Immunoaffinity Research Articles

Multiplexed Quantification of Proglucagon-Derived Peptides by Immunoaffinity Enrichment and Tandem Mass Spectrometry after a Meal Tolerance Test.

Proglucagon-derived peptides (PGDPs), which include glucagon-like peptide (GLP)-1, glucagon, and oxyntomodulin, are key regulators of glucose homeostasis and satiety. These peptide hormones are typically measured with immuno-based assays (e.g., ELISA, RIA), which often suffer from issues of selectivity. We developed a multiplexed assay for measuring PGDPs including GLP-1 (7-36) amide, GLP-1 (9-36) amide, glucagon, and oxyntomodulin by mass spectrometry and used this assay to examine the effect of a meal tolerance test on circulating concentrations of these hormones. Participants fasted overnight and were either given a meal (n = 8) or continued to fast (n = 4), with multiple blood collections over the course of 3 h. Plasma samples were analyzed by microflow immunoaffinity (IA)-LC-MS/MS with an isotope dilution strategy. Assay performance characteristics were examined and established during analytical validation for all peptides. Intra- and interassay imprecision were found to be 2.2%-10.7% and 6.8%-22.5%, respectively. Spike recovery was >76%, and dilution linearity was established up to a 16-fold dilution. Immediately after the meal tolerance test, GLP-1 and oxyntomodulin concentrations increased and had an almost identical temporal relationship, and glucagon concentrations increased with a slight delay. IA-LC-MS/MS was used for the simultaneous and selective measurement of PGDPs. This work includes the first indication of the physiological concentrations and modulation of oxyntomodulin after a meal.

The Role of Isolation Methods on a Nanoscale Surface Structure and its Effect on the Size of Exosomes.

Exosomes are ∼100 nanometre diameter vesicles secreted by mammalian cells. These emerging disease biomarkers carry nucleic acids, proteins and lipids specific to the parental cells that secrete them. Exosomes are typically isolated in bulk by ultracentrifugation, filtration or immunoaffinity precipitation for downstream proteomic, genomic, or lipidomic analysis. However, the structural properties and heterogeneity of isolated exosomes at the single vesicle level are not well characterized due to their small size. In this paper, by using high-resolution atomic force microscope imaging, we show the nanoscale morphology and structural heterogeneity in exosomes derived from U87 cells. Quantitative assessment of single exosomes reveals nanoscale variations in morphology, surface roughness and counts isolated by ultracentrifugation (UC) and immunoaffinity (IA) purification. Both methods produce intact globular, 30–120 nm sized vesicles when imaged under fluid and in air. However, IA exosomes had higher surface roughness and bimodal size population compared to UC exosomes. The study highlights the differences in size and surface topography of exosomes purified from a single cell type using different isolation methods.

Applied Technology in Using HPLC for Determining Aflatoxin Contents in Peanuts

Aflatoxins in grinded peanuts were extracted with 70% methanol and purified with immuno affinity column. In order to enhance the test sensitivity, the Aflatoxin was derived by trifluoroacetic acid. the derived Aflatoxin B1, B2, G1 and G2 were analyzed by HPLC with fluorescence detector. The determination could be completed in 14 minutes. The detect limits of Aflatoxin B1, B2, G1 and G2 were 0.04μg/kg, 0.01μg/kg, 0.20μg/kg and 0.02μg/kg respectively, the recoveries were from 81.0% to 115.0%, the relative standard deviations were less than 8.2%.

Multiplexing Determination of Small Cell Lung Cancer Biomarkers and Their Isovariants in Serum by Immunocapture LC-MS/MS

A multiplex method for the determination of the small cell lung cancer (SCLC) markers progastrin releasing peptide (ProGRP) and neuron specific enolase (NSE) is presented, which involves coextraction by immunoaffinity (IA) beads and codetermination by selected reaction monitoring (SRM). The performance was compared with two IA SRM methods which were recently validated for individual marker determination. The multiplexing method reduces sample volume, handling time per sample, and reagent consumption and shows good linearity, recovery, quantitative measurements, and sensitivity with lower limit of detection (LLOD) values of 7.2 pM (=90 pg/mL) and 4.5 pM (=210 pg/mL) and lower limit of quantitation (LLOQ) values of 24 pM (=300 pg/mL) and 15 pM (=700 pg/mL), for total ProGRP and γ-NSE, respectively. The novel aspect of this approach is the multiplexing of ProGRP and NSE with the additional ability to perform fingerprinting by the selective determination of ProGRP isoform 1, ProGRP isoform 3, and total ProGRP, as well as the α- and the γ-subunit of NSE isoenzymes. Six serum samples from patients with SCLC were analyzed to demonstrate the methods feasibility to simultaneously differ between and individually quantify ProGRP, NSE, and their isoform and isoenzyme variants, respectively. Both the presence of and variation between all the isoforms and isoenzymes, as well as covarying results with the conventional immunometric assays for total ProGRP and γ-NSE, were seen in the analyses of patient serum samples.

