Recent advances in immobilization methods of antibodies on solid supports

Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody-antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures.     

C-Reactive protein-directed immobilization of phosphocholine ligands on a solid surface

The complexes of C-reactive protein (CRP) with its polymerizable phosphocholine ligands adsorbed at the styrene-water interface were polymerized. The molecularly imprinted polymer (MIP) exhibited a binding affinity for CRP comparable to that of immobilized anti-CRP antibody. The determination of human serum CRP using the MIP-based sandwich immunoassay has been demonstrated.     

Superparamagnetic core-shell nanoparticles as solid supports for peptide synthesis

Functional superparamagnetic core-shell nanoparticles are synthesized by a microwave assisted route and can be used as colloidal supports for peptide synthesis in "quasi solution".     

Oriented immobilization of proteins

Immobilized enzymes have found numerous applications in analytical, clinical, environmental and industrial chemistry. However, in most cases, immobilization leads to partial or total loss of activity. It is widely believed that the loss in activity is due to attachment of proteins on the immobilization support through several amino acid residues. This results in a random orientation of the immobilized protein and in increased structural deformation due to multi-point attachment. Several researchers have explored ways to orient proteins on surfaces, such that orderly organization, single point attachment and accessibility of the active site (or binding site) are possible. This article reviews the various approaches available to achieve oriented immobilization of proteins and its applications in several disciplines.     

Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support.

In order to eliminate the kinetic limitation of chymotryptic hydrolysis of proteins due to diffusion, nonporous hydroxyalkyl methacrylate solid support was developed and used for oriented immobilization of chymotrypsin by means of suitable polyclonal antibodies. Nonporous microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in an alcohol-toluene mixture stabilized with cellulose acetate butyrate. The resulting particles were 1.2 microm in diameter and possessed narrow size distribution. After modification with adipic acid dihydrazide they contained 2 micromol of reactive groups available for coupling of anti-chymotrypsin antibodies. Prepared immunosorbent adsorbed 166.7 microg of chymotrypsin per 1 g of dry carrier. Immobilized chymotrypsin retained practically 100% of its native proteolytic activity. Kinetic parameters of catalysis by chymotrypsin immobilized via this way were improved due to the good steric accessibility of the enzyme active site for high-molecular-mass substrates, when digestion of proteins in batch experiments was used.     

Effects on the hydrolysis of native starch and glycogen by a thermostable α-amylase after immobilization on solid supports

A thermostable α-amylase was immobilized on controlled-pore glass beads and contained in enzyme reactors. The optimization of the hydrolytic effect of the immobilized enzyme on glycogen and native starch as substrates was investigated. A pore size of the controlled-pore glass support of about 1489 Å was found to be optimum. The optimum pH was 6.0 for hydrolysis of the substrates. Complete hydrolysis of the substrates to glucose could be accomplished on-line at ambient temperature after passage of a sample through one immobilized α-amylase reactor at pH 6.0 and then through one immobilized amyloglucosidase reactor at pH 4.6.     

Multi-site coordination ligands assembled on organostannoxane supports

The reactions of bis(pyrazol-1-yl) acetic acid LCOOH (1) (L = (Pz)₂CH-) and bis(3,5-dimethylpyrazol-1-yl)acetic acid L′COOH (2) (L = (3,5-Me₂Pz)₂CH-) with organotin oxide (hydroxide) precursors, n-BuSn(O)(OH), n-Bu₂SnO, (n-Bu₃Sn)₂O and (Ph₃Sn)₂O has led to the isolation of several organotin compounds containing bispyrazolyl unit(s) on the periphery of the stannoxane structure [n-BuSn(O)O₂CL]₆ (3), [n-BuSn(O)O₂CL′]₆ (4), [n-Bu₃SnO₂CL]_n (5), [n-Bu₃SnO₂CL′]_n (6), [Ph₃SnO₂CL]_n (7), [Ph₃SnO₂CL′]_n (8), [{n-Bu₂SnO₂CL}₂O]₂ (9) and [{n-Bu₂SnO₂CL′}₂O]₂ (10). Compounds 5, 7, 9 and 10 have been structurally characterized by X-ray crystallography. In the solid state, these compounds possess interesting 3-D and 2-D supramolecular networks as a result of intermolecular C-H?O, C-H?N, C-H?Cl and C-H?π interactions.     

