Increased Sensitivity in Antigen Detection with Fluorescent Latex

Bioconjugate Chem. 2010, 21, 427–435

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Increased Sensitivity in Antigen Detection with Fluorescent Latex Nanosphere-IgG Antibody Conjugates Tao Ji,* M. Catherine Muenker, Rao V. L. Papineni, John W. Harder, Douglas L. Vizard, and William E. Mclaughlin Carestream Molecular Imaging, Carestream Health, Inc., New Haven, Connecticut 06511. Received July 6, 2009; Revised Manuscript Received January 4, 2010

IgG antibodies were conjugated to Kodak X-Sight nanospheres to develop fluorescent-labeled antibodies using two different synthetic routes: one involving the DTT reduction method, and the other involving Traut’s Reagent modification method. These two methods result in different conjugation efficiencies and different performances in antigen detection. Western blotting shows that the nanosphere-IgG antibody conjugates synthesized using the DTT reduction method are more immunospecific than the conjugates synthesized using Traut’s Reagent modification method. In addition, the conjugates synthesized using DTT reduction also show higher antigen detection sensitivity than other commercially available fluorescent-IgG antibody conjugates, including Alexa Fluor, Qdot, and CyDye conjugates.

INTRODUCTION Fluorescent antibodies have been widely used in immunodetectionbothinlife-scienceresearchandinclinicalapplications(1-10). Most of the fluorescent labels used for the labeling of antibodies are organic molecules that suffer from photobleaching and relatively low fluorescence intensity. Efforts to improve the sensitivity of immunodetection by exploring highly fluorescent and photostable labels have gained considerable momentum over the past decade (11-16).1 We have introduced a class of highly fluorescent organic nanospheres, Kodak X-Sight nanospheres (17), for fluorescent labeling of biomolecules. Our nanospheres are approximately 20 nm in diameter and are composed of a cross-linked polymer core coated with a poly(ethylene glycol) derivative that confers over 200 primary amines to the particle periphery. Each nanosphere encapsulates multiple dye molecules in the core, producing bright fluorescence and improved photostability. The primary amine-functionalized poly(ethylene glycol) layer on the surface provides water solubility and allows a variety of biomolecules, such as antibodies, streptavidins, DNA, or enzymes, to be covalently attached through different linking chemistries. Here, we present the conjugation of IgG antibodies to Kodak X-Sight nanospheres and demonstrate the performance of the conjugates in antigen detection. We also address the challenge of quantitative determination of the number of biomolecules attached to the nanospheressa problem typical of any nanoparticle-based conjugate systemsby the development of a simple and sensitive method based on UV spectroscopy. To the best * To whom correspondence should be addressed. Carestream Molecular Imaging, Carestream Health, Inc., New Haven, CT 06511, USA. Phone: (203) 786-5686. Fax: (203) 624-3143. E-mail: [email protected]. 1 Abbreviations: AMAS, N-[R-Maleimidoacetoxy]succinimide ester; BME, β-mercaptoethanol; BMPS, N-[β-maleimidopropyloxy]succinimide ester; DMSO, dimethylsulfoxide; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid disodium salt; NHS, N-hydroxysuccinimide; PVDF, polyvinylidene difluoride; SATA, N-succinimidyl-Sacetylthioacetate; Sulfo-SMCC, sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate; THF, tetrahydrofuran.

of our knowledge, this is the first report on utilizing this method to quantify the number of antibodies attached per nanosphere. A variety of species-specific and highly cross-adsorbed IgG antibodies were successfully conjugated to the nanospheres in four excitation and emission wavelengths ranging from visible to near-infrared (Figure 1). These nanosphere conjugates exhibited increased brightness, high sensitivity, and enhanced signal-to-background ratio in Western blotting assays. We demonstrated up to a 10× improvement in antigen detection overothercommerciallyavailable,spectrallysimilar,fluorescent-IgG antibodyconjugates.Themultiplecolorsofthesenanosphere-antibody conjugates also enable multiplex detection, such as multiplex Western blotting assays, which allows for the detection of several proteins simultaneously on a single blot.

EXPERIMENTAL PROCEDURES Materials. Kodak X-Sight 549 nanosphere, Kodak X-Sight 650 nanosphere, Kodak X-Sight 691 nanosphere, and Kodak X-Sight 761 nanosphere were manufactured by Carestream Health, Inc. All the reagents used for the X-Sight nanosphere synthesis are reagent grade or better unless otherwise specified. Goat anti-mouse, goat anti-rabbit, and goat anti-rat IgG antibodies were purchased from Jackson Immuno Research Laboratories. N-[β-Maleimidopropyloxy]succinimide ester, Traut’s Reagent, BupH phosphate buffer saline (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2), PBS (0.137 M sodium chloride, 0.0027 M potassium chloride, and 0.0119 M phosphates, pH 7.4), Ellman’s Reagent, β-mercaptoethanol, diethyl ether, methanol, and nonfat dry milk were obtained from Thermo Fisher Scientific. Dithiothreitol, dimethyl sulfoxide, sodium phosphate monobasic, sodium phosphate dibasic, sodium chloride aqueous solution (5 M), ethylenediaminetetraacetic acid disodium salt solution (0.5 M), conalbumin, trypsin, poly(ethylene glycol) dimethacrylate (Mn, 875), hydrochloric acid (37%), sodium hydroxide, ethyl acetate, hydrogen chloride, ethylene glycol methyl ether methacrylate, divinylbenzene (technical grade), cetylpyridinium chloride, sodium bicarbonate, tetrahydrofuran, and Dowex 50WX4-200 ion-exchange resin were purchased from Sigma-Aldrich. 10× PBS (0.1 M sodium phosphate, 1.5 M NaCl, pH 7.2) was purchased from Bio-Rad Laboratories. NuPAGE 4-12% BT gel, MOPS SDS running buffer (20×),

