Less is More: A Comparison of Antibody–Gold Nanoparticle

Oct 6, 2017 - The application of the conjugates to the lateral flow immunoassay shows that the antibody concentrations used for the conjugation can be...
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Cite This: Bioconjugate Chem. 2017, 28, 2737-2746

Less is More: A Comparison of Antibody−Gold Nanoparticle Conjugates of Different Ratios Nadezhda A. Byzova, Irina V. Safenkova, Elvira S. Slutskaya, Anatoly V. Zherdev, and Boris B. Dzantiev* A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia S Supporting Information *

ABSTRACT: This comprehensive study is related to gold nanoparticles (GNPs) conjugated with antibodies. The goal of the study is to determine the minimal concentration of antibodies for conjugate synthesis when the conjugates have high antigen-capturing activity. Two systems were studied: gold nanoparticles conjugated with monoclonal antibodies (mAb-GNP) specific to Helicobacter pylori and gold nanoparticles conjugated with polyclonal antibodies (pAb-GNP) specific to mouse immunoglobulins. Several conjugates were synthesized with different GNP-to-antibody molar ratios (from 1:1 to 1:245) through nondirectional and noncovalent immobilization on a surface of GNPs with a diameter of 25.3 ± 4.6 nm. The maximal antigen-capturing activities and equilibrium constants of the conjugates correlate with the formation of a constant hydrodynamic radius of the conjugates for mAb-GNP (GNP to antibody molar ratio 1:58) and with the stabilizing concentration by flocculation curves for pAb-GNP (GNP to antibody molar ratio 1:116). The application of the conjugates to the lateral flow immunoassay shows that the antibody concentrations used for the conjugation can be reduced (below the stabilizing concentration) without losing activity for the mAb-GNP conjugates. The findings highlight that the optimal concentration of antibodies immobilized on the surface of GNPs is not always equal to the stabilizing concentration determined by the flocculation curve.

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with high activity can be obtained).10−13 However, there are established methodological solutions; among these, the main method for concentration choice is based on NaCl-induced flocculation. This means that the minimum antibody concentration is chosen to prevent the aggregation of GNPs in the presence of excess salt (10% NaCl). The method described has been actively used from the pioneer studies on conjugate synthesis14 to present day research.15−17 It is assumed that the protein concentration determined using this method (a stabilizing concentration) is optimal for the synthesis of an active conjugate. Indeed, most studies comparing conjugates synthesized at concentrations higher than the stabilizing concentration show that an increase in protein concentration during synthesis does not lead to an increase of conjugate functional activity.18−20 This also applies to conjugates of GNPs with antibodies, which are widely used in biosensors. Considering that the conjugates for assay are used in solutions containing stabilizing components such as sucrose, bovine serum albumin (BSA), and polyethylene glycol,21 which cover GNP surfaces, the use of antibody concentrations below the stabilizing concentration for synthesis becomes possible. These compounds will in part serve as a protein corona around conjugates of GNPs, which has

INTRODUCTION Conjugates of protein molecules with gold nanoparticles (GNPs) are actively used in various bioanalytical systems and biosensors.1−3 The broad application of the conjugates of GNPs is due to their unique optical and electromagnetic properties.4,5 Another advantage of the conjugates is their simple synthesis; the most common method for synthesizing conjugate proteins immobilized on the GNP surface is nondirectional and noncovalent immobilization by physical adsorption.6,7 Additionally, according to Katz and Willner, this method of conjugation is associated with less instability and inactivation of immobilized proteins that may appear during immobilization by covalent bonding through bifunctional linkers.8 It is well-known that the main active forces of noncovalent immobilization are donor−acceptor interactions involving SH-groups of molecules, hydrophobic interactions involving tryptophan, Coulomb interactions between NH2 groups of lysine, and citrate ions on the surfaces of GNPs.9 However, one of the main conditions for practical use is not known: how to determine the minimal concentration of antibodies for conjugate synthesis when conjugates have a high antigen-capturing activity. The existing data does not allow suggesting a single mechanism of physical adsorption on the surface of GNPs for each protein molecule, which would facilitate choosing the most efficient concentration of immobilized antibodies (defined here as minimal protein concentration at which a conjugate © 2017 American Chemical Society

Received: August 15, 2017 Revised: September 21, 2017 Published: October 6, 2017 2737

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Figure 1. Transmission electron microscopy of GNPs: (A) image of GNPs and (B) histogram of GNP size (n = 198) and fitting by Gauss approximation. The mean diameter is 25.3 nm, and the standard deviation is 4.6 nm.

Figure 2. Flocculation curves of antibodies’ immobilization on the GNP’s surface. Concentration dependencies (four replicates for each measurement) of the absorbance of GNP at 580 nm in the presence of excess salt (10% NaCl) after the addition of different concentrations of the mAb (A) and pAb (B). Arrows indicate the antibody concentrations for the stabilization of the GNP’s surface for each case.

