Quantification of deoxynivalenol in wheat using an immunoaffinity column and liquid chromatography

A simple and accurate method to quantify the mycotoxin deoxynivalenol (DON) in wheat is described. The method uses immunoaffinity chromatography for DON isolation and liquid chromatography (LC) for toxin detection and quantification. Wheat samples are extracted in water, filtered twice and applied to an immunoaffinity column. Following a water wash, DON is eluted from the column with methanol and injected onto an LC system with a UV detector for quantification. Test performance was evaluated in terms of antibody specificity, limit of detection, percentage recovery, precision, column capacity, assay linearity and comparison with the GC-electron-capture detection (ECD) method of Tacke and Casper. Specificity of the immunoaffinity column cleanup procedure was confirmed with only DON (>80%) and its 15-C derivatives (40-50%) being recognized by the antibody while 3-C DON derivatives, nivalenol, T-2 and fusarenon-X did not bind. The limit of detection is at least 0.10 microg/g. Percentage recovery for the entire assay range averages 90% with an average relative standard deviation of 8.3%. Naturally contaminated samples showed comparable precision. Column capacity was determined to be 3.3 microg. The assay showed a high degree of linearity (r2=0.999) and an optimum assay range of 0.10 to 10.0 microg/g. Comparative analysis of 28 naturally or artificially contaminated wheat samples using DONtest-HPLC and the GC-ECD method of Tacke and Casper showed that DONtest-HPLC is a statistically significant predictor of the GC-ECD method (r2=0.982).     

Detection of fungal carbohydrate antigens by high-performance immunoaffinity chromatography using a protein A column with covalently linked immunoglobulin G.

Fungal carbohydrate antigens were analysed by high-performance immunoaffinity chromatography (HPIAC) with immunoglobulin G (IgG) antibodies raised against extracellular polysaccharides of Mucor racemosus. The protein A-IgG complex was covalently bound with dimethyl pimelimidate, which enabled the use of strong acidic buffers to release the tightly bound antigens from the column. Prior to pulsed-amperometric detection, an anion-micromembrane suppressor was used to raise the pH of the effluent to above 12 without dilution. The HPIAC system provides a sophisticated method for the rapid and sensitive detection of antigenic oligomeric carbohydrates in biological samples and is proposed as an alternative to quantitative enzyme-linked immunosorbent assay techniques.     

Drug residue analysis using immunoaffinity chromatography.

The background and applicability of immunoaffinity chromatographic separations and clean-up to drug residue analysis of agricultural commodities is discussed. The uses of antibody specificity for separation and concentration of drug residues are presented. Examples of immunoaffinity chromatography for the determination of residues of (1) nortestosterone and methyl testosterone in swine muscle, urine and bile; (2) chloramphenicol in swine tissue, eggs and milk; (3) clenbuterol in calf urine; (4) zeranol and beta-zearalanolin in calf urine: (5) diethylstilbestrol, dienestrol and hexestrol in calf urine are presented. Further, examples of the successful coupling of immunoaffinity separations with other chromatographic techniques such as gas chromatography and high-performance liquid chromatography are presented.     

Determination of aflatoxin M1 using a dialysis-based immunoaffinity sample pretreatment system coupled on-line to liquid chromatography. Reusable immunoaffinity columns.

A liquid chromatographic column-switching system containing a dialysis unit and an anti-aflatoxin immunoaffinity precolumn (immuno precolumn) is described for the automated determination of aflatoxin M1 in milk samples. Both a flat membrane dialysis unit working according to the flowing donor-flowing acceptor principle and a laboratory made hollow-fibre dialysis unit working according to the stagnant donor-flowing acceptor principle were evaluated. The hollow-fibre unit is superior with respect to repeatability (3% relative standard deviation) and detection limit (10 ng/l for aflatoxin M1 in milk), in spite of the fact that the overall recovery is only 6%. Interfering compounds, which would destroy the activity of the immuno precolumn, are efficiently removed from the system by the dialysis step; a single immuno precolumn can then be used for over 70 milk analyses. No decrease in the performance of either the immuno precolumn or the hollow-fibre dialysis unit is observed.     

