Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.     

General LC-MS/MS method approach to quantify therapeutic monoclonal antibodies using a common whole antibody internal standard with application to preclinical studies

Ligand binding assays (LBAs) are widely used for therapeutic monoclonal antibody (mAb) quantification in biological samples. Major limitations are long method development times, reagent procurement, and matrix effects. LC-MS/MS methods using signature peptides are emerging as an alternative approach, which typically use a stable isotope labeled signature peptide as the internal standard (IS). However, a new IS has to be generated for every candidate, and the IS may not correct for variations at all processing steps. We have developed a general LC-MS/MS method approach employing a uniformly heavy-isotope labeled common whole mAb IS and a common immunocapture for sample processing. The method was streamlined with automation for consistency and throughput. Method qualification of four IgG(2) and four IgG(1) mAbs showed sensitivity of 0.1 μg/mL and linearity of 0.1-15 μg/mL. Quality control (QC) data of these eight mAbs were accurate and precise. The QC performance of the whole molecule labeled IS was better than those of synthetic labeled IS peptides tested. The pharmacokinetic results of two mAbs (an IgG(2) and IgG(1) candidate) dosed in rats were comparable to those of LBA. The general LC-MS/MS method approach overcomes the limitations of current methods to reduce time and resources required for preclinical studies.     

LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma

Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.     

Quantification of NTproBNP in rat serum using immunoprecipitation and LC/MS/MS: a biomarker of drug-induced cardiac hypertrophy

Brain natriuretic peptide (BNP) and N-terminal proBNP (NTproBNP) are well established in the clinic as biomarkers of heart failure. BNP hormone and the inactive NTproBNP are predominantly secreted in the ventricles of the heart in response to pressure overload and, consequently, are being investigated as markers of drug-induced cardiac hypertrophy in rat to support drug development. In the work presented here, an immunoaffinity-based LC/MS/MS assay was developed and validated to measure a selective tryptic fragment of NTproBNP in rat serum. The assay covers the range of 13-329 pg/mL of the tryptic fragment LLELIR, corresponding to 0.1-2.5 ng/mL intact NTproBNP. A stable isotope-labeled version of NTproBNP containing the tryptic fragment LLELI[13C615N1]R was prepared by solid-phase peptide synthesis and was used as an internal standard to minimize assay variability. Due to endogenous NTproBNP present in rat serum, human serum was used as the control matrix, and parallelism between rat and human serum was established by standard addition. Assay accuracy (% RE) and precision (% CV) were measured at three concentrations on each of 4 days and did not exceed 4.2 and 14.5%, respectively. Additionally, study data are presented from the application of this assay in which rats demonstrated a significant increase in NTproBNP serum concentration following administration of an agent known to induce cardiac hypertrophy. In this study, the relationship between serum NTproBNP and cardiac hypertrophy was corroborated by increases in heart weight and magnetic resonance imaging of the test subjects' left ventricle. To our knowledge, this represents the first reported assay for NTproBNP in preclinical species for the assessment of cardiac hypertrophy.     

Development of a monoclonal antibody against ochratoxin A and its application in enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip

A monoclonal antibody (mAb) specific to ochratoxin A (OTA) was produced from a stable hybridoma cell line, 9C9H9, generated by the fusion of P3/NS1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse immunized with OTA-keyhole limpet hemocyanin. The 9C9H9 mAb belongs to the immunoglobulin G1 (kappa chain) isotype. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a competitive indirect ELISA were established for antibody characterization. The concentrations causing 50% inhibition of binding of OTA-horseradish peroxidase to the antibody by OTA, OTB, and OTC were found to be 0.32, 0.17, and 0.28 ng/mL, respectively, in the cdELISA. A sensitive and rapid mAb-based gold nanoparticle immunochromatographic strip was also developed using this mAb. This strip has a detection limit of 5 ng/mL for OTA and can be completed in 10 min. Analysis of OTA in coffee samples revealed that data obtained from immunochromatographic strip were in a good agreement with those obtained from cdELISA. The mAb-based cdELISA and immunochromatographic strip assay established in this study were sensitive and accurate for rapid screening of OTA in coffee samples.     

