Unbound Reagents Research Articles

Plasma extracellular vesicle test sample to standardize flow cytometry measurements

BackgroundExtracellular vesicles (EVs) in body fluids are explored as disease biomarkers, but EV concentrations measured by flow cytometers (FCMs) are incomparable. ObjectivesTo improve data comparability, new reference materials with physical properties resembling EVs and reference procedures are being developed. The validation of new reference materials and procedures requires biological test samples. We developed a human plasma EV test sample (PEVTES) that i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. MethodsThe PEVTES was prepared from human plasma of 3 fasting donors. EVs were immunofluorescently stained with antibodies against platelet-specific (CD61) and erythrocyte-specific (CD235a) antigens or lactadherin. To reduce the concentration of soluble proteins, lipoproteins, and unbound reagents, stained EVs were isolated from plasma by size-exclusion chromatography. After isolation, the PEVTES was filtered to remove remnant platelets. PEVTESs were diluted in cryopreservation agents, dimethyl sulfoxide, glycerol, or trehalose and stored at −80 °C for 12 months. After thawing, stained EV concentrations were measured with a calibrated FCM (Apogee A60-Micro). ResultsWe demonstrate that the developed PEVTES resembles subcellular particles in human plasma when measured using FCM and that the concentrations of prestained platelet-derived, erythrocyte-derived, and lactadherin+ EVs in the PEVTES are stable during storage at −80 °C for 12 months when stored in trehalose. ConclusionThe PEVTES i) resembles subcellular particles in plasma, ii) is ready-to-use, iii) is flow cytometry–compatible, and iv) is stable. Therefore, the developed PEVTES is an ideal candidate to validate newly developed reference materials and procedures.

Abstract 1885: Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality

Abstract Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash࣪ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows. Citation Format: Melvin Lye, Christoph Eberle, Ann Wang, Geoffrey K. Feld, Namyong Kim. Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and cytometry data quality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1885.

Semi and fully automated immunostaining sample preparation platforms improve live leukocyte recovery, reproducibility, and flow cytometry data quality

Abstract Limited innovation in automated cell and organelle sample preparation methodology limits the effectiveness of modern analytical methods, such as single-cell ‘omics, flow and mass cytometry. These techniques traditionally rely on manual centrifugation-based protocols for cell washing and suspension preparation, hampering researchers’ access to the reproducibility and scalability benefits of automation. We have developed a suite of cell suspension preparation systems that enable semi and full automation of cell washing protocols. These Laminar Wash™ technologies robustly, gently, and efficiently remove debris, dead cells, and unbound reagent using laminar flow and liquid handling robotics, rather than turbulent and harsh pelleting-plus-pipetting methods. Adaptation of standard protocols to Laminar Wash automation typically improves repetitive immunostaining processes and workflows, in terms of reduced hands-on time and inter- and intra-operator variability. We demonstrate the superior live cell retention and reproducibility of Laminar Wash over centrifugation in processing murine and humanized mouse peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) for flow cytometry. Furthermore, we show how Laminar Wash improves flow cytometry data quality, in terms of debris removal and separation of immune cell subsets for both PBMCs and TILs. Overall, these results show how Laminar Wash methodology assists in standardizing sample preparation for cytometric analysis, an important and unmet need in cancer immunotherapy discovery and manufacturing workflows.

Multichannel Paper Chip-Based Gas Pressure Bioassay for Simultaneous Detection of Multiple MicroRNAs.