Immunoaffinity clean-up of mycotoxins with organic solvent-free elution

A novel immunoaffinity (IA) clean-up procedure for the isolation of mycotoxins from maize extracts is described. Commonly, mycotoxins are eluted with organic solvents from IA columns after isolation. The new procedure uses water at 70 °C as alternative. The resulting eluate is suitable for direct and complete injection onto a reversed-phase liquid-chromatography column. Evaporation, reconstitution or dilution are not required. The procedure has been tested for a variety of mycotoxins, including deoxynivalenol (DON) zearalenone, T-2 and HT-2 toxins, aflatoxins and ochratoxin A in an array of different matrices. The example of DON in maize is discussed in more detail and evaluated for repeatability (5%, n=10) and recovery (95%, n=10).

Determination of Deoxynivalenol-3-Glucoside in Cereals by Hydrophilic Interaction Chromatography with Ultraviolet Detection

A new hydrophilic interaction liquid chromatography (HILIC) method was developed for the determination of deoxynivalenol-3-glucoside (D3G) in cereals. D3G was extracted with water and cleaned with an immunoaffinity (IA) column, followed by chromatographic separation on a Syncronis HILIC column with acetonitrile-water (90:10, v/v) as the mobile phase, and detected at 220 nm by UV detection. The calibration curve was found to be linear in the range of 0.1–2.0 μg/ml. Limit of detection and limit of quantification were 8 and 25 μg/kg, respectively. The method was applied to the determination of D3G in 14 cereal samples, which were analyzed as well as by LC-MS/MS. The results indicate that the method can be considered as an alternative to LC-MS/MS and could be adopted by nonprofessional analytical labs.

Preparation and evaluation of an immunoaffinity sorbent with Fab′ antibody fragments for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

An immunoaffinity (IA) sorbent with antibody fragments was prepared for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). The antibody fragmentation was evaluated by MALDI-TOF-MS. Fab′ fragments obtained from a polyclonal IgG antibody against Endomorphins 1 and 2 (End1 and End2) were covalently attached to succinimidyl silica particles to prepare the IA sorbent. An IA-SPE-CE–MS methodology was established analyzing standard solutions of End1 and End2 and acceptable repeatability, linearity ranges and LODs (0.5 and 5ngmL−1, respectively) were obtained. The LOD of End1 was slightly better than that previously obtained using an IA sorbent with intact antibodies (1ngmL−1). In human plasma samples, End1 and End2 could be detected at 1 and 50ngmL−1, respectively, which meant an improvement of 100 and 2-fold with regard to the LODs using an IA sorbent with intact antibodies (100ngmL−1).

Characterization and validation of sampling and analytical methods for mycotoxins in workplace air

Mycotoxins are produced by certain plant or foodstuff moulds under growing, transport or storage conditions. They are toxic for humans and animals, some are carcinogenic. Methods to monitor occupational exposure to seven of the most frequently occurring airborne mycotoxins have been characterized and validated. Experimental aerosols have been generated from naturally contaminated particles for sampler evaluation. Air samples were collected on foam pads, using the CIP 10 personal aerosol sampler with its inhalable health-related aerosol fraction selector. The samples were subsequently solvent extracted from the sampling media, cleaned using immunoaffinity (IA) columns and analyzed by liquid chromatography with fluorescence detection. Ochratoxin A (OTA) or fumonisin and aflatoxin derivatives were detected and quantified. The quantification limits were 0.015 ng m(-3) OTA, 1 ng m(-3) fumonisins or 0.5 pg m(-3) aflatoxins, with a minimum dust concentration level of 1 mg m(-3) and a 4800 L air volume sampling. The methods were successfully applied to field measurements, which confirmed that workers could be exposed when handling contaminated materials. It was observed that airborne particles may be more contaminated than the bulk material itself. The validated methods have measuring ranges fully adapted to the concentrations found in the workplace. Their performance meets the general requirements laid down for chemical agent measurement procedures, with an expanded uncertainty less than 50% for most mycotoxins. The analytical uncertainty, comprised between 14 and 24%, was quite satisfactory given the low mycotoxin amounts, when compared to the food benchmarks. The methods are now user-friendly enough to be adopted for personal workplace sampling. They will later allow for mycotoxin occupational risk assessment, as only very few quantitative data have been available till now.

