Affinity Chromatography Method Research Articles

Screening potential ligands of endothelin receptor A from Choerospondias axillaris and evaluation of their drug-like properties by affinity chromatographic methods

Tibetan medicine is traditionally prescribed as crude extracts or mixtures owing to the theoretical basis with cross fertilization from other medical systems like Ayurveda and traditional Chinese medicine. This is challenged to elucidate the action mechanism and material foundation of Tibetan medicine due to lacking a method to confirm the bioactive compounds determining the therapy. This work created a new strategy for screening and evaluating the bioactive compounds against cardiovascular ailments from Choerospondias axillaris. It involved the immobilization of endothelin receptor A (ETAR) by a one-step covalent assay, the screening and identification of the bioactive compounds by ETAR column combined with tandem mass spectrometry, and the evaluation of their drug-like properties by calculating the efficiency indexes using the data collected by frontal analysis and adsorption energy distribution. The immobilized ETAR remained good stability in three weeks in terms of specificity and repeatability. Catechin, pinocembrin, and hyperoside were identified as potential ETAR ligands from Choerospondias axillaris with two types of binding sites on the immobilized receptor. Their association constants on the high and low affinity binding sites were (2.53 ± 0.11) × 105 and (9.94 ± 0.02) × 103 M−1 for catechin, (1.01 ± 0.12) × 106 and (7.40 ± 0.03) × 104 for hyperoside, and (2.05 ± 0.04) × 105 and (2.47 ± 0.09)× 104 M−1 for pinocembrin, respectively. Owing to the highest association constant, hyperoside presented a surface efficiency index of 7.95, and binding efficiency index of 20.7, and the ligand­lipophilicity efficiency of 1.38. These indicated that the three compounds were the main ingredients for the therapy of Choerospondias axillaris, and had potential to become lead compounds for anti-cardiovascular drugs based on drug-ETAR interaction. The immobilized receptor-based strategy is possible to become an alternative for screening and assessing bioactive compounds from Tibetan medicine.

Immunoprecipitation: Variations, Considerations, and Applications.

Immunoprecipitation (IP) refers to methods of affinity chromatography that enrich and/or purify a specific protein from a complex mixture using a specific antibody immobilized on a solid support. Several operations and processes that are dependent on the isolation, concentration, and modification of proteins have seen improvement in their selectivity and separation based on the integration of IP-specific reactions into their workflows. This relatively simple principle has contributed significantly to our understanding of proteins and their behaviors and has become increasingly fundamental to most protein characterization studies today. In this chapter, we review the basic principles of IP and the several factors that influence each stage, and subsequently the success, of an IP experiment. Moreover, variations in application of the IP principle are discussed, and the adaptability of the techniques based on such is highlighted in the provision of two IP workflows to purify a particular protein from an entire cellular proteosome. These workflows cover the preparation and fractionation of crude cellular lysate into individual subcellular fractions, through to both "batch" and "column"-based extractions of the target protein of interest. Protocols for determining the validity of the workflows, and the presence/abundance of the protein of interest, are also briefly described.

Evaluation of interference from 16 hemoglobin variants on hemoglobin A1c measurement by five methods

Hb variants prevalent in China are different from those in other countries. We aimed to assess the interference from Hb variants found in China on HbA1c measurement. All Hb variants were confirmed using Sanger sequencing. HbA1c was measured using a capillary electrophoresis method (Capillarys 3 OCTA), two cation-exchange high-performance liquid chromatography methods (ADAMS HA-8180V and HLC-723 G8 standard mode), an immunoassay (Cobas c501), and a boronate affinity chromatography method (Premier Hb9210). Premier Hb9210 was used as a comparative method. A total of 16 species of Hb variants were identified in 102 variant carriers. The most common variant was Hb E, followed by Hb Q-Thailand, Hb New York and Hb J-Bangkok. Clinically significant interference was observed for the Capillarys 3 OCTA (two Hb variants), ADAMS HA-8180V (seven Hb variants), HLC-723 G8 (14 Hb variants), and Cobas c501 (two Hb variants). The proportion of unacceptable HbA1c results was 13.7% for Capillarys 3 OCTA, 52.9% for HA-8180V, 83.3% for HLC-723 G8, and 3.9% for Cobas c501. Hb variants in China severely affect the accuracy of some commonly used HbA1c methods.

