Antibody modification
Polyclonal rabbit anti-ferritin immunoglobulin G (anti-ferritin IgG) antibodies or IgG antibodies obtained from rabbit serum (IgG) were cationized by the addition of N,N-dimethyl-1,3-propanediamine (DMPDA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling reagent in order to modify carboxyl containing amino acids (aspartic and glutamic acid) with positive charges. EDC-based coupling reactions are highly specific for the covalent coupling of amine to carboxyl groups and proceed by activation of carboxyl group to an ester and formation of an amide bond35. For example, this process has been effectively used to produce supercharged variants of GFP36. Similarly, we modified the acidic residues of primary IgG antibodies from rabbit serum or anti-ferritin IgG antibodies (rabbit) with positively charged DMPDA and then experimentally determined the addition of positive charges by zeta potential measurements and MALDI mass spectrometry. To provide a baseline, rabbit IgG exhibited a zeta potential of +12.7 ± 0.7 mV at neutral pH that is consistent with a prevalence of basic amino acids (198/1320 total) in the primary sequence of rabbit IgG (Fig. 1a)37. Bookkeeping of available acidic residues reveals a rabbit IgG molecule composed of 2 light chains and 2 heavy chains contains a total of 96 acidic amino acids/1320 amino acids that are modifiable37. However, we expect some of these residues will be buried and inaccessible for modification. As a result, we used an empirical approach to determine how many acidic residues are inaccessible within an antibody structure. We selected the human antibody IgG1 b12 as a surrogate model, since its crystal structure is resolved at 2.7 Å (pdb access code 1hzh), contains 122 acidic residues (vs. 98 in rabbit), and shares a conserved structure to rabbit IgG with the exception of a small flexible hinge region38,39. Based on this empirical approach, a residue is regarded as buried if a sphere with a radius of 15 Å is located at the mass center of a residue containing more than 560 heavy atoms (Supplementary Fig. 1). Using this criterion, we found that ASP115, GLU367, and ASP386 (residue number follows FASTA sequences published at pdb databank) within each heavy chain are buried. In total, this accounts for 6 acidic residues buried below the IgG surface that are inaccessible for modification out of a possible 122 available residues. By analogy, we estimate that up to 6 residues within rabbit IgG are inaccessible for modification. Consequently, the cationization of rabbit IgG (C-IgG) was reflected by a much higher zeta potential (+36.3 ± 4.7 mV) and a slight increase in the average diameter (number %) from 9.0 ± 2.7 nm to 12.1 ± 2.8 nm by dynamic light scattering (DLS) (Fig. 1b, Supplementary Fig. 2). MALDI mass spectrometry confirmed the addition of 32 positive charges per C-IgG antibody distributed over the light and heavy chains (Supplementary Fig. 3). Overall, the addition of positive charges represents modifications to only 2.5% of the total antibody, but importantly, results in an increase in surface charge density covalently added on the exterior antibody surface.
a Zeta potential measurements of rabbit IgG antibodies after cationization (C-IgG) and complexation with anions [C-IgG][S]. Error bars represent the standard deviation from three separate measurements. b Dynamic Light Scattering (DLS) plot of rabbit IgG antibodies after cationization (C-IgG) and complexation with anions [C-IgG][S]. Error bars represent the standard deviation from three separate measurements. c Molecular structures of IgG antibodies, IgG with anions of [S] added (labeled [IgG][S]), and [C-IgG][S] antibody liquid obtained after 200 ns of molecular dynamics simulations. d Differential scanning calorimetry (DSC) plot of [C-IgG][S], IgG powder only, and [IgG][S] at a1:4 wt ratio.