10.1021/bc900295v  2010 American Chemical Society Published on Web 02/17/2010

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Figure 1. UV absorption and emission spectra of X-Sight nanospheres. Absorption/emission maxima: X-Sight 549, 549 nm/569 nm; X-Sight 650, 650 nm/573 nm; X-Sight 691, 691 nm/715 nm; X-Sight 761, 761 nm/789 nm.

NuPAGE sample reducing agent (10×), NuPAGE antioxidant, NuPAGE transfer buffer (20×), NuPAGE LDS sample buffer (4×), 4-20% tris-glycine gel, Novex native tris-glycine sample buffer (2×), NativeMark protein standard, tris-glycine native running buffer, Alexa Fluor IgG antibody conjugates, and Qdot IgG antibody conjugates were purchased from Invitrogen. ECL Plex Fluorescent Rainbow Marker, illustra NAP-5 columns, Superdex 200 gel media, and Cy3 goat anti-mouse were purchased from GE Healthcare. Cy5 goat anti-mouse, Cy5.5 goat anti-mouse, and NIH/3T3 whole cell lysate were purchased from Rockland Immunochemicals. Immobilon-FL PVDF membrane, Microcon YM-30, and Microcon YM-100 were purchased from Millipore. Primary IgG antibodies (rabbit antibeta tubulin, mouse anti-beta actin, rat anti-HSC 70) were purchased from Abcam. Magnesium sulfate was purchased from Alfa Aesar. Cysteamine was purchased from TCI America. 2,2-Azobis(N,Ndimethyleneisobutyramidine) dihydrochloride was purchased from Wako Pure Chemical Industries. Spectra/Por dialysis membrane (MWCO 3500) was purchased from VWR. Instrumentation. SDS PAGE gel was imaged on a KODAK Gel Logic 212 imaging system. Western blot images were taken on a KODAK Image Station 4000MM digital imaging system or on a KODAK In-Vivo Multispectral Imaging System FX. UV spectra were measured on a PerkinElmer Lambda 25 UV/ vis spectrometer. Fluorescence spectra were measured on a PerkinElmer luminescence spectrometer LS 50B. Centrifugation was performed on an Eppendorf microcentrifuge 5415R. Chromatography of the conjugates, IgG antibody, and proteins was carried out on a Waters HPLC 2796 equipped with a Superdex 200 column. Buffer Preparation. PBS (0.1 M sodium phosphate, 0.15 M NaCl, pH 7.5), PBS/5 mM EDTA (0.1 M sodium phosphate, 0.15 M NaCl, 5 mM EDTA, pH 8.0), and phosphate buffer/1 mM EDTA (0.1 M sodium phosphate, 1 mM EDTA, pH 8.0) were prepared from sodium phosphate monobasic, sodium phosphate dibasic, NaCl and EDTA. BupH PBS/10 mM EDTA (0.1 M sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2) was constructed from BupH phosphate buffer saline and EDTA. 1× PBS (0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2) was diluted from 10× PBS. Preparation of Fluorescent Nanospheres. The preparation is described briefly here. For a detailed description of the preparation, see U.S. patent application (17) Preparation of Amine-Terminated Poly(ethylene glycol) Macromonomer. PEG dimethacrylate (335 g) in 100 mL of methanol was treated with cysteamine (5.8 g) and diisopropylethylamine (0.08 g). The mixture was stirred at room temperature for 2 days and then concentrated using a rotary evaporator.

The residue was taken up in 1 L of ethyl acetate and extracted with aqueous HCl solution (10%). The aqueous layer was collected, made basic by adding 50% aqueous sodium hydroxide solution, and then extracted with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered, and concentrated. The residue was taken up in anhydrous diethyl ether and treated with gaseous HCl and allowed to stand. The ether was decanted, and the residual dark blue oil was washed with fresh diethyl ether, which was then decanted. The dark blue oil was dried on a rotary evaporator to yield 37 g of the desired product in the form of the hydrochloride salt. 1H NMR (300 MHz, CDCl3): 1.18 (doublet, 3H), 1.93 (broad singlet, 3H), 2.04 (broad singlet, 2H), 2.43-2.77 (broad multiplet, 7H), 3.6-3.7 (broad singlet, -CH2CH2O-), 3.73 (broad triplet, 2H), 3.29 (broad triplet, 2H), 5.56 (broad singlet, 1H), 6.12 (broad singlet, 1H). Synthesis of Nanospheres. Typical example: A 500 mL 3-neck round-bottomed flask was modified with Ace #15 glass threads at the bottom and a series of adapters allowing connection of 1/16 in i.d. Teflon tubing. The flask (hereafter referred to as the header flask) was outfitted with a mechanical stirrer and rubber septum with syringe needle nitrogen inlet. The header was charged with methoxyethyl methacrylate (5.63 g), divinylbenzene (0.63 g, mixture of isomers, 80% pure with remainder being ethylstyrene isomers), amine-terminated poly(ethylene glycol) macromonomer (6.25 g), 2,2-azobis(N,N-dimethyleneisobutyramidine) dihydrochloride (0.06 g), cetylpyridinium chloride (0.31 g), and distilled water (78.38 g). The header contents were brought to pH 5 with 1 N NaOH. A 1 L 3-neck round-bottomed flask outfitted with a mechanical stirrer, reflux condenser, nitrogen inlet, and rubber septum (hereafter referred to as the reactor) was charged with distilled water (159.13 g), 2,2-azobis(N,N-dimethyleneisobutyramidine) dihydrochloride (0.06 g), sodium bicarbonate (0.06 g), and cetylpyridinium chloride (0.94 g). Both the header and the reactor contents were stirred until homogeneous and were bubble degassed with nitrogen for 20 min. The reactor flask was placed in a thermostatted water bath at 60 °C, and the header contents were added to the reactor over 2 h using a model QG6 lab pump. The reaction was allowed to stir at 60 °C for 16 h. The latex was then dialyzed against distilled water for 48 h using a Spectra/Por dialysis membrane (MWCO 3500) and was stirred over 27.5 g Dowex 50WX4-200 ion-exchange resin (sodium form) overnight to afford 312 g of clear latex of 3.26% solids. The volume average diameter of the nanosphere was found to be 20.89 nm with a coefficient of variation of 0.24 by quasielastic light scattering. Loading the Nanospheres with Dyes. For the synthesis of the dyes loaded inside the nanospheres, see patent application (17).

