Chemiluminescence-based detection and comparison of protein amounts adsorbed on differently modified silica surfaces

The biological consequences of protein adsorption on biomaterial surfaces are considered to be of utmost importance for their biocompatibility. A new method based on amino group-labeling coupled to a chemiluminescence reaction for direct determination of proteins adsorbed on material surfaces was employed. This method was used to explore the effects of surface chemistry and surface roughness on protein adsorption in a silicon oxide model system. Corundum sandblasting was applied to silicon wafers to create roughened surfaces while immobilization of fluorocarbon-, hydrocarbon-, and poly(ethylene glycol)-containing silanes produced surfaces of varying wettability. The adsorption behavior of two complex body fluids, human serum and saliva, and of two purified components, human serum albumin and fibronectin, was strongly influenced by the surface parameters. A general tendency to higher amounts of adsorbed protein was found on roughened surfaces and modification with poly(ethylene glycol) or with fluorocarbon moieties reduced protein adsorption. The values obtained with the new method could be confirmed by a colorimetric determination of protein amounts adsorbed on identically modified silica beads and were in accordance with those previously reported utilizing established methods for protein quantification. The presented method, which was methodically simple to perform and allowed the simultaneous measurement of a large number of samples, may be of future value for high-throughput surveying of the protein adsorption characteristics of biomaterials.     

A label-free nanoparticle aggregation assay for protein complex/aggregate detection and study

The detection, analysis, and understanding of protein complexes/aggregates and their formation process are extremely important for biomolecular research, diagnosis, and biopharmaceutical development. Unfortunately, techniques that can be used conveniently for protein complex/aggregate detection and analysis are very limited. Using gold nanoparticle immunoprobes coupled with dynamic light scattering (DLS), we developed a label-free nanoparticle aggregation immunoassay (NanoDLSay) for protein aggregate detection and study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a protein target used routinely in Western blot as a loading control, is demonstrated here as an example. Through this study, we discovered that GAPDH has a strong tendency to form large aggregates in certain buffer solutions at a concentration range of 10-25 μg/ml. The strong light scattering property of gold nanoparticles immunoprobes greatly enhanced the sensitivity of the dynamic light scattering for protein complex/aggregate detection. In contrast to fluorescence techniques for protein complex and aggregation study, the protein targets do not need to be labeled with fluorescent probe molecules in NanoDLSay. NanoDLSay is a very convenient and sensitive tool for protein complex/aggregate detection and study.     

A microfluidic biosensor based on competitive protein adsorption for thyroglobulin detection

We report a microfluidic sensing platform for the detection of thyroglobulin (Tg) using competitive protein adsorption. Serum Tg is a highly specific biomarker for residual thyroid tissue, recurrence and metastases after treatment for differentiated thyroid cancer (DTC). Conventional Tg detection techniques require complicated immobilization of antibodies and need to form a sandwich assay using additional secondary antibodies to enhance the sensitivity. We present a fundamentally different sensing technique without using antibody immobilization on a microfluidic platform. We engineer two surfaces covered by two known proteins, immunoglobulin G (IgG) and fibrinogen, with different affinities onto the surfaces. The microfluidic device offers a selective protein sensing by being displaced by a target protein, Tg, on only one of the surfaces. By utilizing the competitive protein adsorption, Tg displaces a weakly bound protein, IgG; however, a strongly bound protein, fibrinogen, is not displaced by Tg. The surface plasmon resonance (SPR) sensorgrams show that five human serum proteins, albumin, haptoglobin, IgG, fibrinogen and Tg, have different adsorption strengths to the surface and the competitive adsorption of individuals controls the exchange sequence. The adsorption and exchange are evaluated by fluorescent labeling of these proteins. Tg in a protein mixture of albumin, haptoglobin, and Tg is selectively detected based on the exchange reaction. By using the technique, we obviate the need to rely on antibodies as a capture probe and their attachment to transducers.     

