Covalent coupling

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

Gottenbosa B, Henny C, van der Meia, Klatterb F, Nieuwenhuisb P, and Busscher HJ. In vitro and in vivo antimicrobial activity of covalently coupled quaternary ammonium silane coatings on silicone rubber. Biomaterials, 2002, 23, 1417-1423. 

Figure 1. Covalently Coupled Polymer-Drug System.

Dopamine, a neurotransmitter, was covalently coupled, via an amide bond, to a modified polystyrene having A-(2-(3,4-dihydroxyphenyl)ethyl) isonicotinamide units. The dopamine-coupled polymer was coated onto glassy carbon electrodes. In aqueous electrolyte solutions (pH 7), cathodic current caused cleavage of the amide linkage and release of dopamine at potentials more negative than 0.9 V [41]. The chemical scheme for the amide bond cleavage is presented in Figure 18. 

Weetall, H.H. (1993) Preparation of immobilized proteins covalently coupled through silane coupling agents to inorganic supports. Applied Biochemistry and Biotechnology, 41, 157-188. 

Figure 10. Covalent coupling on aldehyde-slides (www.arrayit.com).

Joyce J, Cook J, Chabot D et al (2006) Immunogenicity and protective efficacy of Bacillus anthracis poly-y-D-glutamic acid capsule covalently coupled to a protein carrier using a novel triazine-based conjugation strategy. J Biol Chem 281 4831 -843... 

Figure 2.5 The cisplatin reactive group can covalently couple to methionine-, cysteine-, and histidine-containing peptides or proteins. It also reacts with guanine groups to form a covalent modification on the N7 nitrogen.

Figure 5.24 SADP reacts with amines via its NHS ester end to produce amide bonds. The modified molecule then may be photoactivated to create a nucleophile-reactive dehydroazepine intermediate able to covalently couple with amine-containing compounds.

Many of the applications of dendrimers involve the covalent coupling of other molecules to the dendrimer surface or to points within the branched structure. These attached molecules... 

Primary amine-containing polymeric particles are available from a number of manufacturers and have either aliphatic or aryl amine groups on their surface. Occasionally, a particle type may have secondary or tertiary amines present, but these should be avoided for covalent coupling, as primary amines typically give better reaction yields than secondary amines and tertiary amines are unreactive. 

Maehashi et al. (2007) used pyrene adsorption to make carbon nanotubes labeled with DNA aptamers and incorporated them into a field effect transistor constructed to produce a label-free biosensor. The biosensor could measure the concentration of IgE in samples down to 250 pM, as the antibody molecules bound to the aptamers on the nanotubes. Felekis and Tagmatarchis (2005) used a positively charged pyrene compound to prepare water-soluble SWNTs and then electrostatically adsorb porphyrin rings to study electron transfer interactions. Pyrene derivatives also have been used successfully to add a chromophore to carbon nanotubes using covalent coupling to an oxidized SWNT (Alvaro et al., 2004). In this case, the pyrene ring structure was not used to adsorb directly to the nanotube surface, but a side-chain functional group was used to link it covalently to modified SWNTs. 

---