Increased Sensitivity in Antigen Detection

Typical dye loading example: Under dim lighting, 1.4365 g of a dye stock solution of 0.2784% (w/w) in THF was added to a tinted glass vial and was diluted with THF to a final weight of 10.0 g. The amine-functionalized nanosphere (9.996 g) was added to the vial, and the solution was mixed and concentrated to approximately 40-50% volume on a rotary evaporator. Residual THF was further removed by twice adding 3-5 mL of distilled water and stripping ∼1/4-1/3 of the volatiles. 9.4 g of dye-loaded nanospheres of 4.27% (w/w) solids containing 3.94 × 10-3 mol dye per gram of solid nanospheres was obtained. Fuctionalization of Nanospheres with BMPS. X-Sight 549, 650, 691, and 761 nanospheres, modified with maleimide functional groups, were prepared. In a typical procedure, 549 (40 µL, 32 µM) was added to 60 µL of PBS (0.1 M, 0.15 M NaCl, pH 7.5) and was then added with 6.4 µL (192.2 nmol) of 30 mM BMPS dissolved in anhydrous DMSO. The mixture was stirred at room temperature for 1 h. The maleimide-modified nanospheres were purified on an Illustra NAP-5 column eluted with BupH PBS/10 mM EDTA. ∼500 µL of the purified nanospheres was collected and stored on ice. The concentration of the purified nanospheres, as determined by UV absorbance, was 2.28 µM. The number of maleimide groups attached per nanosphere was quantified by Ellman’s Reagent and is ∼80 maleimide groups per nanosphere. Preparation of SH Groups Containing IgG Antibodies. DTT Reduction Method. Typical example: IgG antibody (0.5 mg) was dissolved in BupH PBS/10 mM EDTA (500 µL) at 1 mg/mL. Five microliters of 1 M DTT dissolved in BupH PBS/ 10 mM EDTA was added to the IgG solution. The mixture was incubated at RT for 1 h on a rotator and transferred to a Microcon YM-30 filter unit. The solution in the Microcon filter unit was concentrated to ∼50 µL by spinning at 13 400 g in an Eppendorf microcentrifuge for 11 min. The filtrate was discarded, and the remaining solution in the filter unit was brought up to 500 µL by BupH PBS/10 mM EDTA and spun again at 13 400 g for 11 min. This concentration-dilution cycle was repeated three more times. Roughly 50 µL of the modified IgG solution was collected. Traut’s Reagent Modification Method. Typical example: IgG antibody (0.5 mg) was dissolved in PBS (0.1 M, 0.15 M NaCl, pH 8.0)/5 mM EDTA buffer (500 µL) at 1 mg/mL. 4.76 µL of 7 mM Traut’s Reagent dissolved in PBS (0.1 M, 0.15 M NaCl, pH 8.0)/5 mM EDTA buffer was added to the above IgG solution. The mixture was incubated at RT for 1 h on a rotator and transferred to a Microcon YM-30 filter unit. The solution in the Microcon filter unit was concentrated to ∼50 µL by spinning at 13 400 g in an Eppendorf microcentrifuge for 11 min. The filtrate was discarded, and the remaining solution in the filter unit was brought up to 500 µL by BupH PBS/10 mM EDTAandspunagainat13 400gfor11min.Thisconcentration-dilution cycle was repeated one more time. Roughly 50 µL of the modified IgG solution was collected. Conjugation of IgG Antibodies to Nanospheres. DTT Reduction Method. Maleimide-functionalized nanospheres (2.28 µM, 255.7 µL) and BupH PBS/10 mM EDTA (70 µL) were added to the above SH group-containing IgG (50 µL) synthesized by DTT reduction method. The mixture was incubated at RT for 2 h on a rotator. The mixture was then transferred to a Microcon YM-100 filter unit, and the volume was reduced to ∼50 µL on an Eppendorf microcentrifuge. The concentrated solution was fractionated on a self-packed Superdex 200 column eluted with 1× PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.2). ∼500 µL of the conjugates was collected. The concentration of the purified nanosphere-IgG antibody conjugates was determined by UV absorbance at λmax to be ∼0.6