Synthesis and characterization of water-free antibody liquid
To form the corresponding antibody liquid, anionic polymer surfactants of poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether [S] are added to the C-IgG component by means of electrostatic complexation in order to balance charges and produce a neutral protein salt. To determine the number of [S] anions that interact with antibody charges, we measured the zeta potential of IgG from rabbit serum or C-IgG upon titration with [S] anions. From the titration plots, we determined ~176 anions of [S] per IgG and ~195 anions per C-IgG antibody are required to balance charges and reach charge neutrality (Supplementary Fig. 4). Collectively, this represents multiple types of antibody-anion interactions that span different ionic binding strengths13. The magnitude of these interactions is influenced by different amino acids (arginine and lysine), type of charge (tertiary and primary amines), and a large assortment of pKa’s. This is reflected by different slopes in the zeta potential titration plots of C-IgG or IgG and lower numbers of complexed anions. Consequently, we added 370 anions of [S] to C-IgG (3:1 wt ratio) to form the [C-IgG][S] conjugate. This ratio was selected based on previous protein liquids and because it represents an excess of anions. An excess was required to reach saturated conjugation of [S] and a full wrapping around C-IgG due to steric influences. After dialysis to remove non-interacting anions, we determined that C-IgG was associated with 305 anions of [S] based on zeta potential of the final material and extrapolation from titration plot (Fig. 1a). By comparison, this is in good agreement with 230 theoretical positive charges on rabbit C-IgG. In addition, DLS showed an increase in the average diameter of C-IgG antibodies (12.1 ± 2.8 nm) in the presence of anionic polymers of [S] to create [C-IgG][S] (64.6 ± 9.6 nm) (Fig. 1b, Supplementary Fig. 2). This larger size is likely attributed to the random association of anionic polymer shells from multiple [C-IgG][S] conjugates and/or a propensity of rabbit IgG to form multimers at high antibody and salt concentrations37. Consequently, the [C-IgG][S] liquid satisfies both of these conditions for formation of multimers. The [C-IgG][S] complex was lyophilized to remove all water content and melted under mild heating to form a viscous translucent liquid. DSC of the [C-IgG][S] conjugate revealed a weak glass transition temperature at ~7 °C and an intense endothermic peak at ~28 °C indicative of melting and similar to other solvent-free protein liquids26. DSC of the lyophilized IgG powder and a partially wrapped IgG (native rabbit IgG antibody with anion at 1:4 wt ratio) showed a broad exotherm and diminished endothermic peaks, respectively (Fig. 1c, d). Notably, the partially wrapped rabbit IgG failed to melt into a liquid due to the lower number of antibody-polymer interactions and presence of multiple exposed regions lacking anions.
The tolerance of antibodies to cationization and complexation with anionic polymers of [S] was determined by measuring the secondary structure and binding affinity of anti-ferritin IgG antibodies. Structurally, the circular dichroism (CD) spectrum of IgG showed a peak with negative ellipticity at 218 nm that is characteristic of a β-sheet structure and consistent with the CD spectra of other IgG’s (Supplementary Fig. 5)40. After cationization and complexation, the CD peak slightly shifted to ~219 nm and lost ellipticity signifying a minor change in secondary structure. In all, these structural changes are similar to other solvent-free protein liquids30. By comparison to anti-ferritin IgG antibodies (KD = 1.2 × 10−10 M), we obtained similar dissociation constants for the [anti-ferritin C-IgG][S] equal to 4.1 × 10−10 M, respectively, using QCM (Supplementary Fig. 6). This confirms that cationization of surface-exposed acidic residues did not affect the binding affinity of [anti-ferritin C-IgG][S] to apoferritin. Also, this suggests that stabilizing anions of [S] dissociate from the [anti-ferritin C-IgG][S] upon reconstitution in aqueous solution and/or are readily displaced by stronger antigen-binding interactions. In nature, antibodies are also commonly modified by means of glycosylation in vivo to increase longevity and stability41. To determine the