HPLC analysis of human epidermal growth factor using immunoaffinity precolumn. I. Optimization of immunoaffinity column

Chromatographia - The preparation of support-coupled antibodies for high-performance immunoaffinity chromatography has been demonstrated. Diol silica had the least non-specific adsorption of all...     

On-line immunoaffinity sample pre-treatment for column liquid chromatography: evaluation of desorption techniques and operating conditions using an anti-estrogen immuno-precolumn as a model system.

An immunoaffinity precolumn (immuno-precolumn) containing an immobilized antibody directed against estrogen steroids, was used as a model system for the evaluation of different desorbing techniques, suitable for on-line coupling to column liquid chromatography (LC). Desorption of estrogen analytes from the immuno-precolumn proved to be impossible with the buffers and chaotropic solutions frequently used in affinity desorption. Micellar solutions are effective in obstructing the antibody-antigen reaction, but their use as desorbing solutions was not found to be practical because of the large interferences introduced into the chromatograms. Desorption with aqueous solutions at elevated temperature, created by microwave action or conventional heating, although effective is not practical in this instance, because the agarose used in this study as the stationary phase for the immuno-precolumn is prone to heat decomposition. The most effective and practical approach is desorption with a methanol-water mixture (95 + 5 v/v). On-line dilution of the eluate allows the concentration of the desorbed analytes using a reversed-phase LC system with subsequent separation and ultraviolet detection. The performance of the system with spiked urine and plasma samples, which were introduced directly into the system, was evaluated and the results were compared with immunoselective desorption.     

An immunoaffinity column for the determination of peanut protein in chocolate.

An immunoaffinity column was prepared from rabbit polyclonal antiserum for the determination of peanut protein from food matrixes. The anti-peanut immunoglobulin G was isolated from antiserum by affinity chromatography on a column coupled with peanut protein and then attached to an AminoLink gel. The column was applied to the determination of peanut protein in chocolate after extraction, immunoaffinity chromatography, and enzyme-linked immunosorbent assay (ELISA). Overall recoveries from chocolate spiked with 0.2-3.2 micrograms/g of peanut protein averaged 77% (range, 72-84%), and the minimum detection limit was 0.1 microgram/g. Chromatography of extracts with the column improved detection limit and eliminated the matrix effect experienced with direct ELISA of chocolate extracts.     

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min⁻¹, respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2 g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L⁻¹ was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.     

Simple and high-yield purification of urine protein 1 using immunoaffinity chromatography: evidence for the identity of urine protein 1 and human Clara cell 10-kilodalton protein.

A simple and high-yield purification procedure for urine protein 1 (UP1) using anti-UP1 immunoaffinity chromatography is described. Pure UP1 was obtained in a final yield of 60.2%, and observed as a single electrophoretic band and as a single peak on reversed-phase high-performance liquid chromatography. The N-terminal amino acid residue of UP1 was found to be glutamic acid, contrary to what was reported previously. Furthermore, the N-terminal sequence of UP1 up to 53 amino acids was confirmed to be identical with that of mature human Clara cell 10-kilodalton protein, which inhibits phospholipase A2 activity.     

Analysis of wine phenolics by high-performance liquid chromatography using a monolithic type column

A polyacrylamide gel crosslinked with allyl-β-cyclodextrin can be used repeatedly for several weeks for the separation of DNA fragments, since bubbles are not generated during a run. Allyl-β-cyclodextrin can easily be synthesized in one step from allylglycidylether and β-cyclodextrin. The plate numbers for DNA fragments, up to about 1500 bp, are high: for the separation of pBR322/HaeIII fragments they were in the range 450 000–1 600 000 m⁻¹. The resolution was almost independent of the concentration of the crosslinker (allyl-β-cyclodextrin) — in sharp contrast to gels crosslinked with N,N′-methylenebisacrylamide.     