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µM. The number of IgG antibodies incorporated per nanosphere was quantified by UV absorbance to be 4.4 IgG antibodies per nanosphere. Traut’s Reagent Modification Method. Maleimide-functionalized nanospheres (2.28 µM, 128 µL) and BupH PBS/10 mM EDTA (120 µL) were added to the above SH group-containing IgG (50 µL) synthesized by Traut’s Reagent modification method. The mixture was incubated at RT for 2 h on a rotator. The mixture was then transferred to a Microcon YM-100 filter unit, and the volume was reduced to ∼50 µL on an Eppendorf microcentrifuge. The concentrated solution was fractionated on a self-packed Superdex 200 column eluted with 1× PBS (10 mM sodium phosphate, 0.15 M NaCl, pH 7.2). ∼500 µL of the conjugates was collected. The concentration of the purified nanosphere-IgG antibody conjugates was determined by UV absorbance at λmax to be ∼0.4 µM. The number of IgG antibodies incorporated per nanosphere was quantified by UV absorbance to be 3.6 IgG antibodies per nanosphere. Determination of the Number of Maleimide Groups Attached per Nanosphere. The quantification was done by taking advantage of the high reactivity of BME with the maleimide group. BME reacts with maleimide group in a 1:1 stoichiometry; thus, the number of maleimide groups was determined by the decrease in bulk BME concentration using Ellman’s Reagent, which reacts with sulfhydryl group to form a colored compound (molar extinction coefficient of 14 150 cm-1 M-1 at 412 nm). Briefly, 200 µL of the maleimide functionalized nanosphere (∼0.5 nmol) was incubated with 10 µL of 10 mM BME in BupH PBS/10 mM EDTA for 1 h at RT. Then, 100 µL of the reaction mixture was taken out and mixed with 850 µL of phosphate buffer/1 mM EDTA (0.1 M sodium phosphate, 1 mM EDTA, pH 8.0) and 50 µL of 10 mM Ellman’s Reagent predissolved in phosphate buffer/1 mM EDTA. The mixture was incubated for 15 min at RT, and the absorbance at 412 nm was recorded. In the control experiment, 10 µL of 10 mM BME in BupH PBS/10 mM EDTA was incubated with 200 µL of BupH PBS/10 mM EDTA without maleimide functionalized nanospheres for 1 h at RT and was analyzed similarly to estimate the total amount of BME added to the reaction mixture. For the maleimide-modified nanospheres synthesized using the method described in this paper, there are generally about 70-80 maleimide groups attached per nanosphere. Determination of the Number of Sulfhydryl Groups Generated on the IgG Antibody. The quantification of the sulfhydryl groups on the IgG after DTT reduction and Traut’s Reagent modification was done using Ellman’s Reagent following the manufacturer’s protocol. Briefly, 1 mg of DTT reduced or Traut’s Reagent modified goat anti mouse IgG was diluted to 950 µL with phosphate buffer/1 mM EDTA (0.1 M sodium phosphate, 1 mM EDTA, pH 8.0) and was incubated

Figure 2. Determination of the ratio (rf) of the absorbance of nanospheres at 280 nm to the absorbance at 650 nm, rf ) A280/A650 ) 0.32/2.65 ) 0.12.


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Scheme 1. Synthesis of Nanosphere-IgG Antibody Conjugates

with 50 µL of 10 mM Ellman’s Reagent predissolved in phosphate buffer/1 mM EDTA for 15 min at RT. The absorbance of the mixture at 412 nm was recorded. As a blank, 50 µL of 10 mM Ellman’s Reagent predissolved in phosphate buffer/1 mM EDTA was incubated with 950 µL of phosphate buffer/1 mM EDTA for 15 min at RT, and the absorbance at 412 nm was recorded. According to the Beer-Lambert law and the extinction coefficient of 14 150 cm-1 M-1 at 412 nm, the number of sulfhydryl groups per IgG was calculated. Generally, there are about 16 sulfhydryl groups per DTT reduced goat anti mouse IgG and 3-4 sulfhydryl groups per goat anti mouse IgG modified by Traut’s Reagent. Quantification of the Number of IgG Antibodies Attached per Nanosphere. Quantification was done according to eq 1. N is the number of IgG antibodies per nanosphere. Cab is the concentration of IgG attached to the nanospheres. Cp is the concentration of the nanospheres in the conjugates. A280 is the absorbance of the conjugates at 280 nm. Amax is the absorbance of the conjugates at λmax. 210 000 is the molar extinction coefficient of the IgG antibody at 280 nm in cm-1 M-1. rf is the ratio of the absorbance of the nanospheres at 280 nm to the absorbance at λmax, e.g., the rf of the 650 nanosphere is 0.12, as shown in Figure 2. k is the slope in M-1 of the standard curve of the nanospheres themselves, generated in a series of known concentrations. The light path in the UV absorbance measurement is 1 cm. The IgG antibody has a typical UV absorbance at 280 nm. According to the Beer-Lambert law, the concentration of IgG antibodies attached to nanospheres could be calculated as C ) A280/ε, where the light path is 1 cm. However, the nanospheres also have some absorbance and scattering at 280 nm (Figure 1). In order to get the net absorbance of the IgG antibodies at 280 nm, the contribution of nanospheres at 280 nm is deducted. In the conjugates, the absorbance of the nanospheres at 280 nm was calculated by Amax × rf; Amax multiplied by rf. The concentration of nanospheres in the conjugates was obtained

by Amax/k. Thus, the number of IgG antibodies attached per nanosphere was determined by the ratio of the concentration of IgG to the concentration of nanospheres in the conjugates. N)