Bartoli indole synthesis on solid supports

[reaction: see text] Bartoli indole synthesis has been performed for the first time on solid supports. Starting from Merrifield resin, immobilization of five nitro benzoic acids was performed. Addition of four different alkenyl Grignard reagents and basic cleavage leads to substituted methyl indole carboxylates in excellent purities. Features of this reaction are the stability of halide groups, ester moieties, and tolerance of o,o'-unsubstituted nitro resins. Heck and Sonogashira reactions are also possible with immobilized indoles.     

Synthesis of diaza-crown ethers on solid supports

Diamines and diiodides derived from diethylene glycol or triethylene glycol are condensed on alumina coated with alkali fluorides in acetonitrile to form diaza-crown ethers in good yield. Comparison of the yields suggests that the contribution of the template effect, if any, is small, and the data reflect rather increasing basicity of the solid supports in passing from LiF to KF on alumina.     

Single-site catalyst immobilization using magnesium chloride supports

Immobilization and activation of a broad range of titanium-, chromium-and nickel-based single-site catalysts for ethylene polymerization has been carried out using supports of type MgCl₂/AlR_n(OEt)_{3 − n} , prepared by reaction of AlR₃ with adducts of magnesium chloride and ethanol. The spherical particle morphology of the support is retained and replicated during catalyst immobilization and polymerization, yielding polyethylenes with controlled particle size and morphology. The single-site nature of these catalysts is also retained, giving polymers with narrow molecular weight distribution. Furthermore, very high catalyst activities can be obtained as a result of a stabilizing effect of the support, which prevents the rapid decay in activity often observed in homogeneous polymerization with these catalysts. The text was submitted by the authors in English.     

Simultaneous multiple peptide synthesis under continuous flow conditions on cellulose paper discs as segmental solid supports

A new approach to the simultaneous chemical synthesis of large numbers of different peptide sequences is described.¹ The particular features of the method presented² are: - cellulose paper discs serve as solid supports for Individual peptide sequences, - the peptide chains are linked to the cellulose via a p-alkoxybenzyl ester anchor cleavable by mild treatment with trifluoroacetic acid in dichloromethane, - the syntheses of peptides are accomplished using N-α-Fmoc protected amino acids, - all reactions are carried out in a simple multi-column continuous flow device with the cellulose discs tightly stacked in column reactors, - as many as 100 discs can be reacted simultaneously with the same amino acid derivative in one reaction column.     

Catalysts with mixed ligands on immobilized supports. Electronic and steric advantages

[reaction: see text] Immobilized dirhodium(II) catalysts having mixed chiral ligands enhance reactivity (AH = azetidinone) and influence stereoselectivity in cyclopropanation and carbon-hydrogen insertion reactions.     

Chemically modified nylons as supports for enzyme immobilization

An isocyanide derivative of nylon, polyisonitrile-nylon (1,2), was used as a starting material whereby, through a series of modification reactions, different chemically reactive functional groups could be introduced on the polyamide backbone. The chemistry employed allowed for considerable flexibility in the choice of procedures for covalent fixation of proteins, all starting from the same chemically reactive parent polymer, polyisonitrile-nylon. Thus, polyisonitrilenylon could be used directly for the immobilization of enzymes via fourcomponent condensation reactions. The isocyanide functional groups of the parent polymer could be transformed, by treatment with bromine, into the strongly electrophilic dibromoisocyanide (—N=CBr^b2) groups. The selectivity of the —N=CBr^b2 group toward the various functional groups present in proteins could be regulated by appropriate control of the pH of the coupling reaction. Dibromoisocyanide-nylon was also further modified into other types of chemically reactive nylon derivatives.     

Palladium-catalyzed synthesis of uridines on polystyrene-based solid supports

In this paper, the solid-phase synthesis of various substituted pyrimidine nucleosides is described starting from 2'-deoxyuridine, which has been attached through a base labile linker to polystyrene resins. The utility of the Pd(0) cross-coupling to functionalized pyrimidine nucleosides is expanded herein to include reactions of resin-supported 5-iodo-2'-deoxyuridine under Sonogashira, Stille, Heck, and Suzuki conditions. Upon cleavage with MeONa, a library of 5-substituted pyrimidine nucleosides was obtained in good (under Sonogashira and Stille conditions) to moderate (under Heck or Suzuki conditions) yields and high purity. Except the Suzuki-type reactions, the presented methods exhibit a significant improvement and facilitate the synthetic procedure with respect to purification and yields (determined after filtration over silica gel).