Sequential ordering among multicolor fluorophores for protein labeling facility via aggregation-elimination based β-lactam probes

Development of protein labeling techniques with small molecules is enthralling because this method brings promises for triumph over the limitations of fluorescent proteins in live cell imaging. This technology deals with the functionalization of proteins with small molecules and is anticipated to facilitate the expansion of various protein assay methods. A new straightforward aggregation and elimination-based technique for a protein labeling system has been developed with a versatile emissive range of fluorophores. These fluorophores have been applied to show their efficiency for protein labeling by exploiting the same basic principle. A genetically modified version of class A type β-lactamase has been used as the tag protein (BL-tag). The strength of the aggregation interaction between a fluorophore and a quencher plays a governing role in the elimination step of the quencher from the probes, which ultimately controls the swiftness of the protein labeling strategy. Modulation in the elimination process can be accomplished by the variation in the nature of the fluorophore. This diversity facilitates the study of the competitive binding order among the synthesized probes toward the BL-tag labeling method. An aggregation and elimination-based BL-tag technique has been explored to develop an order of color labeling from the equimolar mixture of the labeling probe in solutions. The qualitative and quantitative determination of ordering within the probes toward labeling studies has been executed through SDS-PAGE and time-dependent fluorescence intensity enhancement measurements, respectively. The desirable multiple-wavelength fluorescence labeling probes for the BL-tag technology have been developed and demonstrate broad applicability of this labeling technology to live cell imaging with coumarin and fluorescein derivatives by using confocal microscopy.     

Feasibility of protein A for the oriented immobilization of immunoglobulin on silicon surface for a biosensor with imaging ellipsometry

The feasibility of using protein A to immobilize antibody on silicon surface for a biosensor with imaging ellipsometry was presented in this study. The amount of human IgG bound with anti-IgG immobilized by the protein A on silicon surface was much more than that bound with anti-IgG immobilized by physical adsorption. The result indicated that the protein A could be used to immobilize antibody molecules in a highly oriented manner and maintain antibody molecular functional configuration on the silicon surface. High reproducibility of the amount of antibody immobilization and homogenous antibody adsorption layer on surfaces could be obtained by this immobilization method. Imaging ellipsometry has been proven to be a fast and reliable detection method and sensitive enough to detect small changes in a molecular monolayer level. The combination of imaging ellipsometry and surface modification with protein A has the potential to be further developed into an efficient immunoassay protein chip.     

Interactions between protein molecules and the virus removal membrane surface: Effects of immunoglobulin G adsorption and conformational changes on filter performance

Membrane fouling commonly occurs in all filter types during virus filtration in protein‐based biopharmaceutical manufacturing. Mechanisms of decline in virus filter performance due to membrane fouling were investigated using a cellulose‐based virus filter as a model membrane. Filter performance was critically dependent on solution conditions; specifically, ionic strength. To understand the interaction between immunoglobulin G (IgG) and cellulose, sensors coated with cellulose were fabricated for surface plasmon resonance and quartz crystal microbalance with energy dissipation measurements. The primary cause of flux decline appeared to be irreversible IgG adsorption on the surface of the virus filter membrane. In particular, post‐adsorption conformational changes in the IgG molecules promoted further irreversible IgG adsorption, a finding that could not be adequately explained by DLVO theory. Analyses of adsorption and desorption and conformational changes in IgG molecules on cellulose surfaces mimicking cellulose‐based virus removal membranes provide an effective approach for identifying ways of optimizing solution conditions to maximize virus filter performance. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:379–386, 2018     

The effect of azo dyes on heat aggregation of IgG

The mechanism of IgG heat aggregation was studied using IgG aggregates complexed with azo dyes to increase their solubility and stability. Heat dependent and heat independent steps of aggregation were differentiated. On heating IgG at the dye concentration exceeding 100 times that of protein, mainly dimers are formed, as judged from ultracentrifugation and chromatographic analysis, whereas high molecular weight derivatives appear at room temperature when the protein/dye ratio is decreased. The analysis of spectral changes following either the attachment or removal of the dye from IgG aggregates implies that only a part of the dye molecules is bound firmly and directly to the protein binding sites. These dye molecules which are easily removed by adsorption to cellulose or reduced by dithionate but migrate together with IgG aggregates on chromatography and electrophoresis, are supposed to constitute that part of the micelle which extrudes from the binding site and, hence, is fixed indirectly to protein. Various proteins with predominant beta-structure were also found to bind azo dyes when heated.     

