. D., Giddings, J. C., Eds.; Wiley-Interscience: New York, 2000; Chapter 15, pp 225-237. (17) Fromell, K.; Andersson, M.; Elihn, K.; Caldwell, K. D. Colloids Surf., B 2005, 46, 84-91.
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with volumes ranging from 0.25 to 50 µL were added to the test tubes together with 15 µL of reaction cocktail and each mixture was adjusted to a final volume of 200 µL through addition of buffer A. After measurements of the background signal, aliquots of 10 µL of PEP (0.89 mg/mL) were added and the luminescence measured every minute for 5 min. The detection limit for the surface-attached PK-IgY conjugate was then determined from a standard curve with known concentrations of “free” conjugate under otherwise identical measurement conditions. Testing the Selectivity of the Particle ELISA. The 240-nm PS particles were coated with F108-PDS, as described above, and the primary antibody was attached in a manner analogous to that described for BSA (see above). Aliquots of 100 µL of BSA (2 mg/ mL) and 100 µL of C-reactive protein (CRP; 2.56 mg/mL, Chemicon) were added to two tubes, each containing 100 µL of suspended particles surface coated with F108-(anti-BSA)IgY. The samples were incubated on a shaker for 30 min. Unbound material was removed after repeated (3×) centrifugations (14 000 rpm, 20 min) and resuspensions in buffer solution. The PK-IgY conjugate was then added to each tube (BSA and CRP) and placed on a shaker at +4 °C for 30 min. The samples were washed three times, as described before. After the third centrifugation, the particles were resuspended in 155 µL of buffer A. Two wells in a microtiter plate were prepared with 20 µL of luciferase, 5 µL of luciferin, and 10 µL of ADP. The plate was covered with foil and left to incubate for 60 min to remove the ATP impurity. Subsequently, the BSA- and CRP-containing samples described above were each added to one well, and the background was recorded. Aliquots of 10 µL of PEP were added to both wells, and the luminescence was determined using the CCD camera setup described above. RESULTS AND DISCUSSION Validation of Light Emission as a Measure of ATP Concentration. In a series of validation measurements, a luciferase/ luciferin mixture, with the composition given in the Methods section above, was added to each of five concentrations of ATP and the reactions were followed until no detectable light was emitted. This study showed the cumulative number of photons to be a linear function of the ATP concentration. The system was therefore considered to faithfully report the amount of ATP added to the wells. We assume that the ATP produced through phosphorylation of ADP, with PEP and the catalytic assistance of pyruvate kinase, is consumed in a manner identical to that added directly. Preparation of Enzyme-Antibody Conjugate. The ability to produce stable and active conjugates between the antibody and the signal-generating enzyme is of outmost importance in immunoassays. In order to explore the bioluminescence-based ELISA strategy in focus here, a model analyte and its antibody were selected. The choice fell on BSA for which an egg antibody (IgY) with high affinity had been developed by one of us (A.L.), but the same strategy can obviously be used for any other antigen of diagnostic interest. Egg antibodies, IgY, possess some biochemical advantages compared to mammalian antibodies. For instance, they can be used to eliminate assay interference caused by complement activation or rheumatoid factors, a property that is very important 8604 Analytical Chemistry, Vol. 79, No. 22, November 15, 2007
Figure 1. Activity of the SPDP-modified pyruvate kinase resulting from reaction with the coupling reagent in the indicated concentrations
in many immunoassays. In addition, they are easily purified in large amounts from egg yolks.18,19 An anti-BSA IgY was prepared for conjugation by reaction with the bifunctional reagent SPDP as described under Materials above. The enzyme selected here to signal antibody binding is pyruvate kinase, a tetrameric protein with four lysyl -amines and four sulfhydryl groups that all are essential for activity.20 Therefore, care had to be taken to leave as many of these groups as possible intact during the conjugation reaction (see below). Similar concerns regarding the potential loss of activity during conjugation were expressed earlier, albeit in conjunction with a different conjugation chemistry.21 The commercial preparation of pyruvate kinase from rabbit muscle was delivered in freeze-dried form. After dissolution in a 0.02 M PBS buffer of pH 7.4, the enzyme was subjected to gel filtration on a Sephacryl S-300 column coupled to a MALS-RI detector combination for molar mass determination of the eluate. Aside from a minor peak with high molar mass that eluted in the void, the chromatogram showed a well-defined peak with the expected molar mass of 271 kDa. In addition, the commercial sample contained a massive amount of low molar mass material that eluted