effects of cationization and complexation with anions of [S] on binding specificity, we also used an Ouchterlony double diffusion technique to detect formation of an immune complex with anti-ferritin C-IgG or [anti-ferritin C-IgG][S] via immunoprecipitation. Ouchterlony double diffusion is commonly used to detect the formation of an immune complex between antigens and antibodies via immunoprecipitation and to provide insight about the identity of unknown antigens42,43. A typical format for an Ouchterlony assay involves using a central pre-cut well for antibody diffusion surrounded by 6 pre-cut wells for co-diffusion of multiple antigens. Notably, this diffusion format enables the determination of antigen identity with respect to neighboring antigens bearing unknown epitope identities. Similarly, we used this concept of antigen identity to evaluate the effects of antibody modifications on the specificity of binding interactions (e.g., cationization and complexation with anions) by utilizing the central well for antigen diffusion and the surrounding wells to represent different antibody modifications and conditions. Conceptually, the intersection of adjacent immunoprecipitation lines between antigen wells is reliably used to reveal the extent of identity of epitope binding sites on multiple antigens (i.e., fully identical, partially identical, or non-identical) (Supplementary Fig. 7). For example, two intersecting immunoprecipitation lines that cross each other are indicative of antigens that are non-identical; while immunoprecipitation lines that meet and do not cross are classified as being fully identical antigens. Briefly, we obtained a series of thin white lines in the gel signifying antigen recognition and formation of immune complexes. In total, we detected immunoprecipitation complexes with combinations of apoferritin and anti-ferritin C-IgG only, anti-ferritin IgG only, and [anti-ferritin C-IgG][S] liquid at room temperature (Supplementary Fig. 7). For these complexes, the presence of intersecting and non-crossing immunoprecipitation lines confirms the fully identical binding nature of anti-ferritin C-IgG and [anti-ferritin C-IgG][S] with respect to anti-ferritin IgG and binding interactions with apoferritin antigen. In contrast, we did not observe visual precipitation bands for the anions of [S] only, a nonspecific mouse monoclonal antibody against Interleukin 6, and anti-ferritin IgG antibody heated at 100 °C. However, [anti-ferritin C-IgG][S] liquid heated at 100 °C performed similarly to native antibodies confirming the tolerance of antibody ionic liquids to extreme heating.
Thermal stability of water-free antibody liquids
A significant feature of nanoscale protein liquids is their resistance to extreme temperatures due to the absence of water and presence of a dense polymer anion shell protecting the C-IgG. The removal of water eliminates hydrolysis, creates favorable enthalpic contributions, and leads to increased intramolecular binding interactions and forces within the protein interior (i.e., Hydrogen bonding and Van der Waals interactions). Consequently, anti-ferritin IgG, anti-ferritin IgG with [S] anions added, and [anti-ferritin C-IgG][S] were heated to 125 °C and measured for binding to apoferritin by QCM and a dot blot assay. QCM is commonly used to measure antibody-antigen binding, test and validate the performance of immunosensors, and assess the efficacy of antibody stabilizers12,44,45. Using QCM, we precisely quantitated the loss/preservation of binding activity as a function of increasing temperatures by measuring the mass of antigen immobilized on the surface of a gold-coated QCM sensor and the mass of bound antibody. From QCM measurements, we determined binding stoichiometries from the mass ratio of antigen to bound antibody (Supplementary Fig. 8). Consequently, the native aqueous antibody (stored at 4 °C) revealed a binding stoichiometry of ~6 antibodies (anti-ferritin IgG) per apoferritin protein consistent with multi-valent binding to 24 identical protein subunits. This was used as a baseline and reference value to calculate the loss/preservation of binding activity of antibodies after heating at subsequent temperatures. The thermal stability of aqueous anti-ferritin IgG or lyophilized anti-ferritin IgG was determined by individually exposing antibodies to temperatures