[show abstract][hide abstract] ABSTRACT: The scientific community has responded to the misidentification of human cell lines with validated methods to authenticate these cells; however, few assays are available for nonhuman cell line identification. We have developed a multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.
[show abstract][hide abstract] ABSTRACT: Cancer is a heterogeneous disease characterized by changes in the levels and activities of important cellular proteins, including oncogenes and tumor suppressors. Genetic mutations cause changes in protein activity and protein expression levels that result in the altered metabolism, proliferation, and metastasis seen in cancer cells. The identification of the critical biochemical changes in cancer has led to advances in its detection and treatment. An important example of this is the measurement of human epidermal growth factor receptor 2 (HER2), where increased expression occurs in approximately 20-30% of breast cancer tumors. HER2 is a member of the epidermal growth factor receptor family and is an important biomarker expressed on the cell surface. Measurement of the HER2 levels in tumor cells provides diagnostic, prognostic, and treatment information, because a targeted therapeutic is available. The most common methods to measure HER2 levels are immunohistochemistry and in situ hybridization assays. The accurate and reliable measurements of the specific changes in protein biomarkers for detection and treatment of cancer are important challenges. This review is focused on efforts to improve the quantitation and reliability of cancer biomarkers by using standards and reference materials.
[show abstract][hide abstract] ABSTRACT: To transform the linear fluorescence intensity scale obtained with fluorescent microspheres to an antibody bound per cell (ABC) scale, a biological cell reference material is needed. Optimally, this material should have a reproducible and tight ABC value for the expression of a known clinical reference biomarker. In this study, we characterized commercially available cryopreserved peripheral blood mononuclear cells (PBMCs) and two lyophilized PBMC preparations, Cyto-Trol and PBMC-National Institute for Biological Standard and Control (NIBSC) relative to freshly prepared PBMC and whole blood samples. It was found that the ABC values for CD4 expression on cryopreserved PBMC were consistent with those of freshly obtained PBMC and whole blood samples. By comparison, the ABC value for CD4 expression on Cyto-Trol is lower and the value on PBMC-NIBSC is much lower than those of freshly prepared cell samples using both conventional flow cytometry and CyTOF™ mass cytometry. By performing simultaneous surface and intracellular staining measurements on these two cell samples, we found that both cell membranes are mostly intact. Moreover, CD4(+) cell diameters from both lyophilized cell preparations are smaller than those of PBMC and whole blood. This could result in steric interference in antibody binding to the lyophilized cells. Further investigation of the fixation effect on the detected CD4 expression suggests that the very low ABC value obtained for CD4(+) cells from lyophilized PBMC-NIBSC is largely due to paraformaldehyde fixation; this significantly decreases available antibody binding sites. This study provides confirmation that the results obtained from the newly developed mass cytometry are directly comparable to the results from conventional flow cytometry when both methods are standardized using the same ABC approach.
Cytometry Part A 04/2012; 81(7):567-75. · 3.71 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tools for authenticating cell lines are critical for quality control in cell-based biological experiments. Currently there are methods to authenticate human cell lines using short tandem repeat (STR) markers based on the technology and procedures successfully used in the forensic community for human identification, but there are no STR based methods for authenticating nonhuman cell lines to date. There is significant homology between the human and vervet monkey genome and we utilized these similarities to design the first multiplex assay based on human STR markers for vervet cell line identification.
The following STR markers were incorporated into the vervet multiplex PCR assay: D17S1304, D5S1467, D19S245, D1S518, D8S1106, D4S2408, D6S1017, and DYS389. The eight markers were successful in uniquely identifying sixty-two vervet monkey DNA samples and confirmed that Vero76 cells and COS-7 cells were derived from Vero and CV-1 cells, respectively. The multiplex assay shows specificity for vervet DNA within the determined allele range for vervet monkeys; however, the primers will also amplify human DNA for each marker resulting in amplicons outside the vervet allele range in several of the loci. The STR markers showed genetic stability in over sixty-nine passages of Vero cells, suggesting low mutation rates in the targeted STR sequences in the Vero cell line.
A functional vervet multiplex assay consisting of eight human STR markers with heterozygosity values ranging from 0.53-0.79 was successful in uniquely identifying sixty-two vervet monkey samples. The probability of a random match using these eight markers between any two vervet samples is approximately 1 in 1.9 million. While authenticating a vervet cell line, the multiplex assay may also be a useful indicator for human cell line contamination since the assay is based on human STR markers.
