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IgG1, Bovine; ELISA

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[#YSRTPBP003] IgG1, Bovine; ELISA

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(1) Enhanced immune responses to E2 protein and DNA formulated with ISA 61 VG administered as a DNA prime-protein boost regimen against bovine viral diarrhea virus.[TOP]

Pubmed ID :30077483
Publication Date : //
The aim of this study was to develop and test an optimal vaccination strategy against bovine viral diarrhea virus (BVDV) based on the E2 glycoprotein of the BJ1305 strain. To achieve higher E2-specific antibody titers and to broaden the cellular immune response, a plasmid encoding the E2 protein (pcDNA3.1-E2) was constructed and a purified recombinant E2 protein was generated. The E2 protein was emulsified in the adjuvant ISA 61 VG prior to administration. We immunized mice three times with pcDNA3.1-E2 or the recombinant E2 protein or primed twice with pcDNA3.1-E2 and boosted once with the E2 protein. To evaluate the protection against BVDV conferred by the vaccines, the mice were challenged with BVDV strain Oregon C24V after the third immunization. Although all immunized mice developed humoral and cellular immune responses, the E2-specific antibody titers in the DNA prime-protein boost group were significantly higher than those elicited by either the DNA or the protein vaccine. In addition, vaccination with the E2 DNA vaccine induced higher percentages of CD4IFN-γ T cells and CD8IFN-γ T cells among total CD3 T cells than the other regimens. The predominant antibody subclass in the vaccinated mice was IgG1. Serum tumor necrosis factor alpha (TNF-α) levels in the DNA prime-protein boost group were significantly higher after the third immunization than in the other groups. Moreover, the mice treated with the DNA prime-protein boost vaccination regimen acquired protection against BVDV challenge, as shown by a significant reduction of viremia, only minor pathological changes, and a lower viral antigen burden than in the control and solo vaccinated mice. These results demonstrate the potential advantage of a DNA prime-protein boost vaccination approach over a solo vaccination for the prevention of BVDV. The ability of this vaccine strategy to control and eradicate BVD in herds warrants further investigation.

Authors : Cai Dongjie, Song Quanjiang, Duan Cong, Wang Shenghua, Wang Jiufeng, Zhu Yaohong,



(2) Facile preparation of microporous organic polymers functionalized macroporous hydrophilic resin for selective enrichment of glycopeptides.[TOP]

Pubmed ID :30032777
Publication Date : //
A macroporous adsorption resin (MAR) with ∼10 μm diameter was synthesized by seed-swelling polymerization and further modified with a layer of microporous organic polymers (MOP) by "one-pot" solvothermal reaction. The resulting MAR@MOP exhibited high specific surface area of 131.3 m/g, which was higher than that of pristine MAR (57.8 m/g). The contact angle also decreased from 58.8° (MAR) to 24° (MAR@MOP), indicating that the MOP was successfully grafted onto the surface of MAR. The chemical composition of MAR@MOP was confirmed by Fourier-transform infrared spectroscopy, C NMR and element analysis. The enrichment efficiency of MAR@MOP to glycopeptides was demonstrated by trapping N-linked glycopeptides from tryptic digests of human immunoglobulin G (IgG), horseradish peroxidase (HRP) and bovine fetuin. Furthermore, 879 unique N-glycosylation sites in 811 unique glycopeptides sequence mapped to 516 N-glycosylated proteins were identified in three replicate analyses of proteins extracted from mouse liver. Therefore, this hydrophilic MOP-coated adsorbent would be applied in the enrichment and identification of low-abundance N-linked glycopeptides in complicated biological samples.

