Conjugates purification

The resulting product (II) is subsequently coupled to bovine-serum-albumin in a glycerol-w ater mixture in the presence of dicyclohexylcarbodiimide. The mixture is incubated overnight at 4°C, and the protein-hapten complex is dialysed against distilled water thereby causing its purification. Conjugation of the respective barbiturate to the protein carrier, comparison of the barbiturate BGG-conjugate to control BGG-solution and preparation of 14C-pentobarbital sodium are carried out respectively. 

F]fluorobenzaldehyde ([ F]FBA) was prepared within about 30 min in preparative yields of about 50% using solid-phase extraction cartridges for purification. Conjugation of various oxyamine acetate conjugated peptides ( Figs. 42.25 and O 42.26) was carried out in H20/MeOH (pH 2) in 60-80% yield (10 min) and peptide concentrations of 0.5 mm. 

Development of conjugate and peptide vaccines requires the typical organic synthesis process and purification. This is a new area for vaccine technologists. Again, the main concern is to maintain the immunogenicity of the vaccine candidate during the chemical reaction and purification steps. 

The third purification procedure is based on the combination of temperature-triggered aggregation and affinity capture and has been used to not only purify proteins [50-54] but also other molecules [55, 56], and for the removal of pollutants from a solution [57-59]. For this procedure, ELP is conjugated to a capture reagent, which can be done either genetically or chemically. This procedure eliminates the need for cleavage of ELP after purification and introduces the potential to recycle the ELP. 

Figure 17 Conjugation of an amine and a carboxyUc acid via the mixed anhydride method. Although the activated mixed anhydride is stable, it is usually used without purification. Use of low-temperature reactions will limit undesirable side products. Details of the reaction are given in Table 5...

Matejicek D, Houserova P, Kuban V (2007) Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments. J Chromatogr A 1171 (l-2) 80-89... 

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

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