been described in detail by de Poig et al.,22 to reduce falsepositive results. Therefore, it is also important to compare the functional activity of conjugates synthesized below the stabilizing concentration. However, a comparison of conjugates synthesized under such conditions is known to only be applicable in competitive assays23,24 because particular qualities of the competitive assay are associated with a correlation between a decrease in the reactant concentrations (including decreasing molar ratio of the antibody−GNP conjugate) and a decrease in detection limit.23 The use of concentrations below the stabilizing concentration is not common for sandwich assays since increases in the reactant concentrations lead to increases in the detection limit. On the contrary, a high immobilization density of antibodies on the surface of nanoparticles can lead to effects (shielding of binding sites with neighboring immobilized molecules; shielding involving antigens interacting with the nearby immobilized antibody; reduction of Fab-fragment mobility) that have a negative impact on antigen-binding activity.25−27 Moreover, effective conjugates synthesized at antibody concentrations much less than saturation of the surface (asymmetrically functionalized antibody−GNP conjugates) are described.28 Thus, the optimal

antigen-capturing activity of the conjugates can be obtained at concentrations less than those of full surface coverage determined by flocculation. Such results were shown, for example, for conjugates of magnetic nanoparticles with covalent immobilized antibodies.29 The goal of the study was to compare the antigen-capturing activity of the conjugates synthesized using concentrations below the optimal stabilizing concentration. The efficiency of the conjugates obtained with different GNP-to-antibody ratios was determined according to their activities in the sandwich immunoassay systems. This study includes (1) the synthesis of conjugates of different compositions; (2) the determination of stabilizing antibody concentrations for immobilization; (3) the structural characterization of the conjugates synthesized at different concentrations; (4) the functional characterizations of the conjugates synthesized at different concentrations; and (5) a comparison of the conjugates in the sandwich immunoassays where the GNPs are used as labels. To accomplish these tasks, we used monoclonal antibodies (mAb) specific to Helicobacter pylori antigens and polyclonal antibodies (pAb) specific to mouse IgG for conjugation with 2738

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Figure 3. Plots of the hydrodynamic radius of the Ab-GNP vs the concentration of the mAb (A) and pAb (B) used for the conjugation. The average diameter of GNPs determined by the DLS is 32.4 ± 1.8 nm. Arrows indicate the antibody concentrations as the starting points for the formation monolayer on the GNP surface.

The ranges of the concentration dependence, starting from a concentration of 19 μg mL−1 (mAb) and 6 μg mL−1 (pAb), correspond to the completion of the stabilization of the GNP by antibodies. An absorption change at the wavelength of 580 nm (see Figure 2) indirectly indicates the presence of aggregates in the preparation of GNPs, which corresponds to the data we obtained earlier using electron microscopy.19 With the addition of excess salt, conjugates synthesized at antibody concentrations lower than the flocculation point were not stable. However, adding a stabilizing agent to the conjugates allowed the use of concentrations below the stabilizing concentration. To synthesize conjugates, we chose the stabilizing concentration of each antibody (19 μg mL−1 for mAb and 6 μg mL−1 for pAb) and seven concentrations below the stabilizing concentration; for mAb, these concentrations were 10, 5, 3, 1.5, 0.5, 0.3, and 0.1 μg mL−1; for pAb, they were 4, 3, 2, 1, 0.5, 0.3, and 0.1 μg mL−1. These concentrations reflecting different regions of the flocculation curve were used to synthesize the conjugates. These concentrations provide GNP-to-antibody ratios from 1:245 to 1:1 and from 1:77 to 1:1 for mAb and pAb, respectively. The preparations obtained were divided into two series; BSA was added to the final concentration of 0.25% as a stabilizing agent to one preparation (see Materials and Methods) Thus, the first series of the conjugate, containing only a synthesized conjugate and a small fraction of the free antibodies that are not bound to the GNP surface (according to previously published data by Sotnikov et al.35), was used for the correct measurement of the conjugates’ hydrodynamic sizes. The second series of the conjugate contained stabilizing components, and did not contain stabilized free antibodies, making it the most appropriate for determining antigencapturing activity and estimating the analytical potential of conjugates. For all conjugates, the absence of conjugate aggregation was confirmed by absorption spectra similar to the GNP spectrum (Figure S1, B-E, the spectra of the synthesized conjugates). The absorption maxima of all conjugates were from 526 to 530 nm, depending on the concentration of immobilized antibodies and the presence of 0.25% BSA in solution. This shift is consistent with the known influence of immobilized antibodies on the surface plasmon resonance shift of GNPs and does not indicate the GNPs aggregation.36,37

GNPs. The efficiency of conjugates synthesized with different GNP-to-antibody ratios was estimated in a lateral flow immunoassay (LFIA). LFIA is the most common method for GNP conjugates.2,27,30 Moreover, protein adsorption onto nanoparticles and obtained conjugates are key factors for the development of biosensors, LFIA in particular.31,32

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RESULTS AND DISCUSSION Estimation of the Concentrations of GNPs and Conjugates. The citrate-capped GNPs were synthesized, and the size of the GNPs was determined by measuring the diameter of whole particles by dynamic light scattering (DLS) and transmission electron microscopy (TEM). DLS is a more appropriate technique for accurate nanoparticle characterization in a solution (i.e., without use of any support and possible immobilization artifacts).33 According to DLS, the synthesized GNPs were monodispersed and their average diameter was 32.4 ± 1.8 nm. However, the hydrated shell around the GNP contributed to the data of DLS measurements. The impact of TEM to the GNPs’ characterization consisted of data about true dimensions of GNP cores. By TEM, the average diameter of the GNPs was 25.3 ± 4.6 nm, and the form factor (ratio of maximum and minimum axes) was 1.23 ± 0.13 (Figure 1). GNP solutions were characterized in respect to their absorbance by UV−vis spectrophotometry. The maximum of the absorption spectrum of the synthesized GNPs was 526 nm (Figure S1-A) (all tables and figures designated S are found in Supporting Information). The GNP concentration was calculated based on the average size of the particles determined by TEM, and the Au concentration in the solution was determined by inductively coupled plasma mass spectrometry (ICP-MS) to be 0.5 nM (Figure S2 and Table S2 include the calibration curve of Au and primary data from ICP-MS). To determine the stabilizing concentration of mAb and pAb, the flocculation curves for the GNPs were obtained (Figure 2). The addition of 10% NaCl solution results in changes in the electric double layer of GNP and a shift in the equilibrium between the electrostatic repulsion and attraction of particles34 and, correspondingly, the flocculation of the GNPs. The flocculation was observed from 0 to the flocculation point: 19 μg mL−1 (or 127 nM) of mAb or 6 μg mL−1 (or 40 nM) of pAb (see Figure 2). 2739

