Antibody Solution Research Articles

Recombinant human hyaluronidase PH20-mediated dermal spreading activity in mice is not altered by steroids, antihistamines, or salicylic acid.

Drug-drug interaction studies for hyaluronidase safety assessments have evaluated only animal-derived enzyme preparations. We therefore set out to evaluate whether high-dose administration of two antihistamines, a potent corticosteroid, steroid hormone, adrenocorticotropic hormone (ACTH), or salicylic acid would alter the dispersive activity of recombinant human hyaluronidase PH20 (rHuPH20). NCr nu/nu mice were pretreated with diphenhydramine, cetirizine, dexamethasone, estrogen, ACTH, salicylic acid, and/or neutral-buffered saline (NBS). An hour following final pretreatment, dosed mice were anesthetized with ketamine/xylazine and placed in an imaging chamber. A 120mg/mL immunoglobulin G (IgG) solution with 0.3μg/mL IgGDL755 (labeled IgG) was injected intradermally, with/without 2,000U/mL rHuPH20. Fluorescent images of labeled IgG dispersion were acquired≤20min post injection. Dispersion of high-concentration labeled IgG combined with rHuPH20 was greater at all time points vs. antibody alone. At 20min post injection (last time point), the antibody dispersion area was significantly increased with rHuPH20 vs. without rHuPH20 (p≤0.005). The relative percent increase in antibody dispersion with rHuPH20 ranged from 22.8‒106.6% over the 20-min time course, compared with the corresponding non-rHuPH20 treated groups. The area of labeled IgG dispersion was statistically similar between rHuPH20 groups pretreated with an active compound and their paired NBS pretreated controls. The addition of 2,000U/mL rHuPH20 to a high-concentration antibody solution reproducibly incre-ased local antibody dispersion. Systemic pretreatment with diphenhydramine, cetirizine, dexamethasone, estrogen, ACTH, or salicylic acid did not affect the enzymatic spreading activity of rHuPH20, as measured by intradermal dispersion of labeled IgG in mice.

Analysis of Emission Infrared Spectra of Protein Solutions in Low Concentrations

In this work, based on the method of infrared emission spectroscopy, the study of emission spectra of interferon-gamma (IFNγ) solution in a mixture or surrounded by three low-concentration solutions (IFNγ, antibodies to IFNγ, glycine buffer) or water control was performed. First of all, the solutions of low concentrations themselves were studied. It was shown that low-concentration solutions of IFNγ and antibodies to IFNγ had lower emission intensity in three spectral bands near 800, 1,300 and 2000 cm−1 compared to water control. Glycine buffer solution had a radiation level indistinguishable from that of the control. In this work, the effect of adding these low-concentration solutions to IFNγ (1 mg/ml) was compared to the effect of adding water control to IFNγ. All solutions or water were added in 10% (v/v). It was found that adding each of the three test solutions induced an increase in the radiation intensity of the IFNγ solution in the spectral range of 400–1700 cm−1 (compared to the IFNγ solution with control spike). It was also tested whether the radiation of the studied low-concentration solutions surrounding the IFNγ solution (1 mg/ml) affected the IFNγ radiation. The measurement results were compared to the data obtained for IFNγ surrounded by water control. All three solutions were found to exert a distant effect on the IFNγ solution (1 mg/ml), which was manifested in a decrease in the intensity of its radiation near 1,000 and 1,500 cm−1 compared to the control solution of IFNγ. Thus, the emission spectra of low-concentration aqueous solutions were measured for the first time, and differences in the emission spectra of the IFNγ solution depending on low-concentration additives and the environment were shown. The paper interprets the observed differences and discusses possible mechanisms underlying the observed phenomena.