of 25, 50, 75, and 100 °C for 30 min and measuring their binding by QCM to an apoferritin protein-coated sensor (Fig. 2a). Based on these physical states, the aqueous form estimates the pot-life of antibodies, while the lyophilized antibody powder estimates shelf-life and is more comparable to the water-free antibody liquid. At 75 and 100 °C exposures, aqueous anti-ferritin IgG lost 70% and 90% of its binding activity, respectively, to yield a Tm of 65 °C; while the lyophilized form lost 60% of its binding after heating to only 50 °C (Fig. 2a). This loss in binding activity was confirmed by dynamic light scattering (DLS) and revealed that the average size of the aqueous antibody increased from 11 nm to ~250 nm after heating at 100 °C indicative of antibody unfolding and aggregation (Supplementary Fig. 9). By comparison, the water-free antibody liquid form of anti-ferritin C-IgG showed increased stability >100 °C as determined by a loss of only ~10% of its binding activity at 100 °C and ~35% when heated to 125 °C using the same sample for all heating (Fig. 2a). The use of the same sample was necessary because of difficulties in consistently measuring and dispensing accurate amounts of highly viscous antibody liquid. To determine thermal stability at multiple temps using the same sample, [anti-ferritin C-IgG][S] was heated in the water-free state, reconstituted to measure binding, frozen at −80 °C, lyophilized to remove water, exposed to a higher temperature in water-free state, and repeated for all temps. By comparison to controls, the binding affinities are artificially lower due to use of a single sample for all measurements, but notably, demonstrates the reusability of antibody liquids and ability to be cycled between storage in the dry state and reconstituted aqueous forms (Supplementary Fig. 10). In addition, the antibody liquid was subjected to large temperature swings (−80 to 100 °C) in order to simulate extreme conditions.
a Quartz Crystal Microbalance (QCM) binding of anti-ferritin in aqueous and powder forms and [anti-ferritin IgG][S] at 1:4 wt ratio after heating (Left plot). Binding of [anti-ferritin C-IgG][S] after repeated heating of same sample (Right plot). Inset shows QCM binding exp. % Binding was determined from conversion of Δf into an aerial mass (ng/cm2) of adsorbed apoferritin and bound antibody using the Sauerbrey equation and then into a mole ratio. Error bars represent the standard deviation of three separate binding experiments. b Immunoblot binding assay of [anti-ferritin C-IgG][S] at RT and at 100 °C with 10 pM to 1 μM apoferritin developed colorimetrically with GAR-AP secondary antibodies. c Secondary structure changes of IgG and [C-IgG][S] at equilibria and at different temperatures determined by molecular modeling simulations (labeled comp) and experimentally (labeled exp). Error bars from MD simulations are included in plot and determined from 50 snapshots using VMD. Secondary structure content was also experimentally determined by deconvolution of CD spectra for aqueous IgG and by peak fitting the Amide I band in FT-IR spectra (reflection mode) of water-free [C-IgG][S] to known secondary structure vibrational modes. For IgG, only the turn content is represented experimentally.
In parallel, we also used immunoblotting to qualitatively determine binding of antibody liquids of [anti-ferritin C-IgG][S] after heating. For immunoblotting, different concentrations of apoferritin (10 pM to 1 µM) were immobilized on a nitrocellulose membrane, blocked with 1% milk, incubated with [anti-ferritin C-IgG][S] or anti-ferritin IgG antibody, washed extensively with buffer, incubated with a secondary goat anti-rabbit (GAR) antibody conjugated with alkaline phosphatase, washed, and developed using colorimetric substrate. Qualitatively, both the reconstituted [anti-ferritin C-IgG][S] and aqueous anti-ferritin IgG antibody at room temperature showed binding and detection of apoferritin at the lowest concentration of 10 pM (Fig. 2b, Supplementary Fig. 11). However, after heating to 100 °C, only [anti-ferritin C-IgG][S] showed binding to apoferritin (Fig. 2b, Supplementary Fig. 11). Notably, this also confirms that binding of secondary goat anti-rabbit antibodies to the Fc region of [anti-ferritin C-IgG][S] is similarly unaffected by cationization or complexation with anions of [S].