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Figure 4. Dependencies of the conjugates(A) mAb-GNP and (B) pAb-GNPbinding to the antigens from the conjugates’ concentrations.

Table 1. Comparison of the Functional Characterizations of the Conjugates mAb-GNP

mAb concentration at immobilization (μg mL−1)

a

0.1 0.3 0.5 1.5 3 5 10 19

pAb-GNP

KD ± standard deviation (M)

Antigencapturing activity

(1.89 ± 0.12) × 10−9 (9.1 ± 0.9) × 10−10 (6.62 ± 0.29) × 10−10 (1.03 ± 0.18) × 10−10 (2.75 ± 0.3) × 10−11 (2.80 ± 0.2) × 10−11 (3.04 ± 0.2) × 10−11 (3.68 ± 0.3) × 10−11

0.4 0.8 1.1 4.4 26.9 18.5 17.2 14.9

KD ± standard deviation (M) pAb concentration at immobilization (μg mL−1)

0.1 0.3 0.5 1.0 2.0 3.0 4.0 6.0

N/Da (8.93 ± (3.89 ± (1.26 ± (3.38 ± (1.89 ± (1.04 ± (0.57 ±

1.0) × 10−10 0.19) × 10−10 0.16) × 10−10 0.32) × 10−11 0.09) × 10−11 0.22) × 10−11 0.03) × 10−11

Antigencapturing activity 0 0.1 0.3 1.0 2.7 5.7 9.0 21.2

N/D, the constant cannot be determined with the methods used.

therefore, the antibody has an apparently vertical orientation on the GNP surface when antibody concentrations of approximately 7 nm of Rh and higher were used. For mAb, corresponding concentrations began at 3 μg mL−1; pAb concentrations began at 2 μg mL−1. These concentrations are significantly lower than the values chosen according to flocculation dependencies (19 μg mL−1 for mAb and 6 μg mL−1 for pAb, see Figure 2). It can be assumed that the number of antibodies per area unit increases with the increase of antibody concentration (after 3 μg mL−1 for mAb and 2 μg mL−1 for pAb). However, a monolayer of antibodies on the GNP surface was formed at a concentration of 3 μg mL−1 for mAb and 2 μg mL−1 for pAb. This conclusion follows from the conjugate’s diameter being more than the GNP diameter at 14 nm (∼2 sizes of IgG). A similar increase in diameter was reported by James and Driskell,38 who compared the hydrodynamic sizes of conjugates synthesized with different amounts of protein A and mouse IgG. In the presence of 0.25% BSA (which is characteristic of the second series of synthesized conjugates), changes of the conjugate Rh associated with the increase of immobilized antibody concentrations are less visible. In this case, an increase of the Rh of the conjugates to at least 22.9 ± 1.5 nm in the presence of 0.25% BSA in solution occurs at 0.1 μg mL−1 of antibodies (Figure S3). Apparently, at low antibody concentrations (to 2.5 μg mL−1), BSA interacts with the free GNP surface and increases the hydrodynamic sizes of conjugates. This effect does not allow control of the addition of immobilized antibodies in the second series of synthesized conjugates using hydrodynamic parameters.

The precise concentration of each conjugate was determined using the method of ICP-MS, and the data are provided in Table S2. The concentrations obtained were used for further comparisons of conjugates and calculations of their antigencapturing activity. Size Characterization of Antibody-GNP Conjugates by DLS. To obtain the protein layer formation on the surface of the GNPs, 16 conjugates (8 mAb and 8 pAb) were characterized using the DLS method. Figure 3 shows the dependence of the hydrodynamic radius (Rh) on the concentration of antibodies added for conjugation. For both conjugates (with mAb and pAb and without stabilizing components), the dependencies of Rh on the antibody concentration used for synthesis were equal: The radius increased from 16.7 ± 0.6 to 24.4 ± 0.9 nm (mAb) and from 17.4 ± 0.8 to 23.8 ± 1.4 nm (pAb). At the same time, as shown in Figure 3, every dependence has an inflection point joining two linear segments with different tangents of the angle of slope. These inflection points are 3 μg mL−1 for mAb, corresponding to a GNP to antibody ratio of 1:58 and 2 μg mL−1 for pAb, corresponding to a GNP-to-antibody ratio of 1:25. It should be noted that before the inflection point, a small increase in antibody concentration leads to a significant increase in the Rh of the conjugate (see Figure 3), while after the inflection point, an increase in antibody concentration has little influence on the Rh of the conjugate (1.6−1.8 nm increase with an error of ±1.5 nm). The Rh of the GNPs was 16.2 ± 0.9 nm and the antibody immobilization on the GNP surface was added to Rh from 0.5 to 8.2 nm. The diameter of the IgG is approximately 7−10 nm (data from Protein Data Bank); 2740

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Figure 5. Calibration curves for LFIA (n = 3): (A) for the detection of H. pylori antigen AGHPY-0104 with the application of the mAb-GNP conjugate obtained at 19 μg mL−1 of mAb; (B) for detection of anti-B. abortus antibody mAb 7F10 with the application of the pAb-GNP conjugate obtained at 6 μg mL−1 of pAb.