Detection and quantification of anthrax lethal factor in serum by mass spectrometry

The lethal toxin produced during Bacillus anthracis infection is a complex of protective antigen, which localizes the toxin to the cell receptor, and lethal factor (LF), a zinc-dependent endoproteinase whose known targets include five members of the mitogen-activated protein kinase kinase (MAPKK) family of response regulators. We have developed a method for detecting functional LF in serum. Anti-LF murine monoclonal antibodies immobilized on magnetic protein G beads were used to capture and concentrate the LF from serum. The captured LF was exposed to an optimized MAPKK-based peptide substrate, which it hydrolyzed into two smaller peptides. The LF cleavage products were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and quantified by isotope dilution-MS. The entire analytical method can be performed in less than 4 h with detection of LF levels as low as 0.05 ng/mL. The method was used to quantify LF levels in serum from rhesus macaques infected with B. anthracis. Serum samples obtained at day 2 postinfection contained 30-250 ng/mL LF and illustrated the clear potential to detect LF earlier in the infection cycle. This method represents a highly specific and rapid diagnostic tool for early anthrax and has a potential additional role as a research tool for understanding toxemia and effects of medical countermeasures for anthrax.     

LC-MS/MS approach for quantification of therapeutic proteins in plasma using a protein internal standard and 2D-solid-phase extraction cleanup

The bioanalysis of plasma samples generated from in vivo studies of therapeutic proteins is of increasing interesting in the biopharmaceutical industry. The conventional ELISA approach has a long assay development time which can limit use in the early discovery and development of protein-based drugs. In this study, an LC-MS/MS bioassay was developed for the quantification of somatropin and a therapeutic human monoclonal antibody. The assay used bovine fetuin as an internal standard and a two-dimensional solid-phase extraction for the cleanup of the plasma digest. Sample extracts were resolved on an analytical size column using a 6 min LC gradient and analyzed using a triple-quadruple mass spectrometer. The linearity of the assay for somatropin was established from 1 to 1000 microg/mL with accuracy and precision within 15%. This LC-MS approach was also applied to a rat pharmacokinetic study of the therapeutic monoclonal antibody with a lower quantitation limit of 0.5 microg/mL. The LC-MS assay had improved accuracy and precision, and the results from analysis of in vivo study samples showed good agreement with the data obtained with an ELISA. The results from this study indicate that the LC-MS bioassay is a simple and feasible approach for the bioanalysis of therapeutic proteins to support in vivo studies during early drug discovery and development.     

Ultra-sensitive measurements of 11-nor-δ(9)-tetrahydrocannabinol-9-carboxylic Acid in oral fluid by microflow liquid chromatography-tandem mass spectrometry using a benchtop quadrupole/orbitrap mass spectrometer

Oral fluid has been gaining more acceptance as the alternative matrix for forensic toxicology. Currently, Δ(9)-tetrahydrocannabinol (THC) is used as the primary target for detecting cannabis use in oral fluid. Meanwhile, THC carboxylic acid (THCA) in oral fluid is reported as a more reliable marker for cannabis abuse as its presence does not come from passive exposure. An analytical method for simultaneous quantitation of THC and THCA will be efficient for toxicology laboratories. THCA quantitation is challenging due to its very low concentration in oral fluid. Recently reported liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methods achieved sufficient sensitivity but involved complex sample preparation procedures. We aimed to develop a sensitive LC-MS/MS method for simultaneous quantitation of THC and THCA in oral fluid with low-flow liquid chromatography and a Q Exactive mass spectrometer, using offline sample preparation of oral fluid followed by microflow LC with online sample cleanup. The total runtime of the method was 12.5 min. The method had a lower limit of quantitation of 7.5 pg/mL and was linear from 7.5 to 300 pg/mL for THCA. The intra- and interbatch precision of the method ranged from 3.3% to 9.3% for THC and THCA.     