Classification of Extracellular Vesicles Based on Surface Glycan Structures by Spongy-like Separation Media.

Extracellular vesicles (EVs) are lipid bilayer vesicles that enclose various biomolecules. EVs hold promise as sensitive biomarkers to detect and monitor various diseases. However, they have heterogeneous molecular compositions. The compositions of EVs from identical donor cells obtained using the same purification methods may differ, which is a significant obstacle for elucidating objective biological functions. Herein, the potential of a novel lectin-based affinity chromatography (LAC) method to classify EVs based on their glycan structures is demonstrated. The proposed method utilizes a spongy-like monolithic polymer (spongy monolith, SPM), which consists of poly(ethylene-co-glycidyl methacrylate) with continuous micropores and allows an efficient in situ protein reaction with epoxy groups. Two distinct lectins with different specificities, Sambucus sieboldiana agglutinin and concanavalin A, are effectively immobilized on SPM without impacting the binding activity. Moreover, high recovery rates of liposomal nanoparticles as a model of EVs are achieved due to the large flow-through pores (>10 μm) of SPM compared to a typical agarose gel. Finally, lectin-immobilized SPMs are employed to classify EVs based on the surface glycan structures and demonstrate different subpopulations by proteome profiling. This is the first approach to clarify the variation of protein contents in EVs by the difference of surface glycans via lectin immobilized media.

Розподіл серинових протеаз у плазмі крові та підшлунковій залозі за хронічного панкреатиту та онкопатології

The aim of our study was to evaluate the trypsin-like serine proteases (TLPs) distribution between systemic circulation and pancreatic tissue and to investigate the peculiarities of their involvement in the extracellular matrix components degradation in patients with pancreatic pathologies with electrophoretic analysis methods using. Тhe Khmelnitsky Regional Clinical Hospital patients aged 28-89 were selected for this study: 20 people with chronic pancreatitis (group CP); 20 people with pancreatic cancer (group PC); 20 conditionally healthy persons (control). Blood plasma samples and pancreatic tissue homogenates were obtained from all the patients, from which the TLPs fractions were subsequently obtained by the affinity chromatography method. The study showed that TLPs content in the blood plasma of patients with pancreatic pathologies is higher, and in tissue homogenates is lower relative to the values of the corresponding indicators in the control. Disk-electrophoresis using showed that TLPs fractions obtained from the blood plasma of patients of all studied groups contain a lot of high molecular weight (HMW) proteins, while TLPs from the pancreatic tissue homogenates of patients with pancreatic pathologies mainly consists of low molecular weight (LMW) proteins. Enzyme-electrophoresis results showed that all TLPs fractions include enzymes with fibrinogenolytic, gelatinolytic and collagenolytic activity. In plasma, the first were represented by medium molecular weight (MMW) proteins, and the last two groups included a lot of HMW proteins as well as proteins with very high molecular weight. In homogenates, fibrinogenolytic activity was characteristic for LMW proteins only, whereas gelatinases and collagenases were represented by both MMW and LMW proteins. Our results indicate the differences in the TLPs fractions components obtained from blood plasma and pancreatic tissue of patients with investigated pathologies, as well as significant distinctions in the processes of extracellular matrix remodeling under СР and РС.