Figure 6. Influence of mAb-GNP composition on color intensity in the analytical zone of test strips (n = 6): (A) overview of test strips after analysis; 1−8 corresponds to the mAb concentrations used for the conjugate synthesis: 0.1, 0.3, 0.5, 1.5, 3, 5, 10, and 19 μg mL−1; (B) dependence of color intensity on mAb concentration used for the conjugate synthesis. Concentration of antibodies immobilized in the assay is 0.5 mg mL−1, and analyte (H. pylori) concentration is 10 μg mL−1.

Functional Characterizations of the Conjugates. The comparison of the conjugates was performed according to the value of active antibodies immobilized on the GNP surface. For this purpose, an enzyme-linked immunosorbent assay (ELISA) was used, which included the registration of conjugate-binding to immobilized antigens in the plate wells after achieving equilibrium and the determination of the number of active antibodies on the GNP surface using calibration curves for determining mAb and pAb (Figure S4). Antigen-capturing activity of the conjugates equivalent to the native antibody activity was determined in a wide range of concentrations, from 10 pM to 30 nM for mAb and 2 pM to 2 nM for pAb. The dependencies of the antigen-binding of the conjugates (with 0.25% of BSA) on conjugate concentration are shown in Figure 4A and B. It should be noted that, for the first series of conjugate preparations without BSA, accurate quantitative description is hindered due to nonspecific interactions (Figure S5). Based on the analysis of these dependencies, interaction

constants and the antigen-capturing activity of the conjugates were calculated. The equilibrium dissociation constants of the conjugates (KD) of the interaction between the conjugate and the antigen were determined for all conjugates synthesized at different mAb and pAb concentrations, and results are provided in Table 1. For mAb, KD decreased across the range of antibody concentrations (from 0.1 to 3 μg mL−1), reaching 2.75 × 10−11 M; almost no KD changes occurred up to 19 μg mL−1. In the case of pAb, KD decreased across the entire chosen range from 0.1 to 6 μg mL−1, reaching 0.57 × 10−11 M. Thus, KD indicates that the conjugates synthesized at the concentrations of 3 μg mL−1 for mAb and 6 μg mL−1 for pAb are the best for analytical application. At the same time, as seen in Table 1, an increase in antibodies per area unit (at concentrations higher than 3 μg mL−1 for mAb and 2 μg mL−1 for pAb) does not affect KD for mAb and slightly improves KD for pAb. 2741

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Figure 7. Influence of pAb-GNP composition on color intensity in the analytical zone of test strips (n = 6): (A) overview of test strips after analysis; 1−8 corresponds to the pAb concentrations used for the conjugate synthesis: 0.1, 0.3, 0.5, 1, 2, 3, 4, and 6 μg mL−1; (B) dependence of color intensity on pAb concentration used for conjugate synthesis. Concentration of lipopolysaccharide antigen of the B. abortus immobilized in the assay zone is 0.25 mg mL−1 and analyte (mAb 7F10) concentration is 10 μg mL−1.

intensity was different and depended on mAb-GNP conjugates. Staining in the analytical line increased from 0.1 to 3 μg mL−1, after which it remained constant and even decreased at 19 μg mL−1 (Figure 6B). The obtained dependence is similar to the dependence of the hydrodynamic radii of conjugates (see Figure 3A) and complies with the values of KD and antigencapturing activity. These conclusions correspond to the data of our LFIA experiments, in which detection limits of the H. pylori antigen detection were compared with conjugates of different compositions. Limits of detection (appropriate threshold of reliable visual detection) varied very little for conjugates with high mAb concentration (Figure S7-A). Thus, in this system, the most efficient conjugate is mAb-GNP obtained at the antibody concentration of 3 μg mL−1. This value is much lower than the stabilized concentration (19 μg mL−1) and approaches a constant value of hydrodynamic radii for the mAb-GNP conjugates. These findings were confirmed for 5 other antibodies specific to fatty acid binding proteins, troponin I, myoglobin, C-reactive protein, and D-dimer. A 3-to-4-fold decrease of antibody concentrations for conjugates’ syntheses as compared with their stabilizing concentrations did not affect the limit of detection of LFIA (data shown in Table S3). In the pAb-GNP system, binding in the analytical zone was shown by all conjugates except for 0.1 μg mL−1; binding in the control zone was observed for all conjugates (Figure 7A). Staining of the analytical line increased with the increase in immobilized antibody concentrations from 0.1 to 6 μg mL−1 (Figure 7B). However, it is necessary to highlight that the antibody concentration of 2 μg mL−1 corresponded to the formation of a constant hydrodynamic radii of the conjugates for pAb-GNP (see Figure 7B), before which growth of the intensity was greater than it was at the subsequent concentrations ranging from 2 to 6 μg mL−1. Thus, in this system, the most efficient conjugate is pAb-GNP obtained at the antibody concentration of 6 μg mL−1, which corresponds to the stabilizing concentration. However, in LFIA, limits of detection of IgG specific to B. abortus obtained for conjugates with pAb from 2 to 6 μg mL−1 are approximately equal and