A reproducible and high-throughput HPLC/MS method to separate sarcosine from α- and β-alanine and to quantify sarcosine in human serum and urine

While sarcosine was recently identified as a potential urine biomarker for prostate cancer, further studies have cast doubt on its utility to diagnose this condition. The inconsistent results may be due to the fact that alanine and sarcosine coelute on an HPLC reversed-phase column and the mass spectrometer cannot differentiate between the two isomers, since the same parent/product ions are generally used to measure them. In this study, we developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) method that resolves sarcosine from alanine isomers, allowing its accurate quantification in human serum and urine. Assay reproducibility was determined using the coefficient of variation (CV) and intraclass correlation coefficient (ICC) in serum aliquots from 10 subjects and urine aliquots from 20 subjects across multiple analytic runs. Paired serum/urine samples from 42 subjects were used to evaluate sarcosine serum/urine correlation. Both urine and serum assays gave high sensitivity (limit of quantitation of 5 ng/mL) and reproducibility (serum assay, intra- and interassay CVs < 3% and ICCs > 99%; urine assay, intra-assay CV = 7.7% and ICC = 98.2% and interassay CV = 12.3% and ICC = 94.2%). In conclusion, this high-throughput LC-MS method is able to resolve sarcosine from α- and β-alanine and is useful for quantifying sarcosine in serum and urine samples.     

Comparison of MALDI-TOF-MS and HPLC-ESI-MS/MS for Endopeptidase Activity-Based Quantification of Anthrax Lethal Factor in Serum

Diagnosing and treating anthrax at the earliest stage of disease is critical. We developed a method to diagnose anthrax at early stages of infection by detecting anthrax lethal factor (LF) at the attomol/mL level in plasma or serum. This method uses antibody capture and quantification of LF endoproteinase activity by isotope dilution matrix-assisted laser-desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). Many public health laboratories do not use MALDI-TOF-MS; thus, we have adapted the LF method for detection by electrospray ionization (ESI) tandem MS (MS/MS), which allowed comparison of both MS platforms for LF quantification. Calibration curves were linear from 0.05-2.5 ng/mL when measured after 2 h and from 0.005-1.0 ng/mL after 18 h incubation time. The limit of detection was 0.005 ng/mL using a 200 μL sample. The coefficient of variation for quality control samples was 6-12% for both MS platforms. Samples used to perform cross-validation included 158 serum samples from a study in rabbits exposed to anthrax spores by inhalation. Some were treated with anthrax immune globulin before exposure. Concentrations measured by ESI-MS/MS matched those by MALDI-TOF-MS with p = 0.99 (r(2) = 0.997) and -0.25% mean relative difference (±9% standard deviation). This study shows that isotope dilution MALDI-TOF-MS is a robust and precise quantitative MS platform.     

Development of a secondary antibody thio-functionalized microcantilever immunosensor and an ELISA for measuring ginsenoside Re content in the herb ginseng

Ginsenoside Re (GRe) is a major active component of the Chinese medicinal herb ginseng, Panax ginseng . A sensitive and specific monoclonal antibody (mAb), designated as mAb3D6, was generated with a GRe-bovine serum albumin conjugate as an immunogen. Microcantilever immunosensors (MCS), one modified with thiolated anti-GRe antibody and one modified with thiolated goat antimouse immunoglobulin G (IgG), were developed to detect the content of ginsenoside. The MCS immobilized with thiolated goat antimouse IgG had a better sensitivity than the MCS modified with thiolated anti-GRe antibody. The advantage of a secondary antibody thio-functionalized MCS was verified with the anti-paclitaxel mAb. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was also established with mAb3D6. The concentration of analyte producing 50% inhibition and the working range of icELISA were 1.20 and 0.15-16.1 ng/mL, respectively. The icELISA had a cross-reactivity of 89% with ginsenoside Rg1 and less than 3% with other ginsenosides. The icELISA and MCS with thiolated secondary antibody were applied for the determination of GRe in ginseng samples, and the results agreed well with those determined by high-performance liquid chromatography.     