Cloning, Expression, Re-folding, and Purification of Protein Flic (salmonella Enteritidis) with Deletion of Amino Acids 220-320

In order to develop high-performance subunit vaccines, many studies are currently aiming at the development of immunoadjuvants. Flagellin protein (FliC) contained in the Salmonella’s filament is one of the potential candidates for this purpose. However, the high antigenic property of FliC has made it difficult to apply for many vaccines. A variant with the deletion of amino acids from 220 to 320 of flagellin has been proven to have an adjuvant effect with lower immunogenicity. In this present study, we cloned and expressed FliCD220-320 recombinant in Escherichia coli system. The expression of FliCΔ220-320 was induced by IPTG and confirmed by SDS-PAGE and Western Blot probed with 6xHis tag antibody. Results showed that most of the expressed protein is insoluble. The targeted protein was then solubilized, re-folded and purified by affinity chromatography method with the purity of 80 %. With these results, we successfully expressed FliCD220-320 recombinant protein and this is a source for the evaluation and development of immune adjuvants.

Mathematical and experimental validation of an approach for simultaneously determining the binding parameters of two drugs to a receptor

Quantifying drug-protein interactions has a pivotal role in both early phase drug development and clinical processes. Diverse affinity chromatographic methods like nonlinear chromatography can realize such quantification, however, their throughputs are challenged due to the loading of a single ligand during each run. This work derivatized a new equation for simultaneously determining the bindings of two ligands to a protein relying on assumption that the retention factors of the ligands are dependent on their injection amounts. Experimental validation of the derivatization was performed on an immobilized endothelin A receptor (ETAR) column taking ambrisentan, bosentan, and macitentan as injecting solutes. All three ligands presented a decrease in retention times along with increasing moles of injection when they were singly injected into the column. Likewise, negative relationships between the retentions and the injection amounts were observed when co-injection of ambrisentan/bosentan or bosentan/macitentan was performed, thus confirming the assumption of the derivatization. The association constants of ambrisentan, bosentan, and macitentan binding to ETAR were (1.42 ± 0.78)×104, (1.81 ± 0.22)×104, and (1.71 ± 0.41)×104 L/mol when each of them was singly loaded on the column. Such data displayed insignificant changes in four weeks thereby providing a proof of good stability of the column during the period. Co-injections of the two ligand pairs resulted in the association constants of (2.97 ± 0.13)×104 for ambrisentan, (2.51 ± 0.87)×104 for bosentan, and (2.88 ± 0.34)×104 L/mol for macitentan. These results were in good agreement with the calculation when each of the ligands was injected alone into the column and demonstrated little differences from the data by nonlinear chromatography. Owning to the simultaneous analysis of two ligands, the throughput of the proposed method was twofold higher than the typical assays including frontal analysis, zonal elution, and nonlinear chromatography. It is possible to become an alternative for rapid analysis of drug-protein interaction.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42–2.63 × 105 M−1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M−1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07–3.31 × 105 M−1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Application of smart responsive materials in phosphopeptide and glycopeptide enrichment

蛋白质的磷酸化和糖基化作为研究最广泛的两种翻译后修饰(PTMs),在疾病的早期无创诊断、预后和治疗评估中表现出越来越大的潜力。蛋白质的异常磷酸化和糖基化经常被用于临床蛋白质组学研究和疾病相关生物标志物的发现。目前已有多种材料被开发用于磷酸化肽和糖肽的富集研究,其中,智能响应材料由于具有独特的响应特性,已被陆续报道用于磷酸化肽和糖肽的富集。智能响应材料可对外界刺激做出响应,发生结构和性质上的变化,将光、电、热、机械等信号转化为生物化学信号。响应分子是决定智能响应材料响应特性的先决条件,它们在不同刺激条件下(如温度、pH、光、机械应力、电磁场等)的可逆异构化将导致材料的宏观物理和化学性质的动态变化。与传统材料相比,智能响应材料可以可逆地“打开”和“关闭”,具有更好的可调控性。由于引起智能材料响应的刺激信号对其性能具有重要的影响,综述根据施加的刺激种类对智能响应材料进行分类,具体分为外源性响应材料和内源性响应材料,且分别总结了外源性响应材料、内源性响应材料以及内外源共同响应材料在磷酸化肽和糖肽富集方面的工作。此外,综述对智能响应材料在磷酸化肽和糖肽富集方面的发展前景进行了展望,并且提出了智能响应材料在其他蛋白质翻译后修饰方面的应用中存在的挑战。

