scholarly journals Preactivation Crosslinking – An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

scholarly journals Preactivation Crosslinking—An Efficient Method for the Oriented Immobilization of Antibodies

2019 ◽  
Vol 2 (2) ◽  
pp. 35 ◽  
Author(s):  
Barbara Schroeder ◽  
Hoa Le Xuan ◽  
Jule L. Völzke ◽  
Michael G. Weller
Keyword(s):  
Magnetic Particles ◽  
Binding Capacity ◽  
Protein A ◽  
Covalent Immobilization ◽  
High Yield ◽  
Binding Partner ◽  
Oriented Immobilization ◽  
Novel Method ◽  
Antibody Preparations ◽  
Culture Supernatants

Crosslinking of proteins for their irreversible immobilization on surfaces is a proven and popular method. However, many protocols lead to random orientation and the formation of undefined or even inactive by-products. Most concepts to obtain a more targeted conjugation or immobilization requires the recombinant modification of at least one binding partner, which is often impractical or prohibitively expensive. Here a novel method is presented, which is based on the chemical preactivation of Protein A or G with selected conventional crosslinkers. In a second step, the antibody is added, which is subsequently crosslinked in the Fc part. This leads to an oriented and covalent immobilization of the immunoglobulin with a very high yield. Protocols for Protein A and Protein G with murine and human IgG are presented. This method may be useful for the preparation of columns for affinity chromatography, immunoprecipitation, antibodies conjugated to magnetic particles, permanent and oriented immobilization of antibodies in biosensor systems, microarrays, microtitration plates or any other system, where the loss of antibodies needs to be avoided, and maximum binding capacity is desired. This method is directly applicable even to antibodies in crude cell culture supernatants, raw sera or protein-stabilized antibody preparations without any purification nor enrichment of the IgG. This new method delivered much higher signals as a traditional method and, hence, seems to be preferable in many applications.

scholarly journals Affinity sedimentation and magnetic separation with plant-made immunosorbent nanoparticles for therapeutic protein purification

2021 ◽  
Author(s):  
Matthew J McNulty ◽  
Anton Schwartz ◽  
Jesse Delzio ◽  
Kalimuthu Karuppanan ◽  
Aaron Jacobson ◽  
...  
Keyword(s):  
Protein Purification ◽  
Magnetic Separation ◽  
Magnetic Particles ◽  
Binding Capacity ◽  
Therapeutic Protein ◽  
Vector System ◽  
High Yield ◽  
Affinity Capture ◽  
Processing Scheme ◽  
Industry Standards

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via crop cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity of current industry standards by an order of magnitude.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

2020 ◽  
Vol 7 (2) ◽  
pp. 121-133
Author(s):  
Ayesha Akhtar ◽  
Shivakumar Arumugam ◽  
Shoaib Alam
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Affinity Chromatography ◽  
Protein A ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
High Yield ◽  
Staphylococcal Protein A ◽  
Purification Step ◽  
Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

Magnetic Separation of Antibodies with High Binding Capacity by Site-Directed Immobilization of Protein A-Domains to Bare Iron Oxide Nanoparticles

2021 ◽  
Author(s):  
Yasmin Kaveh-Baghbaderani ◽  
Raphaela Allgayer ◽  
Sebastian Patrick Schwaminger ◽  
Paula Fraga-García ◽  
Sonja Berensmeier
Keyword(s):  
Iron Oxide ◽  
Magnetic Separation ◽  
Iron Oxide Nanoparticles ◽  
Binding Capacity ◽  
Protein A ◽  
Oxide Nanoparticles ◽  
High Binding ◽  
A Domains

scholarly journals A novel procedure for the rapid isolation of surfactant protein A with retention of its alveolar-macrophage-stimulating properties

1995 ◽  
Vol 309 (2) ◽  
pp. 551-555 ◽  
Author(s):  
J F van Iwaarden ◽  
F Teding van Berkhout ◽  
J A Whitsett ◽  
R S Oosting ◽  
L M G van Golde
Keyword(s):  
Alveolar Macrophage ◽  
Alveolar Macrophages ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Normal Rat ◽  
Normal Human ◽  
Novel Method ◽  
Alveolar Proteinosis ◽  
Simplex Virus

Previous studies have shown that surfactant protein A (SP-A) derived from alveolar-proteinosis patients activates rat alveolar macrophages. However, it is not known if normal rat, dog and human SP-A can also stimulate alveolar macrophages. As alveolar-proteinosis SP-A has a slightly different structure from ordinary SP-A, it would be possible that the ascribed alveolar-macrophage-stimulating properties of SP-A are restricted to alveolar-proteinosis SP-A. To clarify this issue, we isolated SP-A from normal rat and dog pulmonary surfactants, using the same isolation technique commonly used for the isolation of alveolar-proteinosis SP-A, i.e. by butanol precipitation. In contrast with human alveolar-proteinosis SP-A, rat and dog SP-A obtained thus could not activate rat alveolar macrophages to produce oxygen radicals or enhance the phagocytosis of fluorescein isothiocyanate-labelled herpes simplex virus. However, rat, dog and normal human SP-A isolated by a novel method, involving extraction from pulmonary surfactant by using n-octyl beta-D-glucopyranoside and subsequent purification by cation-exchange chromatography, were able to elicit an oxidative burst in rat as well as normal human alveolar macrophages. In addition, dog and rat SP-A obtained thus stimulated the phagocytosis of herpes simplex virus by rat alveolar macrophages. These findings indicate that normal human, rat and dog SP-A have the same alveolar-macrophage-stimulating properties as human alveolar proteinosis SP-A. Dog and rat SP-A isolated by this novel method had the same Ca(2+)-dependent self-aggregation and lipid-aggregation properties as SP-A isolated by butanol precipitation. The new and milder isolation procedure yielded SP-A of high purity, as judged by SDS/PAGE and ELISA.

