Sol-gel immunoaffinity chromatography for the clean up of ochratoxin A contaminated grains

This paper describes the application of sol-gel immunoaffinity columns for clean up of ochratoxin A contaminated cereal crops. Monoclonal antibodies selective for OTA have been entrapped into the pores of a sol-gel matrix in order to prepare immunoaffinity columns. Different parameters such as amount of entrapped antibodies and loading conditions were optimized to obtain highest possible recoveries of OTA. The method has been found to be a suitable tool in sample preparation prior to HPLC-FLD determination and as selective as conventional commercially available immunoaffinity columns. In the clean up of different cereals mean recoveries of 82±5%, 90±6% and 91±3%, were obtained for wheat, barley and rye, respectively, with sol-gel columns containing 1mg of anti-OTA antibodies. The detection limit (signal-to-noise ratio, 3) was 0.5 μg/kg and the limit of quantification (signal-to-noise ratio, 10) determined to be 1 μg/kg. Sol-gel columns can be reused 7 times without significant loss of recovery. After 10 applications the recovery decreased to approx. 50%.     

Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.     

Online immunoaffinity assay-CE using magnetic nanobeads for the determination of anti-Helicobacter pylori IgG in human serum

About two-thirds of the world's population is infected with Helicobacter pylori (H. pylori). This Gram-negative bacterium is the most important etiological agent of chronic active type B gastritis and peptic ulcer diseases. Conventional methods such as gastric biopsy, ELISA and culture, require a long time for the determination of H. pylori infections. Moreover, the antibodies in human serum sample are capable to react immunologically with the purified H. pylori antigens immobilized on different kinds of support like magnetic nanobeads. In this study, we have developed an online immunoaffinity assay-CE to determine the concentration of anti-H. pylori IgG using magnetic nanobeads as a support of the immunological affinity ligands and an LIF as a detector. The separation was performed in 0.1 M glycine-HCl, pH 2, as the background electrolyte. The linear calibration curve to predict the concentration of H. pylori-specific immunoglobulin G antibodies in serum was produced within the range of 0.12-100 U/mL. The linear regression equation was i = 492.86+96.03 × C(anti-H. pylori), with the linear regression coefficient r(2) = 0.999. The LOD calculated by fluorescence detection procedure was of 0.06 U/mL. The whole assay was done in no more than 35 min and it was entirely automatized. The development of immunoaffinity assay-CE in this study demonstrates that there is a large possibility to introduce nanotechnology in several fields with significant advantages over the classic methodologies. Our proposition comprises the diagnosis and screening field.     

An efficient immunoaffinity chromatographic method for extraction and purification of papaverine from samples of pericarpium papaveris and food products

A highly selective immunoaffinity column was obtained by coupling anti-papaverine polyclonal antibodies to CNBr-activated Sepharose 4B. It was found that the coupling efficiency and performance of the immunoaffinity column were greatly improved by prolonging the coupling reaction time from 3 h at 20 degrees C with shaking to incubation overnight at 4 degrees C after the 3 h shaking reaction. The pH and ionic strength were observed to be the most important factors that influence the binding of papaverine to the immunoaffinity column. Using 0.01 mol/L phosphate buffered saline (PBS, pH 8.3) and methanol-water (80:20, v/v) as the loading and eluting solutions, respectively, papaverine was first retained on the column and then quantitatively eluted out with a mean recovery of 86% at a loading concentration of 1 microg/mL. When applied to real samples of pericarpium papaveris and food products, the established immunoaffinity column showed high efficiency in removing the matrix interferences in the samples and satisfactory recovery results were obtained. The method was useful for extraction and purification of papaverine from related samples.     

Determination of four fluoroquinolones in milk by on-line immunoaffinity capture coupled with reversed-phase liquid chromatography.

An automated, on-line immunoaffinity extraction method was developed for the analysis of 4 fluoroquinolones in milk: ciprofloxacin, difloxacin, enrofloxacin, and sarafloxacin. This method involves analyte extraction using an immunoaffinity capture column containing anti-fluoroquinolone antibodies coupled on-line with reversed-phase column chromatography. Liquid chromatographic analyses were performed by isocratic elution using a mobile phase of 2% acetic acid-acetonitrile (85 + 15) and an Inertsil phenyl column with fluorescence detection at excitation and emission wavelengths of 278 and 444 nm, respectively. No significant interferences from the sample matrix were observed, indicating good selectivity with the immunoaffinity column. Recoveries from fortified raw milk samples (5-50 ppb of each fluoroquinolone) ranged from 72 to 90%, with standard deviations of < or = 8%.     