(A280 - Amax × rf)/210000 Cab ) ) Amax Cp /k (A280 - Amax × rf) × k 210000 × Amax

(1)

SDS PAGE. Different amounts of goat anti-mouse IgG antibody with and without DTT treatment were mixed with NuPAGE LDS sample buffer (4×) and loaded into the wells of a 10-well, 4-12% NuPAGE gel. Five microliters of the ECL Plex Rainbow Marker was loaded as a protein ladder. The gel was run at 150 V in MOPS SDS running buffer for about 1.5 h. Native PAGE. Different amounts of goat anti-mouse IgG antibody with and without DTT treatment were mixed with Novex native tris-glycine sample buffer (2×) and loaded into the wells of a 10-well, 4-20% tris-glycine gel. Five microliters of NativeMark protein standard was loaded as a protein ladder. The gel was run at 150 V in tris-glycine native running buffer for about 5 h. Western Blotting. Protein Electrophoresis for Western Blotting. 3T3 cell lysate was added with a sample reducing agent(10×), boiled at 95 °C for 5 min, and placed immediately on ice. Ten micrograms of the 3T3 cell lysate was loaded per well into a 12-well, 4-12% NuPAGE gel. Five microliters of ECL Plex Rainbow Marker was loaded into the well next to the protein. The gel was run at 100-125 V in MOPS SDS running buffer for about 2 h. Membrane Transfer. The gel was transferred to an Immobilon-FL PVDF membrane at 30 V at 4 °C on a stirrer overnight. Blocking and Detection. The membrane was blocked in PBS with 5% nonfat milk for 1 h. Then, it was incubated in primary antibody diluted in PBS with 5% nonfat milk for 1 h at RT


with gentle shaking. The membrane was washed with PBS 3 times for 5 min each at RT. Then, it was incubated in fluorescent IgG antibody diluted in PBS with 5% nonfat milk for 1 h at RT with gentle shaking. The membrane was washed with PBS 3 times for 5 min each at RT. Then, it was imaged on the Image Station 4000MM or the In-Vivo Multispectral Imaging System FX. Data Analysis of Western Blotting Images. The data were analyzed using KODAK MI Software. The protein band region was selected as a region of interest (ROI). Net intensity is defined as the sum of background-subtracted pixel intensities within the ROI. Background intensity is defined as the median intensity of the perimeter of each ROI. Signal/background is the ratio of net intensity to background intensity.

RESULTS AND DISCUSSION Synthesis of Fluorescent Nanosphere-IgG Antibody Conjugates. The nanosphere-IgG antibody conjugates were synthesized through three steps: modification of nanospheres by a heterobifunctional linker, such as Sulfo-SMCC, BMPS, AMAS, and so forth; introduction of sulfhydryl groups to the IgG antibodies; and the coupling of the IgG antibodies to the nanospheres. The synthetic routes are shown in Scheme 1. The heterobifunctional linker contains an NHS ester moiety that is reactive toward the amino group and a maleimide group that is reactive toward the sulfhydryl group (18). Different starting ratios of linker to nanospheres vs the conjugation efficiency were evaluated. The results show that, if the ratio is too low, the conjugation of the IgG to the nanospheres is not efficient. If the ratio is too high, the solubility of the maleimide-modified nanospheres in aqueous solution decreases tremendously, and some of the nanospheres precipitate out of the solution. It was found that a ratio of linker to nanosphere in the range of ∼100:1 to 150:1 resulted in high conjugation efficiency, little aggregation, and excellent performance of nanosphere-IgG antibody conjugates in Western blotting assays. When the ratio is 150:1, there are generally about 80 maleimide groups attached per nanosphere, and the remaining primary amines on the nanosphere are over 120. There are several different methods to incorporate sulfhydryl groups into antibodies, such as SATA modification (19), Traut’s Reagent modification (20), and DTT reduction. Here, we report the conjugation using two different methods: DTT reduction and Traut’s Reagent modification. DTT was reported to partially reduce the disulfide bonds in the hinge region of the IgG antibody, and the yielded sulfhydryl groups were successfully


