Monoclonals immobilized

Immunoaffinity chromatography utilizes the high specificity of antigen—antibody interactions to achieve a separation. The procedure typically involves the binding, to a soHd phase, of a mouse monoclonal antibody which reacts either directly with the protein to be purified or with a closely associated protein which itself binds the product protein. The former approach has been appHed in the preparation of Factor VIII (43) and Factor IX (61) concentrates. The latter method has been used in the preparation of Factor VIII (42) by immobilization of a monoclonal antibody to von WiHebrand factor [109319-16-6] (62), a protein to which Factor VIII binds noncovalenfly. Further purification is necessary downstream of the immunoaffinity step to remove... 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

Carboxylated silica particles may be coupled with amine-containing ligands, such as proteins, using a carbodiimide reaction with EDC. A similar protocol to that previously described for coupling to carboxylate polymer particles may be used. The following protocol is based on the method of Zhao et al. (2004), which was used for immobilizing monoclonal antibodies to E. coli 0157. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

Donohue-Rolfe, Arthur, David W.K. Acheson, Anne V. Kane, and Gerald T. Keusch. "Purification of Shiga Toxin and Shiga-Like Toxins I and II by Receptor Analog Affinity Chromatography with Immobilized PI Glycoprotein and Production of Cross-Reactive Monoclonal Antibodies." Infection and Immunity 57 (December 1989) 3888-893. 

Factor IX may also be purified by immunoaffinity chromatography, using immobilized anti-IX murine monoclonals. Purification to homogeneity is particularly important in the case of... 

Binding of the monoclonal antibody to the protein A domain would ensue and the immobilized monoclonal antibody would dictate the cell type targeted. 

Fig. 10.15 Virus detection test. Sensor signal (phase change) measured between channel 1 and the reference channel for the immobilization of anti HSV 1 glycoprotein G monoclonal antibody layer on the sensing surface of channel 1 (A HSV i gG) and the binding of HSV 1 particles to this layer (A IISV i). Reprinted from Ref. 28 with permission. 2008 American Chemical Society...

This type of approach is essentially non-competitive and usually requires the use of two monoclonal antibodies directed against two distinct epitopes on the analyte. Other devices have employed a two-stage competitive system in which analyte and labelled analyte compete for antibody in one part of the device. This is followed by transfer of the equilibrium mixture to a separate part of the device where membrane-immobilized antibody removes the unbound labelled material and allows the bound to go through the membrane into the absorbent pad. 

Proteins are frequently powerful immunogens and the availability of specific antibodies, particularly monoclonal antibodies, makes the technique of affinity chromatography very useful in the separation and purification of individual proteins. The technique has been used to purify a wide range of proteins such as hormones, membrane receptors and complement proteins. However, it is not restricted to proteins and is potentially applicable to any immunogenic substance. The availability of suitable antibodies is essential and these may be raised by whole animal polyclonal techniques or by monoclonal cell culture. The former antibodies may need some prior purification before being immobilized. 

CNTs can be functionalized with protein via non-covalent bond (Li et al., 2005 Kim et al., 2003 Mitchell et al., 2002). For example, (beta-lactamase I, that can be immobilized inside or outside CNTs, doesn t change enzyme s activity (Vinuesa and Goodnow, 2002). Taq enzyme can attach to the outside of CNT, and doesn t change its activity (Cui et al., 2004). Peptide with Histidine and Tryptophan can have selective affinity for CNT(Guo et al., 1998). Monoclonal antibody can attach to SWNTs. Protein-modified CNTs can be used to improve its biocompatibility and biomolecular recognition capabilities (Um et al., 2006). For example, CNTs functionalized with PEG and Triton X-100 can prevent nonspecific binding of protein and CNTs. Biotin moiety is attached to the PEG chains Streptavidin can bind specifically with biotin-CNT (Shim et al., 2002). 

B. Johnsson, S. Lofas, G. Lindqurrst, A. Edstrom, R. M. Muller Hillgren, and A. Hansson, "Comparison of Methods for Immobilization to Carboxymethyl Dextran Sensor Surfaces by Analysis of the Specific Activity of Monoclonal Antibodies," Journal of Molecular Recognition JMR, 125-131 (1995). 

Aroxmd the same time, Beckman Instruments (now Beckman Coulter) had begun an array-based product development program focused on the use of modified plastics. Silzel and coworkers (1998) and Matson et al. (2001) of Beckman Coulter were among the first to pursue printing of antibodies onto a plastic surface in a microarray format. Silzel et al. immobilized biotinylated monoclonal antibodies onto an avidin-coated polystyrene surface and performed micro-ELISA-based isotyping of IgG species. Matson et al. 

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