Authors : Li Ya, Wang Hongwei, You Xin, Ma Shujuan, Dong Jing, Wei Yinmao, Ou Junjie, Ye Mingliang,



(3) Fully Human Immunoglobulin G From Transchromosomic Bovines Treats Nonhuman Primates Infected With Ebola Virus Makona Isolate.[TOP]

Pubmed ID :30010950
Publication Date : //
Transchromosomic bovines (Tc-bovines) adaptively produce fully human polyclonal immunoglobulin (Ig)G antibodies after exposure to immunogenic antigen(s). The National Interagency Confederation for Biological Research and collaborators rapidly produced and then evaluated anti-Ebola virus IgG immunoglobulins (collectively termed SAB-139) purified from Tc-bovine plasma after sequential hyperimmunization with an Ebola virus Makona isolate glycoprotein nanoparticle vaccine. SAB-139 was characterized by several in vitro production, research, and clinical level assays using wild-type Makona-C05 or recombinant virus/antigens from different Ebola virus variants. SAB-139 potently activates natural killer cells, monocytes, and peripheral blood mononuclear cells and has high-binding avidity demonstrated by surface plasmon resonance. SAB-139 has similar concentrations of galactose-α-1,3-galactose carbohydrates compared with human-derived intravenous Ig, and the IgG1 subclass antibody is predominant. All rhesus macaques infected with Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05 and treated with sufficient SAB-139 at 1 day (n = 6) or 3 days (n = 6) postinfection survived versus 0% of controls. This study demonstrates that Tc-bovines can produce pathogen-specific human Ig to prevent and/or treat patients when an emerging infectious disease either threatens to or becomes an epidemic.

Authors : Luke Thomas, Bennett Richard S, Gerhardt Dawn M, Burdette Tracey, Postnikova Elena, Mazur Steven, Honko Anna N, Oberlander Nicholas, Byrum Russell, Ragland Dan, St Claire Marisa, Janosko Krisztina B, Smith Gale, Glenn Gregory, Hooper Jay, Dye John, Pal Subhamoy, Bishop-Lilly Kimberly A, Hamilton Theron, Frey Kenneth, Bollinger Laura, Wada Jiro, Wu Hua, Jiao Jin-An, Olinger Gene G, Gunn Bronwyn, Alter Galit, Khurana Surender, Hensley Lisa E, Sullivan Eddie, Jahrling Peter B,



(4) Generation of monoclonal antibodies against 17α-hydroxyprogesterone for newborn screening of congenital adrenal hyperplasia.[TOP]

Pubmed ID :30006291
Publication Date : //
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by the deficiency of one of the five enzymes involved in the biosynthesis of corticosteroids. The most common form of the disease is the lack of 21-hydroxylase which provokes an accumulation of high levels of 17α-hydroxyprogesterone (17-OHP), the main biochemical marker for illness detection. Given the significance of neonatal diagnosis for ensuring a timely treatment to patients suffering from CAH, newborn screening is worldwide performed for the determination of 17-OHP from dried blood spots on filter paper. The non-specificity of antisera employed in immunoassays and the cross-reaction with fetal adrenal hormones produce an overestimation in the 17-OHP quantification. Immunization of mice with 17-OHP-3-(O-carboxymethyl) oxime-bovine serum albumin led to the generation of 15 anti-17-OHP IgG1-and-IgG2b-secreting hybridomas. The 6E2G9 monoclonal antibody presents cross-reactivity values similar to those achieved by rabbit antibodies employed in the solid phase of UMELISA® 17-OH Progesterona Neonatal, assay for the newborn screening of CAH in Cuba. Additionally, the use of 6E2G9 in the evaluation of dried blood spots samples from newborns on filter paper showed a decrease in the mean 17-OHP levels, thus demonstrating it can replace the conventional rabbit antisera.