Homosistein tayini amaçlı QCM-temelli biyosensör

High plasma homocysteine levels can cause an increased risk of cardiovascular, cerebrovascular, and peripheral arterial diseases. Besides this, Alzheimer’s disease and other dementias, osteoporosis, diabetes and renal disease due to folate and B-vitamin deficiency, various drugs or pre-existing atherosclerotic diseases may be the result of high homocysteine levels. The presented research work aimedto perform the detection of Homocysteine (Hcy) by using Quartz Crystal Microbalance (QCM) biosensor. The temperature controlled QCM system was a home-made designed and constructed equipment which can use silver electrod quartz crystal. The modification of silver electrod quartz crystals surfaces was achieved by the surface cleaning process with sodium hydroxide, acetone and methanol in a consecutive manner. Then self-assembled monolayer of cysteamine and chemical coupling of glutaraldehyde (GA) to free end of monolayer was achieved to create the new functional surface in order to complete the formation of spacer arm/ligand. Homocysteine specific recognizing ligand, anti-Homocysteine antibody was immobilized to glutaraldehyde coupled surfaces. The change in resonance frequency values were measured for each modification step. The optimization of dilution ratio of the antibody solution was performed to modified surfaces. The least dilution ratio of antibody, 1/10000 v/v, was determined as optimum antibody ratio. The detection of homocystein was analysed at a detection limit of 0.1 µM and the linear ranges of calibration curves were estimated as 0.1-2.0 µM and 10-50 µM. Homocysteine values indicated good linearities (R2=0.9813 and 0.9875, respectively). The relative standart deviation (RSD %) for precision was calculated as less than 10%. In conclusion, it was found that the detection of homocysteine can be done both in nano- and micro-molar concentration levels. Additionally, designed biosensor showed desired stability and reproducibility. Finally, a new method different from the present methods for the use in the analysis of Hcy was proposed and developed which detects homocysteine by designed QCM technique with a rapid, cheeper and less pretreatment processes. Additionally, homocysteine detection was performed in nano- and micro- molar concentration values.

Adapting virus filtration to enable intensified and continuous monoclonal antibody processing.

Ongoing efforts in the biopharmaceutical industry to enhance productivity and reduce manufacturing costs include development of intensified, linked, and/or continuous processes. One approach to improve productivity and process economics of the polishing step (i.e., anion exchange chromatography) is to preconcentrate the product intermediate using a single-pass tangential flow filtration step before loading on the resin. This intensification of the polishing step consequently leads to changes in product intermediate concentration for subsequent virus filtration operations, potentially impacting filter performance and methods for evaluating viral clearance. The filtrate flux performance of a virus filtration operation was evaluated with monoclonal antibody (mAb) solutions of varying concentrations. These data were used to evaluate the effect on filter sizing for a hypothetical mAb perfusion process. The optimum mAb concentration to minimize the area of the virus filter was a function of the filtration step duration and reflected the competing effects of increasing concentration and decreasing volumetric flux on the membrane productivity. mAb solutions at high and low concentrations were used to evaluate viral clearance with extended filtration times (e.g., 24-72 h) simulating continuous processing conditions. Modifications to more traditional filtration viral clearance study methods were required to avoid experimental artifacts associated with the extended filtration time. No virus passage through the filter was observed under these conditions, similar to previous results for batch processes. These data demonstrate the feasibility of obtaining effective virus removal even when mAb concentration and filtrations times are increased by up to an order of magnitude from current common practices.

In-Use Interfacial Stability of Monoclonal Antibody Formulations Diluted in Saline i.v. Bags.

The use of monoclonal antibodies (mAbs) for the treatment of a variety of diseases is rapidly growing each year. Many mAbs are administered intravenously using i.v. bags containing 0.9% NaCl (normal saline). We studied the aggregation propensity of these antibody solutions in saline and compared it with a low ionic strength formulation buffer. The mAb studied in this work is prone to aggregate, and is known to form a viscoelastic network at the air-solution interface. We observed that this interfacial elasticity increased when formulated in saline. In the bulk, the mAbs exhibited a tendency to self-associate that was higher in saline. We also studied the aggregation of the mAbs in the presence of polysorbate-20, typically added to formulations to mitigate interfacial aggregation. We observed that with surfactants, the presence of salt in the buffer led to a greater mAb adsorption at the interface and resulted in the formation of more particulate aggregates. Our results show that the addition of salt to the buffer led to differences in the interfacial aggregation in mAb formulations, showing that stress studies used to screen for mAb aggregation intended for i.v. administration should be performed in conditions representative of their intended route of administration.

