Optimization of microparticle reagents to collect and detect antibody

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Optimization of microparticle reagents to collect and detect antibody Katily Ramirez, elizabeth campbell, So-Yun Han, Joseph Buehler, Thuong Phan, Hee Young Yoon, Ye Lim Lee, Tanvi Suresh, and Todd A. Sulchek Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.9b01555 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 25, 2019

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Optimization of microparticle reagents to collect and detect antibody Katily Ramirez1, Elizabeth Campbell2, So-Yun Han2, Joseph Buehler2, Thuong Phan2, Hee Young Yoon2, Ye Lim Lee2, Tanvi Suresh2, Todd Sulchek2,3* 1

2

Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta GA 30332

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta GA 30332 3

George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA 30332

Keywords. Microparticles, detection limit, protein G, antibody, hybridomas.

Abstract. Bead reagents are used in a wide number of assays in biosciences and biotechnology to collect and purify antibodies by immobilization. Bead-based immunoassays offer high throughput analysis of multiple antibodies in a single sample. While a variety of antibody-binding moieties on the collection beads have been studied, the physical and material properties of collection beads have not been optimized to isolate specific antibodies over a broad range of concentrations from complex environments containing cells. We present a study of how to optimally use microparticles coated with protein G to collect low concentrations of IgG antibodies from complex solutions. We

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study the impact of bead material, bead size, incubation time, and protein G density to more efficiently collect antibodies and detect specific antibodies via fluorescent antigen labeling. The minimum detectable limit and the minimum incubation time for antibody collection are used as metrics to evaluate the collection parameters. We found larger silica beads can capture more antibodies from a low concentration of sample, with a minimum incubation time of 60 minutes to equilibrium binding, resulting in a minimum detectable concentration of antibodies of 26 nanomolar. We show that simple biophysical optimization of antibody collection reagents can be used to improve the collection of low concentrations of antibodies in complex environments. We demonstrate that the technology may be useful to monitor antibody secretions from hybridoma cultures.

Introduction. Immunoassays and immunosensors utilizing immunoglobulin G (IgG) antibodies are of great importance to numerous applications in the medical diagnosis, pharmaceutical, and clinical chemistry fields.1 Effective isolation and sensing techniques for IgG is pivotal to improve antibody purity, especially from complex conditions.2 New methods to isolate, concentrate, and detect antibody are needed, for example, to analyze the production of antibodies from individual cells or batches of cells, especially in a manner that allow the cells to be viable post-purification. Antibody amounts are commonly detected using the enzyme-linked immunosorbent assay (ELISA). In ELISA, the cells and serum are incubated in an antigen-coated well plate to quantitatively assess the specific antibody in the serum through an enzyme labeled with antiimmunoglobulins.3 But ELISA is time consuming, since it involves multiple incubation steps, costly, and does not provide a high throughput method to accurately multiplex immunoassays simultaneously, especially in a manner that allows secreting cells to remain. Multiplexed microparticle immunoassays use immobilized capture ligands on beads in suspensions.1 The


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principal advantage of bead-based biosensors is the higher protein-capture capacity in comparison to flat 2D surfaces.4 The microparticles can also be biochemically tagged to detect multiple antibody types and can be directly suspended in the serum in proximity to cells without extra preparation steps, and without affecting the cell viability. Applications of microparticle assays can also include the quantitation of protein secretion of cells confined in microenvironments, such as microwells and microdrops, for antibody identification, collection, or secretion rate analysis in a manner that allows the reutilization of cells for cloning and expansion. Bead-based biosensors provide a simple technique to expand the capabilities and reduce costs compared to ELISA.1,5-7 To isolate protein and antibody, the conventional method is column chromatography using a stationary phase functionalized moiety to adsorb antibody. In the case of antibody collection, high affinity binding to the Fab or Fc regions of the antibody can both be used to capture antibodies to then be released by washing under dissociating pH or ionic conditions8. This process requires the sample to be free of particulates and cell debris. Additionally, the required elution pH causes antibody aggregation, alteration of the antibody’s bioactivity, and antibody denaturing which compromise the yield of functional antibodies.9,10 An alternate technique uses microparticles conjugated with immunoglobulin-binding protein, such as protein G, which has a high antibodybinding capability.11,12 The method uses Fc-recognition to immobilize antibody onto the substrate for subsequent tagging by an antigen reporter.9,13-15 The advantage of this approach is higher sensitivity of antibody detection via target labeling antigen due to higher availability of antibody epitopes that result from protein G naturally orienting of the Fab region outward. Since the antigen binding regions of the antibodies are exposed, the antibodies can be recovered and used without washing, leading to a higher-yield production. Moreover, the reaction kinetics of uniformly suspended beads, in which binding can occur in a ubiquitous manner throughout the whole