Identifying potential ligands specifically binding to beta1-adrenoceptor from Radix Aconiti Lateralis Praeparata extract by affinity chromatographic method

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (β1-AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of β1-AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing β1-AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged β1-AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized β1-AR in the analysis of ligand-receptor interaction. The morphology was characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). The chromatographic specificity of the immobilized receptor column was evaluated by determining the association constants of atenolol, esmolol and metoprolol using stepwise frontal analysis plus injection amount-dependent method. The potential ligands binding to β1-AR was screened by collecting the peak with retention time longer than the void time, and identified the collection by reverse phase liquid chromatography coupled with tandem mass spectrometry. The association constants of the three drugs to β1-AR were (3.33 ± 0.29)× 106 M−1, (2.33 ± 0.23)× 106 M−1 and (2.06 ± 0.03)× 106 M−1, indicating a desired specificity of the immobilized receptor for recognizing its ligands. Molecular docking showed that van der Waals, hydrogen bonds, and hydrophobic interactions were the principal interaction forces for the receptor-drug complexes. Benzoylmesaconine was screened as the potential ligand of β1-AR in Radix Aconiti Lateralis Praeparata extract. The association constant of the ligand was (1.06 ± 0.02)× 105 M−1, hinting structural modification may be required before clinical application. The immobilized β1-AR is possible to provide a rapid method for screening potential ligands in herbal extract.

Development of an Allostery Responsive Chromatographic Method for Screening Potential Allosteric Modulator of Beta2-adrenoceptor from a Natural Product-Derived DNA-Encoded Chemical Library.

Allosteric ligands are promising drugs owing to their remote regulations of the orthosteric ligand signaling pathway. There are few allosteric ligands due to the lack of handy and efficacious method for the screening. Herein, we developed an affinity chromatographic method for allosteric ligand screening by immobilizing purified beta2 adrenoceptor (β2-AR) onto macroporous silica gel by a two-point tethering method. The method relies on the occupation of the orthosteric site by an antagonist and the chelation of N-terminal His-tag of the receptor and Ni2+ coated on the gel. The immobilized β2-AR demonstrated the greatest allosteric responsive feature when Cmpd-15 (0.25 μM) was included in the mobile phase. Under the same conditions, the association constants of three agonists (salbutamol, terbutaline, and tulobuterol) reduced to 47%, 19%, and 27% compared with the data without the inclusion of Cmpd-15 in the mobile phase. APF was screened as a potential allosteric modulator of β2-AR by applying the immobilized receptor in a natural product-derived DNA-encoded chemical library (DEL). Relying on these results, we reasoned that the current method has potential in screening allosteric ligands of the receptor. We expect that it is applicable for the discovery of new allosteric binding sites of a target protein and screening allosteric modulators of the other receptors from complex samples.

Effect of hemoglobin, triglyceride, and urea in different concentrations on compatibility between methods used in HbA1c measurement

Abstract Objectives The variability between methods is seen in the existence of hemoglobin (Hb), triglyceride (TG), blood urea nitrogen (BUN) and glucose in the specimens, which can affect the measurement. Therefore, this study aimed to compare boronate affinity chromatography and turbidimetric inhibition immunoassay methods for glycosylated hemoglobin (HbA1c) levels in samples containing different levels of Hb, TG, BUN and glucose. Methods In this study, a total of 412 consecutive samples were used for method comparison. The samples were divided into groups according to HbA1c, TG, BUN, Hb, and glucose levels. Clinically significant changes for HbA1c are defined as ≥0.5%. Results The mean relative difference as % was determined as 1.4% by the Bland–Altman plot of Trinity against Roche. The Passing–Bablok analysis indicated good agreement (r=0.985) between two methods. When compared the group with and without a clinically significant difference, we found differences only between glucose levels (p=0.003). Conclusions High glucose levels may cause a difference between the methods. Therefore, we suggest that a method’s performance and properties should be considered prior to clinical usage.

