Conjugation of a monoclonal antibody with a DTPA modified random copolymer of hydroxyethyl methylacrylate and methyl methacrylate (original) (raw)
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The Chemical Engineering Journal, 1986
Attempts to scale up immunoaffinity separations have highlighted the limitations of the commonly employed CNBr-actiuated agarose gel supports. As an aid to the selection of an alternative configuration, we investigated the behaviour of a monoclonal antibody covalently immobilized to styrene, nylon and acrylic acid using several different chemical methods. The level of uptake of antibody was found to be dependent on the immobilization method used. The apparent antibody-antigen dissociation constant of the immobilized antibody averaged 1.5 X 1 OM6 M. This value was an order of magnitude lower than previously reported results for the same antibody in free solution and was not affected by either the immobilization method or the support polymer used. The different covalent bonds were subjected to commonly encountered eluting agents. Most were stable in at least one solution capable of breaking antibody-antigen bonds although all were highly labile in 4 M KI. The most stable bond in elutant was formed by glutaraldehyde-activated amino groups, followed by bonds formed through N-hydroxysuccinimide esters. Overall, the results suggest that immobilization through glutaraldehydeactivated amino groups, a simple procedure, results in a highly stable bond without adversely affecting antibody immunological reactivity.
Macromolecular Bioscience, 2013
Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.
Antibody orientation enhanced by selective polymer–protein noncovalent interactions
Analytical and Bioanalytical Chemistry, 2009
A unique interaction has been found between protein G' (a truncated recombinant bacterial "alphabet" protein which aligns by noncovalent attachment to the antibody stem) and poly(methyl methacrylate), a thermoplastic polymer substrate, which can be easily fabricated using high-rate processes. Significantly improved orientation efficiency with traditional passive adsorption for this system (termed ALYGNSA) has been achieved as compared to the same assay performed on a polystyrene substrate with protein G'. Results were consistent with an average alignment of 80% of the human immunoglobulin G capture antibody which translated into a 30% to 50% improved alignment over an array of industry standards tested. Laser scanning confocal microscopy confirmed the immunological results. Studies of additional poly(methyl methacrylate) polymer derivatives and protein biolinker (A and AG) combinations have been conducted and have revealed different degrees of antibody alignment. These findings may lead to additional novel noncovalent methods of antibody orientation and greater sensitivity in immunological assays.
Proceedings of the National Academy of Sciences, 1980
The synthetic polypeptide antigen poly(LTyr, LGlu)-poly(DLAl)- -poly(LLys)[T,G)-A- -L] was covalently linked to N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP), which is the minimal adjuvant-active structure that can substitute for Mycobacteria in complete Freund's adjuvant. When injected in aqueous solution into mice, the completely synthetic conjugate elicited significant antibody responses specific to (T,G)-A- -L, whereas (T,G,)-A- -L alone administered under the same conditions did not lead to antibody production. The conjugate was much more efficient in eliciting (T,G)-A- -L responses than was a mixture of DMP and (T,G)-A- -L. One hundred micrograms of MDP mixed with 10 micrograms of (T,G)-A- -L resulted in production of (T,g)-A- -L-specific antibodies. However, the titers obtained were much lower than those observed with 10 micrograms of the conjugate, MDP-(T,G)-A- -L, which contained less than 1 microgram of MDP. MDP was enhanced when the mixture was administered in incompl...
Journal of chromatography. B, Biomedical sciences and applications, 2001
Through their specificity and affinity, antibodies are useful tools in research and medicine. In this study, we investigated a new type of chromatographic method using a thermosensitive polymer for the purification of antibodies against a dextran derivative (DD), as a model. The thermally reversible soluble-insoluble poly(N-isopropylacrylamide)-dextran derivative conjugate, named poly(NIPAAm)-DD, has been synthesized by conjugating amino-terminated poly(N-isopropylacrylamide) to a DD via ethyl-3-(3-dimethylaminopropyl)-carbodiimide. On one hand, this report describes the two steps of poly(NIPAAm)-DD conjugation and characterization. On the other hand, the poly(NIPAAm)-DD conjugate was used as a tool to purify polyclonal antibodies in serum samples from rabbits subcutaneously immunized with the derivatized dextran. Antibodies were purified and quantified by immunoenzymatic assays. Our results indicate that antibodies recognized both DD and poly(NIPAAm)-DD. In contrast, they did not b...
Carboxylated Latexes for Covalent Coupling Antibodies, I
Journal of Colloid and Interface Science, 1995
polymerization are negatively charged in a broad pH range The aim of the present work is to prepare and characterize of 3-10 (4), but their surface charge density is extremely carboxylated latexes (CLs) in order to bind antibodies covalently high (4). At a low ionic strength the antibody cannot peneand efficiently. Carboxylated latexes were prepared by surfactanttrate the double layer of the particle and thus, no coupling free emulsion polymerization using an azo-initiator, which prooccurs (11). The carboxylated latexes prepared by an azovides carboxyl end groups on the latex surface directly. Two latexes initiator have a rigid interface and the surface charge density were characterized using different techniques: particle size by is significantly lower. These types of particles have been electron microscopy and photocorrelation spectroscopy, surface used less in covalently coupling antibodies and antigens to charge density by conductimetric and potentiometric titrations, polymer carriers in the development of particle-enhanced and electrophoretic mobility versus pH and ionic strength (KBr). The colloidal stability of both latexes shows that these carboxylated optical immunoassays. latexes can be used for biomedical applications. Activation of car-In this paper, we report the preparation of surfactant-free boxyl end groups was performed using the carbodiimide method, carboxylated polymer latexes which enables the covalent and the best conditions for covalent coupling of IgG were obtained. coupling of antibodies with a high efficiency rate. The elec
Formation of multimeric antibodies for self-delivery of active monomers
Drug Delivery, 2017
Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.