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels—Alder Bioconjugation

ChemInform ◽  
2004 ◽  
Vol 35 (8) ◽  
Author(s):  
Hallie A. Latham-Timmons ◽  
Andreas Wolter ◽  
J. Shawn Roach ◽  
Rubina Giare ◽  
Michael Leuck
Keyword(s):  
Covalent Immobilization ◽  
Diels Alder ◽  
Novel Method

Secreted surfactant protein A from fetal membranes induces stress fibers in cultured human myometrial cells

2010 ◽  
Vol 298 (6) ◽  
pp. E1188-E1197 ◽  
Author(s):  
Michelle Breuiller-Fouché ◽  
Olivier Dubois ◽  
Mourad Sediki ◽  
Ignacio Garcia-Verdugo ◽  
Nades Palaniyar ◽  
...  
Keyword(s):  
Western Blot ◽  
Conditioned Medium ◽  
Protein A ◽  
Rho Kinase ◽  
Surfactant Protein ◽  
Fiber Formation ◽  
Fetal Membranes ◽  
Myometrial Cells ◽  
Cell Functions ◽  
Culture Supernatants

In the present study, we investigated the ability of human fetal membranes (amnion and choriodecidua) to regulate human maternal uterine cell functions through the secretion of surfactant protein (SP)-A and SP-D at the end of pregnancy. We detected the expression of both SP-A (SP-A1 and SP-A2) and SP-D by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed that human fetal membranes expressed both SP-A and SP-D. By Western blot analysis, we demonstrated that SP-A protein expression was predominant in choriodecidua, whereas the amnion predominantly expressed SP-D. Only the secretion of SP-A was evidenced in the culture supernatants of amnion and choriodecidua explants by immunodot blot and confirmed by Western blot. Exogenous human purified SP-A induced stress fiber formation in cultured human myometrial cells via a pathway involving Rho-kinase. Conditioned medium from choriodecidua and amnion explants mimicked the SP-A effect. Treatment of myometrial cells with SP-A-depleted conditioned medium from choriodecidua or amnion explants failed to change the actin dynamic. These data indicate that SP-A released by human fetal membranes is able to exert a paracrine regulation of F-actin filament organization in myometrial cells.

Novel Method for the Covalent Immobilization of Oligonucleotides via Diels-Alder Bioconjugation

2003 ◽  
Vol 22 (5-8) ◽  
pp. 1495-1497 ◽  
Author(s):  
Hallie A. Latham-Timmons ◽  
Andreas Wolter ◽  
J. Shawn Roach ◽  
Rubina Giare ◽  
Michael Leuck
Keyword(s):  
Covalent Immobilization ◽  
Diels Alder ◽  
Novel Method

Influence Analysis of Sampling Time for Synthesis of Carbon Nanotubes in the V-Type Pyrolysis Flame

2011 ◽  
Vol 221 ◽  
pp. 235-239 ◽  
Author(s):  
Yuan Chao Liu ◽  
Bao Min Sun ◽  
Zhao Yong Ding
Keyword(s):  
Carbon Nanotubes ◽  
Electron Microscope ◽  
Experimental System ◽  
Growth Period ◽  
Sampling Time ◽  
High Yield ◽  
Spray Pyrolysis Method ◽  
Transmission Electron ◽  
Novel Method ◽  
Synthesis Of Carbon Nanotubes

Synthesis of carbon nanotubes from V-type pyrolysis flame is a kind of novel method. It needs simple laboratory equipments and normal atmosphere pressure. The V-type pyrolysis flame experimental system is introduced. Carbon source is the carbon monoxide and heat source is from acetylene/air premixed flame. Pentacarbonyl iron, served as catalyst, is transported by spray- pyrolysis method into the flame. The carbon nanotubes were characterized by scanning electron microscope and transmission electron microscope. This study aims to find the formation rule of carbon nanotubes from the V-type pyrolysis flame in different sampling times. The carbon nanotubes with less impurity and high yield were captured successfully in the V-type pyrolysis flame. The diameter of carbon nanotubes was approximate between 10nm and 20nm, and its length was dozens of microns. When the sampling time was below 3 minutes, the growth of carbon nanotubes came into the preparation growth period. The length of the carbon nanotubes increased gradually and the diameter had no obvious change with the extension of sampling time. When the sampling time was continued to the 5th minute, the growth of carbon nanotubes came into the exuberant growth period. The carbon nanotubes growth was finished within 5minutes. Longer sampling time was meaningless after the carbon nanotubes formation.

Sign in / Sign up

Export Citation Format

Share Document