On-line immunoaffinity column-liquid chromatography-tandem mass spectrometry method for trace analysis of diuron in wastewater treatment plant effluent sample

An on-line immunoaffinity column with liquid chromatography/tandem mass spectrometry (IAC-LC-MS/MS) method for the determination of diuron in water matrices was described. This method used a sol-gel immunoaffinity column (20 mm x 4 mm I.D.) for on-line sample cleanup and enrichment, a monolithic analytical column (100 mm x 4.6 mm I.D.) for separation, and a triple quadrupole mass spectrometer for quantitation. The major challenges for the on-line set-up were discussed. The optimized on-line protocol was emphasized by the fact that low limit of quantitation (LOQ) of 1.0 ng/L was achieved with only 2.5-mL sample. In addition, a satisfactory accuracy ( approximately 90% of recovery) and precision (<6% of relative standard deviation) at 50 ng/L concentration were also obtained. Due to the ability of the sol-gel immunoaffinity column to eliminate matrix effect, the on-line IAC-LC-MS/MS analysis method can reliably determine diuron in wastewater treatment plant effluent sample.     

Simultaneous determination of nandrolone, testosterone, and methyltestosterone by multi-immunoaffinity column and capillary electrophoresis

A multitarget antibody immunoaffinity column was proposed for the purification and enrichment of nandrolone, testosterone, and methyltestosterone from urine. Nandrolone-3-site substituted antigen was designed and synthesized and the polyclonal antibody was prepared with immunizing rabbits. The stationary phase of the immunoaffinity column was synthesized by covalently bonding the antibodies specific to nandrolone, testosterone, and methyltestosterone onto CNBr-actived Sepharose 4B. The analytes of interest were extracted with a methanol/water mixture in one step. The immunoaffinity column showed high affinity and high selectivity to a class of structurally related compounds. The elution was then transferred to a micellar electrokinetic CE system with a running buffer of sodium borate and sodium cholate for separation and determination. Recoveries of the three steroids from complex matrix were 88-94% with RSD values less than 5.2%. Optimization of the immunoaffinity column purification was achieved and the feasibility of the technique for the analysis of steroid hormone was discussed. The results indicated that the combination of multi-immunoaffinity column and CE was an effective technique, which was rapid, simple, and sensitive for the assay of steroids.     

Multiple mycotoxins analysis in animal feed with LC‐MS/MS: Comparison of extract dilution and immunoaffinity clean‐up

The aim of this study was a performance comparison of two clean‐up procedures (dilutions versus immunoaffinity columns) in the simultaneous determination of eight mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1 & B2, ochratoxin A, toxin T‐2 & HT‐2 and zearalenone) in the animal feed. After extraction the analytes were separated on a Kinetex Biphenyl column with a gradient elution using methanol/0.01 M ammonium acetate as a mobile phase and analyzed with the LC‐MS/MS technique. Both of the procedures were validated by analysis of a series of spiked feed samples (n = 6) at three different concentration levels. Better signal to noise ratios were observed for immunoaffinity clean‐up. The recoveries of analyses were in the range 88–110% for the dilution procedure and 78–120% for the immunoaffinity clean‐up. The dilution procedure was more precise (coefficient of variation of the within‐laboratory reproducibility for it was 7.8–22.4% in comparison to 12–35.5% for the immunoaffinity clean‐up. The results show that both procedures fulfilled the requirements for mycotoxin analysis and can be used successfully in multi‐analyte determination. Although the dilution procedure shows better precision and trueness, the immunoaffinity clean‐up procedure can have advantages in more complex feed samples thanks to lower matrix effect and limits of detections.     