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

Role of Free Thiol on Protein Adsorption to Gold Nanoparticles.

Protein-gold nanoparticle (AuNP) bioconjugates have many potential applications in nanomedicine. A thorough understanding of the interaction between the protein and the AuNP is critical to engineering these functional bioconjugates with desirable properties. In this work, we investigate the role of free thiols presented by the protein on the stability of the protein-AuNP conjugate. Human serum albumin (HSA) was modified with 2-iminothiolane (Traut's reagent) to introduce additional thiols onto the protein surface, and three variants of HSA were synthesized to present 1, 5, and 20 free thiols by controlling the molar excess of the chemical modifier. Protein exchange studies on AuNPs were conducted using these HSA species and an IgG antibody which exhibited 10 free thiols. Antibody-AuNP conjugates were synthesized, purified, and dispersed in solutions containing each of the HSA species. No protein exchange was detected with the HSA or modified HSA containing 5 thiols; however, 85% of the antibody was displaced on the AuNP surface by the extensively thiolated HSA presenting 20 free thiols. Furthermore, the impact of the protein adsorption sequence was probed in which each of the HSA species were preadsorbed onto the AuNP and dispersed in a solution of antibody. The antibody fully displaced the HSA with a single thiol from the AuNP within 3 h, required 24 h to completely displace the modified HSA containing 5 thiols, and was unable to displace the modified HSA containing 20 thiols. These results indicate that the number of Au-S interactions governs the binding interaction between the protein and the AuNP. This work provides further insight into the protein-AuNP binding mechanism and identifies important design principles for engineered proteins to optimize bioconjugates.

FTIR Spectroscopy Detects Intermolecular \u03b2-Sheet Formation Above the High Temperature Tm for Two Monoclonal Antibodies

The temperature-dependent secondary structure of two monoclonal IgG antibodies, anti-IGF1R and anti-TSLP, were examined by transmission mode Fourier Transform Infrared (FTIR) spectroscopy. Anti-IGF1R and anti-TSLP are IgG monoclonal antibodies (mAbs) directed against human Insulin-like Growth Factor 1 Receptor for anti-tumor activity and Thymic Stromal Lymphopoietin cytokine for anti-asthma activity, respectively. Differential scanning calorimetry (DSC) clearly indicates both antibodies in their base formulations have a lower temperature protein conformational change near 70 °C (Tm1) and a higher temperature protein conformational change near 85 °C (Tm2). Thermal scanning dynamic light scatting (TS-DLS) indicates a significant particle size increase for both antibodies near Tm2 suggesting a high level of protein aggregation. The nature of these protein conformational changes associated with increasing the formulation temperature and decreasing sucrose concentration were identified by transmission mode FTIR and second derivative FTIR spectroscopy of temperature controlled aqueous solutions of both monoclonal antibodies. The transition from intra-molecular β sheets to inter-molecular β sheets was clearly captured for both monoclonal antibodies using FTIR spectroscopy. Finally, FTIR Spectroscopy was able to show the impact of a common excipient such as sucrose on the stability of each monoclonal antibody, further demonstrating the usefulness of FTIR spectroscopy for studying protein aggregation and formulation effects.

Micro- and macro-viscosity relations in high concentration antibody solutions

Molecular crowding in highly concentrated monoclonal antibody (mAb) solutions results in significant increases in viscosity, which complicates fill-finish steps and patient administration by subcutaneous injection. As viscosity measurements for optimization of the mAb formulation require significant amounts of material not always available in early development, fluorescence correlation spectroscopy (FCS) is evaluated as a potential ultra-low volume technique for viscosity measurement of high concentration protein solutions assuming the Generalised Stokes Einstein relation (GSE) remains valid. Using like-charge fluorescent tracers of different sizes, FCS provided measurements of microviscosities which were compared to the macroviscosity. After parametrising the protein concentration dependence of the viscosity by the exponential coefficient (k) of a simple exponential model, FCS derived k-values of like-size tracer to the crowder followed the same ordering as the macroviscosity derived k-values with respect to solvent conditions. Furthermore, k and the diffusion-derived protein-protein interaction parameter, kD, are linked, and, attractive conditions for mAbs result in a stronger concentration dependence of the viscosity. For tracers and crowders of like-size, a key result is negative deviations from the GSE relation are observed in presence of strong attractive interactions between crowder molecules. These data demonstrate that FCS has application to the screening of high concentration mAb solutions for formulation selection.