used as the attachment sites for dyes and other molecules. The advantage of this method is that the attachment sites were in the hinge regionsaway from the antigen-binding sitessand therefore, the attached molecules did not affect the activity of the IgG antibody (21-25). However, a recent report indicates that DTT completely dissociates the IgG antibody into heavy and light chains, causing the antibody to lose biological activity (28). In the case of SATA and Traut’s Reagent modification, the bifunctional linkers randomly react with the primary amino groups on the antibody; therefore, the antigen-binding regions are also subject to modification. Extensive modification of the antigen-binding regions will cause the antibody to lose activity. Therefore, the ratio of the linker to the antibody needs to be carefully controlled. In this paper, we evaluated both the DTT reduction and Traut’s Reagent modification methods and the biological activity of the yielded conjugates. The goat anti-mouse, goat anti-rabbit, and goat anti-rat IgG antibodies used in the experiments are affinity purified and highly cross-adsorbed polyclononal antibodies that are composed of a mixture of IgG1 and IgG2 subclasses. There are quite a few reports in the literature of using DTT to partially reduce the disulfide bonds in the IgG. The experimental conditions varied from mild (10-100-fold molar excess of DTT to IgG, 30 min reaction at RT or at 37 °C) to more harsh conditions (300-7500-fold molar excess of DTT to IgG, 30 min reaction at RT) (21-25). It was also reported that all the interchain disulfides in IgG were cleaved when 150-1000-fold molar excess of DTT to IgG was used and the reaction was 1-3 h at 37 °C (26, 27). Our experimental conditions reported here show that DTT not only reduced the disulfide bonds in the hinge region, but it also reduced the interchain disulfide bonds between the heavy and light chains, which was indicated by the SDS PAGE results in Figure 3a. This finding is consistent with the some of the reports (26-28). IgG antibodies treated with DTT using the same protocol used in the DTT reduction conjugation were loaded on the gel (Figure 3a, lanes 1-4). Five bands in each lane were detected, of which two major bands were in the lower molecular weight region. These five bands corresponded to a light chain (25 kDa), a heavy chain (50 kDa), a small amount of a half IgG antibody (75 kDa), a whole IgG antibody losing one light chain (125 kDa), and a whole IgG antibody (150 kDa), respectively, while the IgG antibody without DTT treatment showed one main band at 150 kDa (Figure 3a, lanes 5-8). However, SDS PAGE could not deduce whether the antibodies dissociate in the nondenatured condition, because SDS denatures the protein and could dis-

Figure 3. (a) 4-12% SDS PAGE of goat anti-mouse IgG. Lanes 1-4: IgG was treated with 10 mM DTT for 1 h at RT, and then a different amount was loaded on the gel. Lanes 5-8 were loaded with a different amount of IgG without DTT treatment. The gel was run at 150 V for ∼1.5 h. (b) Goat anti-mouse IgG on a 4-20% native PAGE. Lane 1: NativeMark protein standard. Lanes 2-3: IgG was treated with 10 mM DTT for 1 h at RT, and then a different amount was loaded on the gel. Lanes 4-5 were loaded with a different amount of IgG without DTT treatment. The gel was run at 150 V for ∼5 h.


Figure 4. Chromatograms of proteins on HPLC detected at 280 nm. Elution rate: 2 mL/min. Blue curve is the mixture of goat anti-mouse IgG, conalbumin, and trypsin. Pink curve is the goat anti-mouse IgG treated with DTT. 1:1.7 mg/mL of goat anti-mouse IgG, Mw 150 kDa. 2:1.7 mg/mL of conalbumin, Mw 75 kDa. 3:3.0 mg/mL of trypsin, Mw 21 kDa. Mobile phase of blue curve: PBS, pH 7.4. Mobile phase of pink curve: PBS, pH 7.4/10 mM DTT.

sociate heavy and light chains of the antibody treated with DTT. As such, a native PAGE that separates the protein according to the charge, size, and conformation was run, and the result show that DTT reduced antibodies and unreduced antibodies show a similar pattern (Figure 3b), which indicates that the antibody fragments were still held together by noncovalent forces, even though the disulfide linkages were broken. Two bands were detected in both DTT-reduced and unreduced IgG antibodies (Figure 3b), for which the reason is not clear. A similar electrophoresis pattern was observed from more than one IgG antibody preparation. The integrity of the IgG antibodies was also checked by size-exclusion chromatography. In order to establish a running condition that could separate the IgG, half IgG, and the IgG light chain, a mixture of IgG, conalbumin, and Trypsin was used to simulate the mixture of IgG, half IgG, and IgG light chain. Conalbumin is a 75 kDa protein whose size is similar to that of a half IgG, and the molecular weight of trypsin is 21 kDa, similar to an IgG light chain. Three peaks corresponding to IgG, conalbumin, and trypsin, respectively, were detected (Figure 4, blue curve), which indicated that the running condition should be able to separate the IgG antibody, half IgG antibody, and light chain, with the half IgG antibody being eluted out at the similar elution time with that of the conalbumin and the light chain being eluted out at the similar elution time with that of trypsin. In the size-exclusion chromatography of the DTT-reduced IgG antibody, 10 mM DTT was incorporated into the mobile phase to prevent the reformation of the disulfide bridges. One single peak at the same elution time with that of the unreduced antibody corresponding to a molecular weight of 150 kDa was detected (Figure 4, pink curve), indicating that the antibody fragments were still held