Molecular modeling of water-free antibody liquids
We have further investigated the thermal stability and functional mechanism of rabbit [C-IgG][S] at the molecular level using molecular modeling techniques and a surrogate antibody. We have again utilized the human antibody IgG1 b12 (pdb access code 1hzh) to investigate the effect of cationization, complexation with anions of [S], and formation of a water-free antibody liquid on the thermal stability and molecular structure of IgG. Solvating the crystal structure of IgG in TIP3 water (Supplementary Fig. 12), we determined the equilibrated IgG antibody structure after 200 ns of molecular dynamics simulation at 25 °C (Fig. 1c) by monitoring the time evolution of the radius of gyration (Supplementary Fig. 12f). This equilibrated structure of IgG was used as the reference structure to compare the thermal stability under different conditions. First, the equilibrated structure of IgG without modification was further equilibrated at eight different temperatures (25:25:200 °C) using the generalized Born implicit solvent to save simulation time, denoted as IgG in Fig. 2c. Next, we constructed a model to represent the anionic polymer-coated antibody liquid, denoted as [C-IgG][S] in Fig. 2c. In this model, except for the six buried acidic residues, all other acidic residues were cationized from the reference structure (Supplementary Fig 12c). The resulting C-IgG was neutralized by 252 anionic polymer surfactants (Supplementary Fig. 12d) and equilibrated at eight different temperatures (25:25:200 °C). After equilibration, all the end-point structures at different temperatures show that the C-IgG is confined by a continuous polymer surfactant corona of [S] in the absence of free unbound polymer-surfactant molecules. An exemplary wrapping configuration is shown in Fig. 1c. In these structures, conjugation and wrapping do not seem to affect the antibody secondary structure. From the total 16 simulations, we determined the change in secondary structure (β-sheet, turns & coils) of IgG or [C-IgG][S] at each temperature as a means to measure unfolding of antibody structure. When the temperature is higher than 75 °C, the secondary structure of IgG in water undergoes significant structural changes as shown in Fig. 2c. In contrast, the secondary structure of [C-IgG][S] remains stable up to 200 °C and is consistent with the thermal stability of lysozyme at 200 °C22. This stability is also in agreement with our experimental binding observations by QCM and structural analysis of the amide I region by FTIR after heating to 200 °C (Supplementary Fig. 13). In parallel, we also experimentally measured the secondary structures of IgG and [C-IgG][S] vs temperature. To replicate the conditions used for simulations, we used CD and FTIR spectroscopy to measure changes in the secondary structures of aqueous IgG and water-free [C-IgG][S] upon heating (Supplementary Fig. 14). However, it should be noted that the absolute values of secondary structure content differed between measurement techniques and deconvolution methods. This is an expected outcome given that FTIR is more reliable at predicting β-sheets and CD is better at estimating α-helices46. Heating of aqueous IgG showed a loss in β-sheet content and an increase in the number of turns after deconvolution of CD peaks; while peak fitting of the broad amide I band of [C-IgG][S] at ~1640 cm−1 showed minor changes in β-sheet, turns, and coil content upon heating (Supplementary Fig. 14). By comparison, there was a high degree of correlation between experimentally and computationally determined trends in secondary structure vs heating (Fig. 2c). In the case of [C-IgG][S], confinement by the polymer shell was primarily responsible for the extreme thermal stability as also observed by other groups for different solvent-free protein liquids29,47.