Authors : Morejón García Greilys, García de la Rosa Iria, González Reyes Ernesto C, Rubio Torres Anett, Quintana Guerra Joel M, Hernández Marín Milenén, Pérez Mora Pedro L, Feal Carballo Sadys, Lafita Delfino Yesdiley, Pupo Infante Maylín, Castells Martínez Elisa M, Rosabal Poloshkov Alik, Hernández Pérez Liliana,



(5) Concentration of Immunoglobulins in Microfiltration Permeates of Skim Milk: Impact of Transmembrane Pressure and Temperature on the IgG Transmission Using Different Ceramic Membrane Types and Pore Sizes.[TOP]

Pubmed ID :29958476
Publication Date : //
The use of bioactive bovine milk immunoglobulins (Ig) has been found to be an alternative treatment for certain human gastrointestinal diseases. Some methodologies have been developed with bovine colostrum. These are considered in laboratory scale and are bound to high cost and limited availability of the raw material. The main challenge remains in obtaining high amounts of active IgG from an available source as mature cow milk by the means of industrial processes. Microfiltration (MF) was chosen as a process variant, which enables a gentle and effective concentration of the Ig fractions (ca. 0.06% in raw milk) while reducing casein and lactose at the same time. Different microfiltration membranes (ceramic standard and gradient), pore sizes (0.14⁻0.8 µm), transmembrane pressures (0.5⁻2.5 bar), and temperatures (10, 50 °C) were investigated. The transmission of immunoglobulin G (IgG) and casein during the filtration of raw skim milk (<0.1% fat) was evaluated during batch filtration using a single channel pilot plant. The transmission levels of IgG (~160 kDa) were measured to be at the same level as the reference major whey protein β-Lg (~18 kDa) at all evaluated pore sizes and process parameters despite the large difference in molecular mass of both fractions. Ceramic gradient membranes with a pore sizes of 0.14 µm showed IgG-transmission rates between 45% to 65% while reducing the casein fraction below 1% in the permeates. Contrary to the expectations, a lower pore size of 0.14 µm yielded fluxes up to 35% higher than 0.2 µm MF membranes. It was found that low transmembrane pressures benefit the Ig transmission. Upscaling the presented results to a continuous MF membrane process offers new possibilities for the production of immunoglobulin enriched supplements with well-known processing equipment for large scale milk protein fractionation.

Authors : Heidebrecht Hans-Jürgen, Toro-Sierra José, Kulozik Ulrich,



(6) Role of the MAPK/cJun NH-terminal kinase signaling pathway in starvation-induced autophagy.[TOP]

Pubmed ID :29950132
Publication Date : //
Autophagy is required for cellular homeostasis and can determine cell viability in response to stress. It is established that MTOR is a master regulator of starvation-induced macroautophagy/autophagy, but recent studies have also implicated an essential role for the MAPK8/cJun NH-terminal kinase 1 signal transduction pathway. We found that MAPK8/JNK1 and MAPK9/JNK2 were not required for autophagy caused by starvation or MTOR inhibition in murine fibroblasts and epithelial cells. These data demonstrate that MAPK8/9 has no required role in starvation-induced autophagy. We conclude that the role of MAPK8/9 in autophagy may be context-dependent and more complex than previously considered.

Authors : Barutcu Seda Avcioglu, Girnius Nomeda, Vernia Santiago, Davis Roger J,



(7) Conformational and Organizational Insights into Serum Proteins during Competitive Adsorption on Self-Assembled Monolayers.[TOP]

Pubmed ID :29936836
Publication Date : //
Physicochemical interactions of proteins with surfaces mediate the interactions between the implant and the biological system. Surface chemistry of the implant is crucial as it regulates the events at the interface. The objective of this study was to explore the performance of modified surfaces for such interactions relevant to various biomedical applications. Because of a wide range of surface wettability, we aimed to study protein behavior (i.e., conformational changes and their packing) during competitive protein adsorption. Three serum proteins (bovine serum albumin, BSA; fibrinogen, FB; and immunoglobulin G, IgG) were tested for their conformational changes and orientation upon adsorption on hydrophilic (COOH and amine), moderately hydrophobic (mixed and hybrid), and hydrophobic (octyl) surfaces generated via silanization. Modified surfaces were characterized using Fourier-transform infrared spectroscopy, contact angle, and atomic force microscopy (AFM) techniques. Adsorbed masses of proteins from single and binary protein solutions on different surfaces were quantified along with their secondary structure analyses. Maximum adsorbed protein masses were found to be on negatively charged and hydrophobic (octyl) surfaces because of ionic and hydrophobic interactions between protein molecules and surfaces, respectively. Side-on and end-on orientations of adsorbed protein molecules were analyzed using theoretical and AFM analyses. We observed compact and elongated forms of BSA molecules on hydrophilic and hydrophobic surfaces, respectively. We further found a linear increase in the α-helix content of BSA and β-sheet contents of FB and IgG proteins with the increasing side-on (%)-oriented protein molecules on the surfaces. This indicates that side-on orientations of adsorbed FB and IgG lead to the formation of β-sheets. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was employed to quantify the protein types and their ratio in competitively adsorbed proteins on different surfaces. A theoretical analysis was also used to determine the % secondary structures of competitively adsorbed proteins from BSA/FB and BSA/IgG solutions, which very well agreed with experimental results. The competitive protein adsorption from both BSA/FB and BSA/IgG solutions was found to be entropy-driven, as revealed by thermodynamic studies performed using isothermal titration calorimetry.