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solution, are typically faster than a mechanism involving a column in which binding can only occur with the fluid layer directly in contact with the immobilized surface. In order to improve the use of microparticles, guidelines for solving detection problems have been proposed, including problems with non specific binding and antigen and antibody pairing16. This study investigates an antibody collection process utilizing suspended microparticles conjugated with a monolayer of antibody-binding protein G to collect and detect antibody from complex environments containing cells. The study determined that bead size and material properties can be chosen to improve sensitivity and specificity in collecting antibody, especially from complex sample environments containing live cells, and in a manner superior to ELISA. Polystyrene and silica particles of different sizes were functionalized with various concentrations of protein G to optimize the non-specific collection of antibody. Antibody collection properties of each particle were evaluated in complex environments including mixtures containing cells, and supernatant. The optimal concentration of protein G to reach saturation, the minimum antibody detection limit of the particle platform, and the minimum incubation time to achieve sufficient binding of antibodies were determined to engineer a more effective method of isolating and immobilizing antibodies for both practical and research applications. The approach can both detect and isolate IgG antibodies in a mixture of cells. Experimental Section Materials. Silica microparticles were purchased from Bangs Laboratories (Fishers, IN) with diameters of 0.5 μm, 1.0 μm, 2.01 μm, and 4 μm. Carboxylated polystyrene microparticles were purchased with diameters of 0.68 μm, 1.0 μm, 4 μm, and 7.8 μm. Coupling reagents, including (3Aminopropyl)triethoxysilane

(APTES),

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

(EDAC), acetone, phosphate buffered saline (PBS), PolyLink Wash/Storage Buffer, PolyLink


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Coupling Buffer, Dulbecco's Modified Eagle's Medium (DMEM), Iscove’s Modified Dulbecco’s Medium (IMDM), gentamicin, penicillin/streptomycin, L-glutamine 200 mM, 1% sodium pyruvate, concanavalin A Alexa Fluor 657 and concanavalin A Alexa Fluor 488 were purchased from Sigma-Aldrich (St. Louis, MO), fetal bovine serum from Atlanta Biologicals (Atlanta, GA). Rabbit polyclonal antibody to Shipp IgG (FITC), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO). Protein G was purchased from Protein Specialists (East Brunswick, NJ), and human Concanavalin A Antibody ELISA kit from Mybiosource TIB 147 and BCL6 hybridoma were obtained from ATCC and Iowa University, respectively.

Experimental. Preparation of silica and carboxylated polystyrene microparticles. Silica particles were washed with acetone and functionalized with 2% APTES solution in acetone17,18 (Figure 1A) (FIGURE 1A); while polystyrene (PS) particles were washed with PolyLink Coupling Buffer and functionalized with 0.2 mg/mL of EDAC and PolyLink Coupling Buffer (Figure 1B) (FIGURE 1B). Each particle type was then pelleted via centrifugation and excess APTES or EDAC were removed before resuspending in PBS and protein G for 1 hour at room temperature. Excess protein G was washed with PBS and resuspended with BSA Blocking Buffer for 1 hour and washed twice to remove excess of BSA. Rabbit polyclonal antibody was incubated at room temperature before washing the sample twice. The testing of antibody isolation used rabbit polyclonal antibody incubated for one hour at room temperature before washing the sample twice. Determining protein G saturation level. To determine the optimal amount of protein G to collect antibody, APTES-activated 1 μm silica and EDAC-activated polystyrene microparticles were incubated in varying concentrations of protein G (1 μg/μL, 2 μg/μL, 3 μg/μL, 6 μg/μL, 12 μg/μL and 25 μg/μL). Subsequently, particles were incubated with fluorescent antibody and a mean