Magneto immunoassays for Plasmodium falciparum histidine-rich protein 2 related to malaria based on magnetic nanoparticles

Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria are described for the first time by using magnetic micro- and nanoparticles. The covalent immobilization of a commercial monoclonal antibody toward the HRP2 protein in magnetic beads and nanoparticles was evaluated and compared. The immunological reaction for the protein HRP2 was successfully performed in a sandwich assay on magnetic micro- and nanoparticles by using a second monoclonal antibody labeled with the enzyme, horseradish peroxidase (HRP). Then, the modified magnetic particles were easily captured by a magneto sensor made of graphite-epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with the electrochemical magneto immunosensors was successfully evaluated and compared with a novel magneto-ELISA based on optical detection using spiked serum samples. Improved sensitivity was obtained when using 300 nm magnetic nanoparticles in both cases. The electrochemical magneto immunosensor coupled with magnetic nanoparticles have shown better analytical performance in terms of limit of detection (0.36 ng mL(-1)), which is much lower than the LOD reported by other methods. Moreover, at a low level of HRP2 concentration of 31.0 ng mL(-1), a signal of 15.30 μA was reached with a cutoff value of 0.34 μA, giving a clear positive result with a non-specific adsorption ratio of 51. Due to the high sensitivity, this novel strategy offers great promise for rapid, simple, cost-effective, and on-site detection of falciparum malaria disease in patients, but also to screen out at-risk blood samples for prevention of transfusion-transmitted malaria.     

Quantitative method for the analysis of tobacco-specific nitrosamines in cigarette tobacco and mainstream cigarette smoke by use of isotope dilution liquid chromatography tandem mass spectrometry

An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of tobacco specific nitrosamines (TSNA). It utilizes four stable isotope-labeled internal standards instead of two as reported by others. A separate internal standard for each analyte is required to minimize sample matrix effects on each analyte, which can lead to poor analyte recoveries and decreases in method accuracy and precision if only one or two of the internal standards are used, especially for complex sample matrixes and when no sample cleanup steps are performed as in this study. In addition, two ion-transition pairs (instead of one) are used for each analyte for the confirmation and quantification, further enhancing the method's accuracy and robustness. These improvements have led to a new LC-MS/MS method that is faster, more sensitive, and selective than the traditional methods and more accurate and robust than the published LC-MS/MS methods. The linear range of the method was from 0.2 to 250 ng/mL with a limit of detection of each TSNA varied from 0.027 to 0.049 ng/mL. Good correlations between the results obtained by the new method and the traditional method were observed for the samples studied.     

A strategy of plasma protein quantitation by selective reaction monitoring of an intact protein

Immunoassays are used extensively in the quantitative analysis of proteins in plasma, urine, and other biological matrixes to support preclinical and clinical studies. Although immunoassays are both sensitive and rapid, difficulties during development of these assays are compounded by the need to have a specific antibody or antigen to the protein of interest. Furthermore, calibration curves of immunoassays are inherently nonlinear, and the technique often detects many structurally related components in addition to the analyte of interest. We have developed a novel strategy of analyzing protein concentrations in plasma by utilizing 96-well solid-phase extraction and LC-MS/MS detection of the intact protein. This strategy has been successfully applied in method development and assay validation of quantitatively analyzing protein rK5 concentrations in monkey plasma samples. Additional techniques such as precolumn regeneration and column heating were also incorporated into the assay. Total run time for each sample was approximately 15 min. An LLOQ of 99.2 ng/mL from a sample volume of 50 microL, corresponding to only 380 fmol (3.97 ng) of the rK5 analyte being injected onto the analytical column (assuming 100% extraction recovery), was obtained. The validated linear dynamic range was between 99.2 and 52 920.0 ng/mL, with a correlation coefficient (r(2)) ranging from 0.9972 and 0.9994. The intraassay CV for this assay was between 0.6 and 3.8%, and the interassay CV was between 1.7 and 3.2%. Interassay mean accuracies were between 101.5 and 104.7%. The assay has proven rugged and specific and has been employed to generate data in support of preclinical studies. This strategy for rK5 assay could be used for the development of bioanalytical assays to provide preclinical and clinical support for other protein drug candidates and, furthermore, for the validation of biomarkers discovered from proteomic research.     