Simultaneous detection of multi-biomarkers not only enhances the accuracy of disease diagnosis but also improves detection efficiency and reduces cost. It is vital to achieve portable, simple, low-cost, and simultaneous detection of biomarkers for point-of-care (POC) diagnostics in a low-resource setting. Herein, a multichannel paper chip-based gas pressure bioassay was developed for the simultaneous detection of multiple biomarkers by combining multichannel paper chips with a portable gas pressure meter. Four DNA tetrahedral probes (DTPs) were used as capture probes and were immobilized in different detection zones of the paper chips to improve hybridization efficiency and reduce nonspecific adsorption. The formation of a sandwich structure between target microRNAs (miRNAs), the capture probe, and platinum nanoparticles (PtNPs)-modified complementary DNA (PtNPs-cDNA) transformed biomolecular recognition into quantitative detection of gas pressure. Four lung cancer-related miRNAs were detected simultaneously by a portable gas pressure meter. There is a good linear relationship between gas pressure and the logarithm of miRNA concentration in the range of 10 pM to 100 nM. Compared with single-stranded DNA capture probe, the signal-to-noise (S/N) of DNA tetrahedral probes improved more than 3 times. Using ring-oven washing, the unbound reagents in all channels of the paper chip were simultaneously and continuously washed away, leading to a more cheap, simple, and fast separation than magnetic separation. Therefore, it offers a promising multichannel paper chip-based gas pressure bioassay for portable and simultaneous detection of multiple biomarkers.

Droplet-based microfluidic washing module for magnetic particle-based assays.

In this paper, we propose a continuous flow droplet-based microfluidic platform for magnetic particle-based assays by employing in-droplet washing. The droplet-based washing was implemented by traversing functionalized magnetic particles across a laterally merged droplet from one side (containing sample and reagent) to the other (containing buffer) by an external magnetic field. Consequently, the magnetic particles were extracted to a parallel-synchronized train of washing buffer droplets, and unbound reagents were left in an original train of sample droplets. To realize the droplet-based washing function, the following four procedures were sequentially carried in a droplet-based microfluidic device: parallel synchronization of two trains of droplets by using a ladder-like channel network; lateral electrocoalescence by an electric field; magnetic particle manipulation by a magnetic field; and asymmetrical splitting of merged droplets. For the stable droplet synchronization and electrocoalescence, we optimized droplet generation conditions by varying the flow rate ratio (or droplet size). Image analysis was carried out to determine the fluorescent intensity of reagents before and after the washing step. As a result, the unbound reagents in sample droplets were significantly removed by more than a factor of 25 in the single washing step, while the magnetic particles were successfully extracted into washing buffer droplets. As a proof-of-principle, we demonstrate a magnetic particle-based immunoassay with streptavidin-coated magnetic particles and fluorescently labelled biotin in the proposed continuous flow droplet-based microfluidic platform.

Potential and Limitation of Microparticle-Based Immunoassays: A Thermodynamic and Kinetic Study

The fundamental significance of immunoassays in diagnostics and research drives the ongoing quest for improving assay formats, sensitivity and instrument technology. Early on, the advantage of immobilizing antibodies to a surface to allow for separation of bound and unbound reagent was realized in the immunometric (sandwich) assay format. Today, this approach is established in most immunoassays as performed using ELISA (enzyme linked immunosorbent assay), GCSPR (grating-couples surface plasmon resonance) and microparticle-based chemiluminescence. However, antibody immobilization to a surface hampers reaction kinetics and imposes mass transport limitation and sterical hindrance. Theoretical considerations indicate that these effects can be minimized by using a spherical surface underlining why recently developed ultra-high-sensitivity techniques rely on the utilization of microparticles. In the present study, we investigate the potential and limitation of microparticle-based immunoassays in respect to impact of mass transport and surface immobilization on the intrinsic thermodynamic and kinetic properties of antibody-analyte interaction.

A highly sensitive automated flow immunosensor based on kinetic exclusion analysis for determination of the cancer marker 8-hydroxy-2′-deoxyguanosine in urine

A highly sensitive automated flow immunosensor has been developed and validated for the measurement of the cancer marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine. The sensor employed the kinetic-exclusion analytical technology using the KinExA™ 3200 instrument. Various concentrations of 8-OHdG were incubated with a fixed amount of mouse anti-8-OHdG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over a bovine serum albumin conjugate of 8-hydroxyguanosine (8-OHG–BSA) coated onto polymethylmethacrylate beads contained in the observation cell of the KinExA instrument. The free anti-8-OHdG antibody was bound to the immobilized 8-OHG–BSA, however the unbound reagents were removed from the beads bed by flushing the system with phosphate-buffered saline. Fluorescent-labeled secondary antibody was passed rapidly over the beads bed, and the fluorescence was recorded during the flow of the secondary antibody through the beads. A calibration curve was generated by plotting the fluorescence signals that were retained on the beads as a function of 8-OHdG concentrations. The assay limit of detection was 0.05 ng ml−1 and the working range of the assay was 0.05–5 ng ml−1. The analytical recovery of urine-spiked 8-OHdG was 96.05–106.55 ± 3.00–6.40%. The precision of the sensor was satisfactory; relative standard deviation values were 3.28–4.89 and 4.40–6.89% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was found to be superior to the conventional enzyme-linked immunosorbent assay for 8-OHdG. The proposed sensor is anticipated to have a great value in measurement of 8-OHdG where a more confident result is needed.

