Cyanogen bromide particles

This method was used, for example, for the solid-phase immunoassay of thyroxine (affinity chromatography). Various activation methods (CDI, periodate, and cyanogen bromide procedures) were compared with each other for coupling antibodies to magnetizable cellulose/iron oxide solid-phase particles. 211]... 

Figure 14.17 Cyanogen bromide can be used to activate a hydroxyl-particle to a reactive cyanate ester, which then can be used to couple amine-containing ligands.

Cyanogen bromide can be used to activate hydroxyl groups on particles to create reactive cyanate esters, which then can be coupled to amine-containing ligands to form an isourea bond (Figure 14.17). CNBr activation also can produce cyclic imidocarbonate groups, which are less reactive than the cyanate ester, but can form imidocarbonate bonds. The exact reactive species formed by the reaction is dependent on the structure of the hydroxylic support being activated (Kohn and Wilchek, 1982). 

Add a drop of the cyanogen bromide solution at a time to the particle suspension with constant mixing at room temperature. The entire solution should be added to the particles over the course of about 10 seconds. 

Several other methods are proposed, such as use of coulomb force between anionic DNA and cationic particle (7), use of biospecific affinity between biotin-labeled DNA and avidin-carrying particle (8), etc. Other methods for immobilization of DNA on the particle include reactions using cyanogen bromide, glutaraldehyde, and cyanuric chloride (9). 

Techniques for the coupling of antibodies to cellulose particles or tubes were initially unreliable, time-consuming, and hazardous as they involved toxic reagents such as cyanogen bromide. A simple and convenient method is now available which employs I,I-carbonyldi-imidazole to couple antibody to microcrystalline cellulose. 

Cyanogen bromide (CNBr)-activated insoluble matrices, such as microcrystalline cellulose particles, or paper disks, can be chemically... 

Van Leemputten E and Horisberger M. Immobilization of enzymes on magnetic particles. Biotechnol. Bioeng. 1974 16 385-396. lost R, Miron T, and Wilchek M. The mode of adsorption of proteins to aliphatic and aromatic amines coupled to cyanogen bromide-activated agarose. Biochim. Biophys. Acta 1974 362 75-82. 

The most common method of attachment of the ligand to the matrix involves treatment of the matrix with cyanogen bromide (CNBr) at pH 11. The reaction conditions and the relative proportion of the reagents will determine the number of ligand molecules which can be attached to each matrix particle. The procedure for CNBr activation and ligand coupling is outlined below. 

Normally, the immobilization of heparinase to agarose catalyst particles is terminated after 4-5 h because greater than 85% of the initial heparinase is bound (49). Based on a cyanate ester stability study, the cyanate ester concentration drops to only 88% of its initial value. For modeling purposes, the cyanate ester concentration was assumed constant. In addition, because of its small size relative to the large molecular weight cutoff (1.5 x 106 daltons) of the catalyst particle, cyanogen bromide (MW 106) should diffuse rapidly into the particle and uniformly activate the matrix. 

The results of one of the first multistep enzyme systems to be immobilized were presented in 1970 by Mosbach and Mattiasson (3). They covalently bonded hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) to individual polymer particles via the cyanogen bromide reaction. Using a solution containing glucose, ATP and NADP" ", they demonstrated that the coimmobilized enzymes formed product... 

The polymer particles formed in Problem 7.11 are suspended in water adjusted to pH 10.5 with NaOH and activated at 25°C with 10 mg of cyanogen bromide per mimiiter of suspension. After 15 min, it is diluted with equal volume of eold 0.1 Af borate buffer at pH 8.5 and 4°C. The immunolatex conjugates thus prepared are known to bind antigens. Explain why this occurs. 

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