Affinity chromatography of milk sIgA-1 preparation from first donor on DNA-cellulose and ATP-Sepharose.

<p>sIgA-1 preparation was chromatographed on DNA-cellulose (A) and ATP-Sepharose (B) in standard conditions: (–), absorbance at 280 nm; symbols correspond to the relative catalytic activities (RA) in the hydrolysis of DNA (▵), ATP (○), oligosaccharides (×) as well as phosphorylation of lipids (▪) and polysaccharides (⋄) tightly bound with sIgAs. Depending on the RA and reaction analyzed, the reaction mixtures were incubated for 0.5–2 h and then the RAs were normalized to the standard conditions and the RA of the fraction with the highest activity was taken for 100%. The average error in the initial rate determination from two experiments in each case did not exceed 7–10%. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#s4" target="_blank">Materials and methods</a> for other details.</p

Affinity chromatography of milk pIgGs on two different resins.

<p>Various IgG fractions were separated using DNA-cellulose (A) and ATP-Sepharose (B): (–), absorbance at 280 nm; symbols correspond to the relative catalytic activities (RA) in the hydrolysis of DNA (▵), ATP (○), oligosaccharides (×); phosphorylation of lipids (▪) and polysaccharides (⋄) tightly bound with IgGs. Depending on the RA and reaction analyzed, the reaction mixtures were incubated for 0.5–2 h and then the RAs were normalized to the standard conditions and the RA of the fraction with the highest activity was taken for 100%. The average error in the initial rate determination from two experiments in each case did not exceed 7–10%.</p

The dependencies of the absorbance (A₄₅₀) on the concentration of tested IgG preparation.

<p>The analysis was performed using a direct (A) and sandwich (B) ELISA. For a direct analysis, the tested IgG preparations were adsorbed on ELISA strips, then mouse Abs against human kappa-IgGs or lambda-IgGs (anti-kappa-Abs and anti-lambda-Abs) were added, and finally the conjugate of polyclonal rabbit anti-mouse IgGs with horseradish peroxidase (HRP) was used (C). For a sandwich analysis, anti-kappa-Abs and anti-lambda-Abs were adsorbed on ELISA strips, then tested IgG preparations were added, and finally the conjugate of HRP with mouse Abs against human kappa-IgGs or lambda-IgGs (anti-x-Ab-HRP) were used (B). Anti-lambda-Abs (or anti-lambda-Abs and anti-lambda-Ab-HRP) were used for the analysis of the content of IgGs having affinity only for anti-lambda-Ab-Sepharose (▪), for anti-kappa-Ab-Sepharose (□), and for both of these Sepharoses (▴), while anti-kappa-Abs (or anti-kappa-Abs and anti-kappa-Ab-HDP) were used for the analysis of preparations having affinity only for anti-kappa-Ab-Sepharose (•), for anti-lambda-Ab-Sepharose (○), and for both of these Sepharoses (▾). (⋄), a control mixture containing no analyzed IgGs (A and B). The RAs of lambda-IgGs, kappa-IgGs, and kappa-lambda-IgGs in the catalysis of different chemical reactions (C). The RA of the preparation with the highest activity in each reaction analyzed was taken for 100%. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042942#s4" target="_blank">Materials and Methods</a> for other details.</p

SDS-PAGE analysis of FITC-modified pIgG_mix formation.

<p>The analysis was performed after non-labeled pIgG_mix incubation with preparations of separated FITC-modified heavy (A, B) and light chains (C, D) of Abs. The relative fluorescence of proteins (A and C) and their position on the gel (B and D; Coomassie staining) were analyzed. Before electrophoresis, FITC-heavy-chains were incubated alone (lanes 1, A and B), in the presence of the IgG_mix and GSH (lanes 2, A and B) or in the presence of the IgG_mix, GSH and human milk plasma (lanes 3, A and B). Lane 4 (B) corresponds to human milk plasma incubated alone. FITC-L-chains were incubated alone (lanes 1, C and D), in the presence of the IgG_mix and GSH (lanes 2, C and D) or in the presence of the IgG_mix, GSH, and human milk plasma (lane 3 C and D). The arrows (lane 4, D) indicate the positions of molecular mass markers. For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042942#s4" target="_blank">Materials and methods</a>.</p

Affinity chromatography of non-modified and FITC-modified pIgG_mix on DNA-cellulose.

<p>(–) and (–), absorbance of IgGs at 280 nm before and after modification of IgGs with FITC, respectively; the bars correspond to the relative fluorescence of FITC-IgG_mix fractions (A). Analysis of a relative efficiency of specific-molecule exchange under different conditions between non-modified IgG_mix and FITC-IgG_mix having different affinity for DNA-cellulose (B–D). Before chromatography, the IgG_mix eluted from DNA-cellulose by 0.15 M NaCl (0.15 M-IgG_mix) were incubated with FITC-IgG_mix eluted by 1.5 M NaCl (1.5 M-FITC-IgG_mix) in the presence of TBS and GSH (B); 0.5 M-IgG_mix and 1.5 M-FITC-IgG_mix (C) or 0.5 M-FITC-IgG_mix +1.5 M-IgG_mix (D) were incubated in the presence of TBS containing GSH and human milk plasma.</p

Sequential affinity chromatography of sIgA-2 and sIgA-3 preparations on casein-Sepharose and lipid-resin.