Another functional characteristic to compare conjugates is the antigen-capturing activity of the conjugate. The calculated values of the antigen-capturing activity of the conjugates are summarized in Table 1 (the method of linear fits for determining antigen-capturing activity of the conjugate is described in Materials and Methods, and the obtained linear fits are provided in Figure S6. The maximal antigen-capturing activities of the conjugates were found at 3 μg mL−1 for mAb and at 6 μg mL−1 for pAb. The maximal antigen-capturing activities and equilibrium constants of the conjugates correlate with the formation of a constant hydrodynamic radius of the conjugates for mAb-GNP (GNP-to-antibody molar ratio equals 1:58) and with the stabilizing concentration by flocculation curves for pAb-GNP (GNP-to-antibody molar ratio equals 1:116). The results obtained show that the mAb concentration can be reduced for their conjugations with GNPs without losing the activity of the conjugates. To confirm this hypothesis experimentally, we used 8 mAb-GNP and 8 pAb-GNP conjugates in LFIA and compared the registered binding in these test systems. Efficiency of the Conjugates in the Sandwich LFIA. On the basis of synthesized conjugates, the following LFIA test systems with the sandwich format were prepared: (1) mAbGNP conjugates were tested to determine H. pylori; (2) pAbGNP conjugates were tested to determine mouse IgG (through the example of IgG specific to B. abortus). At the first stage, both LFIA test systems included a conjugate synthesized in accordance with the optimal values on the flocculation curve (19 and 6 μg mL−1 for mAb and pAb, respectively). The dependencies of staining in the assay zone on the concentration of the determined analyte in the sample are provided in Figure 5. For both systems, the upper plateau starts at the level of 10 μg mL−1 of the corresponding antigen. Thereby, this concentration of antigens was chosen to compare the conjugates synthesized with different Ab-to-GNP ratios. The content of both analytes was fixed and equal to 10 μg mL−1. In the mAb-GNP system, all conjugates showed binding in the analytical and control zones (Figure 6A). However, the line 2742

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Mdi Easypack (Advanced Microdevices, India) kits of membranes were used for the LFIA. Preparation of GNPs. GNPs were prepared according to the protocol described by Frens.39 A 1% gold chloride hydrate solution (1.0 mL) was added to deionized water (97.5 mL). The reaction mixture was brought to the boiling point, and then a 1% sodium citrate solution (1.5 mL) was added with stirring. The reaction mixture was boiled for 25 min, cooled, and stored at 4−6 °C. Synthesis of Ab-GNP Conjugates. Synthesis of Ab-GNP conjugates was carried out as described by Hermanson.16 Prior to the conjugation to GNP, the antibody was dialyzed against a 1000-fold excess of 10 mM Tris-HCl buffer, pH 9.0, for 2 h at 4 °C. Then 0.2 M potassium carbonate solution was added to the GNP solution (OD520 = 1.0) until pH 9.0 was reached. A GNP solution with OD520 = 1.0 (1.0 mL portions) was added to solutions (0.1 mL) of antibodies (mAb or pAb) in concentrations from 1 to 250 μg mL−1. The mixtures were stirred and incubated at room temperature for 10 min. Then a 10% NaCl solution (0.1 mL) was added to each sample, the mixtures were stirred for 10 min, and OD580 was measured. Then flocculation curves were plotted as dependencies of the absorbance at 580 nm in the presence of 10% NaCl against the antibody concentration. To synthesize Ab-GNP conjugates, antibodies at chosen concentrations were mixed with the GNP solution. The mixture was incubated with stirring at 20−22 °C for 30 min, and preparations were divided into two parts; BSA was added to one final concentration of 0.25%. The preparations including BSA were separated from unbound proteins by centrifugation at 8000 g for 30 min. After removal of the supernatant, the precipitate was resuspended in 10 mM Tris-HCl buffer, pH 9.0, containing 0.25% BSA and 0.05% Tween-20. The spectra of the GNPs and their conjugates were recorded with a Biochrom Libra S60 double beam spectrophotometer (Biochrom, UK). Transmission Electron Microscopy. GNPs were dropped onto a grid (300 mesh, Pelco International, USA) coated with polyvinyl formal support film. The images were obtained with a CX-100 electron microscope (Jeol, Japan) at an accelerating voltage of 80 kV and a magnification of 3,300,000×. They were digitalized with Image Tool software (University of Texas Health Science Center in San Antonio, USA). To approximate TEM data about distribution of GNPs’ diameters, one-peak Gauss approximation was used. The fitting was calculated by OriginPro 9.0 software (Origin Lab, USA). Estimation of GNPs and Conjugate Concentrations. Au concentrations in the solutions were obtained by ICP-MS. The ICP-MS measurements were carried out with a quadrupole ICP-MS instrument Aurora M90 (Bruker Corp., USA) equipped with a MicroMist low-flow nebulizer. A series of Au standard solutions (0.1−5.0 ppb in 1% HCl [v/v]) were prepared before each experiment. Scandium was used as the internal standard, eliminating the fluctuations coming from the measuring conditions. The optimal operating parameters are summarized in the Supporting Information (Table S1). All samples were prepared in triplicate. Quantum software (Bruker Corp., v 3.1 b1433) was used for data collection and processing. In the calculations of the concentration of GNPs, the density of gold was equal to 19.3 g cm−3, and the initial assumption was that the volume of one particle is 4/3·πr3, where r is half the sum of the half-mean major and half-mean minor axis lengths obtained by transmission electron microscopy.

significantly lower than for conjugates with poor pAb concentration (Figure S7−B). In this way, increasing the GNP surface coverage and saturation of the monolayer coverage does not lead to proportionate growth of active antibodies, which is essential for immunoassays. Apparently, crowding effects (shielding of binding sites with neighboring immobilized molecules, reduction of Fab-fragment mobility, etc.) at higher coverage of the conjugates are more powerful than is commonly assumed at the development of sandwich immunoassays. The application of the conjugates to the LFIA confirmed that the antibody concentrations used for the conjugation can be reduced without losing conjugate activity.

