Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma

High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components that the control as indicated by acrylamide gel electrophoresis in the presence of urea. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.     

Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.     

Isolation of Salmonellae and Shigellae from an Artificial Mixture of Fecal Bacteria

Numerous selective media, available commercially, act by suppressing "normal" bacterial inhabitants of the intestine while permitting the growth of so-called pathogenic representatives of the family Enterobacteriaceae. This investigation attempts to evaluate the action of Salmonella-Shigella (SS) agar, xylose lysine desoxycholate (XLD) agar, and hektoen enteric (HE) agar. Salmonellae and shigellae, isolated from clinical material, were mixed in various ratios with escherichiae, Klebsiella-Enterobacter-Serratia group bacteria, and members of the tribe Proteeae, also of clinical origin. Several of the mixtures were plated in multiple dilutions on the three media. Stools in preservative were also used for evaluation of the media after the addition of definite numbers of the pathogenic bacteria. Results indicate that SS agar suppresses the shigellae along with the autochthonous members of Enterobacteriaceae. XLD and HE agars readily permit the recovery of shigellae as well as salmonellae. This recovery is not obscured by the higher yield of other species obtained with these media.     

Isolation of Salmonellae from Pork Carcasses

Four hundred and twenty pork carcasses from four abattoirs were examined for the presence of salmonellae by use of swabbing-enrichment techniques and contact plate methods. Carcasses from only one abattoir were found to be contaminated by swabbing-enrichment (23.3%) and contact plate (17.9%) methods. The area of the skin side of the ham, near the anal opening, was determined to be the area to examine for isolating salmonellae from pork carcasses with the greatest frequency. The most frequently isolated species of salmonellae in this study were Salmonella derby, S. anatum, S. typhimurium, and S. indiana.     

Use of an immunoaffinity column for tetrachlorodibenzo-p-dioxin serum sample cleanup

Covalently linking 1-amino-3,7,8-trichlorodibenzo-p-dioxin with either keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) provided antigens that generated antibodies in chickens. Competitive ELISA analysis demonstrated that the antibodies isolated from egg yolk (IgY) bound with 1,3,7,8-tetrachlorodibenzo-p-dioxin (1,3,7,8-TCDD). The antibodies were linked to CNBr-Sepharose to generate an immunoaffinity column. Radiolabeled 1,3,7,8-TCDD in a 0.05% Tween 20 solution was retained by the column and could be eluted by increasing the Tween 20 concentration. The binding efficiency for 10.7 ng per ml gel matrix ranged from 85 to 97%. Immunoaffinity columns generated by this method did not effectively bind ¹⁴C-1,3,7,8-TCDD from serum samples. Diluting the serum 1:20 with 0.05% Tween 20 increased the binding efficiency. Alternately, ethanol–hexane extraction followed by solid phase extraction on a carbon column using a fat removal protocol also provided an appropriate preaffinity column cleanup for serum samples. After this preaffinity column cleanup, spiked serum samples applied to the immunoaffinity column showed binding efficiencies of over 90%.     

Specificity of an immunoaffinity column for odorant-binding protein from bovine nasal mucosa

The odorant-binding protein from bovine nasal mucosa, purifiedby fast protein liquid chromatography, has been used to elicitpolyclonal antibodies in a New Zealand white rabbit and to preparean affinity Sepharose 4B column. The antibodies have been purifiedthrough the affinity column and used to prepare an immunoadsorbentfor specific and efficient purification of bovine odorant-bindingprotein. The immunoadsorbent does not bind proteins of comparablemolecular weight, including the olfactory marker protein, whichare localized in the primary olfactory pathway. Furthermore,the immunoadsorbent does not bind the odorant-binding proteinpresent in the nasal mucosa of other animal species.     

Analysis of deoxynivalenol and de-epoxy-deoxynivalenol in animal tissues by liquid chromatography after clean-up with an immunoaffinity column

Mycotoxin Research - A method for the determination of deoxynivalenol (DON) and its metabolite de-epoxy-deoxynivalenol (de-epoxy-DON) in blood serum, urine, faeces/digesta and bile fluid is...     

Isolation of Salmonellae by Selective Motility Systems

Salmonellae were isolated from naturally infected swine and human feces by means of selective migration through semisolid enrichment (SM) media in modified “U” tubes. Comparative studies showed that recovery of Salmonella by SM techniques was equal or superior to that of standard procedures employed in two routine diagnostic laboratories. Primary Salmonella isolations from SM enrichment were relatively free from normal fecal bacteria and often pure. The SM method required only 15% of the media and correspondingly less time and skill than the usual methods for isolating Salmonella.     

Isolation of melatonin by immunoaffinity chromatography

A single-step, highly specific and easy-to-use method was developed for isolation and purification of melatonin from complex biological matrices. Polyclonal antibodies highly specific against melatonin (with cross-reactivities with related compounds below 0.02%, except for 6-hydroxymelatonin) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of immunoaffinity gel. Melatonin recovery by the immunoaffinity method was approximately 95%, allowing single-step processing of samples prior to electrospray HPLC-MS analysis (with detection limit 10 fmol). The method was successfully used for determining melatonin in human serum and turned out to be better than the non-specific solid-phase extraction published earlier.     