<p>After chromatography of sIgA-2 and sIgA-3 preparations on ATP-Sepharose (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#pone-0048756-g003" target="_blank">Figs 3C and 3D</a>, respectively), fractions 10–18 having high affinity to ATP-Sepharose were combined and chromatographed on casein-Sepharose (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#pone-0048756-g004" target="_blank">Figs 4A and 4B</a>, respectively). Panel C demonstrates chromatography on lipid-resin of a mixture of fractions 9–17 corresponding to sIgA-2 and sIgA-3 eluted from casein-Sepharose (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#pone-0048756-g003" target="_blank">Figs 3C and 3D</a>): (–), absorbance at 280 nm; relative catalytic activities (RA) in the hydrolysis of DNA (▵), phosphorylation of casein (□) and lipids tightly bound with Abs (▪). Depending on the RA and reaction analyzed, the reaction mixtures were incubated for 0.5–2 h and then the RAs were normalized to the standard conditions and the RA of the fraction with the highest activity was taken for 100%. The average error in the initial rate determination from two experiments in every case did not exceed 7–10%. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#s4" target="_blank">Materials and methods</a> for other details.</p

Sequential affinity chromatography of sIgA-2 and sIgA-3 preparations on phenyl-Sepharose and DNA-cellulose.

<p>Hydrophobic chromatography of sIgA_mix (equimolar mixture of Abs from five donors) on phenyl-Sepharose using reverse gradient of NaCl concentration (A). Chromatography of fractions 0–70 ml (B) and 160–190 ml (C) eluted from phenyl-Sepharose on DNA-cellulose: (–), absorbance at 280 nm; relative catalytic activities (RA) in the hydrolysis of DNA (Δ) and maltoheptaose (x) as well as phosphorylation of oligosaccharides (◊) and lipids (▪) tightly bound with Abs. Depending on the RA and reaction analyzed, the reaction mixtures were incubated for 0.5–2 h and then the RAs were normalized to the standard conditions and the RA of the fraction with the highest activity was taken for 100%. The average error in the initial rate determination from two experiments in each case did not exceed 7–10%. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#s4" target="_blank">Materials and methods</a> for other details.</p

Affinity chromatography of milk pIgGs on anti-kappa-L-Sepharose.

<p>The chromatography was performed under the conditions of over-saturation of the affinity capacity of the sorbent (A) and re-chromatography of the preparation eluted by acidic buffer (peak 2) on anti-lambda-L-Sepharose (B) under the conditions of the excess of the affinity sorbent. After incubation of a mixture of equal amounts of purified lambda- and kappa-IgGs for 24 h, lambda+kappa-IgG_mix was subjected to standard affinity chromatography on Protein G-Sepharose (C) and gel filtrated (data not shown). Then lambda- and kappa-IgGs were separated by affinity chromatography of lambda+kappa-IgG_mix on anti- lambda-L-Sepharose (D). The preparation of lambda-IgGs purified on anti-lambda-L-Sepharose was rechromatographed on anti-kappa-L-Sepharose (E), while kappa-IgGs on anti-lambda-L-Sepharose (F). In all cases: (–), absorbance at 280 nm (A₂₈₀).</p

Affinity chromatography of non-modified and FITC-modified sIgA_mix on DNA-cellulose:

<p>(–) and (- - -), absorbance of sIgA_mix at 280 nm before and after modification of sIgAs with FITC, respectively. Analysis of a relative efficiency of specific-molecule exchange under different conditions between non-modified sIgA_mix and FITC-sIgA_mix having different affinity for DNA-cellulose; relative fluorescence (RF) is shown by the bars (B–D). Before chromatography, the sIgA_mix eluted from DNA-cellulose by 0.6 M NaCl (0.6M-IgG_mix, Panel A) were incubated for 24 h with FITC-IgA_mix eluted by 8 M urea (8M-FITC-IgA_mix, Panel A) in the presence of TBS and GSH (B), TBS + human milk plasma (C), and with this buffer containing both GSH and human milk plasma (D). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048756#s4" target="_blank">Materials and methods</a> for other details.</p

The relative content of IgGs of different subclasses in the preparations of total non-fractionated milk IgGs, and in specific lambda-IgGs, kappa-IgGs, and kappa- lambda-IgGs^*.

*<p>Average data for IgGs from five different donors of milk are given.</p>**<p>For each fraction, a mean of tree repeats is used.</p