In situ synthesis and unidirectional insertion of membrane proteins in liposome-immobilized silica stationary phase for rapid preparation of microaffinity chromatography

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with KD values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl4-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Purification of Therapeutic Antibodies Using the Ca2+-Dependent Phase-Transition Properties of Calsequestrin.

Affinity chromatography utilizing specific interactions between therapeutic proteins and bead-immobilized capturing agents is a standard method for protein purification, but its scalability is limited by long purification times, activity loss by the capturing molecules and/or purified protein, and high costs. Here, we report a platform for purifying therapeutic antibodies via affinity precipitation using the endogenous calcium ion-binding protein, calsequestrin (CSQ), which undergoes a calcium ion-dependent phase transition. In this method, ZZ-CSQ fusion proteins with CSQ and an affinity protein (Z domain of protein A) capture antibodies and undergo multimerization and subsequent aggregation in response to calcium ions, enabling the antibody to be collected by affinity precipitation. After robustly validating and optimizing the performance of the platform, the ZZ-CSQ platform can rapidly purify therapeutic antibodies from industrial harvest feedstock with high purity (>97%) and recovery yield (95% ± 3%). In addition, the ZZ-CSQ platform outperforms protein A-based affinity chromatography (PAC) in removing impurities, yielding ∼20-fold less DNA and ∼4.8-fold less host cell protein (HCP) contamination. Taken together, this platform is rapid, recyclable, scalable, and cost-effective, and it shows antibody-purification performance superior or comparable to that of the standard affinity chromatography method.

Study of native SMAC protein production in the pUbiq expression system: Molecular cloning, biosynthesis and molecular modelling

BackgroundIn the process of recombinant protein biosynthesis affinity tags are efficient tools to achieve the expected purity and yield during the purification steps. Nonetheless these tags might alter enzyme specificity and activity, therefore in functional assays it is recommended to use authentic or native proteins. Several ubiquitin fusion systems have been developed for E. coli-based recombinant protein expression that provide high levels of expression, with simple purification, and allow the production of various proteins with authentic N-terminus for subsequent applications. ResultsIn the present research, we describe an ubiquitin fused bacterial biosynthetic system (pUbiq) for the production of the native Second mitochondria-derived activator of caspases (SMAC) recombinant protein. Using this system, the recombinant protein is expressed with an ubiquitin-decahistidine fusion partner, then purified from the cell-forming proteins by affinity chromatography. The fusion partner is then removed by proteolytic digestion, resulting the native structure of the recombinant protein without unnecessary amino acid residues. Following proteolysis, another affinity chromatography method is used to separate the native protein from the fusion partner and the proteolytic enzyme. The folding of the protein of interest was verified by a pull-down assay. ConclusionsBased on our results, the presented pUbiq system was successfully applied in the production of native SMAC recombinant protein, where the affinity tag required for purification was completely removed. Our study suggests that the ubiquitin-fusion technology will be useful for enhancing expression and purification of native and authentic proteins for structural and functional studies as well as for therapeutic uses.How to cite: Salamona P, Orbána CK, Molnár-Nagy K, et al. Exon based amplified polymorphism (EBAP): A novel and universal molecular marker for plants. Electron J Biotechnol 2022;56. https://doi.org/10.1016/j.ejbt.2022.01.002.