Creation and characterization of water-free antibody liquid-based plastic materials
Besides exhibiting thermal stability, solvent-free protein liquids have also been shown to display unconventional solubility properties in organic solvents and anhydrous ionic liquids that deviate from natural proteins26,33,48,49,50,51,52. Generally, natural proteins possess an inability to function and operate in non-physiological and harsh environmental conditions (organic solvents, high temperatures) because of poor solubility, denaturation, and aggregation53,54. However, the ability to access the biological activity of proteins in organic solvents, anhydrous ionic liquids, and/or in biologically incompatible materials (e.g., plastics) is an appealing property that presents new opportunities for biomolecules. For example, solvent-free protein liquids of M13 bacteriophage, glucosidase, and α-chymotrypsin showed enhanced solubility and thermal stability in anhydrous ionic liquids;33,49,50,51 while in another example, peroxidase and hydrolase enzymes wrapped with random heteropolymers demonstrated increased solubility and stability in toluene55. Similarly, we examined the universal solubility, high-temperature processibility, and compatibility of [C-IgG][S] with inert high molecular weight polymeric plastics to create bioactive thermoplastics with biorecognition capability. To demonstrate universal solubility of [C-IgG][S], we reconstituted [C-IgG][S] or rabbit IgG powder in water, toluene, chloroform, and acetone (Fig. 3a). In all, the lyophilized IgG powder was only soluble in water; while [C-IgG][S] was soluble in all solvents tested.
a Solubility of IgG powder (control) or [C-IgG][S] in water (W), toluene (Tol), Chloroform (Chl), and acetone (Ac). b Top view image of hot glue blended with [GAR-C-IgG-Au][S], molded into a single rectangular block, and then cross-sectioned into three pieces (illuminated from below on a glass slide with AFRL written). The three red block pieces are shown with cross-sectioned area facing upward and bottom illumination. UV-Vis plot of [GAR-C-IgG-Au][S] in glue or GAR-IgG-Au in water. Inset shows block dimensions. c Colorimetric assay of hot glue cylinder (control) and molded forms of [C-IgG][S]/glue after incubation with GAR-AP and addition of colorimetric substrate (Left column). Chemiluminescence of the cross-sectioned hot glue cylinder (control) and cross-sectioned forms of [C-IgG][S]/glue after incubation with GAR-HRP and addition of chemiluminescent substrate (Right column). Scale bar equals 6 mm. d Images of well bottoms from a 96-well plate with a normal surface or with the bottom replaced with [C-IgG][S] glue. e Binding assay of antibody glued well bottoms with multiple concentrations of GAR-HRP. Chemiluminescence was measured for each well using a microplate reader. Error bars represent standard deviation from triplicate measurements. f Sandwich binding assay of [anti-ferritin C-IgG][S] in glue with apoferritin and anti-ferritin antibody conjugated to HRP. Image shows chemiluminescence of control and antibody glue with arrows indicating location of glue pieces in tube.
Next, we selected a hot melt adhesive (e.g., hot glue sticks) from a large set of commercial plastics because of its high viscosity when melted as well as multiple uses and applications. Because of the general incompatibility with biological materials (e.g., insolubility in water and high melting temperature), hot glue represents a challenging and non-traditional thermoplastic for the incorporation of antibodies. Initially, we created a simple version of hot glue by combining poly(ethylene-co-vinyl acetate), glycerol ester of gum rosin, and paraffin wax in a 2:1:1 wt ratio. The labmade hot glue exhibited comparable melting, viscosity, adhesion, and consistency to commercial-grade products (SurebonderTM cool shot mini glue sticks) (Supplementary Fig. 15). For processing antibodies with plastic, we melted a piece of commercial hot glue (mp = ~90 °C) in the presence of cationized goat anti-rabbit IgG conjugated to 15 nm Au particles with [S] to yield [C-GAR-Au][S], [C-IgG][S] from rabbit serum, or [anti-ferritin C-IgG][S] (~10 wt%) on a hot plate at ~140 °C until flowing, blended both materials together to reach homogeneity, and solidified into the new material by cooling (Supplementary Fig. 16). The glue blended antibody materials were remelted and molded in the shape of a rectangular block and arbitrary forms using appropriate PDMS molds to produce desired shapes (Fig. 3). As a control, we also blended hot glue with lyophilized rabbit IgG powder (unmodified) at ~10 wt% and observed the presence of clumps due to incompatibility (Supplementary Fig. 17). To show the uniformity of antibody liquids in glue, we exploited the optical properties of [C-GAR-Au][S] to create red rectangular blocks (Fig. 3b). By UV-Vis, the plasmon resonance of gold nanoparticles from [C-GAR-Au][S] in glue showed similar peak shapes and wavelengths to unmodified GAR-Au in water (Fig. 3b); while EDAX mapping of the cross-sectioned rectangular block pieces showed the presence of evenly distributed gold signals (Supplementary Fig. 18). Also, the cross-sectioned blocks of [C-GAR-Au][S]/glue showed no leaching of the gold-antibody conjugate after 5 days in water, thus confirming that the gold-antibody conjugate is physically entrapped inside the polymeric hot glue.