An automated flow immunosensor based on kinetic exclusion analysis for measurement of a free β-subunit of human chorionic gonadotropin in serum

An automated immunosensor assay has been developed and validated for measurement of the β-subunit of human chorionic gonadotropin (β-hCG). The assay employed the flow kinetic-exclusion analytical technology of the KinExA™ 3200 instrument. Our KinExA™ consisted of a capillary flow observation cell with a microporous screen that maintained a compact antigen-coated bead bed, polymethylmethacrylate (PMMA) beads coated with β-hCG. Various concentrations of β-hCG were incubated with a fixed amount of mouse anti-β-hCG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over the β-hCG-coated PMMA beads. Anti-β-hCG antibody not bound to the soluble β-hCG was captured by the β-hCG immobilized on the beads. The unbound reagents were removed from the bead bed by subsequent flushing with phosphate-buffered saline (PBS). The beads were then subjected to brief contact with a fluorescent-labeled secondary antibody, followed by a second PBS flush. The fluorescence was monitored and recorded during the flow of the fluorescent-labeled antibody through the bead bed. The fluorescence signal retained on the beads was replotted versus β-hCG concentrations to generate a calibration curve. The concentrations of β-hCG in the samples were then obtained by interpolation on the curve. The assay limit of detection was 0.2 ng mL−1. This highly sensitive automated system allowed rapid and reliable quantification of β-hCG without any matrix effect; analytical recovery of serum-spiked β-hCG was 96.9–105.4 ± 3.54–6.84%. The precision of the sensor was satisfactory; the relative standard deviation (RSD) was 3.57–5.61 and 4.23–6.81% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was found to be superior to the reported enzyme-linked immunosorbent assays for β-hCG. The automated analysis by the sensor facilitated the processing of a large number of specimens, and the new sensor-based assay is anticipated to have a great value in measurement of β-hCG.

In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

AbstractThis unit describes the selection of aptamers from a pool of single‐stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.Curr. Protoc. Nucleic Acid Chem. 40:9.3.1‐9.3.27. © 2010 by John Wiley & Sons, Inc.

In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.

Bioelectrochemical immunoassay of polychlorinated biphenyl

A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP–PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP–PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP–PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 10 pg mL −1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.

Magnetic beads-based bioelectrochemical immunoassay of polycyclic aromatic hydrocarbons

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3,3′,5,5′-tetramethylbenzidine (TMB) and hydrogen peroxide (H 2O 2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL −1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

AbstractA fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H2O2) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L−1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.

An Immunologically Cryptic Epitope of Plasmodium falciparum Circumsporozoite Protein Facilitates Liver Cell Recognition and Induces Protective Antibodies That Block Liver Cell Invasion

Circumsporozoite, a predominant surface protein, is involved in invasion of liver cells by Plasmodium sporozoites, which leads to malaria. We have previously reported that the amino terminus region (amino acids 27-117) of P. falciparum circumsporozoite protein plays a critical role in the invasion of liver cells by the parasite. Here we show that invasion-blocking antibodies are induced by a polypeptide encoding these 91 amino acids, only when it is presented in the absence of the rest of the protein. This suggests that when present in the whole protein, the amino terminus remains immunologically cryptic. A single reactive epitope was identified and mapped to a stretch of 21 amino acids from position 93 to 113. The epitope is configurational in nature, since its recognition was affected by deleting as little as 3 amino acids from either end of the 21-residue peptide. Lysine 104, the only known polymorphic position in the epitope, affected its recognition by the antibodies, and its conversion to leucine in the protein led to a substantial loss of binding activity of the protein to the hepatocytes. This indicated that in the protein, the epitope serves as a binding ligand and facilitates the interaction between sporozoite and hepatic cells. When considered along with the observation that in its native state this motif is immunologically unresponsive, we suggest that hiding functional moieties of the protein from the immune system is an evasion strategy to preserve liver cell binding function and may be of importance in designing anti-sporozoite vaccines.

