|
|
|
|
|
|
|
|
|
|
| Simultaneous Detection of Trisomies 13, 18, and 21 with Multiplex Ligation-Dependent Probe Amplification-Based Real-Time PCR [Molecular Diagnostics and Genetics] | |
BACKGROUND:
Trisomies 13, 18, and 21 account for the majority of chromosomal aneuploidies detected in prenatal diagnosis. Diagnosis of these trisomies relies mainly on karyotype analysis. Several molecular methods have been developed for trisomy detection, but performance or throughput limitations of these methods currently constrain their use in routine testing.
METHODS:
We developed multiplex ligation-dependent probe amplification–based real-time PCR (MLPA/rtPCR) to simultaneously detect these 3 trisomy conditions with a single reaction. We applied the method to DNA isolated from 144 blinded clinical samples that included 32 cases of trisomy 21, 11 cases of trisomy 18, 1 case of trisomy 13, and 100 unaffected control samples; results were compared with karyotype analysis.
RESULTS:
As judged by the results of the karyotype analysis, MLPA/rtPCR correctly detected all 44 cases of trisomy in the analysis of the blinded clinical samples. The method was able to detect a change in chromosome dosage as low as 1.2-fold.
CONCLUSIONS:
This novel PCR-based technology simultaneously identified 3 types of trisomy in a single reaction and accurately detected trisomy with mosaicism, while reducing assay times and costs compared with conventional methods. The MLPA/rtPCR approach may have applicability in noninvasive prenatal diagnosis with maternal blood samples.
| | 8/27/2010 12:02:05 PM |
|
|
|
|
|
| Identifying Prenatal Cannabis Exposure and Effects of Concurrent Tobacco Exposure on Neonatal Growth [Drug Monitoring and Toxicology] | |
BACKGROUND:
Cannabis is the most frequently used illicit drug among pregnant women, but data describing the effects of prenatal cannabis exposure and concurrent nicotine and cannabis exposures on neonatal growth are inconsistent. Testing of meconium, the first neonatal feces, offers objective evidence of prenatal cannabis exposure, but the relative ability of meconium testing and maternal self-report to identify affected neonates remains unclear.
METHODS:
Eighty-six pregnant women provided detailed self-reports of daily cannabis and tobacco consumption throughout pregnancy. Cannabinoids and tobacco biomarkers were identified in oral fluid samples collected each trimester and quantified in meconium at birth.
RESULTS:
Cannabis-using women were significantly more likely to also consume tobacco, and smoked similar numbers of cigarettes as non–cannabis-using tobacco smokers. As pregnancy progressed, fewer women smoked cannabis and those who continued to use cannabis reported smoking a smaller number of cannabis joints, but positive maternal oral fluid tests cast doubt on the veracity of some maternal self-reports. More neonates were identified as cannabis exposed by maternal self-report than meconium analysis, because many women quit cannabis use after the first or second trimester; meconium was more likely to be positive if cannabis use continued into the third trimester. Cannabis exposure was associated with decreased birth weight, reduced length, and smaller head circumference, even after data were controlled for tobacco coexposure.
CONCLUSIONS:
Prenatal cannabis exposure was associated with fetal growth reduction. Meconium testing primarily identifies prenatal cannabis exposure occurring in the third trimester of gestation.
| | 8/27/2010 12:02:05 PM |
|
| Evaluation of Nonenzymatic Posttranslational Modification-Derived Products as Biomarkers of Molecular Aging of Proteins [Review] | |
BACKGROUND:
During their biological life, proteins are exposed in a cumulative fashion to irreversible nonenzymatic, late posttranslational modifications that are responsible for their molecular aging. It is now well established that these damaged proteins constitute a molecular substratum for many dysfunctions described in metabolic and age-related diseases, such as diabetes mellitus, renal insufficiency, atherosclerosis, or neurodegenerative diseases. Accordingly, the specific end products derived from these reactions are considered potentially useful biomarkers for these diseases.
CONTENT:
The aim of this review is to give an overview of nonenzymatic posttranslational modifications of proteins and their influence in vivo, take inventory of the analytical methods available for the measurement of posttranslational modification–derived products, and assess the potential contribution of new technologies for their clinical use as biological markers of protein molecular aging.
SUMMARY:
Despite their clinical relevance, biomarkers of posttranslational modifications of proteins have been studied only in the context of experimental clinical research, owing to the analytical complexity of their measurement. The recent implementation in clinical chemistry laboratories of mass spectrometry–based methods that provide higher specificity and sensitivity has facilitated the measurement of these compounds. These markers are not used currently by clinicians in routine practice, however, and many challenges, such as standardization, have to be confronted before these markers can be used as efficient tools in the detection and monitoring of long-term complications of metabolic and age-related diseases.
| | 8/27/2010 12:02:05 PM |
|
| Intrapersonal and Populational Heterogeneity of the Chemokine RANTES [Proteomics and Protein Markers] | |
BACKGROUND:
Current immunoassays for the chemokine RANTES (regulated on activation, normal T-cell expressed and secreted) are not tailored for specific isoforms that exist endogenously, despite the fact that variants with modified activity are known to exist. This is surprising in view of this protein’s ubiquitous increased presence in many diseases and that the 2 established isoforms are truncated by enzymes also correlated to disease. An in-depth population survey of RANTES heterogeneity in the context of multiple diseases via a mass spectrometric immunoassay (MSIA) may resolve this issue.