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fluorescent intensity (MFI) was determined for each particle using flow cytometry (BD C6 Accuri, Franklin Lakes, NJ). One site-specific binding association model was employed to find the maximum specific binding in MFI (Bmax), equilibrium binding constant (K), and the minimum concentration of protein G necessary to achieve saturation on the particle surface. Evaluating antibody collection using protein G coated particles. To test the collection of immunoglobulin with protein G particles, APTES-functionalized 4μm silica particles were incubated for 1 hour with 10 μg/μL of protein G and BSA as a protein control. A particle control that was uncoated was also tested. Particles were then incubated with 2 μg/μL of fluorescent antibody for 1 hour, washed with PBS, and the MFI was determined using flow cytometry. An uncoated sample with no antibody added was also tested. Significance of each sample was calculated through one-way ANOVA. Determining the effect of microparticle size on detectable antibody. To find the minimum amount of detectable antibody collected by our particle platform, we incubated protein G functionalized beads with a varied concentration of fluorescent antibody. We determined the minimum detection limit of antibody defined as the lowest concentration with a significantly higher mean fluorescence intensity (MFI) than control with no incubated fluorescent antibody. Functionalized silica and polystyrene microparticles (1 μm and 4 μm) were incubated with 10 μg/μL of Protein G for 1 hour prior to antibody incubation. Protein G particles were then incubated for 1 and 4 hours with varied concentration of fluorescently labeled IgG antibody of 0 μg/μL (control), 0.005 μg/μL, 0.05 μg/μL, 0.5 μg/μL, 1 μg/μL, 2 μg/μL and 4 μg/μL. The MFI was measured using flow cytometry and significance of each variable was determined by nonparametric multiple t-tests for each concentration (GraphPad Prism, La Jolla, CA). The minimum detection limit was determined from one-site specific binding model and by single variable


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ANOVA. Significance of each sample was calculated through one-way ANOVA and post-hoc multiple comparison tests with a Dunnett correction in which the 0 μg control for each individual condition was compared to the successive data points. Verifying the efficiency of antibody collection. To verify the protein G particles efficiently collect antibody, we incubated particles with samples containing prepared concentrations of antibody and evaluate the supernatant after incubation for any remaining antibody. Protein G particles were incubated with 4 and 0.5 μg/μL antibody for one hour at room temperature. The supernatant containing the remaining unbound antibody was then provided to additional protein G silica particles for two hours at room temperature and the mean fluorescent intensity (MFI) of bound antibody was determined using flow cytometry in comparison to a negative control containing particles in PBS. The percentage of particles with MFI above the control was determined as a measure of amount of antibody in the second incubation. Determining the detectable antibody concentration and number of antibodies bound to the particles. The molarity and number of the antibody and particle-bound antibody particle solution were calculated based on the equations of molarity for one-phase association: 𝑀𝑤⁄ 𝑚𝐴𝑏 𝑀= (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1) 𝑉𝑝 + 𝑉𝑃𝑔 + 𝑉𝐴𝑏