[show abstract][hide abstract] ABSTRACT: Cardiac troponin I (cTnI) is considered the 'gold standard' cardiac biomarker. However, the result comparability of commercial cTnI immunoassays is still lacking despite the availability of NIST Standard Reference Material, SRM 2921 (human cardiac troponin). To facilitate the standardization of the cTnI immunoassays, a secondary reference material consisting of a panel of three cTnI-positive human serum pools is proposed by the IFCC Working Group on Standardization of Troponin I. The objective of this study is to develop measurement procedures for the characterization of the future secondary reference material using a pooled cTnI-positive serum sample as a development model.
We used magnetic beads coupled with 6 different anti-cTnI monoclonal antibodies that bind specifically to different amino acid sequence regions of the cTnI molecule to immunoprecipitate cTnI proteins from the pooled cTnI-positive serum sample followed by sensitive detection using a fluorescent Western blot.
The degradation of cTnI in the pooled sample was detected and the concentration of cTnI was determined.
We demonstrated the utility of this measurement procedure in support of the development of the proposed secondary cTnI-positive, serum-based reference material.
Clinica chimica acta; international journal of clinical chemistry 01/2011; 412(1-2):107-11. · 2.54 Impact Factor
[show abstract][hide abstract] ABSTRACT: In this study, the first steps in the development of a secondary reference measurement procedure (RMP) 'higher metrological order measurement procedure' to support the cardiac troponin I (cTnI) standardization initiative is described. The RMP should be used to assign values to serum-based secondary reference materials (RMs) without analytical artifacts causing bias. A multiplexed bead-based assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were used to identify the optimum monoclonal antibody pair (clones 560 and 19C7) for the RMP. Using these antibodies, an ELISA-based procedure was developed to accurately measure the main cTnI forms present in blood. The proposed RMP appears to show no bias when tested on samples containing various troponin complexes, phosphorylated and dephosphorylated forms, and heparin. The candidate assay displayed suitable linearity and sensitivity (limit of detection, 0.052 μg/L) for the measurement of the proposed cTnI secondary RMs. Preliminary comparison data on patient samples with a commercial cTnI assay are also provided to support the suitability of RMP for value assignment to RMs. Full validation and final assessment of the RMP will be performed through transferability and inter-comparison studies.
Clinical Chemistry and Laboratory Medicine 11/2010; 48(11):1603-10. · 3.01 Impact Factor
[show abstract][hide abstract] ABSTRACT: Virus reference materials are needed to develop and calibrate detection devices and instruments. We used electrospray differential mobility analysis (ES-DMA) and quantitative amino acid analysis (AAA) to determine the particle concentration of three small model viruses (bacteriophages MS2, PP7, and phiX174). The biological activity, purity, and aggregation of the virus samples were measured using plaque assays, denaturing gel electrophoresis, and size-exclusion chromatography. ES-DMA was developed to count the virus particles using gold nanoparticles as internal standards. ES-DMA additionally provides quantitative measurement of the size and extent of aggregation in the virus samples. Quantitative AAA was also used to determine the mass of the viral proteins in the pure virus samples. The samples were hydrolyzed and the masses of the well-recovered amino acids were used to calculate the equivalent concentration of viral particles in the samples. The concentration of the virus samples determined by ES-DMA was in good agreement with the concentration predicted by AAA for these purified samples. The advantages and limitations of ES-DMA and AAA to characterize virus reference materials are discussed.
Journal of chromatography. A 07/2009; 1216(30):5715-22. · 4.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cell-based assays for measuring ribosome inhibition by proteins such as the plant toxin ricin are important for characterizing decontamination strategies and developing detection technologies for field use. We report here an assay for ricin that provides a response that is relevant to the mechanism of ricin activity and permits a much faster readout than the commonly used assays for cytotoxicity. The assay relies on the response of an engineered reporter cell line that was produced by stably transfecting Vero cells to express green fluorescent protein (GFP) under the control ofa cytomegalovirus (CMV) promoter. The results of the GFP-based assay were compared with the assay results from three commercially available cytotoxicity assays. The GFP assay reports a sensitive response to ricin after 6 h of treatment while the other assays require a 24-h incubation. Unlike the other assays, monitoring cellular GFP on a per-cell basis allows detection of reduced ribosome activity before significant cell death occurs, and the results are not convoluted by the numbers of cells being assayed.
