Use of an affinity immunosorbent for the isolation of lactoferrin from breast milk serum

An affinity or biospecific method of isolating an iron-binding glycoprotein — lactoferrin — from breast milk is described which permits in a single chromatographic operation the isolation of lactoferrin homogeneous according to the results of electrophoresis in polyacrylamide gel. The affinity method of isolating lactoferrin is based on the use of a specific immunosorbent consisting of antibodies to lactoferrin covalently conjugated with cyanogen-bromide-activated Sepharose 4B.Scientific-Research Institute of Hematology and Blood Transfusion Ministry of Health of the Uzbek SSR, Tashkent. Translated from Khimiya Priordnykh Soedinenii, No. 6, pp. 749–750, November–December, 1986.     

Immobilised peptide displaying phages as affinity ligands. Purification of lactoferrin from defatted milk

An affinity purification procedure for the direct purification of lactoferrin from defatted (skimmed) milk has been developed. The procedure is based on using selected phage clones expressing a peptide with high binding affinity for lactoferrin which were covalently coupled to macroporous poly(dimethylacrylamide) monolithic column. Large pore size (10-100 microm) of macroporous poly(dimethylacrylamide) makes it possible to couple long (1 microm) phage particles as ligands without any risk of blocking the monolithic column. Bound lactoferrin was eluted using 1M NaCl with a purity of >95%. The technique presents a good alternative to conventional immunoaffinity chromatography for purification of a protein of interest from complex samples due to (i) the robustness of the system in terms of recovery and ligand leakage and (ii) economical aspect in terms of low ligand cost.     

Two ion-exchange chromatographic methods for the isolation of antibacterial peptides from lactoferrin. In situ enzymatic hydrolysis on an ion-exchange membrane

Two ion-exchange chromatographic methods are reported for the rapid isolation of antibacterial peptides from lactoferrin (LF). Using the first method, a pepsin hydrolysate of LF was fractionated by bead-based cation-exchange chromatography. After removal of weakly bound material by washing with ammonia, highly purified lactoferricin-B (LFcin-B) was obtained in a single step by elution with 2 M NaCl. Some other cationic peptides, copurified as minor components, were also characterised by N-terminal sequencing, mass spectrometry and antibacterial activity determination. With the second method, cheese whey was filtered through a cation-exchange membrane, and the selectively bound LF was directly hydrolysed in situ with pepsin. Inactive LF fragments were washed off the membrane with ammonia, and a fraction enriched in LFcin-B was obtained by further elution with 2 M NaCl. The membrane method is more rapid and offers several economic advantages.     

Development of an enzyme-linked immunosorbent assay for gentamicin in human blood serum

An enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of gentamicin in human blood serum was developed. Peculiarities of the adsorption on the microtitre plate surface of the gentamicin-protein conjugate were investigated. Different conditions of the competition stage of the analysis were studied and conditions for gentamicin monitoring in human blood serum in the clinical range were optimized. The matrix effect on the assay results, the specificity of the analytical system and the stability of the reagents were examined. The method permits gentamicin concentrations to be determined in human blood serum, diluted 1/1000, in the linear range from 1 to 30 ng/mL. The assay is characterized by high sensitivity (0.5 ng/mL), good reproducibility (CV < 12%) and good correlation with PFIA (r = 0.943). Received: 17 June 1997 / Revised: 30 September 1997 / Accepted: 2 October 1997     

Development of an enzyme-linked immunosorbent assay for gentamicin in human blood serum

An enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of gentamicin in human blood serum was developed. Peculiarities of the adsorption on the microtitre plate surface of the gentamicin-protein conjugate were investigated. Different conditions of the competition stage of the analysis were studied and conditions for gentamicin monitoring in human blood serum in the clinical range were optimized. The matrix effect on the assay results, the specificity of the analytical system and the stability of the reagents were examined. The method permits gentamicin concentrations to be determined in human blood serum, diluted 1/1000, in the linear range from 1 to 30 ng/mL. The assay is characterized by high sensitivity (0.5 ng/mL), good reproducibility (CV < 12%) and good correlation with PFIA (r = 0.943). Received: 17 June 1997 / Revised: 30 September 1997 / Accepted: 2 October 1997     

An affinity probe for isolation of abscisic acid-binding proteins

An affinity probe has been developed for isolation of receptor proteins that bind the plant hormone abscisic acid (ABA). The structural features required for biological activity have been preserved, and the probe has been demonstrated to bind to known ABA-binding proteins.     

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.     

Bovine lactoferrin purification from whey using Yellow HE‐4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatography methods need to be developed for specific and low‐cost protein purification. Here, we describe a novel affinity process with the dye Yellow HE‐4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low‐cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.     

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared. They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid. The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells. For the latter enzyme, a 11,000-fold purification was achieved in one step.     

Lactoferrin from human breast milk and from neutrophil granulocytes. Comparative studies of isolation, quantitation, characterization and iron binding properties

The isolation and properties of lactoferrin from human breast milk and from neutrophilic granulocytes were investigated. Human breast milk lactoferrin was purified by means of heparin-sepharose or Cibacron Blue affinity chromatography. Quantitative recovery using these two methods was comparable but Cibacron Blue affinity chromatography allowed for isolation of a more homogenous protein. Lactoferrin could only be isolated from human neutrophilic granulocytes by sequential use of antibody affinity followed by non-specific affinity chromatography. Both breast milk lactoferrin and granulocyte lactoferrin were separated into apo and iron-rich species by SDS polyacrylamide gel chromatography. Iron binding is accompanied by a conformational change in tertiary structure associated with more rapid electrophoretic migration. The isoelectric point of both human breast milk lactoferrin and human granulocyte lactoferrin is 5.5-6.2. Both types of lactoferrin have similar iron binding properties with release of iron from the one binding site occurring at pH 5.2-6.0 while the other binding site holds on to iron down to pH 3.6-3.2. Despite the high affinity for iron the percentage saturation of native lactoferrin is low, that for breast milk lactoferrin averaging 12-25% and that for granulocyte lactoferrin less than 10%.     