𝑁𝐴𝑏

𝑀𝑤⁄ 𝑚𝐴𝑏 ) 𝑁𝐴 = (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 2) 𝑁𝑃 (

𝑁𝐴𝑏 𝑠 =

𝐶𝑎𝑏 𝑉𝑆 𝑁𝐴 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 3) 𝑀𝑤 𝑁𝑃


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in which M is the molarity of the antibody suspension, MW is the molecular weight of the antibody, mAb is the weight of the antibody added, Vp is the initial volume of the particle suspension, VPG is the volume of protein G added, VAb is the volume of antibody solution added, NAb is the estimated number of antibody molecules per particle, N A is Avogadro’s number, NP is the total number of particles in the suspension, NAbs is the number of antibody molecules per particle from cell supernatant, 𝐶𝑎𝑏 is the concentration of antibody determined by ELISA, and VS is the volume of cell supernatant. Equations 1 and 3 were derived from the equation of molarity and the definition of molecular mass, while equation 2 provides the number of molecules per particles based on the definition of molecular mass divided by the total number of particles in the solution. A one-site specific binding model in GraphPad was used to describe antibody-protein G binding on the particle surface.

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Sample 7 Sample 8 Sample 9 Table 1.

Material & Diameter (μm) Silica 0.54 Silica 1.0 Silica 2.01 Silica 4 Polystyrene 0.68 Polystyrene 1.0 Polystyrene 2.01 Polystyrene 4.08 Polystyrene 7.32 Overview of particle samples

SA/particle (μm2) # of particles (x106) 0.916 3.141 12.692 49.764 1.452 3.141 15.06 52.296 168.334 normalized to total

520 150 37 9.52 326 151 31 9.06 2.81 surface area. The columns include

surface area per particle and number of particles.

Comparison between silica and polystyrene microparticles of different sizes to efficiently collect antibodies at low concentration. Silica and carboxylate polystyrene microparticles of various sizes


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were functionalized with 10μg of protein G to identify which particle material and size optimized antibody collection and detection signal. Particle material is important as the number of functionalization sites and binding affinities for both protein G, and thus for IgG antibody, are material-dependent.18 In order to accurately compare particles of different sizes, conditions were normalized to a total bead surface area of 474 mm2 per condition. Table 1 (TABLE 1) shows the estimated surface area of each microparticle, and number of beads needed to have a total surface area of 474 mm2. Calculations were based according to the manufacturer’s specifications20. As an unbiased approach to evaluate the effect of particle size, small and large particles were mixed together to determine their relative collection of antibody from the same sample (Table 2) (TABLE 2). In these tests, a constant level of protein G is maintained by fixing a total surface area of particles at 474 mm2 for each condition. As with the antibody detection limit procedure, all experimental groups were conjugated with 10 μg/μL of protein G followed by incubation of 0.05mg/mL of antibody for 1 hour. Particles were then analyzed using flow cytometry to measure MFI and the signal to noise (SNR), defined as the MFI of the sample / MFI of the control, calculated. Multi-variable ANOVA was employed to determine the significance of particle size and material. Polystyrene

Silica

Sample 1 Sample 2 Sample 3 Sample 4 # of Size # of Size # of Size # of particles (μm) particles (μm) particles (μm) particles (x106) (x106) (x106) (x106) 0.68 326 0.68 326 0.54 520 0.54 520 4.08 9.06 7.32 2.81 2.01 37 4 9.52 Table 2. Volume of each particle size added to mixed-particle size samples. Size (μm)

Determining the optimal binding time of protein G functionalized microparticles for different antibody concentrations. Fluorescent antibody was added to protein G functionalized silica 4 μm