Counter-current chromatography separation scaled up from an analytical column to a production column

An analytical separation was performed on an analytical J-type counter-current chromatography (CCC) instrument using a 5.4 ml column, with a 1 ml/min mobile phase flow rate. This separation had a resolution of 0.69 and was achieved in 10 min. The same separation was performed using two 2300 ml columns connected in series at a flow rate of 850 ml/min using a production scale J-type centrifuge. This production scale separation was also obtained in 10 min with a resolution of 0.71. This represents an 850 times increase in productivity. This paper presents these separations and the underlying scale up theory.     

Determination of aflatoxins in Chinese medicinal herbs by high-performance liquid chromatography using immunoaffinity column cleanup Improvement of recovery

Although analytical methods are available for the determination of aflatoxins in medicinal herbs, none of them can be applied satisfactorily to all sample matrices. The difficulty arises from the complex chemical composition of the herbs. Recovery is generally low by using immunoaffinity column cleanup due to the acidity of the water extractive leading to a weakened binding affinity. As a solvent for dilution and neutralization, phosphate buffer saline is useful for certain herbs but not for others that have high acidity. The problem can be solved by using 0.1 M phosphate buffer, which has a higher buffering capacity and eliminates sodium chloride. The modified method was validated by the analysis of a certified reference material and shown to be useful for the determination of aflatoxins in herbal samples of high acidity.     

Selective clean-up method using an immunoaffinity column following radioimmunoassay of prostaglandin F2 alpha in biological fluids.

A selective clean-up method using an immunoaffinity column followed by radioimmunoassay (RIA) was developed for determining prostaglandin F2 alpha (PGF2 alpha) in human urine and plasma. Polyclonal antibody raised against PGF2 alpha, obtained from rabbits, was coupled to a tresyl-activated support based on a synthetic hydrophilic resin, TSKgel Tresyl-Toyopearl 650M, and used as the stationary phase for the immunoaffinity column. A human urine or plasma sample was introduced to this column, and PGF2 alpha was eluted with methanol-water (50:50, v/v) after the column had been washed. The eluate was subjected to competitive RIA for PGF2 alpha. The cross-reactivities of the RIA to a number of endogenous prostanoids, except PGD2, were negligible and the sensitivity was 4 pg/tube (p less than 0.05), giving a detection limit of 40 pg/ml when 1 ml of plasma or urine was available. The recoveries of plasma and urine samples were 98-108% and 96-106%, respectively, and their assay variances were 7-23%. The concentrations of endogenous PGF2 alpha in plasma and urine used here were estimated to be 72 and 98 pg/ml, respectively. This method should be very useful for various biological samples because of its good specificity, sensitivity, reliability and reproducibility.     

Isolation of an interleukin 2-binding receptor from activated lymphocytes by high-performance immunoaffinity chromatography.

Isolation of a lymphokine-binding receptor, from activated lymphocyte membranes, can be achieved by high-performance immunoaffinity chromatography (HPIAC), using immobilized antibodies against human interleukin 2 (IL-2), as the ligand and natural IL-2 as the receptor probe. Activated lymphocytes were reacted with IL-2, sonically disrupted and their membranes solubilized, prior to passage through the HPIAC column. The IL-2 acted as an efficient receptor probe, which helped to maintain the integrity of the receptor during the isolation procedure and also acted as an attachment antigen for the immunoaffinity ligand. Recovery of the bound receptor was achieved by dissociation of the receptor-antigen-immobilized ligand complex by the action of chaotropic ions and collection of the released receptor from the column effluent during the elution phase of the separation.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

Purification of pediocin PA-1 by immunoaffinity chromatography

A one-step purification procedure for purifying pediocin PA-1, a class IIa bacteriocin produced by Pediococcus acidilactici UL5, was developed based on column immunoaffinity chromatography with specific antipediocin PA-1 polyclonal antibodies coupled to cyanogen bromide-activated SepharoseTM. About 13.3 microg/mL purified pediocin PA-1 was obtained from 15 mL P. acidilactici UL5 culture supernatant, as measured by enzyme-linked immunosorbent assay. The specific activity and average recovery of the eluted pediocin PA-1 were about 6602 AU/mg and 53.3%, respectively. This is the first report of successful purification of pediocin PA-1 by immunoaffinity using pediocin PA-1-specific polyclonal antibodies.     