Effects of the Non-Neutralizing Humanized Monoclonal Anti-Adrenomedullin Antibody Adrecizumab on Hemodynamic and Renal Injury in a Porcine Two-Hit Model.

Adrenomedullin is a vasoactive peptide that improves endothelial barrier function in sepsis, but may also cause hypotension and organ failure. Treatment with a non-neutralizing monoclonal anti-adrenomedullin antibody showed improvement in murine sepsis models. We tested the effects of the humanized monoclonal anti-adrenomedullin antibody Adrecizumab in a porcine two-hit model of hemorrhagic and septic shock.In this randomized, blinded study 12 German Landrace pigs were bled to half of baseline mean arterial pressure for 45 min. Sepsis was induced using an Escherichia coli clot placed into the abdominal cavity 6 h after hemorrhagic shock. Animals received either 2 mg/kg BW anti-adrenomedullin antibody or vehicle solution immediately after sepsis induction. After 4 h, resuscitation was initiated using balanced crystalloids and noradrenalin to maintain a central venous pressure of 8 to 12 mm Hg, a mean arterial pressure ≥ 65 mm Hg, and a ScvO2 ≥70% for another 8 h. Hemodynamic parameters, laboratory parameters, and kidney histology were assessed.The amount of volume resuscitation was significantly lower and significantly less animals developed a septic shock in the antibody-treated group, compared with the vehicle group. Kidney histology showed significantly lower granulocytes in both cortex and medulla in antibody-treated animals, while the remaining four kidney measures (serum creatinine and urine output and cortical and medullary injury in histopathology) did not reach the significance levels. After induction of sepsis, plasma adrenomedullin increased immediately in both the groups, but increased quicker and more pronounced in the antibody group.In this two-hit shock model, treatment with an anti-adrenomedullin antibody significantly increased plasma adrenomedullin levels, while significantly less animals developed septic shock and renal granulocyte extravasation was significantly reduced. Thus, therapy with Adrecizumab may provide benefit in sepsis, and clinical investigation of this drug candidate is warranted.

Deterministic Lateral Displacement-Based Separation of Magnetic Beads and Its Applications of Antibody Recognition.

This work presents a magnetic-driven deterministic lateral displacement (m-DLD) microfluidic device. A permanent magnet located at the outlet of the microchannel was used to generate the driving force. Two stages of mirrored round micropillar array were designed for the separation of magnetic beads with three different sizes in turn. The effects of the forcing angle and the inlet width of the micropillar array on the separating efficiency were studied. The m-DLD device with optimal structure parameters shows that the separating efficiencies for the 10 μm, 20 μm and 40 μm magnetic beads are 87%, 89% and 94%, respectively. Furthermore, this m-DLD device was used for antibody recognition and separation among a mixture solution of antibodies. The trajectories of different kinds of magnetic beads coupled with different antigens showed that the m-DLD device could realize a simple and low-cost diagnostic test.

Surface Plasmon Resonance Signal Amplification Using Secondary Antibody Interaction for Illegal Compound Detection

The development of highly selective and sensitive surface plasmon resonance (SPR) immunoassay for the rapid detection of illegal compound using secondary antibody interaction labelled nanoparticle was conducted. For the construction of SPR sensor surface, the illegal compound (clenbuterol) was immobilized as antigen onto gold succinimide-terminated monolayer to perform amide-coupling reaction. In order to avoid non-specific reaction, the blocking agent (ethanol amine) was injected to the SPR system. Furthermore, indirect competitive inhibition method was employed in the detection of clenbuterol. In this work, the antibody solution (PBS solution containing clenbuterol antibody) premixed with a sample solution (PBS solution containing antigen) before the injection into the sensing system. As the premixed solution flowed over the sensor surface, the SPR senses the dielectric constant change at the interface due to the binding of the unreacted primary antibody to antigen-immobilized on the sensor surface. After this primary antibody detection, secondary antibody was injected to the SPR sensor surface. Here, we compared the signal difference of secondary antibody injection labelled Au nanoparticles (d = 40 nm). For the regeneration of the sensor surface, 0.1 M NaOH was used, so primary and secondary antibodies could be detached from the sensor surface. According to the indirect competitive inhibition method, it was found that the sensitivity for clenbuterol detection was enhanced from 2.5 ppt to 0.07 ppt.