Moreover, we examined the binding ability of [C-IgG][S] in hot glue. To date, surface binding on plastics is achieved by simple adsorption processes or conventional surface immobilization techniques of biomolecules on polypropylene via standard functionalization/coupling chemistries56. However, beyond surface functionalization of plastics, the poor compatibility of natural proteins has prevented successful integration/blending with plastics without the use of methylene diphenyl diisocyanate for soy protein isolate57 or silica encapsulation of DNA to facilitate increased miscibility with polycaprolactone58. In a similar example, partially shielded enzymes buried in silica were shown to exhibit enhanced tryptic activity with large protein substrates59. After processing and molding, [C-IgG][S] or lyophilized rabbit IgG powder embedded in hot glue was assayed for binding and homogeneity by incubating antibody/hot glue molds with secondary antibodies raised against rabbit IgG (Goat Anti-Rabbit conjugated with alkaline phosphatase GAR-AP) or horse radish peroxidase (GAR-HRP)). As a result, the molded forms of [C-IgG][S]/hot glue showed binding of GAR-AP over its entire surface upon addition of the colorimetric substrate; while hot glue only remained colorless (Fig. 3c). Binding of the secondary antibody to the plastic confirms that the Fc regions of [C-IgG][S] embedded within the hot glue polymer are accessible to large macromolecules. By comparison, the lyophilized antibody powder blended with hot glue showed a lack of binding to GAR-HRP by chemiluminescence (Supplementary Fig. 19) and an absence of silver staining compared to [C-IgG][S] in glue (Supplementary Fig. 20).
To determine the presence and binding activity of [C-IgG][S] throughout the hot glue, we cross-sectioned the glue stick only and molded bioplastic materials and measured binding using GAR-HRP antibodies. In the cross-sectioned plastic materials, the HRP-conjugated antibodies should only bind to the interior area of the new unexposed glue given that the IgG antibodies near the glue surface were already blocked by bound GAR-AP antibody conjugates. As expected, only the cross-sectioned plastic pieces containing [C-IgG][S] showed a chemiluminescence signal indicative of binding to GAR-HRP (Fig. 3c). In total, these results confirm that the [C-IgG][S] liquid is uniformly blended throughout the entire hot glue material and biologically active. To achieve miscibility with plastic, [C-IgG][S] features an amphiphilic wrapping polymer that contains a hydrophilic and hydrophobic region. In these materials, the charged hydrophilic end of the anionic polymer interacts directly with the cationized antibody surface, while the hydrophobic tail is exposed to the hydrophobic environment of high molecular weight poly(ethylene-co-vinyl acetate) polymers or organic solvents. Because of these favorable hydrophobic interactions, the miscibility of [C-IgG][S] with melted hot glue was significantly increased. ATR-FTIR of antibody glue revealed a shifted peak associated with the H-C-O bending modes of vinyl acetate (Supplementary Fig. 21)60. This suggests [C-IgG][S] interacts primarily with the vinyl acetate region of poly(ethylene-co-vinyl acetate) in hot glue. In addition, the amide I region of [C-IgG][S] in glue showed an increase in β-sheet structure relative to the water-free [C-IgG][S] material only due to the hydrophobic nature of hot glue.