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CONCLUSIONS Ab-GNP conjugates of different compositions were studied. It was shown that changes in the hydrodynamic size, KD, antigencapturing activity, and efficiency of the immunoassay correlates to the range of conjugates with different GNP-to-immobilized mAb molar ratios. At the same time, the mAb concentration usually recommended for conjugate synthesis and obtained by the flocculation curve does not satisfy the maximum functional properties of the conjugates. The mAb concentration for conjugation with GNPs can be reduced by about six times. However, for pAb, the most efficient pAb-GNP conjugate was obtained at the stabilizing antibody concentration. Thus, at nondirectional and noncovalent immobilization Ab on the GNP surface by physical adsorption, the optimal antibody concentration should be chosen for each individual case, and it does not always coincide with the high concentration determined on the basis of the flocculation curve. These factors should be considered when immunoassay systems are developed based on GNPs.

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MATERIALS AND METHODS Chemicals and Materials. The monoclonal anti-H. pylori antibody, clone ABHPY-0404; H. pylori antigen AGHPY-0104; goat anti-mouse immunoglobulins (GAMI); and donkey antigoat immunoglobulins (DAGI) were from Arista Biologicals (USA). The monoclonal anti-H. pylori antibody, clone 939 (mAb 939), was from the Russian Research Center of Molecular Diagnostics and Therapy (Russia). The lipopolysaccharide antigen (LPS) of the Brucella abortus and the monoclonal anti-B. abortus antibody, clone 7F10 (mAb 7F10), were from National Center for Biotechnology (Kazakhstan). The Au standard ICP-MS (999 ± 2 mg mL−1 in 5% HCl [w/ w]) came from Fluka, Switzerland; ICP-MS internal standards of Li6, Sc, Y, In, Tb (100 ± 0.5 μg mL−1 in 2% HNO3 [w/w]) came from Bruker Daltonics Chemical Analysis, USA; hydrochloric acid (38% w/w, ultrapure) came from Reactivcomponent, Russia. The BSA, sodium citrate, nitric acid (70% w/w, purified by redistillation), sucrose, sodium azide, and 3,3′,5,5′-tetramethylbenzidine dihydrochloride (TMB) were from Sigma (USA). The gold chloride hydrate was from Fluka (Buchs, Switzerland). The Tween-20 was from MP Biomedicals (USA). The polyvinyl formal was from SPI Supplies (USA). All other chemicals (salts and solvents of analytical grade) were from Khimmed (Russia). The solutions of GNPs and its conjugates with antibodies were prepared using deionized water (Milli-Q, Millipore, USA, 18.2 M Mom cm−1 at 25 °C). 2743

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

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Bioconjugate Chemistry

Equilibrium dissociation constants of the conjugates (KD) were determined using the dependencies of the binding level on the conjugate’s concentration, according to the equation

These measurements were carried out on the equipment of the Shared-Access Equipment Centre “Industrial Biotechnology” of Federal Research Center “Fundamentals of Biotechnology” Russian Academy of Sciences (Moscow, Russia). Dynamic Light Scattering. DLS from GNP preparations and Ab-GNP conjugates was recorded with a Photocor instrument (Photocor Instruments, USA) equipped with a helium−neon laser (632.8 nm). The hydrodynamic radius distribution of GNPs and their conjugates with antibodies was obtained at 25 °C by measuring the intensity of scattered light at an angle of 90°. The results of the measurements were processed using DynaLS v 2.5.2 (Alango, Israel). All GNPs were analyzed at the initial concentration; the preparations of antibody-GNP conjugates were analyzed at a concentration corresponding to A520 = 1.0. Each experiment was repeated ten times. Biotinylation of Antibodies. Biotinylation was carried out as described by Hermanson.16 The antibodies were dialyzed against a 1000-fold volume of 50 mM potassium phosphate, pH 7.4, 0.14 M NaCl (PBS), for at least 14 h at 4 °C. Then, biotinamidohexanoic acid N-hydroxysuccinimide ester (10 mg mL−1, in dimethyl sulfoxide) was added to the antibodies in a 20:1 M ratio. The mixture was incubated for 1 h at room temperature under intensive stirring and then dialyzed against PBS overnight. Determination of the Equilibrium Dissociation Constant and Antigen-Capturing Activity of the Conjugate. The antigen-capturing activity of the conjugate and the antibody content in the Ab-GNP conjugate was determined by ELISA. The captured H. pylori antigen AGHPY-0104 or anti-B. abortus antibody mAb 7F10 were coated by adding 50 μL of 1.0 or 0.2 μg mL (respectively) in PBS to each plate well and incubating at 4 °C overnight. The microplate wells were washed 4 times using PBS with 0.05% Triton X-100 (PBST). Then, for calibration curves, 50 μL of mAb or pAb (from 0.1 to 100,000 pM) in PBST were added into the wells. For conjugate characterization, 50 μL of Ab-GNP conjugate (from 0.1 to 1000 pM) soluble in PBST was added into the wells. The microplate was incubated for 1 h at 37 °C and then washed 4 times with PBST. To determine the antigen-capturing activity of the monoclonal antibody−gold nanoparticle conjugate (mAbGNP), 50 μL of anti-mouse immunoperoxidase conjugate (1:3000 dilution in PBST) was added, and this mixture was incubated for 1 h at 37 °C. To determine the antigen-capturing activity of the polyclonal antibody-gold nanoparticles conjugate (pAb-GNP), 50 μL of biotinylated DAGI (1.0 μg mL−1) in PBST was added into each well. The microplate was incubated for 1 h at 37 °C. After washing the microplate as described above, 50 μL of streptavidin−peroxidase conjugate (dilution of 1:1000 in PBST) was added into the wells and incubated for 1 h at 37 °C. After washing, the peroxidase activity for all experiments was equally determined. The substrate (100 μL of 0.4 mM TMB solution in 40 mM sodium citrate buffer, pH 4.0, containing 3 mM H2O2) was added to each well. After incubation at room temperature for 15 min, the reaction was terminated by the addition of 1 M H2SO4 (50 μL), and the absorbance at 450 nm was measured using a microplate photometer Zenyth 3100 (Anthos Labtec Instruments, Austria). The mAb and pAb content in the Ab-GNP conjugates was calculated using the calibration curve obtained for free mAb and pAb as a standard.