Ellipsometric study of immunologic reactions on the surface of silicon

Adsorption of human serum albumin (HSA), egg albumin (EA), immunoglobulin G (IgG) and immunologic reactions occurring between them on silicon surface were studied by ellipsometry. Adsorption of HSA, IgG and antibodies on the monolayer of antigen is monomolecular in their isoelectric points and can be depicted by Langmuir's equation. Adsorption of EA is polymolecular apparently because of great tendency of the protein to aggregation in aqueous solutions. Comparison of the magnitudes of the protein monolayer thickness and areas per adsorbed molecule with their linear dimensions indicate that they preserve their native conformation. This allows an evaluation of the maximum number of the active sites (as approximately four) on the antigen surface accessible for antibodies and the adsorption constants for specific and nonspecific adsorption of IgG.     

Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling

Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.     

人IgG标记金纳米棒并用于抗人IgG抗体的检测

目的：研究金纳米棒（GNRs）IgG生物学标记及其在抗人IgG检测中的应用。方法：利用种子生长法制备GNRs,用巯基十一酸（MUA）对GNRs端头邀111妖晶面进行化学修饰,MUA提供的羧基可与人IgG结合;抗人IgG与活化的GNRs反应引起GNRs表面等离子体共振（SPR）特征变化,通过读取SPR值判断免疫反应的结果。结果：合成了不同长径比（AR）的GNRs,成功地将人IgG标记于GNRs（AR=3.7）的端头;利用标记后的GNRs对抗人IgG进行检测,其SPR最大吸收峰发生9nm红移,检测灵敏度可达纳摩尔量级。结论：基于人IgG-抗人IgG免疫反应建立了GNRs用于免疫检测的方法,为GNRs用于免疫检测进而研制免疫传感器奠定了基础。     

Protein A磁性纳米颗粒载体的制备及应用

本研究采用本课题组合成的表面氨基化磁性纳米微球,首先通过化学共价交联制备了葡萄球菌Protein A磁性纳米微球载体（SPA-MP）,并探讨了载体制备的优化条件。然后根据生物分子特异性亲合作用原理,在外加磁场的定向控制下,通过亲和吸附、清洗和解吸附等操作,探讨了SPA-MP载体在抗体分离纯化领域的应用可行性。载体制备优化实验结果显示,通过改变蛋白质浓度、交联剂浓度和交联剂活化时间可以制备不同表面密度的SPA-MP载体。300 μg SPA,2.5% (V/V)戊二醛浓度和3小时的活化时间可以获取表面密度高达35 mg SPA/g磁性纳米微球的载体。此外,应用结果显示每克SPA-MP磁性微球载体可以结合高达14 mg 的CD25抗体,同时可有效地分离纯化人抗血清样品中的IgG抗体。     

Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene.

The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation. However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation. Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures. The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene. The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently. CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate. The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol. The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form. The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.     

Aggregation of ligand-modified liposomes by specific interactions with proteins. II: Biotinylated liposomes and antibiotin antibody

The aggregation of biotinylated phospholipid vesicles (liposomes) cross-linked by antibiotin IgG was studied experimentally and theoretically. The liposomes were either low density liposomes that contained 0.4 mol% biotinylated phospholipid ( approximately 100 exposed biotin molecules per liposome), or high density liposomes that contained 2.7 mol% biotinylated phospholipid ( approximately 1000 exposed biotin molecules per liposome). The solution turbidity and mean particle size measured by quasi-elastic light scattering (QLS) were monitored throughout the aggregation. Three different lots of antibiotin antibodies, each with different association constants and binding heterogeneities, were used. The antibody binding characteristics affected the aggregation rates. The aggregation kinetics were analyzed using a model based on the Smoluchowski theory of aggregation, fractal concepts of aggregate microstructure, and Rayleigh and Mie light scattering theory. The experimental conditions of liposome concentration, protein concentration, and ligand density under which aggregation occurred correlated well with calculated sticking probabilities based on isotherms describing the adsorption of antibiotin antibody to the liposomes. These results are compared with prior observations made when avidin was used as the cross-linking protein. (c) 1996 John Wiley & Sons, Inc.     

Influence of surface and protein modification on immunoglobulin G adsorption observed by scanning force microscopy.