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together by noncovalent interactions under native conditions, even though the interchain disulfide bonds were cleaved. Therefore, with the DTT reduction method, the IgG antibodies attached to the nanospheres were still whole antibodies (Scheme 1). In the DTT reduction conjugation approach, the feed ratio of IgG antibody to nanosphere was 5.7 to 1, assuming there was no loss of the antibody after DTT reduction and purification. The corresponding conjugation efficiency and yield were determined by UV absorbance measurement according to eq 1. The results showed that the number of IgG antibodies attached was ∼4.4 whole antibodies. The conjugation efficiency was also checked on an HPLC equipped with a Superdex 200 column. The chromatogram showed that the peak corresponding to the unconjugated antibody was very small; integration of the peak showed that ∼90% of the antibodies were conjugated to the nanospheres (Figure 5a). Traut’s Reagent is very stable in neutral buffers. The hydrolysis is slow compared with the amine reaction rate. Therefore, thiolation with Traut’s Reagent does not require a large excess of reagent like SATA. It was reported that, for IgG molecules, a 10-fold molar excess of Traut’s Reagent would yield the antibody molecules modified with at least three sulfhydryl groups. Different stoichiometries of Traut’s Reagent to IgG antibody (5:1 and 10:1) and IgG antibody to nanosphere (5.7:1 and 11:1) vs the conjugation efficiency and the performance of the conjugates in Western blotting assays was investigated. We found that the overall conjugation efficiency was low relative to the DTT reduction conjugation approach. When 10:1 ratio of Traut’s Reagent to IgG antibody was used, a 5.7:1 of antibody-to-nanosphere ratio yielded 2 antibodies attached per nanosphere, and a 11:1 of antibody-to-nanosphere ratio yielded 3.6 antibodies attached per nanosphere, as calculated from eq 1. The low conjugation efficiency was also confirmed by HPLC analysis (Figure 5b). Western blotting results also showed that the 10:1 ratio of Traut’s Reagent to antibody did not adversely affect the antibody function compared with 5:1 ratio of Traut’s Reagent to antibody. Western Blotting of the Nanosphere-IgG Antibody Conjugates. The performance of the conjugates synthesized through both the DTT reduction and Traut’s Reagent modification method and other commercially available fluorescent-IgG antibody conjugates was investigated by Western blotting assays (Figures 6-9). In order to obtain an accurate comparison, all the Western blotting experimental conditions were kept identical. For example, the blots used for the comparison were transferred from the same gel to avoid the variation in the amount of protein transferred from different gels. The blots were blocked in the same blocker and incubated in the same primary antibody solution, and so forth. After being probed with the fluorescent antibodies, the blots in comparison were placed side by side on

Figure 5. Chromatograms of the X-Sight 650 nanosphere-goat anti-mouse IgG conjugate mixture on HPLC detected at 280 nm. Mobile phase: PBS, pH 7.4. (a) DTT reduction method. (b) Traut’s Reagent modification method, Ab: nanosphere ) 11:1.


Figure 6. Western blot image of the X-Sight 650 nanosphere-goat anti-mouse IgG conjugates. Each blot contains two duplicated protein bands and a protein ladder on the right-hand side. The concentration of the conjugates in Western blotting assay was 3 nM. Conjugate 1 was synthesized using the Traut’s Reagent modification method and has 3.6 antibodies attached per nanosphere. Conjugate 2 was synthesized using DTT reduction method and has 4.4 antibodies attached per nanosphere. Image was scanned for 4 min using excitation/emission filter set 630 nm/700 nm. Data analysis was done using KODAK MI software. The net intensity, background intensity, and signal/background of conjugate 2 was 2.2-fold, 0.8-fold, and 2.8 fold, respectively, of that of conjugate 1.

the imaging system, and the images were acquired using the optimal excitation/emission filter sets for each fluorescent antibody. In addition, each of the blots was made to contain two duplicate protein bands, and the detected intensity was averaged in the data analysis to minimize the error. The results show that the conjugate synthesized through the DTT reduction method has much better performance than that synthesized through Traut’s Reagent modification method, even though they both have about four antibodies attached per nanosphere, and the concentration of both conjugates in the Western blotting assay was the same (Figure 6). The image analysis shows that the net intensity and background intensity of the conjugates synthesized through the DTT reduction method is about 2× and 0.8×, respectively, of the net intensity and background intensity of the conjugates synthesized through Traut’s Reagent modifica-


tion method, and hence the signal-to-background ratio is about 2.8×. It has been demonstrated that nonspecific binding of the nanospheres to the PVDF membrane has contributed substantially to the background intensity in the Western blotting (data not shown). We speculate that the IgG antibody attached to the nanospheres helps to suppress the nanosphere/membrane interaction. The more antibodies that are attached per nanosphere, the lower the background intensity. A smaller difference in the background intensity between conjugates 1 and 2 further indicates that both conjugates have similar amounts of antibody attached per nanosphere. The conjugates synthesized through the DTT reduction method shows higher net intensity, which suggests that the antibodies in the conjugates are more active. The higher activity might be due to the nanospheres’ attachment to the hinge region of the antibody, i.e., remote from the antigen binding sites and having less effect on the antibody activity. In the DTT reduction method, the thiol groups in the hinge region cleaved by DTT are more accessible to nanospheres than those in the other regions because the hinge region of the antibody is more flexible, which allows the lateral and rotational movement of the Fab fragments (29). With the Traut’s Reagent modification method, the nanospheres’ attachment sites on the antibodies are not controlled. The nanospheres could be attached to the antigen binding sites, which results in the antibodies losing activity. The comparison of the performance of the conjugates synthesized through the DTT reduction method with Alexa Fluor IgG antibody conjugates shows that the performance is related to the concentration of the conjugates. Both the signal-tobackground ratio and the net intensity increase with increasing concentration from 1.5 nM to 6 nM (Figure 7). For lower wavelength conjugates (Figure 7a,b), the nanosphere conjugates at a concentration of 1.5 nM have comparable

Figure 7. Western blot images of Alexa Fluor IgG antibody conjugates vs the X-Sight nanosphere-IgG antibody conjugates at different concentrations. The images were taken on an Image Station 4000MM. (a) The image was scanned for 25 s using an excitation/emission filter set 515 nm/570 nm. (b) The image was scanned for 30 s using an excitation/emission filter set 610 nm/670 nm. (c) The image was scanned for 3 min using an excitation/ emission filter set 730 nm/790 nm. Each blot contains two duplicated protein bands and a protein ladder on the right-hand side. The concentration of the conjugates in the Western blotting assay ranged from 1.5 nM to 6.7 nM. The net intensity and signal/background of X-sight conjugates and Alexa Fluor conjugates were analyzed using MI software. X-Sight/Alexa (net intensity): the ratio of the net intensity of X-Sight nanosphere conjugates to that of Alexa Fluor conjugates. X-Sight/Alexa (signal/background): the ratio of the signal to background of X-Sight nanosphere conjugates to that of Alexa Fluor conjugates.