Method for the determination of vitamin K homologues in human plasma using high-performance liquid chromatography-tandem mass spectrometry

We report here the development of a precise and sensitive method for the determination of vitamin K homologues including phylloquinone (PK), menaquinone-4 (MK-4), and menaquinone-7 (MK-7) in human plasma using HPLC-tandem mass-mass spectrometry with atmospheric pressure chemical ionization (LC-APCI-MS/MS). The method involves the use of stable isotope (18)O-labeled internal standard compounds, which were synthesized in our laboratory, and the selection of a precursor and product ion with a MS/MS multiple reaction monitoring method. The average intraassay and interassay variation values for PK, MK-4, and MK-7 were <10%. Average spiked recoveries from authentic compounds added to normal human plasma samples for PK, MK-4, and MK-7 were 98-102%. Mean plasma concentrations of PK, MK-4, and MK-7 from healthy subjects (n = 20) were 1.22 +/-0.57, 0.39 +/- 0.46, and 6.37 +/- 7.45 ng/mL, respectively. We conclude that this novel LC-APCI-MS/MS method should be useful for the evaluation of vitamin K status in postmenopausal women and elderly subjects and provides useful information for the treatment and prevention of osteoporosis with vitamin K.     

Absolute quantification of monoclonal antibodies in biofluids by liquid chromatography-tandem mass spectrometry

The development of a quantification method for monoclonal antibodies in serum has been accomplished by high-performance liquid chromatography multiple reactions monitoring mass spectrometry. A human monoclonal antibody (HmAb) was used as the model protein for method development and validation. A peptide from the CDR3-region of its heavy chain was selected and used for quantifying the entire mAb. This signature peptide served as a template for the internal standard. Prior to mass spectrometric analysis approximately 50% of the total serum protein content was removed by albumin depletion. The accuracy of the method ranged between 99 and 112% in cynomolgus monkey serum. The intra-assay coefficient of variation (CV) was lower than 4% at 4 microg/mL and 200 microg/mL HmAb (n = 3). The CV at 400 microg/mL corresponded to 9% (n = 3). In addition, the interassay variation was investigated in a male cynomolgus serum pool and in a female cynomolgus serum pool. The CV for the male cynomolgus pool at 4 microg/mL HmAb was 7% (n = 3). The CV obtained from the female pool was 8% (n = 3), at 4 microg/mL. The dynamic range of the method was 3 orders of magnitude. After albumin depletion of 25 microL of serum, a lowest limit of quantification of 2 microg/mL HmAb was reached in both human and cynomolgus monkey samples.     

Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap

A method is presented to quantify intermediate-abundance proteins in human serum using a single-quadrupole linear ion trap mass spectrometer-in contrast, for example, to a triple-quadrupole mass spectrometer. Stable-isotope-labeled (tryptic) peptides are spiked into digested protein samples as internal standards, aligned with the traditional isotope dilution approach. As a proof-of-concept experiment, four proteins of intermediate abundance were selected, coagulation factor V, adiponectin, C-reactive protein (CRP), and thyroxine binding globulin. Stable-isotope-labeled peptides were synthesized with one tryptic sequence from each of these proteins. The normal human serum concentration ranges of these proteins are from 1 to 30 microg/mL (or 20 to 650 pmol/mL). These labeled peptides and their endogenous counterparts were analyzed by LC-MS/MS using multiple reaction monitoring, a multiplexed form of the selected reaction monitoring technique. For these experiments, only one chromatographic dimension (on-line reversed-phase capillary column) was used. Improved limits of detection will result with multidimensional chromatographic methods utilizing more material per sample. Standard curves of the spiked calibrants were generated with concentrations ranging from 3 to 700 pmol/mL using both neat solutions and peptides spiked into the complex matrix of digested serum protein solution where ion suppression effects and interferences are common. Endogenous protein concentrations were determined by comparing MS/MS peak areas of the endogenous peptides to the isotopically labeled internal calibrants. The derived concentrations from a normal human serum pool (neglecting loss of material during sample processing) were 9.2, 110, 120, and 246 pmol/mL for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively. These concentrations generally agree with the reported normal ranges for these proteins. As a measure of analytical reproducibility of this single-quadrupole assay, the coefficients of variance based on 12 repeated measurements for each of the endogenous tryptic peptides were 17.0, 25.4, 24.2, and 14.0% for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively.     

Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry

The toxin ricin is a biological weapon that may be used for bioterrorist purposes. As a member of the group of ribosome-inactivating proteins (RIPs), ricin has an A-chain possessing N-glycosidase activity which irreversibly inhibits protein synthesis. In this paper, we demonstrate that provided appropriate sample preparation is used, this enzymatic activity can be exploited for functional ricin detection with sensitivity similar to the best ELISA and specificity allowing application to environmental samples. Ricin is first captured by a monoclonal antibody directed against the B chain and immobilized on magnetic beads. Detection is then realized by determination of the adenine released by the A chain from an RNA template using liquid chromatography coupled to tandem mass spectrometry. The immunoaffinity step combined with the enzymatic activity detection leads to a specific assay for the entire functional ricin with a lower limit of detection of 0.1 ng/mL (1.56 pM) after concentration of the toxin from a 500 microL sample size. The variability of the assay was 10%. Finally, the method was applied successfully to milk and tap or bottled water samples.     

Microfabricated renewable beads-trapping/releasing flow cell for rapid antigen-antibody reaction in chemiluminescent immunoassay

A renewable flow cell integrating a microstructured pillar-array filter and a pneumatic microvalve was microfabricated to trap and release beads. A bead-based immunoassay using this device was also developed. This microfabricated device consists of a microfluidic channel connecting to a beads chamber in which the pillar-array filter is built. Underneath the filter, there is a pneumatic microvalve built across the chamber. Such a device can trap and release beads in the chamber by "closing" or "opening" the microvalve. On the basis of the pneumatic microvalve, the device can trap beads in the chamber before performing an assay and release the used beads after the assay. Therefore, this microfabricated device is suitable for "renewable surface analysis". A model analyte, 3,5,6-trichloropyridinol (TCP), was chosen to demonstrate the analytical performance of the device. The entire fluidic assay process, including beads trapping, immuno binding, beads washing, beads releasing, and chemiluminesence signal collection, could be completed in 10 min. The immunoassay of TCP using this microfabricated device showed a linear range of 0.20-70 ng/mL with a limit of detection of 0.080 ng/mL. The device was successfully used to detect TCP spiked in human plasma at the concentration range of 1.0-50 ng/mL, with an analytical recovery of 81-110%. The results demonstrated that this device can provide a rapid, sensitive, reusable, low-cost, and automatic tool for detecting various biomarkers in biological fluids.     

Preparation of a single-chain variable fragment and a recombinant antigen-binding fragment against the anti-malarial drugs, artemisinin and artesunate, and their application in an ELISA

Two different recombinant antibodies, a single-chain variable fragment (scFv) and an antigen-binding fragment (Fab), were prepared against artemisinin (AM) and artesunate (AS) and were developed for use in an enzyme-linked immunosorbent assay (ELISA). The recombinant antibodies, which were derived from a single monoclonal antibody against AM and AS (mAb 1C1) prepared by us, were expressed by Escherichia coli cells and their reactivity and specificity were characterized. As a result, to obtain sufficient signal in indirect ELISA, a much greater amount of a first antibody was needed in the use of scFv due to the differences of the secondary antibody and conformational stability. Therefore, we focused on the development of the recombinant Fab antibodies and applied it to indirect competitive ELISA. The specificity of the Fab was similar to that of mAb 1C1 in that it showed specific reactivity toward AM and AS only. The sensitivity of the icELISA (0.16 μg/mL to 40 μg/mL for AM and 8.0 ng/mL to 60 ng/mL for AS) was sufficient for analysis of antimalarial drugs, and its utility for quality control of analysis of Artemisia spp. was validated. The Fab expression and refolding systems provided a good yield of high-quality antibodies. The recombinant antibody against AM and AS provides an essential component of an economically attractive immunoassay and will be useful in other immunochemical applications for the analysis and purification of antimalarial drugs.