Rapid separation of solasodine glycosides by an immunoaffinity column using anti-solamargine monoclonal antibody

Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column. This revised version was published online in August 2006 with corrections to the Cover Date.     

Isolation of Salmonellae from Sewage with a New Procedure of Enrichment

Forty samples of sewage on Moore's swabs were examined for the presence of salmonellae. They were first pre-enriched in buffered peptone water. From each pre-enrichment, three enrichments were made: (1) in a new, considerably modified, formula of Rappaport medium (R 10) incubated at 43 °C (R 10/43 °C), (2) in the usual formula (R25) of the same medium at 37 °C (R25/37 °C) and (3) in Muller-Kauffmann's tetrathionate broth at 43 °C (MK/43 °C). Practically the same numbers of swabs were found positive by the first two enrichment procedures, 38 and 39 respectively, while only 17 were found positive by the MK procedure. The R10/43 °C method was superior to the two other procedures; it yielded 103 strains of salmonellae as against 82 with the second Rappaport procedure, and only 25 with the MK/43 °C technique. A similar observation was made concerning the frequency of isolation of different serotypes by the three procedures; the number of the isolated serotypes was 24, 19 and 11, respectively. The new R 10/43 °C method of enrichment had also a much stronger inhibitory effect on the competing bacteria than the two other procedures of enrichment used.     

Preparation of an immunoaffinity column and its application in sample cleanup for methandrostenolone residues detection

Methandrostenolone (MA) is a steroid used as veterinary medicine on stockbreeding to promote animal growth. The use of MA has been strictly regulated because of its harmful effect on consumers. This paper describes the production of polyclonal antibody (pAb) against MA, the preparation of immunoaffinity column (IAC) and its potential application to the selective extraction of MA residues from animal tissue and feed samples. The produced pAb exhibited good sensitivity to MA with an IC(50) value of 5.6 ng/mL. The cross-reactivity values of the antibody with MA structurally related compounds of testosterone propionate (TP) and trenbolone (TR) were lower than 0.6%. By coupling the produced antibody with CNBr-activated Sepharose 4B, an IAC was prepared. 2% methanol and 80% methanol were selected as loading and eluting solution by optimization. The maximum capacity of the column for MA was approximately 334 ng/mL gel. The average recovery of 20, 40 and 60 ng/mL MA standard solutions from IACs was 97.9% with the relative standard deviation (RSD) among columns of 6.7%. After 3 times of repeated usage, the column capacity and recovery rate still remained 82.0% and 92.6% respectively. The IACs were then challenged with MA-fortified animal tissue and feed samples, recoveries of MA were found to be in the range of 83.5-99.7%.     

Multi-analyte analysis of biological fluids with a recycling immunoaffinity column array.

A system for isolating and measuring up to 30 analytes in a single biological sample is described. The system is based on recycling a pre-labeled sample through an array of capillary immunoaffinity columns, each packed with glass beads, coated with a different antibody, thus enabling each column to isolate and extract a single analyte. Detection of the bound analytes is achieved by laser-induced fluorescence (LIF), using a laboratory-built scanning detector coupled to a fiber-optic spectrometer. The array can be regenerated up to 200 times, provided a suitable temperature is maintained. The individual immunoaffinity columns are able to bind between 2.9 and 3.6 ng of analyte, depending upon the individual column, with lower limits of detection (LOD) in the order of 1.6-2.8 pg/ml. The inter- and intra-assay coefficients of variation (CV) for all 30 columns in the array were less than 6.03+/-0.33 at analyte concentrations of 100 pg/ml. Comparison to standard enzyme-immunoassays demonstrated r(2) values in the range of 0.9151-0.9855 when analyzed by least-squares linear regression.     

Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples

An immunoaffinity column (IAC) was developed by covalently coupling polyclonal antibodies against estrogenic bisphenols to CNBr-activated Sepharose 4B. The IAC showed high affinity for bisphenol A, while phenol was barely retained. Proteins in the sample matrix showed little nonspecific adsorption on the column. The best binding solvent for bisphenol A was found to be 0.01 mol l(-1) phosphate-buffered saline (PBS) and the optimal operating temperature was 4 degrees C. The bound bisphenol A could be quantitatively recovered by 1 ml of methanol-water (80:20) with an average recovery of 91.8% and a relative standard deviation of 7.1% (n=6). The immunoaffinity column has been successfully used for the isolation and purification of bisphenol A from serum samples.     

Isolation of nitrotyrosine-containing peptides by using an insoluble-antibody column

Antibodies to nitrotyrosine were prepared in goats or rabbits by injecting a nitrotyrosine-protein conjugate. The antibodies were purified by using a protein that had been nitrated with tetranitromethane. These antibodies were used to isolate nitrotyrosine-containing peptides from nitrated lysozyme. The nitrotyrosine-containing peptides were thus purified (55% yield) in one step and the positions of nitration in lysozyme were found to be at tyrosine-20 and tyrosine-23. This method is of general applicability for the determination of the position of nitrotyrosine in proteins.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.