Immunoaffinity extraction

The primary sample extract is subsequently subjected to cleanup using several different approaches including conventional liquid-liquid partitioning, diphasic dialysis, solid-phase extraction, immunoaffinity chromatography, and... 

M Carvajal, F Mulholland, RC Garner. Comparison of the EASI-EXTRACT immunoaffinity concentration procedure with the AOAC CB method for the extraction and quantitation of aflatoxin B, in raw ground unshelled. J Chrom 511 379-383, 1990. 

It is very probable that the SLM technique will become widely utilized for selective and sensitive extractions (immunoaffinity-based ISLM design). Moreover, the ESTM technique based on equilibrium SLM is likely to be extensively investigated in the context of speciation studies in environmental samples, with the major focus being on characterizing the interaction of analytes and macromolecules (such as HAs). Furthermore, the SLM format is well suited for studying different types of transport mechanisms. 

DNA isolation from any tissue of interest after compound administration followed by (1) addition of stable isotope-labeled internal standard for quantification, (2) hydrolysis/ digestion of DNA, (3) enrichment of DNA adducts of interest (e.g., solid-phase extraction, immunoaffinity chromatography, or DNA repair enzymes),... 

Aflatoxin Ml in milk No previous extraction Immunoaffinity prior to LC coupled to fluorescence detector 0.005 pgr 70-95... 

Analytical techniques Charcoal column chromatography Chromatography Coleus forskohlii Lorskolin ELISA GLC HPLC HPTLC Hydrotropic extraction Immunoaffinity chromatography Microwave-assisted extraction Molecular simulation TLC... 

Immunoaffinity extraction combined on-line with LC in conjunction with MS (108 -110) or tandem MS (111, 112) has also been demonstrated for the determination of analytes in biological fluids. Obviously, such systems offer a very high... 

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... 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

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. 

Fig. 5 Sequential steps involved in an immunoaffinity extraction procedure...

Immunoaffinity procedures can be performed either on-line or off-line, and can be coupled to chromatographic systems [ 118,119] or even to immunoassays [120]. Many examples can be found in the literature regarding the use of immunoaffinity extraction of drugs and pharmaceuticals from biological matrices, as well as of organic pollutants such as pesticides from environmental samples [115,121-124]. 

Also the production of specific Abs for PCDDs/PCDFs has been directed toward the development of immunoaffinity procedures [248,249]. Shelver et al. reported several works regarding the use of IAC to selectively extract and analyze these compounds from complex matrices such as milk or serum [250-253]. Moreover, a separation of very similar dioxin congeners (i.e., 1,3,7,8-TCDD and 2,3,7,8-TCDD) was also examined [254]. 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

A technique called on-line immunoaffinity CE has been presented (45) that was also coupled to MS. However, in this setup the affinity principle is used to extract the analyte from a complex matrix in a microchamber affinity device prior to CE separation. Therefore, it cannot be considered ACE. 

Delaunay, N., Pichon, V, and Hennion, M. C., Immunoaffinity solid-phase extraction for the trace-analysis of low-molecular-mass analytes in complex sample matrices. Journal of Chromatography. B, Biomedical Sciences and Applications 745(1), 15-37, 2000. 

Three forms of purified FSH are available. Urofollitropin, also known as uFSH, is a purified preparation of human FSH that is extracted from the urine of postmenopausal women. Virtually all the LH activity has been removed through a form of immunoaffinity chromatography that uses anti-hCG antibodies. Two recombinant forms of FSH (rFSH) are also available follitropin alfa and follitropin beta. The amino acid sequences of these two products are identical to that of human FSH. They differ from each other and urofollitropin in the composition of the carbohydrate side chains. The rFSH preparations have a shorter half-life than preparations derived from human urine but stimulate estrogen secretion at least as efficiently and, in some studies, more efficiently. The rFSH preparations are considerably more expensive. 

A continuing source of concern is the operating pressure of an immunoaffinity column. Excessively pressures will generate shear-type forces that could cause destruction of the antibody-support bond and lower the efficiency of the column. In general, pressures should not exceed the value of 0.34. IO Pa to prevent loss of immobilized antibody (167). This condition meets the major restriction of all immunoaffinity cleanup procedures, which is the need to use aqueous extracts. Analytes extracted from the original sample with organic solvents cannot be... 

Immunoaffinity cleanup was first applied in drug residue analysis for the determination of chloramphenicol in swine muscle tissue by LC (113). The lAC column was prepared using monoclonal antibodies originally developed for an enzyme-linked immunosorbent assay (ELISA) method (171) specific for chloramphenicol. Meat samples were extracted with water, and a concentrated phosphate buffer was added to the filtered extracts before immunoaffinity cleanup. A phosphate buffer was used in the washing process, whereas chloramphenicol was eluted from the column with a glycine/sodium chloride solution of pH 2.8. For subsequent LC analysis, this eluate was extracted with ethyl acetate, evaporated, and reconstimted in the mobile phase. The same analytical scheme was later successfully applied for the determination of chloramphenicol in eggs and milk as well (170, 172). 

For reliable identification of a residue, detailed information about the molecular structure of the analyte is essential. The total information about the molecular structure of the analyte is the sum of the information derived from each individual analytical step of tire method. Frequently used selective analytical steps based on chromatography or immunoaffinity, provide more or less general indirect information. For example, solid-phase extraction (SPE) cleanup followed by liquid chromatography/ultraviolet detection (LC/UV) has been suggested for screening and quantification of ivermectin residues in liver, but presumptive positive samples can be confirmed by derivatizing an aliquot of the SPE eluate and reanalyzing the fluorescent derivative of ivermectin in an LC-fluorescence system (17). 

Following initial sample extraction, the primary extract must frequently be subjected to some kind of further cleanup including liquid-liquid partitioning, diphasic dialysis, solid-phase extraction, matrix solid-phase dispersion, immunoaffinity chromatography cleanup, liquid chromatography cleanup, or online trace enrichment. In some instances, some of these procedures are used in combination in order to attain higher purification levels. 

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