Single-molecule immunosorbent assay as a tool for human immunodeficiency virus-1 antigen detection

Ultrasensitive detection and quantification of viral antigen with a novel single-molecule immunosorbent assay (SMISA) was achieved. Antigen from human immunodeficiency virus type 1 (HIV-1), the major etiological agent of acquired immune deficiency syndrome, served as the screening target in this study. The target molecule was sandwiched between a polyclonal capture antibody and a monoclonal detector antibody. The capture antibody was covalently immobilized on (3-glycidoxypropyl) trimethoxy silane-modified glass slides. The detector antibody was conjugated with fluorescent Alexa Fluor 532 labeled secondary antibody prior to being used as a probe for the antigen. Imaging was performed with a total internal reflection fluorescence single-molecule detection system. This technique is demonstrated for detecting HIV-1 p24 antigen down to 0.1 pg/mL with a dynamic range of over four orders of magnitude. A Langmuir isotherm fits the molecule count dependence on the target concentration. The target antigen was further tested in 20% human serum, and the results showed that neither sensitivity nor dynamic range was affected by the biological matrix. SMISA is therefore a promising approach for the early diagnosis of viral induced diseases.     

Sol-gel glass as a new support matrix in immunoaffinity chromatography

A new type of immunoaffinity matrix, based on the encapsulation of antibodies in a sol-gel glass network was investigated in order to extract traces of 1-nitropyrene from water. In opposite to generally used support matrices, the sol-gel does not need an activation or derivatization step to immobilize antibodies. Also no bacteriostatic agent must be added for the storage because due to the narrow network (<100 Å) no bacteria or proteolytic enzymes can diffuse to the entrapped antibodies. Easy preparation (one-step) under mild conditions (in buffer and always at 4°C) within a short time (about 2 min) makes the sol-gel method attractive as an inexpensive immobilization technique for immunoaffinity chromatography. The sol-gel glass was used to develop an immunoadsorber for 1-nitropyrene (1-NP). No unspecific adsorption of 1-NP on the new sorbent material was found and no antibody leaching under harsh elution conditions. Traces of 1-NP were enriched with excellent recoveries and determined with on-line reduction in HPLC, followed by fluorescence detection.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70th birthday     

Analytical aspects of veterinary drug residues

A survey is given with respect to recent developments in the analysis of veterinary drug residues in food of animal origin. The application of solidphase extraction, immunoaffinity and matrix solid-phase dispersion as rapid clean-up and concentration techniques is discussed. Attention is paid as well to automated methods such as FAST-LC and immunoassays.     

The new genotypic human calicivirus isolated in Seoul

A new type of human calicivirus (HuCV) showing the classic cup-shaped surface morphology was identified in the stool sample from a child with symptoms of acute gastroenteritis in Seoul, Korea (SK virus). Genomic RNA was extracted directly from the stool sample, and the nucleotide sequence of 3.2 kb of the 3' end of SK virus was determined from cDNA. This region spanned sequences from the RNA-dependent RNA polymerase (RDRP) region in the open reading frame 1 (ORF1) to the 3' poly A tail. The non-structural and capsid protein coding sequences were fused in a single ORF as observed in Manchester type (Genogroup III). However, ORF2 of Manchester virus was missing in SK virus. In RDRP region, SK virus showed amino acid and nucleotide identities of 74-75% and 68-69% respectively, with those of Manchester virus, while showed 34-46% and 55-60% identities respectively with those of other human caliciviruses. However, capsid protein of SK virus showed a partial (29-46%) amino acid identity with those of other caliciviruses including Manchester type. The closest resemblance in amino acid (97-99%) and nucleotide sequence (85-86%) identities were found in RDRP region with Vanderbijlpark and Pretoria isolates recently found in South Africa. These results suggest that SK virus together with Vanderbijlpark and Pretoria isolates belong to a new type different from Manchester virus.     