in the total volume of the column. A quick test for kinase activity indicated that both the void and the main peaks were active, while the third peak was without activity. The propensity to aggregate makes measurements of enzyme kinetic parameters difficult to interpret. In preparation for antibody coupling, the kinase was reacted with SPDP in different concentrations. As seen in Figure 1, the enzymatic activity was somewhat perturbed by interaction with SPDP, although the activity remaining after contact with up to 3 mM reagent was still quite acceptable. By contrast, a 10-fold higher concentration of the reagent led to an unacceptable reduction in activity. Given the importance of both thiols and amines for kinase activity, the SPDP reagent with its reactivity to both groups had to be added with considerable care. The degree of substitution with 2-pyridyl disulfide residues was estimated from absorbance measurements after cleavage with DTT. The result is shown in Table 1. To enable conjugation of the kinase to the analyte-specific antibody, this component was also activated with SPDP. A subsequent reductive cleavage with DTT removed pyridyl groups from the IgY-attached SPDP linkers and laid bare the free thiols (18) Larsson, A.; Sjo ¨quist, J. Comp. Immunol. Infect. Dis. 1990, 13, 199-201. (19) Carlander, D.; Larsson. A. Uppsala J. Med. Sci. 2001, 106, 189-195. (20) Flashner, M.; Hollenberg, P. F.; Coon, M. J. J. Biol. Chem. 1972, 24, 81148121. (21) Gadow, A.; Fricke, H.; Strasburger, C. J.; Wood, W. G. J. Clin. Chem. Clin. Biochem. 1984, 22, 337-347.
Figure 2. Activity of different PK preparations in solution. The pyridyl disulfide-modified enzyme (PK‚PDS) is seen to display an activity largely comparable to the free enzyme (PK), while the antibody conjugate (PK-IgY) is somewhat less active. Table 1. Relationship between Concentration of Coupling Reagent and Number of Functional Groups Introduced per Enzyme Molecule conc SPDP (mM)
PDS groups/ PK molecule
1 3 10 30
4.5 4.4 5.6 18
that enabled the coupling of kinase to antibody. From the activity evaluation in Figure 2 it is clear that the conjugation reaction under the chosen conditions proceeds with only minor impairment of the kinase activity. We find conjugates prepared in this manner to be quite stable and withstand storage in solution in a refrigerator at +4 °C for over 1 week without significant losses of activity. Determination of Ability To Bind Antigen. In order to precisely quantify the conjugate’s ability to bind its antigen, if trapped on a surface, a test material was prepared consisting of BSA molecules attached to PS latex particles with a uniform diameter of 240 ( 1 nm. The BSA antigen was attached to Pluronic-coated nanoparticles, as described in the Materials section, to mimic the arrangement in a POCT system under development, where the capture antibody will be coupled to the nanoparticles. The Pluronic F108-PDS-coated nanoparticles are ideal platforms for antigen presentation as they offer high mobility of the attached molecules while minimizing steric hindrance to binding. This, in turn, facilitates capturing of antigen from complex samples such as blood or saliva. Thiolated BSA was attached to the coated particles whose various layers, i.e., Pluronic and BSA, each were quantified by SdFFF as outlined under Methods. The different steps in the buildup of this particulate test material are illustrated in Figure 3a. SdFFF fractograms depicting each step, and the corresponding mass determinations for the bare particles and their coating layers, are presented in Figure 3b. From the SdFFF analyses, one finds each PS particle to contain an average of 700 ( 50 BSA molecules (mean of three analyses). Equally sized aliquots of such characterized BSA particles were then exposed to a dilution series of the antibody-kinase conjugate and allowed to form the enzymatically active antigen-antibody complex during a 2-h incubation. Each aliquot was subsequently washed three times by centrifugation
Figure 3. (a) Schematic illustration of the particle coating process. (b) Mass determination by SdFFF. The elution curves represent (---) bare PS particles, (--) Pluronic F108-PDS-coated particles, and (---) particles with immobilized BSA.
at 14 000 rpm followed by supernatant removal, whereupon it was assayed for enzymatic activity. This test, which is illustrated in Figure 4, allowed a determination of the amount of conjugate needed to saturate the particulate antigen load. Here the resulting amount was 700 nmol/7.5 × 1010 BSA-containing particles. Although the curved surface of the nanoparticles increases access to surface-attached ligands and reduces steric hindrance to binding,22 larger amounts of conjugate than expected must be added to fully saturate the surface. Most likely the conjugates formed bigger complexes of more than two molecules, as suggested by the SEC-MALS result referred to initially. Since both (22) Huang, S. C.; Swerdlow, H.; Caldwell, K. D. Anal. Biochem. 1994, 222, 441-449.