Rapid detection of fluoroquinolone residues in aquatic products based on a gold-labeled microwell immunochromatographic assay

Abstract Objectives Fluoroquinolones (FQs) are widely used in aquaculture, and their residues have caused many problems threatening human health. Here, this study aims to develop a colloidal gold immunochromatographic strip based on gold-labeled microwells to screen the residues of FQs on site. Materials and methods The Protein A Magarose Beads affinity chromatography method was adopted to purify the ascites against FQs. By using a strategy of heterologous coating antigen, different coating antigens are applied to detect FQs. The gold-labeled microwell immunochromatographic assay was used to improve the sensitivity of the test strip by the advanced reaction of antigen and antibody. Results The antibodies were verified to be of high purity up to 99%, and the titer reached 1:1 024 000. The combination (enoxacin-OVA and the antibody) detected the 4 banned FQs (pefloxacin, PEF; norfloxacin, NOR; lomefloxacin, LOM; ofloxacin, OFL) with the 50% inhibiting concentration (IC50) values ranging from 1.3 to 2.1 ng/mL and cross-reactions ranging from 67.3% to 106.1%. The analysis of spiked crucian carp, silver carp, grass carp, and shrimp samples showed that the limit of detection for PEF, NOR, LOM, and OFL was 4 μg/kg. A comparative study with liquid chromatography tandem mass spectrometry (LC-MS/MS) demonstrated that the assay provided an effective screening tool for the rapid detection of FQs residues. Conclusions The results indicated that the test strip can realize full coverage recognition of the 4 banned FQs and has good accuracy, specificity, reproducibility, and stability; therefore, they are more suitable for rapid detection of FQs in aquatic products.

Purification of Recombinant Galectins from Different Species Using Distinct Affinity Chromatography Methods.

Galectins are lectins having the capacity to recognize β-galactose-containing glycan structures and are widely distributed among various taxa. However, the exact physiological and biochemical functions mediated by galectins that necessitate their wide occurrence among diverse species have not yet been delineated in a precise manner. Purification of recombinant galectins in active form is a fundamental requirement to elucidate their biological function. In this chapter, we are describing methods to recombinantly express and purify galectins using three different methods of affinity purification, i.e., lactosyl-Sepharose chromatography for fungal galectin Coprinopsis cinerea galectin 2 (CGL2), nickel-chromatography for histidine-tagged human galectin-7, and glutathione-Sepharose chromatography for Glutathione S-transferase-tagged (GST-tagged) human galectin-7. Step-by-step instructions are provided for obtaining the above-mentioned recombinant galectins that retain carbohydrate-binding activity and are suitable for conducting biochemical experiments.

Purification of Porcine Circovirus Type 2 Using an Affinity Chromatography Based on a Neutralizing Monoclonal Antibody against Viral Capsid Protein.

Porcine circovirus type 2 (PCV2) is a DNA virus without an envelope. The viral particle is icosahedral and has a diameter of approximately 17 nm. In order to obtain the purified virus, a broad-spectrum monoclonal antibody 3A5 against PCV2 was coupled to CNBr-activated SepharoseTM 4B, and an affinity chromatography was established for PCV2 purification. A total of 6.5 mg of purified PCV2a/LG with 97% purity was obtained from 120 mL of the viral culture medium, and only PCV2 was detected by electron microscopy. No significant changes in the antigenic characteristics of the purified virus were detected by a capture enzyme-linked immunosorbent assay (ELISA). Furthermore, the titer of the purified PCV2 was 100 times higher than that of the unpurified virus. This affinity chromatography method was also used to purify PCV2b/LN590516 and PCV2d/SD446F16, and the purified viruses were detected by electron microscopy, capture ELISA, and virus titration, respectively. The results showed that these two strains can be successfully purified, but the yield is lower than that of the PCV2a strain. In addition, the purified virus could be used to study the viral adsorption and invasion of PK15 cells using indirect immunofluorescence assays. A large number of PCV2 signals were detected to transfer from the cellular surface to the periphery of the nucleus of the PK15 cells after 30 min of adsorption of the PCV2 to the PK15 cells. The affinity chromatography is a simple and convenient tool to obtain PCV2 with high purity. It could be applied for virus structure analysis, antibody preparation, and viral adsorption and invasion research.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma

The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.

Recent Advances in Supramolecular Affinity Separations: Affinity Chromatography and Related Methods.

Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.