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particles at concentrations of 0.05 μg/μL, 0.5 μg/μL and 1 μg/μL and incubated for: 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 150 minutes, 180 minutes, and 240 minutes. Particles were analyzed with flow cytometry. A one-phase association binding model (GraphPad Prism) was employed to find the saturating incubation time. Multiple comparison ANOVA was used to determine statistical significance of the variables. Culture of hybridoma cells. TIB 147 and BCL6 hybridomas were used to produce two types of antibodies for testing the collection and detection of specific antibodies using protein G microparticles in cell supernatants and within cell cultures. TIB 147 and BCL6 hybridoma cells produces anti-concanavalin A and anti-BCL6, respectively. TIB 147 cells were cultured in complete media composed of DMEM supplemented with 10% fetal bovine serum and 0.1% penicillin/streptomycin. BCL6 cells were cultured in IMDM, 20% fetal bovine serum, 1% Lglutamine 200mM, 1% sodium pyruvate 100Mm, 0.1% gentamicin 50mg/ml, and 0.1% penicillin/streptomycin. BCL6 hybridomas were bought from Iowa University (Iowa University Hybridoma Bank, Iowa City, IA) and TIB147 cells were purchased from ATCC (Manassas, VA). Both cells were grown in a humidified incubator at 37°C with 5% CO2. Cells were expanded in cell culture flasks over three days for a final concentration of 1x106/mL. For antibody secretion analysis, cells were incubated for a period of 12 hours in a 48 well plate under cell culture conditions. Comparing Concanavalin A antibody detection in cell supernatants using Protein G microparticles and ELISA. To test the ability of the protein G microparticles to detect antibody from cell supernatants, different numbers of TIB 147 cells (10, 1x102, 1x103, 1x104 and 1x106), which secrete antibody binding concanavalin A, and 1x106 BCL6 cells were incubated overnight in a 48 well plate. Cell culture supernatant was centrifuged at 3000 rpm for 15 minutes to collect


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cells and cell debris and the supernatant pipetted and tested. Comparison of antibody detection by ELISA and protein G particles was performed by adding the number of microparticles to the collection well with a surface area equal to the well plate bottom (0.36 cm2), representing the area of reaction using ELISA. From Table 1 (TABLE 1), 1x105 particles with a total surface area of 0.49 cm2 was chosen. Fewer particles (1x103) and longer incubation time were also tested to increase the collection of antibodies per bead. The cell supernatant was incubated with 1x105 and 1x103 silica 4 μm Protein G beads for 1 hour and overnight in a vertical rotator at room temperature. The beads were then washed with PBS and resuspended with 100 μL of 1% BSA Blocking Buffer for 1 hour and washed twice to remove excess of BSA. Anti-concanavalin A was detected through the binding of 10 μg/μL fluorescent FITC concanavalin A for 1 hour at room temperature for specific antibody labeling. Particles were analyzed with flow cytometry and the signal to noise ratio (SNR) was calculated as the MFI of the sample / MFI of the control condition consisting of Protein G particles in cell culture medium. Similarly, the SNR of the ELISA method was calculated as the optical density (O.D.) of the sample normalized by the mean O.D of three blank controls. The O.D. of the blank control was established by coating the ELISA well with culture media. Significance of ELISA and Protein G beads results were evaluated by one-way ANOVA and post-hoc multiple comparison test with a Dunnett correction, comparing controls with successive data points. Testing specificity of concanavalin A antibody collection by labeling using fluorescent concanavalin A. BCL6 and TIB147 cells were incubated overnight with 1x103 Silica 4 um Protein G particles in 200 uL of DMEM media using 29 mm Glass Bottom Dishes in a humidified incubator at 37 °C with 5% CO2. FITC Concanavalin A at a concentration of 10 μg/μL was then added for 1 hour incubation at 37 oC to specifically label the concanavalin A antibody bound to


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beads. BCL6 cells, which produce BCL6 antibody, were tested as control cells to evaluate the specificity of the antibody labeling and detection process. We thus expect to see low FITCConcanavalin A fluorescence signal in conditions testing BCL6 cells. Cells and particle images were obtained using a Zeiss laser scanning confocal microscope (Zeiss LSM 510 VIS Confocal Microscope). The MFI of concanavalin A was determined for 24 particles using the ZEN 2.3 SP1 (Zeiss) software. SNR was defined as the ratio of the MFI of the particle to the mean MFI of control particles measured in PBS. A t test was performed to find the significance of the difference between the groups. Calculating the collection of antibody at different distances from the nearest cell. Since the protein G particles were untargeted and did not frequently attach to cells, we hypothesized that the antibody collection may be dependent upon distance from nearest cell. An incubation of 1x10 4 TIB147 cells and 4 um Protein G particles were cultured for 72 hours in a humidified incubator at 37°C with 5% CO2 with 200 uL of DMEM media in a 29 mm Glass Bottom Dish coated with Polylysine, to prevent the movement of the sample. To stain the collected antibody, APC Concanavalin A was added at 10 μg/mL for 1 hour and images were acquired and analyzed using confocal laser microscopy (Zeiss LSM 510 VIS). The mean fluorescent intensity from the cell was excluded in cases where the antigen bound to cell and the analyzed particle was attached to the cell. The distance of 40 particles to the closest cell and the MFI of APC concanavalin A were determined. The distance to the closest cell was calculated using the ZEN 2.3 SP1 (Zeiss) software. The signal to noise (SNR) was defined as the ratio of the MFI of the particle to the mean MFI of negative control particles measured in PBS. The values were binned by the distance to nearest cell and the significance of each bin was calculated using one-way ANOVA on ranks (Kruskal-Wallis