Lab-on-a-Chip System for Developing and Fluorescence Imaging a Three-Dimensional Model of Pancreatic Islets Under Flow Conditions

Introduction Diabetes mellitus is a group of metabolic diseases characterized by elevated blood sugar resulting from a defect in the production of insulin by pancreatic β-cells or tissue insensitivity for this peptide hormone [1]. The most common form of the disease is diabetes mellitus type 2 (insulin-dependent), which is diagnosed in about 80% of all patients [2]. In patients with this type of diabetes, both the efficiency and the secretion of insulin are disturbed [3]. For this reason, it is very important to thoroughly understand the kinetics of insulin release in response to various conditions, which can help develop effective therapy. The most important cells for this disease are insulin secreting β-cells and their antagonist - glucagon-secreting α-cells [4]. In our research, we focused on imitate the pancreatic islet structure, which can be a universal model for testing the impact of various environmental factors on disease development. The idea of using a Lab-on-a-chip system for three-dimensional islet cells culture came from the possibility of mimicking in vivo conditions. This can give a wider and more accurate view of the pancreatic islets function than the cell culture in static conditions. Lab-on-a-chip system In our research we present a Lab-on-a-chip system in which a pancreatic “pseudoislet” model will be developed. This system is composed of biocompatible, non-toxic and transparent materials, such as: poly(dimethylsiloxane) (PDMS) and thin glass. Thanks to this, it is possible to conduct cell culture and observe the results in various types of microscopes. The geometry of Lab-on-a-chip system consist of two elliptical cell culture chambers, one for the test sample and second for the reference sample. In in each of the chambers there are 15 round microtraps. Each of the microtraps is made of 6 micropillars. This solution forces the dense packing of cells and their aggregations. The geometry of the developed system is consistent with the culture wells on a standard multi-well plate, which allows measurements in a multi-well plate reader. Methods All experiments were performed using two commercially purchased pancreatic islets cell lines: β-cells (INS-1E) and α-cells (α-TC1-6). Appropriate α-cells and β-cells ratios were selected to reflect the morphology and composition of the pancreatic islet found in the body. The cells were introduced through the inlet to the Lab-on-a-chip system and placed for 24h in an incubator to form spherical aggregates (“pseudoislets”). In the next stage immunostaining protocol using primary and secondary antibody solutions was developed. Due to the use this method and confocal microscope it was possible to confirm the correct distribution of cells in the obtained aggregate. To confirm cells viability previously prepared solution of Calcein AM and Propidium Iodide were introduced through the microsystem inlet with a flow rate 5µL/min for 10 min. After 10 min incubation (37oC, 5% CO2) the viability of the cells was determined using fluorescence microscope. Proliferation of the ells was measured using spectrofluorimetric multi-well plate reader after incubation with AlamarBlue solution. The “pseudoislets” were then stimulated with various glucose concentrations (imitation of health and disease state). After this, the standard ELISA test procedure was adapted to the microfluidic conditions to examine the insulin secretion profile from the obtained “pseudoislets”. Results and Conclusions It was confirmed that 15 spherical α and β-cell aggregates with diameters of 160-180 µm were obtained in one culture chamber using the method described earlier. As was expected the participation of β-cells in the core and α-cells on the periphery of the aggregate was confirmed by the use of immunostaining method. Moreover, the compatibility of the designed microsystem with the multi-well plate reader allowed to monitor cell proliferation and viability in situ during the culture. A high viability after 24 and 48 hours of culture in the microchip was confirmed and was 97% and 95% respectively. The cells maintained their morphology, function and high level of proliferation for up to 48 hours of culture. Moreover, in the developed model health and disease conditions were simulated and the level of insulin secreted from this model was determined using the ELISA test. At this stage, a research model corresponding to the model in vivo conditions was obtained. The next step will be to develop a method for analyzing insulin secretion in real time (such as Surface Plasmon Resonance). This study presents basic research and in the future, this model could be utilized to simulate diabetes, testing new drugs and therapy in diabetes mellitus treatment.