Understanding the Role of Immunoaffinity-Based Mass Spectrometry Methods for Clinical Applications

Both ligand binding assays (LBA)2 and mass spectrometry (MS) methods are widely used in routine clinical analysis. In recent years, an interesting trend has emerged which blends these 2 techniques to combine the selectivity of immunoaffinity (IA) extraction with the specificity of MS detection (IA-MS). The brief communication by Laha and coworkers in the current issue of Clinical Chemistry , entitled “Characterizing Antibody Cross-reactivity for Immunoaffinity Purification of Analytes prior to Multiplexed Liquid Chromatography–Tandem Mass Spectrometry,” is an excellent example of the power of this combined approach (1). These authors, who have had previous experience with multiplexed liquid chromatography–tandem MS (LC-MS/MS) vitamin D analysis (2), showed that the limited specificity of an anti–1α,25-dihydroxyvitamin D capture antibody could be used with LC-MS/MS to deliver a sensitive, highly specific assay for 5 vitamin D–related metabolites. Because of differing affinities of the capture antibody for the various metabolites and their wide range in concentrations (1000-fold), Laha et al. performed a detailed binding assessment by LC-MS/MS using synthetic standards to verify the suitability of their approach. Moreover, because 2 known interfering metabolites were shown not to bind to the antibody, the authors were able to use a shorter chromatographic run time. The report by Laha et al. highlights the growing awareness about the need for highly specific assays in clinical biomarker analysis. Historically, lipids have been measured by competitive LBA formats. However, accumulating evidence has raised concerns about the specificity of these approaches, and several assays have been replaced by more specific LC-MS/MS methods. Testosterone analysis is probably the most widely recognized example of this trend (3). Still, although LC/MS/MS is a well-established tool for clinical biomarker analysis, comparatively few IA-MS applications have appeared for small molecules (4), and the uptake of IA-MS utilization is uncertain. Interestingly, the situation is entirely different for …

Lab on a Chip: Scandinavia

Examples of early Scandinavian papers on microfluidics, miniaturized sensors and integrated systems date back to the early to mid-90s. At that time, the emergence of lab-on-a-chip systems and micro-Total-Analysis-Systems was driven on the one side by analytical chemists trying to incorporate and miniaturize all steps of an analytical process. Here, separation methods and, in particular capillary electrophoresis, played a central role in those early days. Coming from another angle, engineers with experience in semiconductor technology, microfabrication and fluid mechanics were also investigating novel ways to manipulate fluids in confined spaces and apply this to problems in chemistry, biology and medicine. Both ‘‘camps’’ have a strong tradition in Scandinavia, which is why it is hardly surprising that Scandinavian researchers at academic institutions, as well as in companies, were involved in the establishment of the field of lab-on-achip from the get-go. Across the Scandinavian territory a number of nucleation points can readily be found. In particular, the Uppsala region in Sweden pioneered early developments in industry, headed by Pharmacia Biotech, which had a research unit at the time doing very early developments in hot embossed polymer microchannels for microfluidic manipulations. This later gave rise to Gyros AB, developing centrifugal microfluidics-based assays and sample preparation. Also, the SPR technology and the commercialization of immuno affinity interaction instrumentation was pioneered in Uppsala by Biacore. Since then, many other strongholds, from Aalto University in Finland to the centers in the Oslo area and Trondheim in Norway, to KTH, Chalmers, Linkoping and Lund in Sweden and on to DTU in Denmark, have been established and continue to thrive. An important milestone for the consolidation of LOC research in Scandinavia and an acknowledgement of the research quality in the region was certainly the MicroTAS conference in Malmo in 2004, the major international conference on miniaturized systems for application in the life sciences, at that time attended by about 750 people. Department of Measurement Technology and Industrial Electrical Engineering, Lund University, Lund, Sweden. E-mail: thomas.laurell@elmat.lth.se Department of Micro and Nanotechnology, Technical University of Denmark, Lyngby, Denmark. E-mail: joerg.kutter@nanotech.dtu.dk

Preparation and evaluation of an immunoaffinity sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry

In this study, we explored a procedure for the preparation of an immunoaffinity (IA) sorbent for the analysis of opioid peptides by on-line immunoaffinity solid-phase extraction capillary electrophoresis–mass spectrometry (IA-SPE-CE–MS). We followed a site-specific antibody immobilization approach based on the covalent attachment of the oxidized antibodies through their carbohydrate moieties to hydrazide silica particles, using a polyclonal antibody against Endomorphin 1 and 2 (End1 and End2). The main features of the IA sorbent were studied, such as the amount of hydrazide groups and antibodies attached onto oxidized diol silica particles. Once the procedure was optimized, standard solutions of End1 and End2 were used in order to establish the IA-SPE-CE–MS methodology. Acceptable repeatability, reproducibility and linearity range values were obtained for the proposed methodology. The limits of detection (LODs) of 1ngmL−1 were approximately 100-fold better than those obtained by CE–MS. Selectivity of the IA sorbent was good but some cross-reactivity against Dynorphin A (1–7) was observed when a mixture of several opioid peptides was analyzed. Human plasma samples spiked with End1 and End2 were also analyzed and both peptides could be detected down to 100ngmL−1.

Tumor microenvironment derived biomarkers for the early detection of pancreatic cancer (PDAC).

Abstract e14591 Background: The important role played by activated pancreatic stellate cells (aPSCs) in PDAC has only been recently appreciated. By shaping the tumor microenvironment, APSCs contribute to tumor growth, metastasis and drug resistance. Innovative stable isotope labeling with amino acids in cell culture (SILAC) and multiple reaction monitoring (MRM)-MS provide tools for interrogating human serum samples for aPSC derived biomarkers of PDAC. 600 proteins secreted by aPSCs were previously characterized (Wehr, 2010). Selected proteins were shown to be overexpressed in pancreatic tumors. An immortalized PSC cell line was SILAC labeled; secreted proteins were collected from activated cells and used as labeled internal standards. An MRM-MS method was developed to quantitate the 40 most abundant secreted aPSC proteins in serum. Internal standard proteins were added to human serum samples prior to immunoaffinity (IA) removal of abundant serum proteins, trypsin digested and analyzed by liquid chromatography (LC)/MRM-MS. Powered primary endpoint: characterize the sensitivity and specificity of biomarker panel in a case control study of 10 patients with PDAC and 10 healthy controls. Secondary endpoint: biological pathway analysis of discriminating biomarkers. 183 unique peptides were identified for the selected 40 aPSC proteins. 69 peptides, representing 36 proteins, were reproducibly detected by MS. During method development, a prototypical biomarker candidate, MMP2, was monitored; Western blot and MS analysis showed that MMP2 was not lost by IA processing, and removal of abundant proteins was required for detection of MMP2 by MS. The method was found to be linear over a range of 4-100 fmol/µL media, and met precision criteria of

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300μl oligosorbent (24nmol DNA) showed a linear (r=0.999) behaviour in the range of 0.4–500ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5–50ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r=0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.

Depletion of highly abundant proteins in blood plasma by hydrophobic interaction chromatography for proteomic analysis

The proteomic analysis of plasma is extremely complex due to the presence of few highly abundant proteins. These proteins have to be depleted in order to detect low abundance proteins, which are likely to be of biomedical interest. In this work it was investigated the applicability of hydrophobic interaction chromatography (HIC) as a plasma fractionation method prior to two-dimensional gel electrophoresis (2DGE). The average hydrophobicity of the 56 main plasma proteins was calculated. Plasma proteins were classified as low, medium and highly hydrophobic through a cluster analysis. The highly abundant proteins showed a medium hydrophobicity, and therefore a HIC step was designed to deplete them from plasma. HIC performance was assessed by 2DGE, and it was compared to that obtained by a commercial immuno-affinity (IA) column for albumin depletion. Both methods showed similar reproducibility. HIC allowed partially depleting α-1-antitrypsin and albumin, and permitted to detect twice the number of spots than IA. Since albumin depletion by HIC was incomplete, it should be further optimized for its use as a complementary or alternative method to IA.

Optimization of antibody immobilization for on‐line or off‐line immunoaffinity chromatography

The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended.