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test) and Dunn’s multiple comparison test, where the group of 0 μm (adjacent to a cell) was compared with the successive groups.

Results. Determining protein G saturation level. Particles were prepared with different concentration of protein G and antibody. Following the one-site specific binding model, Figure 1C and D (FIGURE 1C and D) shows the point at which the fluorescent signal of collected antibody plateaus at a protein G concentration of 10 μg/μL. The results showed that 10 μg/μL of protein G saturates silica particles that have 474 mm2 of total surface area.

A

B

C

D


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Figure 1. Protein G conjugation optimization. Schemes for protein G conjugation to (A) silica and (B) carboxylated polystyrene particles. One-site specific binding model fit to measured secondary antibody signal at different concentrations of protein G conjugated to (C) silica and (D) polystyrene particles. BSA coated particles do not collect antibody. Figure S1(SUPPLEMENTARY FIGURE 1) shows that the protein control (BSA coated) and the uncoated particle control produced significantly less antibody collection than the Protein G particles. Protein G particle size improves the minimum detection limit of antibody. Figure 2 (FIGURE 2) shows the signal that results from incubation of different concentrations of fluorescent antibody with protein G particles of different sizes. The minimum detection limit of captured antibody was determined to be the condition with the lowest concentration of antibody resulting in a statistically significant difference between the control condition (significance shown by green stars). The results indicate that larger particle size significantly increased the signal resulting from antibody capture (p < 0.001). Figure S2 (SUPPLEMENTARY FIGURE 2) shows the percentage of nonbound antibodies after incubation with the 4 μm Protein G microparticles incubated with 4 μg/μL and 0.5 μg/μL antibodies, calculated by measuring the MFI of particles incubated with supernatant containing the non-bound antibodies. The results show that for particles incubated with 0.5 μg/μL, an undetectable amount of antibodies remain in the supernatant; while in particles incubated with 4 μg/μL, approximately 75% of the starting antibody remain in the supernatant and 25% collected by the particles. Thus, we calculate from Figure 2 (FIGURE 2) that the minimum detection limit for 1 μm silica and for 1 and 4 μm polystyrene microparticles, is 0.5 μg antibody per μL, corresponding to a concentration of 260 nM and resulting in approximately 1x105 IgG molecules/particle, from Equation 2. For the 4 μm silica microparticles, the minimum detection


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limit is an antibody concentration of 0.05 μg/μL, corresponding to a concentration of 26 nM. From Equation 2, we estimate that approximately 1x103 IgG molecules/particle can be detected and, indicating that large particles can detect lower concentration of antibodies. Multiple t-tests analysis was performed for antibody capture for different microparticle sizes, (Figure 2 (FIGURE 2) shown in black). A significant statistical difference was observed for all the different concentrations.

A

B

Figure 2. Minimum antibody detection. Fluorescent secondary antibody signal was measured for 1 μm and 4 μm (A) silica and (B) polystyrene particles after 1 hour incubation and compared to their respective 0 μg/μL control. Statistical analysis between 1 μm and 4 μm particles is represented by black stars, while the control and successive data points to determine minimum detection is represented by green stars. *, ** and **** indicate p

Optimization of microparticle reagents to collect and detect antibody

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