On-Site and Daily Stress-Measurement Using Gold-Linked Electrochemical Immuno Assay for IgA Determination

[Introduction]Mental stress is closely connected with our physical and mental wellness. In particular, depression may be associated with stress. Therefore, stress measurement can contribute to assess our lifestyle and increase our quality of life. However, objective stress-measurement is not established. To establish objective stress-measurement, an individual must indicate the stress level by obtaining numerous data continuously throughout the day. Therefore, the measurement method which can be suitable for this condition is needed. In this paper, we detect the secretory Immunoglobulin A (sIgA), which is the candidate of salivary stress-markers, with original electrochemical immunoassay: Gold-Linked Electrochemical Immuno Assay (GLEIA). This biosensor is based on a sandwich-type immunosensor, and adopt the electrochemical method to detect the reduction peak from Au nanoparticles linked to the secondary antibody. GLEIA is convenient and cost-effective that only requires low sample volume (10 µL). In addition, all devices that GLEIA needs are minimal such as note PC and portable potentiostat and printed electrode. Therefore, GLEIA method shows various advantages suitable for stress measurement as compared with other sIgA detection methods. In this paper, we aim to the on-site and continuous measurement of salivary IgA in our daily life, to determine the various concentration of sIgA in standard solution and artificial saliva using GLEIA.[Experimental]Primary Anti-IgA Antibody solution was dropped onto the working electrode for immobilizing the antibody by physical adsorption. After, the Au nanoparticle solution was mixed with the phosphate buffer (50 mM, pH=8.3). The anti-IgA to dissolve the 5 mM of phosphate buffer add the Au nanoparticle solution. For the preparing of secondary antibody modified Au nanoparticle, the antibody was conjugated with Au nanoparticle under the pH=8.3 condition and kept for 10 min at room temperature. Then, 10% BSA in PBS buffer and 1% PEG in PBS buffer were added in preparing Au solution. Finally, the concentration was adjusted for OD520=6.0 by centrifuge and exclude the supernatant. The Au Anti-IgA conjugate was diluted by 3 times with 50 w/v of trehalose (OD520=2) and dripping 5µL of this solution in the immunomodule. Then the immunomodule was dried in vacuum condition for 5 minutes. Dried immunomodule was stored at 4 ℃ until use. Different concentrations of the IgA antigen solution (0, 10, 60, 150, 300 ng/mL) were made by diluting in PBS. For the detection of Antibody-Antigen reaction, the IgA solutions were mixed in prepared immunomodule, After the 1 minute, and 1.4 µL of the solution was placed on immunosensor to incubate for 1 hour, after rinsing with PBS, and eliminate the PBS solution with the air gun. The direct redox reaction was performed in 2M KCl solution (20 µL) covering the entire three-electrode zone at room temperature.[Results and Discussion]We obtained the linear response to relate the concentration of sIgA (10-300 ng/mL) in D-PBS buffer with the artificial-saliva which includes salivary inorganic salt and typically glycoprotein (mucin). Figure 1 shows the differential pulse voltammogram of GLEIA with IgA in artificial saliva (a) and calibration curve (b). In addition, the calibration curve shows the linear range within 10-300 ng/mL and the correlation coefficient (R2) of the calibration curve for this relationship was 0.963. The peak current intensity is decreased from in standard solution. It may prevent the Antigen-Antibody reaction from artificial saliva which pH is nearly 8.5. Generally, the antibody shows the best affinity in pH= 6.5~8.4 condition. However, these results show that GLEIA is suitable for salivary IgA detection because the salivary pH is mainly neutral. Furthermore, we evaluate the selectivity of the immunosensor by comparing it with the reduction peak between IgA= 300ng/mL or 0 ng/mL and interference protein such as α-amylase, human serum albumin, immunoglobulin G (IgG), lysozyme and mucin. We acknowledge GLEIA shows the signal increasing within the existence of interference proteins. This may be the affinity of antibody and non-specific reaction between antibody and the proteins. More importantly, we recognize that the reduction peak is increasing with the concentration of sIgA in the existence of interference proteins. Therefore, GLEIA can be applied for stress measurement using salivary sIgA that can be used in daily life anywhere and anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva.nd anytime whenever necessary. In the future, we try to detect the sIgA in real saliva for on-site stress measurement using GLEIA and to integrate the various immunosensors for stress-markers in saliva. Figure 1