METHODS:
We developed an MSIA for RANTES and endogenous variants apparent in human plasma. Samples from multiple cohorts of individuals (type 2 diabetes, congestive heart failure, history of myocardial infarction, and cancer patients) were run in parallel with samples from healthy individuals (239 people total). We used 230 µL of plasma per individual and tabulated relative percent abundance (RPA) values for identified isoforms.
RESULTS:
We detected at least 19 variants, including the dipeptidyl peptidase IV (DPP-IV)–truncated variant. The majority of variants were unreported in the literature. Identifiable modifications included N- and/or C-terminal truncations, oxidation, glycation, and glycosylation. We observed statistically significant differences in RPA values for multiple variants between disease cohorts and recognized prospective disease-specific protein profiles for RANTES.
CONCLUSIONS:
Because of widespread interest in the clinical value of RANTES, the protein diversity established here may aid in the design of future, fully quantitative assays. Equally important, an inclusive qualitative understanding of RANTES heterogeneity may present new insights into the relationship between RANTES and disease.
| | 8/27/2010 12:02:05 PM |
|
|
|
| Dry-Reagent Double-Monoclonal Assay for Cystatin C [Proteomics and Protein Markers] | |
BACKGROUND:
Cystatin C is a low molecular weight cysteine proteinase inhibitor whose plasma or serum concentrations have been shown to be better correlated with glomerular filtration rate than serum creatinine concentrations. Routine assays for cystatin C are based on use of polyclonal antibodies and immunoturbidimetric and nephelometric designs. This study aimed to develop a double-monoclonal immunoassay for cystatin C.
METHODS:
We tested functionality of 42 2-site antibody combinations involving 7 monoclonal antibodies with recombinant and plasma cystatin C. We developed a heterogeneous assay using 2 antibodies selected to give the best analytical performance. The assay used a dilution step and was based on a dry-reagent, all-in-one immunoassay concept with time-resolved fluorometry. The assay was performed on an automated immunoanalyzer in single wells that contained all the required assay components. We used heparin-derived plasma samples for methodological evaluation of the assay.
RESULTS:
From a relative epitope map involving 7 cystatin C–specific antibodies, we selected a pair of antibodies for a 2-site sandwich-type dry-reagent assay. Total assay time was 15 min, and 10 µL of a 100-fold diluted sample was used. The analytical detection limit (background + 3SD) and functional detection limit (CV 20%) were 0.01 mg/L and 0.02 mg/L, respectively. Within-run and total assay imprecision were <4.7% and <5.6% (at 0.84–3.2 mg/L), respectively, and plasma recoveries of added cystatin C were 94%–110%. Regression analysis with the Roche particle-enhanced immunoturbidimetric method yielded the following (SD): slope, 1.391 (0.029); y-intercept, –0.152 (0.045) mg/L; Syx=0.294 mg/L (n=131).
CONCLUSIONS:
The developed assay enables rapid and reliable measurement of cystatin C.
| | 8/27/2010 12:02:05 PM |
|
| Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen [Proteomics and Protein Markers] | |
BACKGROUND:
Detection limit challenges associated with measuring low-abundance protein biomarkers can be addressed with hybrid immunoaffinity–mass spectrometric assays, such as antipeptide antibody capture followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Popular assay formats use magnetic bead–based immunoaffinity enrichment and nanoflow LC-MS/MS or high-flow immunoaffinity chromatography coupled online to conventional LC-MS/MS. As a proof of principle, we describe a novel online immunoaffinity LC-MS/MS configuration that combines high-flow peptide immunoaffinity enrichment and nanoflow LC-MS/MS.
METHODS:
We configured and validated an assay for the measurement of total pepsin/pepsinogen from human saliva that uses a pepsinogen standard. Saliva was heat-inactivated to quench residual enzymatic activity and then digested with endoproteinase AspN. Online immunoaffinity enrichment using an antipeptide antibody directed against the pepsin C-terminal sequence, DRANNQVGLAPVA, was linked to nanoflow liquid chromatography and selected reaction monitoring mass spectrometry. We used the assay to measure pepsin/pepsinogen concentrations in human saliva from presumed healthy volunteers.
RESULTS:
Heat inactivation at 100 °C for 25 min stabilized the target peptide. The final assay had <15% interassay relative error and <15% interassay CV across a range of 4.08–2980 pmol/L human pepsinogen (0.165–120 µg/L). Low but quantifiable signals were observed in some samples from presumed normal healthy volunteers ranging from 4.3 to 16.6 pmol/L (0.17–0.67 µg/L) total salivary pepsin/pepsinogen.
CONCLUSIONS:
This assay approach provides a high-sensitivity platform for protein bioanalysis in the low picomolar range. It bears the potential to deliver additional data on the salivary occurrence of pepsin/pepsinogen with greater confidence than previously.
| | 8/27/2010 12:02:05 PM |
|
|
|