Scanning force microscopy has been used successfully to produce images of individual protein molecules. However, one of the problems with this approach has been the high mobility of the proteins caused by the interaction between the sample and the scanning tip. To stabilize the proteins we have modified the adsorption properties of immunoglobulin G on graphite and mica surfaces. We have used two approaches: first, we applied glow discharge treatment to the surface to increase the hydrophilicity, favoring adhesion of hydrophilic protein molecules; second, we used the arginine modifying reagent phenylglyoxal to increase the protein hydrophobicity and thus enhance its adherence to hydrophobic surfaces. We used scanning force microscopy to show that the glow discharge treatment favors a more homogeneous distribution and stronger adherence of the protein molecules to the graphite surface. Chemical modification of the immunoglobulin caused increased aggregation of the proteins on the surface but did not improve the adherence to graphite. On mica, clusters of modified immunoglobulins were also observed and their adsorption was reduced. These results underline the importance of the surface hydrophobicity and charge in controlling the distribution of proteins on the surface.     

Elucidation of acid-induced unfolding and aggregation of human immunoglobulin IgG1 and IgG2 Fc

Understanding the underlying mechanisms of Fc aggregation is an important prerequisite for developing stable and efficacious antibody-based therapeutics. In our study, high resolution two-dimensional nuclear magnetic resonance (NMR) was employed to probe structural changes in the IgG1 Fc. A series of (1)H-(15)N heteronuclear single-quantum correlation NMR spectra were collected between pH 2.5 and 4.7 to assess whether unfolding of C(H)2 domains precedes that of C(H)3 domains. The same pH range was subsequently screened in Fc aggregation experiments that utilized molecules of IgG1 and IgG2 subclasses with varying levels of C(H)2 glycosylation. In addition, differential scanning calorimetry data were collected over a pH range of 3-7 to assess changes in C(H)2 and C(H)3 thermostability. As a result, compelling evidence was gathered that emphasizes the importance of C(H)2 stability in determining the rate and extent of Fc aggregation. In particular, we found that Fc domains of the IgG1 subclass have a lower propensity to aggregate compared with those of the IgG2 subclass. Our data for glycosylated, partially deglycosylated, and fully deglycosylated molecules further revealed the criticality of C(H)2 glycans in modulating Fc aggregation. These findings provide important insights into the stability of Fc-based therapeutics and promote better understanding of their acid-induced aggregation process.     

Lysozyme microencapsulation within biodegradable PLGA microspheres: urea effect on protein release and stability

Lysozyme was encapsulated within biodegradable poly(D, L-lactide-co-glycolide) microspheres by a double emulsion solvent evaporation method for studying its release mechanism associated with protein stability problems. When urea, a protein unfolding agent, was added into the incubation medium lysozyme release rate from the microspheres increased with the increase in urea concentration. The enhanced lysozyme release was attributed to the suppression of protein aggregation, to the facilitated diffusion of unfolded lysozyme by an efficient reptile motion of unfolded protein molecules through porous channels in microspheres, and to the largely decreased extent of nonspecific protein adsorption onto the enlarged surface area of degrading polymer microspheres in the presence of urea. Encapsulating lysozyme in an unfolded form within PLGA microspheres was attempted by using urea as an excipient. This new urea-based formulation exhibited a more sustained lysozyme release profile than the control formulation, and released lysozyme from the microspheres showed a much less amount of lysozyme dimer population while maintaining a correct conformation after refolding in the incubation medium. This study provides new insights for the formulation of protein encapsulated PLGA microspheres.     

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Therapeutic non-hinge-modified IgG4 molecules form bispecific hybrid antibodies with endogenous human IgG4 molecules via a process known as Fab-arm exchange (or called half molecule exchange). Analysis of the bispecific hybrids is critical for studies of half molecule exchange. A number of analytical methods are available to detect IgG4 hybrids. These methods mostly necessitate labeling or alteration of the model IgG4 molecules, or rely on time-consuming immunoassays and mass spectrometry. In addition, these methods do not allow isolation of hybrid antibodies. We report here the only analytical method to date that relies on chromatographic separation for detection of hybrids formed from intact antibodies in their native forms using pembrolizumab as an example. This method employs a mixed-mode chromatography using a Sepax Zenix SEC-300 column to separate a bispecific hybrid from the parental antibodies. The simultaneous quantitative monitoring of the newly formed hybrid and parental antibodies was achieved by UV absorption and/or protein fluorescence. The bispecific hybrid antibodies were purified with the same method for further biochemical characterization. The method has allowed monitoring of half molecule exchange between a human serum IgG4 and a tested IgG4 molecule, and has been implemented for the analysis of in vitro as well as in vivo samples.     

