Immunoaffinity chromatography

Protein A chromatography Lectin affinity chromatography Immunoaffinity chromatography... 

The aqueous or organic extract obtained at this step of analysis may be a very dilute solution of the analyte(s) of interest. It may also contain coextractives, which, if allowed in the final extract, will increase the background noise of the detector, making it impossible to determine trace level concentrations of the analyte(s). To reduce interferences and concentrate the analyte(s), the primary sample extracts are subjected to some kind of cleanup including liquid-liquid partitioning, solid-phase extraction, matrix solid-phase dispersion, online trace enrichment, affinity chromatography, immunoaffinity chromatography, and ultrafiltration. In many instances, more than one of these procedures may be used in combination to increase extract purification. 

The primary sample extract is subsequently subjected to cleanup using several different approaches including conventional liquid-liquid partitioning, solid-phase extraction, liquid chromatography, immunoaffinity chromatography, and supercritical fluid extraction cleanup. In some instances, more than one of these purification procedures can be applied in combination for better results. 

Affinity chromatography (immunoaffinity, metal affinity, chiral)... 

Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

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... 

For detection residue amounts of tetracyclines in dairy products widely used methods FIPLC, immunoaffinity chromatography, kinetic spectrophotometry, which are expensive and complicated. 

W. Haasnoot, M. E. Ploum, R. J. A. Paulussen, R. Schilt and F. A. Huf, Rapid determination of clenbuterol residues in urine by high perfoi mance liquid cliromatography with on-line automated sample processing using immunoaffinity chromatography , 7. Chromatogr. 519 323-335 (1990). 

G. S. Rule, A. V. Moi dehai and J. Henion, Determination of carbofuran by on-line immunoaffinity cliromatography with coupled-column liquid chromatography/mass spectrometiy . Aim/. Chem. 66 230-235 (1994). 

The low selectivity of the SPE columns currently in use can be increased with more selective sorbents such as the immunosorbents, which have been quite extensively used in SPE-LC (72). Immunoaffinity-based solid-phase extraction (lASPE) sorbents have also been used in coupled gas chromatography for determining... 

Figure 13.20 GC-FID chromatograms of an exuact obtained by (a) SPE and, (b) lASPE of 10 ml of municipal waste water, spiked with 1 p.g 1 of seven s-triazines (c) represents a blank mn from lASPE-GC-NPD of 10 ml of EIPLC water. Peak identification is as follows 1, ati azine 2, terbuthylazine 3, sebuthylazine 4, simetiyn 5, prometiyn 6, terbutiyn 7, dipropetiyn. Reprinted from Journal of Chromatography, A 830, J. Dalliige et al, On-line coupling of immunoaffinity-based solid-phase exUaction and gas chi-omatography for the determination of 5-triazines in aqueous samples , pp. 377-386, copyright 1999, with permission from Elsevier Science.

Human interleukin 2, a 133-residue protein, has been separated into multiple molecular forms by selective immunoaffinity chromatography and chromatofocusing. Most of the heterogeneity has been attributed to variations in glycosylation of the threonine residue in position 3 of the polypep-... 

Specific extraction methods are used to prepare the analyte for immunoassay by freeing the analyte fromboth specific and nonspecific interferences. Supercritical fluid extraction has been used to decrease the amount of solvent waste generated. Solid-phase extraction has gained popularity, and many different supports are available. One promising extraction and concentration method is immunoaffinity chromatography, which will be addressed later. 

E. Martlbauer, R. Dietrich, and E. Usleber, Immunoaffinity chromatography as a tool for the analysis of antibiotics and sulfonamides, in Veterinary Drug Residues, Food Safety, ed. WA. Moats and M.B. Medina, American Chemical Society, Washington, DC, Chapter 13, pp. 121-131 (1995). 

E. M. Thurman and C. Batian, Determination of atrazine and atrazine mercapture in drinking water samples and in urine using immunoaffinity SPE with positive ion spray HPLC/MS , Presented at the 15th Symposium on Liquid Chromatography/Mass Spectrometry, Montreux, Switzerland, November 9-10, 1998. 

Downham, M., Busby, S., Jefferis, R., and Lyddiatt, A., Immunoaffinity chromatography in biorecovery an application of recombinant DNA technology to generic adsorption processes, /. Chromatogr., 584, 59, 1992. 

Another potential disadvantage of an immunoaffinity separation is the assumed abundance of the purified antigen in sufficient quantities to immobilize on a chromatography support. Protein antigens should be immobilized at densities of at least 2-3 mg/ml of affinity gel to produce supports of acceptable capacity for binding antibody. Often, the antigen is too expensive or scarce to obtain in the amounts needed. 

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