We exploited the adhesive properties of antibody glue to join two pieces of a microfuge tube together. In this application, the glued microfuge pieces produced a water-tight seal; while at the same time, imparted antigen binding to the glued interface by containing embedded [C-IgG][S] (Supplementary Fig. 22). To assess activity, GAR-HRP antibodies were added to the glued tube and incubated to promote binding. After binding and washing, addition of luminol/peroxide produced a chemiluminescent signal localized to the glued interface (Supplementary Fig. 22). Similarly, we replaced the well bottoms of a conventional 96-well plate with antibody glued bottoms (Fig. 3d). To construct well bottoms with glue, we punched out the existing plastic surfaces of wells, pressed and molded melted antibody glue over wells to form a water-tight seal, and then cooled glue to form solid bottoms. In this 96-well format, a microplate reader was used to measure the surface binding of antibody glued wells. For binding measurements, we incubated the set of glued wells with different concentrations of GAR-HRP, washed, and measured chemiluminescence according to ELISA protocols. Consequently, we obtained an exponential increase in chemiluminescence for antibody glued wells upon increasing concentration of secondary antibody (Fig. 3e) and only a slight increase in fluorescence for glued bottoms only. In a final example, we replaced the liquid form of [C-IgG][S] from rabbit serum displaying broad binding specificity with an antigen-specific primary antibody [anti-ferritin C-IgG][S] to determine the binding range of antibodies in hot glue. [anti-ferritin C-IgG][S] liquid blended with hot glue or hot glue only (no antibodies) were incubated with apoferritin antigen, washed, and then interrogated with anti-ferritin antibodies conjugated with HRP in a sandwich assay format. Addition of luminol/H2O2 produced a strong chemiluminescent signal for the antibody glue indicative of apoferritin binding to the antibody/glue surface (Fig. 3f). In all, we have tested the Fc and Fv regions of an antibody embedded in hot glue for binding and demonstrated that both regions remain functional for binding.
Conclusion
In total, we converted thermally labile primary IgG antibodies into stable water-free antibody liquids. For example, [anti-ferritin C-IgG][S] retained >60% binding activity after repeated heating cycles up to 125 °C and displayed partial binding even after heating to 200 °C. This represents a significant improvement over other stabilization methods for antibodies that include encapsulation, immobilization, and/or use of stabilizing excipients. In addition, we expect this process to be broadly applicable to all antibody isotypes (IgM, IgY, IgG) and impart similar stabilization at high temperatures; however, optimization will be needed for each isotype. In a separate and parallel study, evaluation of the therapeutic potential of antibody liquids revealed cytotoxicity at high concentrations against several common cell lines61. Consequently, antibody liquids will require improvements in their design and/or replacement of stabilizing anions in order to be suitable for therapeutic applications.
For diagnostics, we exploited the thermal stability, universal solubility, and material compatibility properties of antibody liquids to create bioactive antibody plastics and glues using high molecular weight hot melt adhesives. Notably, antibody embedded plastics are in a “ready to use” bioactive format and do not require removal of proteins/biomolecules from plastic unlike DNA stored in polycaproplactone. As described above, DNA encapsulated in SiO2 and dispersed in polycaprolactone requires extraction by tetrahydrofuran and purification for utilization. Also, the high loading and homogenous blend of antibodies embedded throughout plastic offer the ability to harness, replicate, and access biological activity from anywhere within a plastic structure by cross-sectioning into multiple pieces and/or remolding into new forms to generate a renewable surface. The ability to generate renewable bioactive surfaces on demand is advantageous for the manufacturing of multi-use diagnostics vs. single-use disposable devices, lateral flow assays, and responsive packaging that is capable of sensing antigens or pathogens. In addition, we envision antibody-embedded plastics for use in crack detection, whereby damage to the plastic surface would result in the exposure of active antibodies as demonstrated above.