Authors : Hasan Abshar, Waibhaw Gyan, Pandey Lalit M,



(8) Experimental and bioinformatic approach to identifying antigenic epitopes in human α- and β-enolases.[TOP]

Pubmed ID :29922723
Publication Date : //
Human α- and β-enolases are highly homologous enzymes, difficult to differentiate immunologically. In this work, we describe production, purification and properties of anti-α- and anti-β-enolase polyclonal antibodies. To raise antibodies, rabbits were injected with enolase isoenzymes that were purified from human kidney (α-enolase) and skeletal muscle (β-enolase). Selective anti-α- and anti-β-enolase antibodies were obtained by affinity chromatography on either α- or β-enolase-Sepharose columns. On Western blots, antibodies directed against human β-enolase, did not react with human α-isoenzyme, but recognized pig and rat β-enolase. To determine what makes these antibodies selective bioinformatic tools were used to predict conformational epitopes for both enolase isoenzymes. Three predicted epitopes were mapped to the same regions in both α- and β-enolase. Peptides corresponding to predicted epitopes were synthesized and tested against purified antibodies. One of the pin-attached peptides representing α-enolase epitope (the C-terminal portion of the epitope 3 - SPDDPSRYISPDQ) reacted with anti-α-enolase, while the other also derived from the α-enolase sequence (epitope 2 - NVIKEKYGKDATN) was recognized by anti-β-enolase antibodies. Interestingly, neither anti-α- nor anti-β-antibody reacted with a peptide corresponding to the epitope 2 in β-enolase (GVIKAKYGKDATN). Further analysis showed that substitution of E with A in α-enolase epitope 2 peptide lead to 70% loss of immunological activity, while replacement of A with E in peptide representing β-enolase epitope 2, caused 67% increase in immunological activity. Our results suggest that E is essential for preserving immunologically active conformation in epitope 2 peptidic homolog, while it is not crucial for this epitope's antigenic activity in native β-enolase.

Authors : Pietkiewicz Jadwiga, Danielewicz Regina, Bednarz-Misa Iwona S, Ceremuga Ireneusz, Wiśniewski Jerzy, Mierzchala-Pasierb Magdalena, Bronowicka-Szydełko Agnieszka, Ziomek Edmund, Gamian Andrzej,



(9) A new monoclonal antibody against rhamnogalacturonan II and its application to immunocytochemical detection of rhamnogalacturonan II in Arabidopsis roots.[TOP]

Pubmed ID :29912643
Publication Date : //
Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly.

Authors : Zhou Ye, Kobayashi Masaru, Awano Tatsuya, Matoh Toru, Takabe Keiji,



(10) Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, CMab-5.[TOP]

Pubmed ID :29872736
Publication Date : //
CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, CMab-5 (IgG, kappa), recognized both CD44s and CD44v3-10. CMab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that CMab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the CMab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Authors : Yamada Shinji, Itai Shunsuke, Nakamura Takuro, Yanaka Miyuki, Kaneko Mika K, Kato Yukinari,