MEK Is a Key Modulator for TLR5-induced Interleukin-8 and MIP3α Gene Expression in Non-transformed Human Colonic Epithelial Cells

Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.

In situ activation of caspases and serine proteases during apoptosis detected by affinity labeling their enzyme active centers with fluorochrome-tagged inhibitors.

Activation of caspases is the key event of apoptosis. To detect this event in situ we applied fluorochrome-labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA are fluorescein- or sulforhodamine-conjugated peptide-fluoromethyl ketones that covalently, with 1:1 stoichiometry, bind to enzymatic centers of caspases; the specificity is provided by the peptide sequence of amino acids. Similarly, we applied fluorescent inhibitors of serine proteases (FLISP) to detect active sites of the latter enzymes. Exposure of live cells to FLICA of FLISP led to uptake of these ligands and their binding to activated caspases or active sites of serine proteases; the unbound reagents were removed by cell rinse. Only cells undergoing apoptosis were labeled with FLISP or FLICA. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA or FLISP allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases; two proteins reacting with FLISP were about 57 and 60 kDa, which suggests that they are novel enzymes. Detection of caspases or serine proteases activation can be combined with other markers of apoptosis or cell cycle for multiparametric analysis by flow or laser scanning cytometry. Being caspase inhibitors, FLICA arrest the process of apoptosis and prevent cell disintegration. The stathmo-apoptotic assay was developed, therefore, to obtain cumulative apoptotic index over a long period of time and estimate a rate of cell entry into apoptosis for cell populations.

A simple, rapid method for demonstrating bacterial flagella.

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.

In Vitro Selection of RNA Aptamers to a Protein Target by Filter Immobilization

This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially function as protein inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate a ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.

A Relatively Simple Test for the Direct Determination of the Cysteine Content in Photographic Gelatin Using a Thiol-Specific Fluorogenic Reagent

This report describes the development and validation of a direct measurement of cysteine in photographic gelatin using a fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole 4-sulphonate (SBD-F). This unique analytical approach greatly simplifies the measurement of cysteine without sacrificing the quality of the information obtained. No complicated acid hydrolysis or difficult high-performance liquid chromatographic separations are required. SBD-F is water soluble and thiol specific. SBD-Fhas many excellent features which allow direct labeling of the cysteine thiol in gelatin solutions under controlled laboratory conditions. At phi 8.0 the reaction kinetics arc complete in 1 hour at 60 °C. Additionally, the probe is not naturally fluorescent and thus no separation step is required for removal of unbound reagent. The analytical test is both simple and rapid. This method allows determination of cysteine content by fluorescence emission in less than 2 hours.

Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system

We report a rapid and reproducible method to quantify total DNA at picogram levels. Two high-affinity DNA-binding proteins are used to construct a sandwich assay and a semiconductor sensor is used for quantitation. Single-stranded DNA-binding protein (SSB) from Escherichia coli is conjugated with a linker molecule, biotin, for specific capture of the DNA complex onto a membrane. Monoclonal anti-DNA antibody is conjugated with an enzyme, urease, for signal generation. To detect DNA, a sample is denatured to form single-stranded DNA and then incubated with a reagent containing both DNA-binding protein conjugates and streptavidin. After incubation of the reagent with the DNA sample for 1 h at 37°C to form a complex of streptavidin-biotin-SSB-DNA-anti-DNA-urease, the mixture is filtered through a biotin-coated nitrocellulose membrane which binds the streptavidin component of the complex. The unbound reagent is washed off the membrane, and then the captured DNA complex is detected with a ligh-addressable potentiometric sensor which measures the pH change catalyzed by the urease in the complex. This assay can detect 2 pg of DNA with a quantitation coefficient of variation of less than 10% in the range 10 to 200 pg.