Old but not obsolete: an enhanced high-speed immunoblot.

The immunoblotting technique (also known as western blotting) is an essential tool used in biomedical research to determine the relative size and abundance of specific proteins and protein modifications. However, long incubation times severely limit its throughput. We have devised a system that improves antigen binding by cyclic draining and replenishing (CDR) of the antibody solution in conjunction with an immunoreaction enhancing agent. Biochemical analyses revealed that the CDR method reduced the incubation time of the antibodies, and the presence of a commercial immunoreaction enhancing agent altered the affinity of the antibody, respectively. Combination of the CDR method with the immunoreaction enhancing agent considerably enhanced the output signal and further reduced the incubation time of the antibodies. The resulting high-speed immunoblot can be completed in 20 min without any loss in sensitivity. Further, the antibodies are fully reusable. This method is effective for both chemiluminescence and fluorescence detection. Widespread adoption of this technique could dramatically boost efficiency and productivity across the life sciences.

Coarse-Grained Molecular Dynamics Simulations for Understanding the Impact of Short-Range Anisotropic Attractions on Structure and Viscosity of Concentrated Monoclonal Antibody Solutions.

Understanding protein-protein interactions in concentrated therapeutic monoclonal antibody (mAb) solutions is desirable for improved drug discovery, processing, and administration. Here, we deduce both the net protein charge and the magnitude and geometry of short-ranged, anisotropic attractions of a mAb across multiple concentrations and cosolute conditions by comparing structure factors S(q) obtained from small-angle X-ray scattering experiments with those from molecular dynamics (MD) simulations. The simulations, which utilize coarse-grained 12-bead models exhibiting a uniform van der Waals attraction, uniform electrostatic repulsion, and short-range attractions between specific beads, are versatile enough to fit S(q) of a wide range of protein concentrations and ionic strength with the same charge on each bead and a single anisotropic short-range attraction strength. Cluster size distributions (CSDs) obtained from best fit simulations reveal that the experimental structure is consistent with small reversible oligomers in even low viscosity systems and help quantify the impact of these clusters on viscosity. The ability to systematically use experimental S(q) data together with MD simulations to discriminate between different possible protein-protein interactions, as well as to predict viscosities from protein CSDs, is beneficial for designing mAbs and developing formulation strategies that avoid high viscosities and aggregation at high concentration.

Clogging of microfluidic constrictions by monoclonal antibody aggregates: role of aggregate shape and deformability.

The formation of aggregates in solutions of monoclonal antibodies is difficult to prevent. Even if the occurrence of large aggregates is rare, their existence can lead to partial or total clogging of constrictions in injection devices, with drastic effects on drug delivery. Little is known on the origin and characteristics of such clogging events. Here we investigate a microfluidic model system to gain fundamental understanding of the clogging of constrictions by monoclonal antibody aggregates. Highly concentrated solutions of monoclonal antibodies were used to create protein aggregates (larger than 50 microns) using mechanical or heat stress. We show that clogging occurs when aggregates reach the size of the constriction and that clogs can in some cases be released by increasing the applied pressure. This indicates the important role of protein aggregate deformability. We perform systematic experiments for different relative aggregate sizes and applied pressures, and measure the resulting flow-rate. This allows us to present first in situ estimates of an effective Young's modulus. Despite their different shapes and densities, we can predict the number of clogging events for a given constriction size from the aggregate size distribution measured by Flow Imaging Microscopy (MFI). In addition our device can detect the occurrence of very rare big aggregates often overlooked by other detection methods.

