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Antibody purification

Tarang Sharma, Chandresh Sharma, Anurag Sankhyan, Sanjiv Pal Bedi, Shinjini Bhatnagar, Navin Khanna, Vikas Gautam, Sunil Sethi, Sudhanshu Vrati, Ashutosh Tiwari
Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever...
June 21, 2018: Immunologic Research
Jadwiga Pietkiewicz, Regina Danielewicz, Iwona S Bednarz-Misa, Ireneusz Ceremuga, Jerzy Wiśniewski, Magdalena Mierzchala-Pasierb, Agnieszka Bronowicka-Szydełko, Edmund Ziomek, Andrzej Gamian
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...
September 2018: Biochemistry and Biophysics Reports
Jonathan Visentin, Lionel Couzi, Claire Dromer, Martine Neau-Cransac, Gwendaline Guidicelli, Vincent Veniard, Karine Nubret-le Coniat, Pierre Merville, Carmelo Di Primo, Jean-Luc Taupin
Human leukocyte antigen (HLA) donor-specific antibodies are key serum biomarkers for assessing the outcome of transplanted patients. Measuring their active concentration, i.e. the fraction that really interacts with donor HLA, and their affinity could help deciphering their pathogenicity. Surface plasmon resonance (SPR) is recognized as the gold-standard for measuring binding kinetics but also active concentrations, without calibration curves. SPR-based biosensors often suffer from non-specific binding (NSB) occurring with the sensor chip surface and the immobilized targets, especially for complex media such as human serum...
June 7, 2018: Biosensors & Bioelectronics
Julien Valton, Valerie Guyot, Bijan Boldajipour, Cesar Sommer, Thomas Pertel, Alexandre Juillerat, Aymeric Duclert, Barbra Johnson Sasu, Philippe Duchateau, Laurent Poirot
CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety...
June 12, 2018: Scientific Reports
Lan Zhao, Wei Wei, Yongdong Huang, Rongyue Zhang, Kai Zhu, Dongxia Hao, Zhiguo Su, Guanghui Ma
Protein A chromatography has been a popular method for purification of therapeutic monoclonal antibodies (mAb). Protein A chromatographic media using alkali-resistant rProtein A ligands from site-directed coupling method have been pursued for both high dynamic capacities and excellent stabilities. However, the mechanism of rProtein A leaking under cleaning-in-place (CIP) conditions is not very clear and difficulties have been commonly encountered when improving the media's chromatographic performance. We investigated the chromatographic performance of site-directed coupled rProtein A chromatographic media during CIP procedure...
June 9, 2018: Journal of Immunological Methods
Wenyue Guan, Romain Guyot, Frédéric Flamant
Understanding the transcriptional function of thyroid hormone receptors implies a precise analysis of their interactions with chromatin and other protein components of the cells. We present here two protocols that are routinely used in our laboratory. The first co-immunoprecipitation procedure allows addressing the capacity of proteins to form stable multiprotein complexes with TRs in cells. The chromatin affinity purification enables us to define the sites occupied by TRs on chromatin. In this case the lack of high quality antibodies is circumvented by introducing an N-terminal tag in TR, with unspecific affinity for immunoglobulins...
2018: Methods in Molecular Biology
Patrick P Komane, Pradeep Kumar, Thashree Marimuthu, Lisa C du Toit, Pierre P D Kondiah, Yahya E Choonara, Viness Pillay
The complete synthesis, optimization, purification, functionalization and evaluation of vertically aligned multiwalled carbon nanotubes (VA-MWCNTs) was reported for potential application in dexamethasone delivery to the ischemic brain tissue. The conditions for high yield were optimized and carbon nanotubes functionalized and PEGylated prior to dexamethasone loading. Morphological changes were confirmed by SEM and TEM. Addition of functional groups to MWCNTs was demonstrated by FTIR. Thermal stability reduced following MWCNTs functionalization as demonstrated in TGA...
June 10, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Marie Godar, Kim Deswarte, Karl Vergote, Michael Saunders, Hans de Haard, Hamida Hammad, Christophe Blanchetot, Bart N Lambrecht
BACKGROUND: Asthma is a chronic inflammatory airway disease in which innate and adaptive immune cells act together to cause eosinophilic inflammation, goblet cell metaplasia (GCM) and bronchial hyperreactivity (BHR). In clinical trials employing biologicals against interleukin (IL)-4Rα or IL-5, only a subset of moderate-to-severe asthmatics responded favorably, suggesting that distinct pathophysiological mechanisms are at play in subgroups of patients, called endotypes. However, the effect of multiple cytokine blockade using bispecific antibodies (Ab) has not been tested...
June 8, 2018: Journal of Allergy and Clinical Immunology
F Verachi, Z Percario, P Di Bonito, E Affabris, C Amici, L Accardi
In Human Papillomaviruses- (HPV-) associated carcinogenesis, continuous expression of the E6 oncoprotein supports its value as a potential target for the development of diagnostics and therapeutics for HPV cancer. We previously reported that the I7 single-chain antibody fragment (scFv) specific for HPV16 E6, expressed as an intrabody by retroviral system, could inhibit significantly the growth of cervical cancer cells in vitro and was even able to reduce tumor development in experimental HPV-related cancer models...
2018: BioMed Research International
Sara Kanje, Raminta Venskutonytė, Julia Scheffel, Johan Nilvebrant, Karin Lindkvist-Petersson, Sophia Hober
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa , is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid (EDTA). Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified...
