The results indicate that the reduction in morbidity associated with formula supplementation is of sufficient magnitude to be of public health significance. The use of these preparations provides an effective method for the prevention of diarrheal diseases.

Whey is a suitable source of immunoglobulins and lactoferrin to enrich infant formulas. Gel filtration on Sephacryl S-300 and on Fractogel TSK HW-55 was used to isolate immunoglobulins from colostral whey, acid whey, and Cheddar cheese whey. The SDS-PAGE and immunoelectrophoresis techniques indicated that the purity of the fractions from fractionation on Sephacryl S-300 was better than that by fractionation on TSK HW-55 column. Biological activity of fractions from the Sephacryl S-300 column as assessed by immunochemical analysis was 99, 83.3, and 92% for colostral, acid, and sweet wheys. The well-proven antimicrobial agent, lactoferrin, was isolated from sweet whey by heparin-attached Sepharose. Lactoferrin selectively adsorbed to the column was subsequently eluted with 5 mM Veronal-HCl containing .5 M NaCl, pH 7.4. Purity of the isolated protein was confirmed by SDS-PAGE and immunoelectrophoresis.

Different adsorption and chelating chromatographic methods were used to isolate immunoglobulins and lactoferrin from cheese whey. Among three adsorption solid supports (silica, controlled pore glass, and alumina), controlled pore glass showed the highest adsorption of immunoglobulins; however, its capacity was low. 1,4-Butanediol diglycidyl etheriminodiacetic acid on Sepharose 6B was loaded with copper ion and used for the same purpose. Of the two peaks eluted using pH gradient, the first yellowish peak was rich in lactoferrin and the second was rich in Ig. The purity of IgG in the Ig rich fraction as indicated by radial immunodiffusion was 77.2 and 53.0% for acid whey and Cheddar cheese whey, respectively. The capacity of the column was high; a 25-ml copper charged column could absorb Ig from 1 L of cheese whey. Modification of histidine residues in Ig with diethyl pyrocarbonate almost completely eradicated the adsorption, implicating the coordination compound formation between histidine in Ig and Cu on the chelating column as the adsorption mechanism. Enzyme-linked immunosorbent assays of the Ig thus separated demonstrated their binding activity against lipopolysaccharides extracted from Escherichia coli, Salmonella typhimurium, and Bordetella parapertussis.

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Metal chelate interaction chromatography was used to separate Ig, transferrin, and albumin from blood serum and blood plasma. A column was packed with iminodiacetic acid: 1,4-butanediol diglycidyl Sepharose 6B or Sephacryl S-300 and loaded with copper, zinc, nickel, or cobalt ion. Radial immunodiffusion assay indicated that Ig-rich fractions of blood serum obtained from Zn-, Ni-, Co-, and Cu-loaded columns contained 23.2, 81.3, 79.4, and 98.1% active IgG, respectively. Transferrin was recovered from the second peak. When the same conditions of metal chelate interaction chromatography were used for blood plasma, hemoglobin tended to bind strongly to the Cu-loaded column and was eluted only with 50% ethanol. Modification of histidine residues in Ig and transferrin with diethyl pyrocarbonate almost completely destroyed their binding ability to the column. Immunoglobulin G separated showed antilipopolysaccharide antibody activity against Escherichia coli, Salmonella typhimurium, and Bordettella parapertussis.