Development of Blood Donation Activities in Kyrgyzstan and General Implications for Blood Donation

Objectives: This article is aiming to give an essential informative principle about the development of blood donation activities in Kyrgyzstan. Background: The sequence of blood donation procedure steps before donation, during donation, and after donation was shown and discussed. Methods/Materials: Rhesus factor determination in donors was achieved by “COLICO” method which is based on the use of monoclonal antibodies solution. Results: According to the blood donation activity of donors in Osh, Kyrgyzstan for the 2018 year, it was found that gratuitous donors who donated blood in 6 months were 269, in 9 months were 366, and in 1 year were 499 people. Relative donors who donated blood in 6 months were 3480, in 9 months were 5100, and in 1 year were 6701 people. Active donors donated blood in 6 months were 12, in 9 months were 14 and in 1 year were 17 people. Conclusion: No agglutination was observed in any of the plates for the first blood group; agglutination was observed with anti-A monoclonal antibody solution for the second blood group; agglutination was observed with anti-B monoclonal antibody solution for the third blood group; agglutination was observed with both of anti-A and anti-B monoclonal antibody solutions for the fourth blood group.

Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO)

We have developed an electrospray mass spectrometry method which is capable to determine antibody affinity in a gas phase experiment. A solution with the immune complex is electrosprayed and multiply charged ions are translated into the gas phase. Then, the intact immune-complex ions are separated from unbound peptide ions. Increasing the voltage difference in a collision cell results in collision induced dissociation of the immune-complex by which bound peptide ions are released. When analyzing a peptide mixture, measuring the mass of the complex-released peptide ions identifies which of the peptides contains the epitope. A step-wise increase in the collision cell voltage difference changes the intensity ratios of the surviving immune complex ions, the released peptide ions, and the antibody ions. From all the ions´ normalized intensity ratios are deduced the thermodynamic quasi equilibrium dissociation constants (KDm0g#) from which are calculated the apparent gas phase Gibbs energies of activation over temperature (ΔGm0g#T). The order of the apparent gas phase dissociation constants of four antibody – epitope peptide pairs matched well with those obtained from in-solution measurements. The determined gas phase values for antibody affinities are independent from the source of the investigated peptides and from the applied instrument. Data are available via ProteomeXchange with identifier PXD016024. SignificanceITEM - TWO enables rapid epitope mapping and determination of apparent dissociation energies of immune complexes with minimal in-solution handling. Mixing of antibody and antigen peptide solutions initiates immune complex formation in solution. Epitope binding strengths are determined in the gas phase after electrospraying the antibody / antigen peptide mixtures and mass spectrometric analysis of immune complexes under different collision induced dissociation conditions. Since the order of binding strengths in the gas phase is the same as that in solution, ITEM – TWO characterizes two most important antibody properties, specificity and affinity.

The prediction of the operating conditions on the permeate flux and on protein aggregation during membrane processing of monoclonal antibodies

The lack of available material during early stage bioprocess development poses numerous processing challenges such as limiting the number of full-scale experiments. Extended fundamental process understanding could be gained with the use of an ultra scale-down (USD) device using as little as 1.7 mL per experimental run. The USD system is used to predict diafiltration and ultrafiltration/diafiltration (UF/DF) performance of a pilot-scale tangential flow filtration (TFF) system, fitted with a flat-sheet cassette, operating at 500-fold larger scale. Both systems were designed by maintaining a volumetric loading of 8.1 L of feed per m2. Permeate flux was predicted for monoclonal antibody solutions with the USD system across a range of transmembrane pressure drops, feed concentrations and flow conditions during diafiltration, and desired retentate concentrations during UF/DF operations. The resulting USD data were in good agreement with the pilot-scale TFF when scaled based on similar shear rates over the membrane surface. Little change in soluble aggregates was observed in both systems but there were significantly higher increases in product turbidity in the USD system. A correlation was established to relate turbidity increase based on the volume fraction of high shear stress zone for USD systems and various pilot-scale TFF systems. The correlation was extended to encompass the processing time and concentration for a wide range of membrane processing challenges in both scales.