Ji et al.

Figure 8. Western blot images of the X-Sight nanosphere-goat anti-mouse IgG conjugates vs CyDye-goat anti-mouse IgG conjugates. The images were taken on an Image Station 4000MM. (1) The image was scanned for 1 min using excitation/emission filter set 515 nm/570 nm. (2) The image was scanned for 1 min using an excitation/emission filter set 635 nm/700 nm. (3) The image was scanned for 1 min using an excitation/emission filter set 635 nm/700 nm. Each blot contains two duplicated protein bands and a protein ladder on the right-hand side. The concentration of the conjugates in Western blotting assay was 3 nM. The net intensity and signal/background of X-sight conjugates and CyDye conjugates were analyzed using MI software. X-Sight/CyDye (net intensity): the ratio of the net intensity of X-Sight nanosphere conjugates to that of CyDye conjugates. X-Sight/CyDye (signal/background): the ratio of the signal to background of X-Sight nanosphere conjugates to that of CyDye conjugates.

Figure 9. Multiplex Western blot of the X-Sight nanosphere-IgG antibody conjugates vs Qdot IgG antibody conjugates. Blots were probed with the mixture of rabbit anti-HSP60, mouse anti beta actin, and rat anti-HSC 70 primary antibodies followed by the mixture of X-Sight nanosphereIgG antibody conjugates or the mixture of Qdot-IgG antibody conjugates. Each blot contains two duplicated protein bands and a protein ladder on the right-hand side. Images were captured for 30 s using an optimal excitation/emission filter set for each of the antibody conjugates on the In-Vivo Multispectral Imaging System FX and then overlaid in Photoshop. The concentration of the conjugates in the Western blotting assay was 2 nM. The net intensity and signal/background of X-sight conjugates and Qdot conjugates were analyzed using MI software. X-sight 549/Qdot 565: net intensity, 4.2; signal/background, 4.0. X-sight 650/Qdot 705: net intensity, 2.9; signal/background, 2.1. X-sight 761/Qdot 800: net intensity, 6.3; signal/ background, 1.4.

performance with Alexa Fluor conjugates at a concentration of 6.7 nM. At similar concentration of the conjugates, the nanosphere conjugates (6 nM) have much higher signal-tobackground ratio and net intensity than Alexa Fluor conjugates (6.7 nM). The X-Sight 761 nanosphere conjugates show higher brighteness and sensitivity than Alexa Fluor 750 conjugates, even at a much lower concentration (1.5 nM) (Figure 7c). Figure 8 shows that our nanosphere conjugates also have higher sensitivity than CyDye conjugates at all comparable wavelengths at matched concentration (3 nM) of the conjugates. It is noteworthy that our nanosphere conjugates not only exhibit higher signal-to-background ratio but also a much higher net intensity. In other words, our nanosphere conjugates were much brighter than Alexa Fluor and CyDye conjugates. Figure 7 shows that the X-Sight 549 and 650 nanosphere conjugates are ∼4 times brighter than Alexa Fluor conjugates, and the X-Sight 761 nanosphere conjugates are up to 29 times brighter than Alexa Fluor conjugates. The advantage of relative higher brightness is that the acquisition time for the detection of a certain amount of signal is relatively shortened.

Quantum dots have been widely used in biological applications because of their superior brightness and photostability. The performance of our nanosphere-IgG antibody conjugates and spectrally similar Qdot IgG antibody conjugates in Western blotting assays was also evaluated (Figure 9). Multiplex Western blotting of the X-Sight nanospheres and Qdot conjugates is shown in Figure 9. The images were taken using the optimal filter sets for the X-Sight nanospheres and Qdots conjugates, respectively. The optimal excitation/emission filter sets were evaluated based on the signal-to-background ratio. Figure 9 shows that all of the X-Sight nanosphere conjugates have better performance than the counterparts of Qdot conjugates; they show not only higher signal-to-background ratio, but also much higher net intensity.

CONCLUSIONS Two different coupling chemistries, BMPS-DTT and BMPSTraut’s Reagent, were evaluated for the synthesis of fluorescent nanosphere-IgG antibody conjugates. Native PAGE and size exclusion chromatography indicate that the DTT reduction


conditions (used herein) minimally disturb the antibody conformation, minimizing the effect upon biological activity of the antibody, even though the disulfide bridges in the antibody were cleaved. Moreover, BMPS-DTT chemistry gives higher conjugation efficiency, and the yielded conjugates show better performance in antigen detection. Side-by-side comparisons with other commercially available fluorescent IgG antibodies, like Alexa Fluor, Qdot, and CyDye antibody conjugates, show that the X-Sight nanosphere conjugates synthesized through BMPSDTT chemistry provide increased brightness and higher signalto-background in the detection of antigens.

ACKNOWLEDGMENT This article would not have materialized without the support of the Molecular Imaging Division at Carestream Health, Inc.

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