Increased Throughput for Low-Abundance Protein Biomarker Verification by Liquid Chromatography/Tandem Mass Spectrometry

Low-abundance protein quantification has historically been performed using ligand binding techniques. However, due to the time and cost associated with developing enzyme-linked immunosorbent assay (ELISA), mass spectrometric approaches are playing an increasingly important role. Protein quantification at or below the nanogram per milliliter level using liquid chromatography/tandem mass spectrometry (LC/MS/MS) typically utilizes an immunoaffinity (IA) enrichment step such as immunoprecipitation. In order to maximize mass spectrometry (MS) sensitivity, protein enrichment is followed by a proteolytic cleavage step used to generate a surrogate peptide with better mass spectrometric properties. Unlike ELISA, IA-LC/MS/MS is a serial technique that can require up to 3 days for a single batch analysis due to lengthy incubation and digestion steps. This report describes the use of immunoprecipitation in 96-well ELISA format (IPE) and microwave-assisted protein digestion to reduce the time required to perform LC/MS/MS protein analyses to within a single day. The utility of this approach was investigated through its application to previously published LC/MS/MS protein assays from our laboratory for two cardiotoxicity biomarkers, Myl3 and NTproBNP. Using commercially available antibodies, IPE and microwave-assisted digestion were used to repeat intraday validations for these markers, and intraday precision (%CV) and accuracy (%RE) did not exceed 11% or 3% for either assay, respectively. Additionally, lower limits of quantification of 100 pg/mL (NTproBNP) and 0.95 ng/mL (Myl3) were achieved.

Investigating the biomarker potential of glycoproteins using comparative glycoprofiling — application to tissue inhibitor of metalloproteinases-1

Cancer-induced alterations of protein glycosylations are well-known phenomena. Hence, the glycoprofile of certain glycoproteins can potentially be used as biomarkers for early diagnosis. However, there are a substantial number of candidates and the techniques for measuring their biomarker potential are limited, calling for new methods. Here, we have investigated the cancer marker potential of the glycoprofile of tissue inhibitor of metalloproteinase-1 (TIMP-1) using a method for comparative glycoprofiling. Glycoprofiles were obtained from plasma TIMP-1 of five healthy donors and five colorectal cancer (CRC) patients showing increased amounts of TIMP-1. Furthermore, the TIMP-1 glycoprofiles of media from two colon cancer cell lines (CCC) and a prostate cancer cell line were determined as disease references. TIMP-1 was purified from IgG-depleted samples using immuno affinity and gel electrophoresis and the glycoprofiling was performed using glycopeptide enrichment and mass spectrometry. The heterogeneous glycoprofiles of TIMP-1 were found to be highly conserved among the healthy donors, proving an ideal candidate marker and showed high reproducibility of the method. Numerous CCC-specific TIMP-1 glycans were observed illustrating cancer-induced changes. Unexpectedly, quantitation revealed that the glycoprofiles of healthy donors and CRC patients varied minimally. Considering the increased CRC TIMP-1 levels and the observed CCC-specific glycans, the lack of variation indicates that the increased amount of CRC TIMP-1 is not a direct product of the cancer cells. Hence, the TIMP-1 glycoprofile holds no biomarker potential for CRC when using plasma as the sample origin. This study clearly illustrates that the technique is capable of performing individualised site-specific glycan analysis and representing a new tool for biomarker investigation of low-abundant glycoproteins.

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti-EPO-antibody coupled immunoaffinity (IA) extraction yielded low (25-40%) recovery. Albumin-depletion using antialbumin-antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre-extraction of spiked plasma using hydroxyapatite (HTP)-ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS-PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS-PAGE, for peptide fingerprinting using MALDI-TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS-PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS-PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose-response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS-PAGE improved the quality of the MALDI-TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.

Statistical optimization of immunoaffinity purification of hepatitis B surface antigen using response surface methodology

Response surface methodology (RSM) was successfully applied to find an elution condition enhancing the performance of immunoaffinity (IAF) purification of hepatitis B surface antigen (HBsAg), conducted in batch chromatography. The immunosorbent efficiency measured as HBsAg eluted per milliliter of immunosorbent was selected as the response. Two numerical independent variables were studied: concentration of potassium thiocyanate (KSCN) and ethanol, and a categorical variable formed by two levels of sodium deoxycholate under defined ionic strength (DCS/NaCl). A three-level factorial design of RSM was developed and gets a reduced cubic model which accurately fits the data ( R 2 ≈ 0.97). RSM predicted an optimal region where the response could allow maximum values. At the optimal condition 1.7 M KSCN, 30% (v/v) ethanol, and 0% (w/v) DCS and 1 M NaCl a response of 582 ± 11 μg/ml (559 ± 52 by the model), wherein the measured response does not differs statistically with the maximum experimental value. At optimized condition, a recovery of HBsAg of ∼94% of the adsorbed antigen was obtained, representing a ∼69% more HBsAg than the previously reported. Due to less accumulation of HBsAg this optimum could increase the immunosorbent life in a multiple cycle's operation.

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.