Classification and characterization of therapeutic antibody aggregates

A host of diverse stress techniques was applied to a monoclonal antibody (IgG(2)) to yield protein particles with varying attributes and morphologies. Aggregated solutions were evaluated for percent aggregation, particle counts, size distribution, morphology, changes in secondary and tertiary structure, surface hydrophobicity, metal content, and reversibility. Chemical modifications were also identified in a separate report (Luo, Q., Joubert, M. K., Stevenson, R., Narhi, L. O., and Wypych, J. (2011) J. Biol. Chem. 286, 25134-25144). Aggregates were categorized into seven discrete classes, based on the traits described. Several additional molecules (from the IgG(1) and IgG(2) subtypes as well as intravenous IgG) were stressed and found to be defined with the same classification system. The mechanism of protein aggregation and the type of aggregate formed depends on the nature of the stress applied. Different IgG molecules appear to aggregate by a similar mechanism under the same applied stress. Aggregates created by harsh mechanical stress showed the largest number of subvisible particles, and the class generated by thermal stress displayed the largest number of visible particles. Most classes showed a disruption of the higher order structure, with the degree of disorder depending on the stress process. Particles in all classes (except thermal stress) were at least partially reversible upon dilution in pH 5 buffer. High copper content was detected in isolated metal-catalyzed aggregates, a stress previously shown to produce immunogenic aggregates. In conclusion, protein aggregates can be a very heterogeneous population, whose qualities are the result of the type of stress that was experienced.     

Effective novel dissociation methods for intact protein: heat-assisted nozzle-skimmer collisionally induced dissociation and infrared multiphoton dissociation using a Fourier transform ion cyclotron resonance mass spectrometer equipped with a micrometal electrospray ionization emitter

Heating of a nano-electrospray ionization (nanoESI) source can improve the dissociation efficiency of collisionally induced dissociation (CID) methods, such as nozzle-skimmer CID (NS-CID) and infrared multiphoton dissociation (IRMPD), for large biomolecule fragmentation. A metal nanoESI emitter was used due to its resistance to heating above 250 degrees C. This novel method for the dissociation of large biomolecular ions is termed "heat-assisted NS-CID" (HANS-CID) or "heat-assisted IRMPD" (HA-IRMPD). Multiple charged nonreduced protein ions (8.6 Da ubiquitin, 14 kDa lysozyme, and 67 kDa bovine serum albumin) were directly dissociated by HANS-CID and HA-IRMPD to effectively yield fragment ions that could be assigned. The fragment ions of ubiquitin by HANS-CID can be analyzed by tandem mass spectrometry (MS/MS) using sustained off-resonance irradiation CID (SORI-CID) and IRMPD. In addition, a native large protein, immunoglobulin G (IgG, 150 kDa), was efficiently dissociated by HA-IRMPD. The product ions that were obtained reflected the domain structure of IgG. However, these product ions of IgG and lysozyme were not dissociated by MS/MS using the same heating energetic methods such as IRMPD and SORI-CID.     

Electrostatic layer-by-layer nanoassembly on biological microtemplates: platelets

Platelets were coated with 78-nm silica nanoparticles, 45-nm fluorescent nanospheres, or bovine immunoglobulin G (IgG) through layer-by-layer assembly by alternate adsorption with oppositely charged linear polyions. Sequential deposition on platelet surfaces of cationic poly(dimethyldiallylammonium chloride) and anionic poly(styrene sulfonate) was followed by adsorption of nanoparticles or immunoglobulins. Nano-organized shells of platelets were demonstrated by transmission electron microscopy and fluorescence microscope images. Bovine IgG was assembled on platelets, as verified with anti-bovine IgG-FITC labeling. Localized targeting of anti-IgG shelled platelets was also demonstrated. An ability to coat blood cells with nano-organized shells can have applications in cardiovascular research and targeted drug delivery.