Development of a fast and simple immunochromatographic method to purify alpha 1-acid glycoprotein from serum for analysis of its isoforms by capillary electrophoresis

Alpha 1-acid glycoprotein (AGP) is a very heterogeneous glycoprotein presenting several isoforms due to variations in its polypeptidic and glycosidic moieties. Differences in AGP isoforms between healthy and diseased individuals have been related to different pathological situations such as cancer or cardiovascular diseases, among others. Capillary electrophoresis study of the role of AGP isoforms as biomarkers requires prior purification of AGP from biological samples. Current AGP purification methods are time- and labour-consuming, and generally they have not been proven to be compatible with capillary electrophoresis analysis. In this work, different methods for AGP purification from human serum are developed and compared. The applicability of acidic precipitation and immunoaffinity chromatographic methods for AGP purification are studied. Two different immunoaffinity approaches are employed; in the first one, interferents present in the AGP sample are captured and removed, and in the second one, AGP is retained in a house-made anti-AGP column, being in this way isolated from the rest of interferents of the sample. Best results in AGP purification from human serum to be analyzed by capillary zone electrophoresis (CZE) were obtained when acidic purification was combined with immunoaffinity chromatography (IAC) employing the house-made anti-AGP column. The method was shown not to alter the proportion of AGP peaks due to isoforms existing in AGP samples. The applicability of this fast and easy purification method developed for analyzing by CZE isoforms of AGP from natural serum samples by CZE is demonstrated.     

Multi-immunoaffinity chromatography: a simple and highly selective clean-up method for multi-anabolic residue analysis of meat

A method for the detection of nortestosterone (NT) in bovine muscle at levels below 1 microgram/kg is described, based on enzymatic digestion of the sample, clean-up by immunoaffinity chromatography after defatting and detection by gas chromatography-mass spectrometry (selected-ion monitoring). The immunoaffinity matrix was prepared after combining the isolated immunoglobulin G fractions from a rabbit antiserum raised against NT and methyltestosterone (MT). Its capacity per millilitre of gel was approximately 10 ng for each of the two steroids. Results for samples containing 0.1 microgram/kg NT and above are described. It is concluded that for multi-residue analysis of samples of muscle at levels as low as 0.1 microgram/kg, multi-immunoaffinity chromatography is a very suitable method of sample clean-up. For purposes of quantification the trideuterated internal standard [16,16,17 alpha-2H3] nortestosterone was synthesized.     

免疫亲和富集-荧光光谱法检测牛血清白蛋白的研究

本文通过戊二醛法合成免疫磁球(IMMs),采用荧光电镜、透射电镜和振动样品磁强计对其进行表征。合成的IMMs为单分散的球形结构,粒径约为510 nm,具有超顺磁性(饱和磁化强度为40.44 emu/g)。基于此,以牛血清白蛋白(BSA)作为蛋白质模型,建立了免疫亲和富集-荧光光谱法用于目标蛋白质的分离与检测。考察并优化了影响免疫亲和富集的因素。在最佳实验条件下,IMMs对目标蛋白质的最大吸附容量为107.7μg/g,可重复使用10次以上。将该方法应用于人血清基质中BSA的测定,加标回收率不低于89.3%,由基质辅助激光解吸电离飞行时间质谱仪测得分离后的BSA纯度较高。结果表明,该方法适用于人血清样品中蛋白质的选择性分离与检测。     

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.     

Faster isolation of recombinant factor VIII SQ, with a superporous agarose matrix.

A superporous agarose matrix was compared with a corresponding homogenous matrix in the isolation of recombinant factor VIII SQ (r-VIII SQ) by immunoaffinity chromatography. As a reference, the commercially available Sepharose FastFlow, used for a similar purification in the industry, was also evaluated. Breakthrough curves are described for flows between 50 and 400 cm/h with pre-purified r-VIII SQ and with cell culture broth. The superporous gel gave the best performance and a 1000-fold purification was obtained in a one-step procedure. The superporous matrix made it possible to increase the throughput about four-fold, presumably due to its better mass transfer properties. The importance of the ligand distribution profile is discussed based upon immunofluorescence microscopy data.     

Applications and Recent Developments in the use of Antibodies for Analysis

ABSTRACT

There are many compounds that require analysis ranging from pesticide levels in corn to disease markers in human patients. There are copious challenges to be met when measuring analytes such as the matrix in which they are to be determined, the amounts present, the cost and the rapidity of the result required. Enzyme immunoassays, immunoaffinity chromatography, immunomagnetic polymerase chain reaction, flow cytometry and immunobiological biosensors have all characteristics that can enhance analytical techniques. Antibody-based methods have found applications in a large number of diverse areas such as food and water analysis, clinical diagnosis and therapeutics The structure and modes of production of antibodies and antibody-based derivatives is described and their applications in analysis critically examined.