Determination of zearalenone in cereal grains, animal feed, and feed ingredients using immunoaffinity column chromatography and liquid chromatography: interlaboratory study

A method using immunoaffinity column chromatography (IAC) and liquid chromatography (LC) for determination of zearalenone in cereal grains, animal feed, and feed ingredients was collaboratively studied. The test portion is extracted by shaking with acetonitrile-water (90 + 10, v/v) and sodium chloride. The extract is diluted and applied to an immunoaffinity column, the column is washed with water or phosphate-buffered saline or methanol-water (30 + 70, v/v), and zearalenone is eluted with methanol. The eluate is evaporated, the residue is dissolved in mobile phase and analyzed by reversed-phase LC with fluorescence detection. The presence of zearalenone can be confirmed using an alternate excitation wavelength or diode array detection. Twenty samples were sent to 13 collaborators (8 in Europe, 2 in the United States, one in Japan, one in Uruguay, and one in Canada). Eighteen samples of naturally contaminated corn, barley, wheat, dried distillers grains, swine feed, and dairy feed were analyzed as blind duplicates, along with blank corn and wheat samples. The analyses were done in 2 sample sets with inclusion of a spiked wheat control sample (0.1 mg/kg) in each set. Spiked samples recoveries were 89-116%, and for the 18 naturally contaminated samples, RSDr values (within-laboratory repeatability) ranged from 6.67 to 12.1%, RSDR values (among-laboratory reproducibility) ranged from 12.5 to 19.7%, and HorRat values ranged from 0.61 to 0.90.     

Controlled oxidative protein refolding using an ion-exchange column

Column-based refolding of complex and highly disulfide-bonded proteins simplifies protein renaturation at both preparative and process scale by integrating and automating a number of operations commonly used in dilution refolding. Bovine serum albumin (BSA) was used as a model protein for refolding and oxido-shuffling on an ion-exchange column to give a refolding yield of 55% after 40 h incubation. Successful on-column refolding was conducted at protein concentrations of up to 10 mg/ml and refolded protein, purified from misfolded forms, was eluted directly from the column at a concentration of 3 mg/ml. This technique integrates the dithiothreitol removal, refolding, concentration and purification steps, achieving a high level of process simplification and automation, and a significant saving in reagent costs when scaled. Importantly, the current result suggests that it is possible to controllably refold disulfide-bonded proteins using common and inexpensive matrices, and that it is not always necessary to control protein-surface interactions using affinity tags and expensive chromatographic matrices. Moreover, it is possible to strictly control the oxidative refolding environment once denatured protein is bound to the ion-exchange column, thus allowing precisely controlled oxido-shuffling.     

Alkaline treatment of the cellulose fiber affecting membrane column behaviour for high-performance immunoaffinity chromatography

The original cellulose fibers and those treated by alkaline solution were both used to prepare the acrylic membranes. The two kinds of membranes were packed into the columns for high-performance immunoaffinity chromatography by the immobilization of protein A on them. It was observed that the alkaline treatment of the cellulose fiber decreased the pressure resistance of the membrane to the mobile phases and greatly increased the accessible volume to the proteins, but affected the adsorption capacity of human IgG on the protein A membrane columns less. There is little difference between those two kinds of membranes on the adsorption capacities of HIgG, which means that the alkaline treatment of the cellulose fiber only significantly changes the void volume inter-membrane, and the porosity and surface area of membrane less. Alkaline treatment of the cellulose fiber reduced the membrane-column efficiency significantly. Some typical examples for the immunoaffinity analysis of IgG from human and dog plasma on the protein A membrane columns are illustrated.