High-performance liquid affinity chromatography for the purification of immunoglobulin A from human serum using jacalin

A high-performance liquid affinity chromatographic method for the purification of serum immunoglobulin A (IgA) using a jacalin column is described. The automated procedure takes about 2 with minimal manipulation. The yields of the isolated IgA and of its IgG and IgM contamination were studied by enzyme-linked immunosorbent assay (ELISA) of 30 sera. Purity was assured by immunoelectrophoresis. The ratio of IgA1 to total IgA was unchanged after purification, as verified by ELISA. The results showed that greater than 90% IgA could be recovered with less than 0.5% total IgG and greater than 2.0% total IgM remaining in the fractions containing purified IgA.     

Use of steroid glycosides for the affinity chromatography of cholesterol

A number of sorbents for the sorption of cholesterol has been synthesized from eight saponins of the spiro- and furostanol series immobilized on amino-Silochrome. The most effective have proved to be a sorbent containing capsicosin. Amino-Silochrome, and also sorbents containing digitonin, F-gitonin, and purpureagitoside cause hemolysis. The sorption capacity of the sorbents obtained falls on passing from saponins of the spirostanol series to saponins of the furostanol series, and also with a decrease in the length of the oligosaccharide moiety of the saponins. M. V. Lomonosov Moscow State University. 2nd Moscow State Medical Institute, Institute of Chemistry, Academy of Sciences of the Moldavian SSR, Kishenev. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 652–655, September–October, 1980.     

Polysaccharide-modified iron oxide nanoparticles as an effective magnetic affinity adsorbent for bovine serum albumin

The magnetic separation technique based on magnetic iron oxide nanoparticles (MNPs) has potential applications in protein adsorption and purification, enzyme immobilization, cell sorting, nucleic acid detachment, and drug release. However, the naked MNPs are often insufficient for their hydrophilicity, colloidal stability, and further functionalization. To overcome these limitations, chitosan was firstly carboxymethylated and then covalently conjugated on the surface of the MNPs ranging in size from about 5 to 15 nm, which were prepared by co-precipitating iron (II) and iron (III) in alkaline solution and then treating under hydrothermal conditions. It was found that such modification did not result in the phase change of the MNPs, and the resultant modified nanoparticles were still superparamagnetic. In particular, the colloidal stability of MNPs in aqueous suspension was improved after the surface modification. By investigating the adsorption of bovine serum albumin (BSA) on the modified MNPs, it was observed that the adsorption capacity of the BSA on the modified MNPs increased rapidly within several minutes and then reached the maximum value at about 10 min. The adsorption equilibrium isotherm could be fitted well by the Langmuir model. The medium pH affected greatly the adsorption of the BSA. The maximum adsorption of the BSA occurred at the pH value close to the isoelectric point of the BSA, with a saturation adsorption amount of 94.45 mg/g (25 °C). For the BSA feed concentration of 1.017 mg/ml, a high desorption percentage of 91.5% could be achieved under an alkaline condition (pH 9.4).     

Determination of levofloxacin (the Levorotatory Stereoisomer of Ofloxacin) in milk by an indirect enzyme-linked immunosorbent assay

In this work we obtained polyclonal antibodies for levofloxacin. We synthesized conjugates of levofloxacin with cationized BSA for immunization and with ovalbumin for development of an ELISA for the detection of levofloxacin. The method is characterized by a range of detectable concentrations of 0.03 ng/mL to 0.41 ng /mL and the limit of detectable concentrations is 0.01 ng/mL. We tested the 28 fluoroquinolones for cross reactivity and only ofloxacin (145%), marbofloxacin (82%), ofloxacin in its dextrorotatory form (68%), rufloxacin (67%), and garenoxacin (24%) had cross reactivity. The optimized ELISA technique allowed the detection of levofloxacin in milk from 0.33 ng/mL to 3.34 ng/mL. The recoveries were in the range of 89.5–102% with a relative standard deviation of 3%. We tested 45 real samples of milk purchased in local stores; for 5 of them the results were positive (near 1 ng/mL).     

A novel molecularly imprinted method with computational simulation for the affinity isolation and knockout of baicalein from Scutellaria baicalensis

A novel molecularly imprinted polymer (MIP) was synthesized by precipitation polymerization with baicalein (BAI) as the template and used as solid‐phase extraction (SPE) adsorbent, aiming at the affinity isolation and selective knockout of BAI from Scutellaria baicalensis Georgi (SB). We used computational simulation to predict the optimal functional monomer, polymerization solvent and molar ratio of template to functional monomer. Characterization and performance tests revealed that MIP exhibited uniform spherical morphology, rapid binding kinetics, and higher adsorption capacity for BAI compared with nonimprinted polymer (NIP). The application of MIP in SPE coupled with high‐performance liquid chromatography to extract BAI from SB showed excellent recovery (94.3%) and purity (97.0%). Not only the single BAI compound, but also the BAI‐removed SB extract was obtained by one‐step process. This new method is useful for isolation and knockout of key bioactive compounds from herbal medicines. Copyright © 2015 John Wiley & Sons, Ltd.