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Figure 4. Particle ELISA: 3.75 × 1010 particles with the composition PS-F108-BSA to which additions of antibody conjugate PK-IgY (antiBSA) have been made. The formed complex is assayed after careful washing.
Figure 6. Testing the selectivity of the particle ELISA using the conjugates. The thick line represents the BSA sample and the thin line the negative control. The intensity is in arbitrary units.
Figure 5. (a) BSA-coated particles in different amounts, assayed following additions of one and the same amount of antibody conjugate (700 nmol). (b) Light emission curves representative of the amounts of conjugate associated with the data points in Figure 6a.
Testing the Selectivity of the Particle ELISA Using the Conjugates. The selective capture ability of the surface-attached IgY antibody was examined in a particle ELISA. Here the capture antibody was immobilized on the 240-nm PS particle surface via the F108-PDS linker, as described for the BSA-antigen above. After washing, the particles were exposed to solutions containing either BSA, for which they have high affinity, or another serum protein, namely, CRP, which was used as a negative control. After capture and the subsequent exposure to the PK-IgY conjugates, the particles were assayed for enzymatic activity. This time, the light signal was detected using the CCD camera described above under Validation. As seen from the results in Figure 6, there was a significant difference in intensity between those particles that had specifically taken up BSA and those that had been exposed to the negative control protein (CRP). While there is some unspecific uptake of the negative control protein, the IgY antibodies are in general highly specific, but like most proteins, they show some avidity to other proteins present in high concentrations.
the IgY and the PK themselves are large proteins (MW 180 and 237 kDa, respectively), these complexes are by their nature quite bulky and therefore hindered in their finding and binding to the antigen. Determination of Limit of Detection. A suspension was prepared in which the particles had exactly the composition of those having reached the saturation level in Figure. 5. From this suspension, a dilution series was made and assayed for bioluminescence intensity in the same manner as in Figure 5. As expected, the signal intensity decreased upon dilution of the suspension, reaching a level insignificantly different from the background. In the absence of luciferase inhibitors, the oxidation of luciferin occurs with a light intensity that is proportional to available ATP.23 An inspection of Figure 5 shows the limit of detection for this system (3 × CV) to be 2.5 pmol of particlebound BSA and 13.5 fmol of PK-IgY conjugate. On average, there are 22 BSA molecules/PK-IgY conjugate. This ratio is somewhat surprising, given that the PK-IgY conjugate is only about six times larger than the BSA molecule; it suggests that some conjugates containing aggregated PK may have found their way to the antigen, blocking further access, and therefore reducing the number of bound conjugates. However, the significant and stable signals generated by the 3.75 × 108 particles are a strong support for the particulate platform approach to immunosensing. (23) Lundin, A.; Thore, A. Anal. Biochem. 1975, 66, 47-63.
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CONCLUSIONS In the present study, it is demonstrated that polystyrene nanoparticles are well suited to serve as solid phase in the presentation of antigens for sensitive bioluminescence-based quantification. Even bulky pyruvate kinase-conjugated antibodies can readily bind to antigens on such particles and generate the ATP that energizes the light-producing oxidation of luciferin to oxyluciferin. With the detection geometry and under the biochemical conditions employed here, the limit of detection (3 × CV) for the bioluminescent assay lies at 15 fmol of the PK-IgY conjugate prepared under the conditions reported here, Since the coupling occurs through formation of a disulfide bridge, and since the kinase has essential thiols in its active sites, the coupling must be performed with minimal additions of the bifunctional reagent SPDP. The resulting conjugate is relatively stable and can be used even several weeks after preparation when stored refrigerated. The bioluminescence reaction gives a reproducible signal with low detection limits and a light generation that begins immediately after sample application. The detection instrumentation needed for measurement is relatively simple and easy to use, which makes it suitable for “point-of-care testing” (POCT) techniques. To mimick the design of an immunoassay for POCT under development, we chose BSA as a model protein to be coupled to a solid phase consisting of nanoparticles. In the present work, this has proven to be an arrangement highly suitable for the quantification of antigen with
second antibodies of the PK-IgY conjugate type used here. Even when other antigen-antibody systems are being used, the detection limit should not deviate from the femtomole concentration range for the signal-generating enzyme observed here.
the aim of developing a strategy for point-of care diagnosis. Discussions with Dr. Arne Lundin of Biothema, AB, are gratefully acknowledged. We also thank the students in our Biomaterials course, who were working with us during their laboratory practice.
ACKNOWLEDGMENT This work has enjoyed much appreciated support from the Swedish Science Council (VR) through grant 621-2004-4157. It is one part in a program supported by Uppsala Bio (VINNOVA) with
Received for review July 17, 2007. Accepted August 15, 2007. AC0715118
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