Affinity extraction

D. Stevenson, B. A. Abdul Rashid, and S. J. Shahtaheri, Immuno-affinity extraction, in N. J. K. Simpson, ed., Solid-Phase Extraction Principles, Techniques, and Applications, Marcel Dekker, New York, 2000, pp. 349-360. 

A Low-Cost Aqueous Two-Phase System for Affinity Extraction... 

In order to maximize our recovery and purification, several PEG-triazine dye derivatives were tested for their binding of yeast alcohol dehydrogenase in the maltodextrin based ATPS. The screening experiment, illustrated in Figure 3, showed that under our conditions the best extraction would be made with PEG-FGF as the affinity ligand. All subsequent affinity extractions use this ligand. 

SZLAGETAL. Low-CostAqueous Two-Phose System for Affinity Extraction 81... 

Figure 5. Recovery of YADH activity as a function of pH. Optimum pH for recovery is in the range 4.8-5.2. Phase volume ratio was 1.38. Because of the rapid denaturation of YADH at pH values below 5, all subsequent affinity extractions were carried out at a pH between 5.0 and 5.1.

Ghenciu EG, Beckman EJ. Affinity extraction into carbon dioxide. 1. Extraction of avidin using a biotin-functional fluoroether surfactant. Ind Eng Chem Res 1997 36 5366-5370. 

Szlag DC, Giuhano A, and Snyder SM, A low-cost aqueous two phase system for affinity extraction. In Hamel JFP, Hunter JB, and Sikdar SK, eds. Doivnstream Processing and Bioseparation, Recovery and Purification of Biological Products. ACS Symp. Ser. 419, ACS Press, Washington, 1990 pp. 71-86. 

Other. Leaching, or solid extraction, is a similar process in which solute(s) are removed from a solid by a liquid mass-separating agent for which they have an affinity. Extraction is typically analyzed as an equilibrium-limited separation. 

Human peripheral blood-derived DCs take up therapeutic proteins via macropinocytosis or receptor-mediated endocytosis, degrade them proteolyti-cally, and load the resulting peptide fragments onto HLA class II molecules, provided that the latter contain an appropriate epitope and resist downstream quality control by the peptide editor HLA-DM (Vogt et al., 2005). The second step foresees cell lysis, affinity extraction of HLA-peptide complexes, and acid elution of bound peptides. This procedure provides a mixture of both selfepitopes from the DC proteome and potential epitopes derived from the therapeutic protein. To separate mixtures of 1500-2000 distinct peptides with maximal resolution, the so-called Multidimensional Protein Identihcation Technology is employed (Kropshofer and Spindeldreher, 2005). Peptides leaving the HPLC capillary upon separation are sprayed directly into the orifice of an ion trap mass spectrometer for sequence determination. 

Affinity extraction is another approach that can be used for solute detection. In this method, an affinity column is used for the removal of a specific solute or group of solutes from a sample prior to their determination by a second method. This employs the same operating scheme as shown in Figure 1, but now involves combining the affinity column either offline or online with some other method for the actual quantitation of analytes. This often involves the use of antibodies as ligands, but other binding agents can also be employed. 

Nelson, B.C., Pfeiffer, C.M., Margolis, S.A., and Nelson, C.P., 2003. Affinity extraction combined with stable isotope dilution LC/MS for the determination of 5-methyltetrahydrofolate in human plasma. Analytical Biochemistry. 313 117-127. 

The fundamentals of affinity chromatography can be taken from the literature [2-5]. Since affinity-based analytical methods are extremely dependent on the analytes and their respective binding molecules, it is not possible to go into much detail with regard to practical advice. It has to be mentioned that affinity chromatography is rarely a true chromatographic technique, and in most cases has to be considered to be a selective extraction method. Hence, the term affinity extraction or immunoaffinity extraction should be preferred where appropriate. 

Fig. 3. Affinity extraction for the sample preparation of urine for the analysis of PAH metabolites. Comparison with conventional CIS SPE. (Figure courtesy of Prof Dietmar Knopp).

I n Fig. 3, two chromatograms are compared, which show the difference between sample preparation with classical Cjg SPE and immunoaffinity extraction (afEnity SPE). The main metabolite, 1-hydroxypyrene, can be examined with both methods all other metabolites, which occur in lower concentrations, carmot be quantified without affinity purification. The detection limit with affinity extraction can be improved even further ifa concentrating Cjg-SPE is subsequently applied. Eigure 3 clearly shows the improvement that can be achieved with affinity purification. Furthermore, it is much easier to identify new metabolites that have not been identified before because the chromatogram contains only very few peaks. Hyphenation with mass spectrometry would be particularly suitable to achieve a structural analysis. 

Cai, J. Henion, On-line immimo-affinity extraction-coupled column capillary liquid chromatography/tandem mass spectrometry Trace analysis of LSD analqis and metabolites in human urine. Anal. Chem. 68 (1996) 72-78. 

J Li, EJ Beckman. Affinity extraction into CO2 2. Extractions of heavy metals into CO2 from low-pH aqueous solutions. Ind Eng Chem Res 37 4768-4773, 1998. Anon. Environmentally sound alternative may help dry cleaning industry. J Environ Health 58 37-39, 1995. 

The fractions containing cyciase activity were dyalized against buffer A, combined and concentrated and further purified by affinity coior with increasing NaCI molarity steps. Abietadiene cyciase was eluted principally from 1 to 1,5M NaCI. The affinity extract was dyalized and then subjected to FPLC gel permeation. At this stage of the fractionation, 85-fold... 

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