June 7, 2018: Journal of Molecular Biology
Julie Robinson, David Roush, Steve Cramer
Although a platform process has been established for purification of antibodies, a deep, fundamental understanding of how these molecules interact with chromatography resins has yet to be developed. The increasing prevalence of antibody-related therapeutics and associated purification challenges further motivate research into these molecular level interactions. The objective of this work is to understand the nature (i.e. size and properties) of preferred protein-ligand binding regions for large, multi-domain molecules such as antibodies...
May 28, 2018: Journal of Chromatography. A
Nataliya Balashova, Alexander Giannakakis, Angela C Brown, Evan Koufos, Roland Benz, Tsutomu Arakawa, Hsin-Yao Tang, Edward T Lally
A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His6 ) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic...
June 4, 2018: Gene
Ryoichi Mori, Katsuya Tanaka, Isao Shimokawa
Inflammation at a wound site is essential for preventing infection. However, misregulated inflammation leads to pathologies of the healing process, including chronic non-healing wounds and scarring. MicroRNAs (miRNAs) are key regulators of the inflammatory response and tissue repair, acting by translational processing of target mRNAs. In the final step of miRNA processing, Argonaute 2 (Ago2)-bound mature miRNA complexes bind to target mRNAs and inhibit their translation. A variety of wound healing-related miRNAs have been identified and their misregulation likely contributes to wound pathologies, including scarring and chronic healing...
June 5, 2018: Development, Growth & Differentiation
Pranveer Singh
Recent advances in Recombinant antibody technology / Antibody Engineering has given impetus to the genetic manipulation of antibody fragments that has paved the way for better understanding of the structure and functions of immunoglobulins and also has escalated their use in immunotherapy. Bacterial expression system such as Escherichia coli has complemented this technique through the expression of recombinant antibodies. Present communication has attempted to optimize the expression and refolding protocol of single chain fragment variable (ScFv) and single chain antigen binding fragment (ScFab) using E...
July 2018: Biochemistry and Biophysics Reports
Fortunato Ferrara, Martin Kolnik, Sara D'Angelo, Frank M Erasmus, Daniela Vorholt, Andrew R M Bradbury
The study of the biology and function of B cells, or the dissection and in vitro creation of enormous recombinant antibody repertoires, requires the isolation of large numbers of pure CD19+ B cells. The StraightFrom® Leukopak CD19 MicroBead Kit was recently introduced as a fast and robust kit to isolate human CD19+ B cells. This uses paramagnetic microbeads conjugated to high-affinity anti-CD19 monoclonal antibodies to bind B cells in leukapheresis (Leukopak) samples. The overall purity of the isolated cells, together with the characterization of the different CD19+ subclasses, was assessed by flow cytometry using a recombinant (REAffinity) antibody panel, revealing that the method allowed the recovery of over 93% of CD19+ B cells without any pre-purification step...
June 2, 2018: New Biotechnology
Luis Alberto Estrada-Figueroa, José Alfredo Díaz-Gandarilla, Verónica Ivonne Hernández-Ramírez, María Maylen Arrieta-González, Carlos Osorio-Trujillo, José Luis Rosales-Encina, Toledo-Leyva Alfredo, Patricia Talamás-Rohana
Cyclooxygenase-2 (COX-2) is an enzyme responsible of prostaglandins production, such as prostaglandin E2 (PGE2 ), an immune response modulator that regulates the immune system to inhibit Th1 and to promote Th2 cytokines production. Many parasites modulate their host immune response through PGE2 effects; however, in parasites, only one protein with COX activity has been described, the α-actinin of Entamoeba histolytica. Prostanoids production has been reported in some species of Leishmania but not the enzymes responsible of their production...
June 1, 2018: Biochimie
Jordan B Fishman, Eric A Berg
Caprylic acid has been used to enrich IgG from serum, ascites, and cell culture supernatants by precipitating the non-IgG serum proteins. By precipitating all of the unwanted serum proteins rather than the antibodies, the tendency of antibodies to aggregate when precipitated is avoided. This method should not be used with antibody sources that contain low concentrations of antibody, such as many cell culture supernatants, owing to the potential loss of high-affinity antibodies, which may be bound by the caprylic acid...
June 1, 2018: Cold Spring Harbor Protocols
Jordan B Fishman, Eric A Berg
Before the widespread availability and use of Protein A (rabbit) or Protein G (rodent) for the purification of IgG, the use of an ammonium sulfate "cut" was the standard method to isolate IgG and other serum proteins. The addition of ammonium sulfate reduces the effective solubility of proteins through direct competition for binding sites on the surface of the protein. The resulting precipitated proteins can be isolated by centrifugation. An ammonium sulfate concentration between 40% and 50% results in the precipitation of IgG from most species, and thus 50% is usually used...
June 1, 2018: Cold Spring Harbor Protocols
Frank Z Stanczyk, Xia Xu, Patrick M Sluss, Louise A Brinton, Katherine A McGlynn
Higher circulating estradiol levels are generally obtained using conventional radioimmunoassays (RIA) compared to liquid chromatography/tandem mass spectrometry (LC-MS/MS) assays, and this has been attributed to the presence of estradiol metabolites that cross-react with the antibody used in the RIA. This study aimed to determine which estradiol metabolites may contribute to this effect. LC-MS/MS analysis was performed on 70 serum samples from premenopausal women, after purification by extraction and Celite column partition chromatography as would be used prior to conventional RIA for estradiol...
May 28, 2018: Clinica Chimica Acta; International Journal of Clinical Chemistry
Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Tetsuo Sato, Shizuka Hayashi, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Takahiro Kusakabe
Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs...
May 31, 2018: Journal of General Virology
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