Assay and Drug Development Technologies 07/2009; 7(4):356-65. · 1.90 Impact Factor
[show abstract][hide abstract] ABSTRACT: A recombinant mouse interleukin-4 (IL-4) and three different purified rat antimouse IL-4 monoclonal antibodies (Mab) with different clonalities were employed as a model system. This system was used to examine monoclonal antibody effectiveness using both conventional and high-throughput measurement techniques to select antibodies for attaining the most sensitive detection of the recombinant IL-4 through the "sandwich-type" immunoassays. Surface plasmon resonance (SPR) measurements and two high-throughput methods, suspension arrays (also called multiplexed bead arrays) and forward-phase protein microarrays, predicted the same capture (BVD4-1D11) and detection (BVD6-24G2) antibody pair for the most sensitive detection of the recombinant cytokine. By using this antibody pair, we were able to detect as low as 2 pg/mL of IL-4 in buffer solution and 13.5 pg/mL of IL-4 spiked in 100% normal mouse serum with the multiplexed bead arrays. Due to the large amount of material required for SPR measurements, the study suggests that the multiplexed bead arrays and protein microarrays are both suited for the selection of numerous antibodies against the same analyte of interest to meet the need in the areas of systems biology and reproducible clinical diagnostics for better patient care.
Journal of Proteome Research 01/2008; 6(12):4720-7. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel. Reactions of the disinfectants resulted in a decrease in the native fluorescence that was measured in real time in a noninvasive manner. We compared the inactivation of two well-characterized model enzymes to the behavior of ricin. The model enzymes studied were lysozyme, a small basic enzyme stabilized with internal disulfide bonds, and heart-muscle-type lactate dehydrogenase (LDH), a large protein composed of four subunits. The biological activities of the model enzymes were measured in parallel with their fluorescence. Gel electrophoresis showed a large number of modifications of the proteins caused by the disinfectants reflected in changes in mobility and the formation of higher-order aggregates. Size-exclusion chromatography showed that the disinfectants did not break down the subunit structure of ricin but instead resulted in an increased size and heterogeneity of the protein. Size-exclusion chromatography of LDH indicated that the subunits were dissociated and that higher-order aggregates were also formed. Bleach caused a rapid inactivation of biological activity correlated with a rapid decrease in the fluorescence. Monochloramine required much higher concentrations for significant effects and the kinetics of the reactions were slow, with half-life values of the decrease on the order of minutes. Each protein showed individual differences in responses to the disinfectants, but there was a consistent correlation between the loss of fluorescence and the decrease in biological activity. These results indicate that the monitoring the fluorescence is a useful process with limitations that can be used to monitor the inactivation of toxins using disinfectants.
[show abstract][hide abstract] ABSTRACT: The molar absorption coefficient of ricin in phosphate-buffered saline (PBS) at 279 nm was measured as (93,900+/-3300) L mol(-1) cm(-1). The concentration of ricin was determined using amino acid analysis. The absorption spectrum of ricin was interpreted in terms of 69% contribution from absorption by tryptophan residues and 31% contribution from absorption by tyrosine residues. The total dipole strength of the ricin band at 280 nm was determined to be (147+/-8) D2 and was consistent with the combined dipole strengths of 10 tryptophan ([11.7+/-1.0] D2) and 23 tyrosine ([1.4+/-0.2] D2) residues. The structure of ricin was used to determine the coupling of the tryptophan residues in ricin. The maximum interaction energy was found to be 424 cm(-1)/epsilon while the average interaction between any two pairs of tryptophan residues was approximately 18 cm(-1)/epsilon. In this study, epsilon is the dielectric constant inside the protein. The fluorescence from ricin, excited at 280 nm, was dominated by fluorescence from tryptophan residues suggesting the presence of energy transfer from tyrosine to tryptophan residues. The absorbance and fluorescence of ricin increased slightly when ricin was denatured in a high concentration of guanidine. Irreversible thermal unfolding of ricin occurred between 65 degrees C and 70 degrees C. (D=3.3364*10(-30) Cm, not SI unit, convenient unit for the magnitude of the electric dipole moment of molecules.).
Photochemistry and Photobiology 01/2007; 83(5):1149-56. · 2.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.
[show abstract][hide abstract] ABSTRACT: A biofilm reactor was constructed to monitor the long-term growth and removal of biofilms as monitored by the use of a quartz crystal microbalance (QCM) and a novel optical method. The optical method measures the reflectance of white light off the surface of the quartz crystal microbalance electrode (gold) for determination of the biofilm thickness. Biofilm growth of Pseudomonas aeruginosa (PA) on the surface was used as a model system. Bioreactors were monitored for over 6 days. Expressing the QCM data as the ratio of changes in resistance to changes in frequency (DeltaR/Deltaf) facilitated the comparison of individual biofilm reactor runs. The various stages of biofilm growth and adaptation to low nutrients showed consistent characteristic changes in the DeltaR/Deltaf ratio, a parameter that reflects changes in the viscoelastic properties of the biofilm. The utility of white light reflectance for thickness measurements was shown for those stages of biofilm growth when the solution was not turbid due to high numbers of unattached cells. The thickness of the biofilms after 6 days ranged from 48 mum to 68 mum. Removal of the biofilm by a disinfectant (chlorine) was also measured in real time. The combination of QCM and reflectance allowed us to monitor in real time changes in the viscoelastic properties and thickness of biofilms over long periods of time.
