Characterization of Lactoferrin (LF) from Colostral Whey Using Anti‐LF Antibody Immunoaffinity Chromatography

Abstract

Lactoferrin (LF) in colostral whey was isolated by anti-LF immunoglobulin in yolk (IgY)-Sepharose 4B immunoaffinity chromatography, and parameters such as binding capacity (q m ) and dissociation constant (K d , 3 10 -6 M) of this immunoaffinity gel for LF were discussed. Purification folds for colostral whey I (from colostrum collected within 6 d of postpartum) and colostral whey II (from colostrum collected within 1 d of postpartum) by anti-LF IgY-immunoaffinity chromatography were 135.80 and 103.60, respectively. The recovery for LF in the same colostral whey sample by anti-LF IgY-immunoaffinity chromatography was 82 to 99 %. q m of anti-LF IgY-immunoaffinity gel for LF in colostral whey I and whey II were 0.372 and 0.272 mg LF/mL wet gel, respectively. K d of anti-LF IgY-immunoaffinity gel for LF in colostral whey I was 1.594 × 10 -6 M and II was 1.587 3 10 -6 M.

Determination of lactoferrin in bovine milk, colostrum and infant formulas by optical biosensor analysis

“…50m g mL À1 ), illustrating a significant physiological response to lactogenesis. These biosensor-SPR based results obtained from a single lactating cow are consistent with data based on alternative techniques, which have variously reported a dramatic reduction ranging from 1500-5000 mg mL À1 in colostrum to 20-350 mg mL À1 in milk (Senft & Klobasa, 1973;Pakkanen & Aalto, 1997;Fox & McSweeney, 1998;Tu et al, 2002;Turner et al, 2003).…”

“…The mean lactoferrin content in consumer cows' milk has been estimated by this technique as 170 mg mL À1 (range: 150-210 g mL À1 ; n: 10), a level consistent with the 20-750 mg mL À1 range reported using other techniques (Jensen, 1995;Pakkanen & Aalto, 1997;Fox & McSweeney, 1998;Steijns & van Hooijdonk, 2000;Tu, et al, 2002;Palmano, & Elgar, 2002;Farr et al, 2002;Fox & Kelly, 2003;Lindmark-Ma˚nsson, Fonden, Pettersson, 2003;Turner et al, 2003;Chen & Mao, 2004). The described SPR-immunoassay also reveals that lactoferrin levels expressed in raw milk from individual animals are more variable, probably dependent on genotype, diet, somatic cell status and collection time (Turner et al, 2003;Chen & Mao, 2004).…”

“…Such techniques should, however, be distinguished from methods intended for the determination of biological activity. Ion-exchange, gel permeation, adsorption and immunoaffinity chromatographic techniques have been utilised for the isolation of lactoferrin isolates from milk or whey (Ekstrand & Bjo¨rck, 1986;Hahn, Schulz, Schaupp, & Jungbauer, 1998;Tu, Chen, Chang, & Chang, 2002;Zhang, Nakano, & Ozimek, 2002;Zhang, Nakano, & Ozimek, 2003). Analytical chromatographic techniques for bovine whey proteins have been reviewed (Gonzalez-Llano, Polo & Ramos, 1990;Strange, Malin, Van Hekken, & Basch, 1992;Elgar et al, 2000), while several chromatographic techniques for the quantitation of lactoferrin in milk and whey have also been reported (Monti, Fumeaux, Barrios-Larouze, & Jolles, 1984;Law, Leaver, Banks, & Horne, 1993;Francis, Regester, Webb, & Ballard, 1995;Palmano & Elgar, 2002).…”

Isolation of bovine serum albumin from whey using affinity chromatography

“…We found several reports on affinity chromatography for protein purification from whey (for example, Ben Ounis, Gauthier, Turgeon, Roufik, & Pouliot, 2008;Chen, Guo, Guan, & Liu, 2007;Puerta, Jaulmes, De Frutos, DiezMasa, & Vidal-Madjar, 2002;Tu, Chen, Chang, & Chang, 2002). Heparin affinity chromatography can, for example, be used to isolate protein fractions or lactoferrin.…”

Isolation and purification of lactoferrin and immunoglobulin G from bovine colostrum with serial cation-anion exchange chromatography

“…Chromatography is another technology widely used in isolation of high purity LF and IgG. Some examples of chromatographic separation are ion-exchange chromatography [3,4], hydrophobic interaction chromatography [5,6], adsorption chromatography [7,8], gel permeation chromatography [9], affinity chromatography [10,11] and immunoaffinity chromatography [12,13]. Compared with the other chromatographic methods, ion-exchanger chromatography has the advantages of low cost, reduced steps, continuous feed-in, and easy to scale-up.…”