R eq =

KA × [Conjugate] × R max 1 + KA × [Conjugate]

(1)

where Req is the level of binding; Rmax is the maximum observed level of binding; [Conjugate] is the concentration of the conjugate; and KA is equilibrium association constant at KD−1. The data were processed by the steady-state affinity fitting using BIAevaluation software v 4.1 (GE Healthcare, USA). The antigen-capturing activity of the conjugate was determined based on the assumption that for each conjugate, regardless of its concentration, the antibody-per-GNP ratio is constant and expressed by the equation

p*[Ab] =N [GNP]

(2)

where N corresponds to the number of antibodies per GNP; p is a coefficient considering the portion of the GNP surface, which is not sterically available for determining Ab, and is equal for all conjugates; [Ab] is the concentration of the antibody determined according to the calibration curve for each concentration of the conjugate; and [GNP] is the concentration of the conjugate added to the system. For calculations, we used the region of low conjugate concentrations so that the number of free antibodies could be neglected. Accordingly, for the linear dependence of [Ab] on [GNP], the slope tangent corresponds to N/p (the antigencapturing activity of the conjugate). Preparation of Lateral Flow Test Strips. Immunoreagents were immobilized on the membranes of the test system using an IsoFlow automated dispenser (Imagene Technology, USA). The mAb-GNP conjugates were immobilized on a pad in a dilution corresponding to OD520 = 6.0 (16 μL per centimeter of the pad width). The test zone was formed using the mAb 939 (0.5 mg mL−1 in PBS) and the control zone with the use of GAMI (0.25 mg mL−1 in PBS). The solutions were stabilized, and 2.0 μL of the solution was applied per centimeter of the working membrane. The pAb-GNP conjugates were immobilized on a pad in a dilution corresponding to OD520 = 6.0 (16 μL per centimeter of the pad width). The test zone was formed using the B. abortus LPS (0.25 mg mL−1 in 0.1 M sodium citrate buffer, pH 4.1) and the control zone with the use of DAGI (0.125 mg mL−1 in PBS). The solutions were stabilized and 2.0 μL of the solution was applied per centimeter of the working membrane. The pads and working membranes thus prepared were dried in air at 20−22 °C for at least 20 h. The multimembrane composite was assembled and then cut into 3.5-mm-wide strips with an Index Cutter-1 automated guillotine cutter (A-Point Technologies, USA). Lateral Flow Assay and Data Processing. The assay was performed at room temperature. The test strip was dipped into a tested solution (50 μL) for 1 min and then placed on a horizontal surface. Ten minutes after beginning the assay, the result was checked, a digital image of the test strips was obtained with a CanoScan LiDE 90 scanner, and the integrated intensities of the color in the test and control zones were calculated as described previously by Safenkova et al.19 2744

DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746

Article

Bioconjugate Chemistry

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(8) Katz, E., and Willner, I. (2004) Integrated nanoparticlebiomolecule hybrid systems: synthesis, properties, and applications. Angew. Chem., Int. Ed. 43, 6042−6108. (9) Zhang, S. Y., Moustafa, Y., and Huo, Q. (2014) Different interaction modes of biomolecules with citrate-capped gold nanoparticles. ACS Appl. Mater. Interfaces 6, 21184−21192. (10) Pan, H., Qin, M., Meng, W., Cao, Y., and Wang, W. (2012) How do proteins unfold upon adsorption on nanoparticle surfaces? Langmuir 28, 12779−12787. (11) Miranda, E. G. A., Tofanello, A., Brito, A. M. M., Lopes, D. M., Albuquerque, L. J. C., de Castro, C. E., Costa, F. N., Giacomelli, F. C., Ferreira, F. F., Araujo-Chaves, J. C., and Nantes, I. L. (2016) Effects of gold salt speciation and structure of human and bovine serum albumins on the synthesis and stability of gold nanostructures. Front. Chem. 4, 13. (12) Dewald, I., Isakin, O., Schubert, J., Kraus, T., and Chanana, M. (2015) Protein identity and environmental parameters determine the final physicochemical properties of protein-coated metal nanoparticles. J. Phys. Chem. C 119, 25482−25492. (13) Scaletti, F., Feis, A., Centi, S., Pini, R., Rotello, V. M., and Messori, L. (2015) Tuning the interactions of PEG-coated gold nanorods with BSA and model proteins through insertion of amino or carboxylate groups. J. Inorg. Biochem. 150, 120−125. (14) Geoghegan, W. D. (1988) The effect of three variables on adsorption of rabbit IgG to colloidal gold. J. Histochem. Cytochem. 36, 401−407. (15) Wiriyachaiporn, N., Maneeprakorn, W., Apiwat, C., and Dharakul, T. (2015) Dual-layered and double-targeted nanogold based lateral flow immunoassay for influenza virus. Microchim. Acta 182, 85−93. (16) Hermanson, G. T. (2013) Bioconjugate Techniques, 3rd ed.; Academic Press, Boston. (17) Di Nardo, F., Baggiani, C., Giovannoli, C., Spano, G., and Anfossi, L. (2017) Multicolor immunochromatographic strip test based on gold nanoparticles for the determination of aflatoxin B1 and fumonisins. Microchim. Acta 184, 1295−1304. (18) Thobhani, S., Attree, S., Boyd, R., Kumarswami, N., Noble, J., Szymanski, M., and Porter, R. A. (2010) Bioconjugation and characterisation of gold colloid-labelled proteins. J. Immunol. Methods 356, 60−69. (19) Safenkova, I., Zherdev, A., and Dzantiev, B. (2012) Factors influencing the detection limit of the lateral-flow sandwich immunoassay: a case study with potato virus X. Anal. Bioanal. Chem. 403, 1595−1605. (20) Pollitt, M. J., Buckton, G., Piper, R., and Brocchini, S. (2015) Measuring antibody coatings on gold nanoparticles by optical spectroscopy. RSC Adv. 5, 24521−24527. (21) Chun, P. (2009) Colloidal gold and other labels for lateral flow immunoassays, in Lateral Flow Immunoassay (Wong, R., and Tse, H., Eds.) pp 1−19, Humana Press, Totowa, NJ. (22) de Puig, H., Bosch, I., Carre-Camps, M., and Hamad-Schifferli, K. (2017) Effect of the protein corona on antibody-antigen binding in nanoparticle sandwich immunoassays. Bioconjugate Chem. 28, 230− 238. (23) Zvereva, E. A., Byzova, N. A., Sveshnikov, P. G., Zherdev, A. V., and Dzantiev, B. B. (2015) Cut-off on demand: adjustment of the threshold level of an immunochromatographic assay for chloramphenicol. Anal. Methods 7, 6378−6384. (24) Lu, Y., Peterson, J. R., Luais, E., Gooding, J. J., and Lee, N. A. (2015) Surface epitope coverage affects binding characteristics of bisphenol-a functionalized nanoparticles in a competitive inhibition assay. J. Nanomater. 2015, 756056. (25) Vertegel, A. A., Siegel, R. W., and Dordick, J. S. (2004) Silica nanoparticle size influences the structure and enzymatic activity of adsorbed lysozyme. Langmuir 20, 6800−6807. (26) Wu, X. Y., and Narsimhan, G. (2008) Effect of surface concentration on secondary and tertiary conformational changes of lysozyme adsorbed on silica nanoparticles. Biochim. Biophys. Acta, Proteins Proteomics 1784, 1694−1701.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00489. Optimal operating parameters of ICP-MS measurements and data of ICP-MS measurements; absorption spectra of GNPs and antibody−GNP conjugates; hydrodynamic radii of the conjugates; concentration dependences for antigen-capturing activities of the antibodies and conjugates; dependences of the detection limit on the composition of the conjugates (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel./fax: +7-(495)-954-31-42. ORCID

Irina V. Safenkova: 0000-0002-3621-4321 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Russian Science Foundation (project no. 14-14-01131). The authors are extremely grateful to Prof. P. G. Sveshnikov (Russian Research Centre of Molecular Diagnostics and Therapy, Moscow, Russia) for the production of the monoclonal anti-H. pylori antibody.

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ABBREVIATIONS Ab−GNP, antibody−gold nanoparticles conjugate; BSA, bovine serum albumin; DAGI, donkey anti-goat immunoglobulins; GAMI, goat anti-mouse immunoglobulins; GNPs, gold nanoparticles; IgG, Immunoglobulin G; LFIA, lateral flow immunoassay; LPS, lipopolysaccharide antigen; mAb, monoclonal antibody; mAb−GNP, monoclonal antibody−gold nanoparticles conjugate; pAb, polyclonal antibody; pAb− GNP, polyclonal antibody−gold nanoparticles conjugate; PBS, 50 mM potassium phosphate, pH 7.4, 0.14 M NaCl; PBST, PBS with 0.05% Tween-20; TEM, transmission electron microscopy; TMB, 3,3′,5,5′-tetramethylbenzidine dihydrochloride

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DOI: 10.1021/acs.bioconjchem.7b00489 Bioconjugate Chem. 2017, 28, 2737−2746