Journal of Microbiological Methods 10/2006; 66(3):449-59. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: We employed ovalbumin (a simulant used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, dynamic range, linearity, and reproducibility of forward-phase array results in comparison to suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of less than 1 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were close to 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for enzyme-linked immunosorbent assays (ELISAs) in the literature. We used ovalbumin samples with two different purities, 38.0% and 76.0% (w/w), as determined by polyacrylamide gel electrophoresis (PAGE). These samples were used to evaluate the effect of impure samples on detection. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays were not as consistent and may indicate that this format may not give as reliable data with impure samples. Knowledge of the advantages and disadvantages of the two proteomic methods would allow their more rational use in clinical diagnosis.
Journal of Proteome Research 08/2006; 5(7):1770-5. · 5.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although strong fluorescence makes the R-phycoerythrin (R-PE) proteins increasingly useful in biological and clinical assays, they are subject to nonlinear effects including transitions to collective dark states and photodegradation, which complicate quantitative applications. We report measurements of R-PE fluorescence intensity as a function of incident power, duration of illumination and temperature. Emission intensity in the band at 570 nm is proportional to incident power for low power levels. At higher incident power, the emission at 570 nm is smaller than expected from a linear dependence on power. We propose that R-PE undergoes both reversible emission cessation on a millisecond time scale attributed to transitions to a collective dark state, and irreversible photodegradation on a time scale of minutes. Singlet oxygen scavengers such as dithiothreitol and n-propyl gallate have protective effects against the latter effect but not the former. Electrophoretic analysis of irradiated solutions of R-PE indicates that significant noncovalent aggregation is correlated with photodegradation. A multistate model based on fluorescence measurements and geometric analysis is proposed for the fluorophores in R-PE. The phycobilin fluorophores are partitioned into three groups: the phycourobilins (PUB) absorbing at 490 nm, one group of phycoerythobilins (PEB) absorbing at 530 nm (PEB-530) and another group of PEB absorbing at 560 nm (PEB-560). The two processes that result in the loss of fluorescence intensity are most likely associated with the PEB-560 group.
Photochemistry and Photobiology 01/2006; 82(3):635-44. · 2.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: In order to rationally select and design probes for real-time PCR, we have determined the influence of the overhang region of the complementary strand on the resulting fluorescence from a hybridising probe. A series of target oligonucleotides, each with a unique 3' overhang (4 bases), was hybridised to either 5' fluorescein (FAM)- or Alexa-488-labelled probes, and the changes in fluorescence properties were monitored. We found that the number of guanine bases in the overhang region of the target oligonucleotides was proportional to the amount of fluorescence quenching observed for both the FAM and Alexa-488 dyes. FAM appeared to be more sensitive to guanine-induced quenching with three and four guanine bases resulting in greater than a twofold decrease in the quantum yield of the fluorophore compared to the no-overhang target. In addition, we found that adenine bases caused fluorescence quenching of the Alexa-488-labelled probe, whereas the FAM-labelled probe appeared insensitive. The quenching data, generated with the steady-state fluorescence measurements, displayed a linear correlation with that obtained using a fluorescent thermal cycler, suggesting the applicability to real-time PCR measurements. Anisotropy data from the series of duplexes correlated with the fluorescence quantum yield, suggesting that quenching was accompanied by increased dye mobility.
[show abstract][hide abstract] ABSTRACT: We succeeded in using 40 nm FRET (fluorescence resonance energy transfer) microspheres conjugated to antibodies as the fluorescent reporters to perform the multiplexing suspension array measurements on two simulants of biological threats, ricin (A chain) and a crude spore preparation of Bacillus globigii (Bg). The microspheres were impregnated with two types of fluorophores in equal number (approximately 140 fluorophores in total per microsphere) and displayed bright PE-like fluorescence via a fluorescence resonance energy transfer mechanism. Activated microspheres (aldehyde groups) were directly coupled to antibodies and used to form sandwich-type immunoassays in a suspension array. For the crude preparations of Bg, the assay sensitivity using antibody-conjugated microspheres is an order of magnitude higher than that using the conventional fluorescent reporter, R-phycoerythrin (PE). Using the microspheres, Bg at the concentration of 5 ng/mL can be easily detected. For ricin, the assay sensitivity was similar to that obtained using PE as the reporter, but washing the reaction mixtures resulted in the fluorescence signals that were 2-3 times higher compared to those using PE. Ricin at a concentration of 1 ng/mL can be readily identified. Importantly, the two simulants do not interfere with each other in the multiplexing experiments. The 40 nm FRET microspheres are a new sensitive alternative as fluorescent reporters for detection in suspension arrays.