Technology platforms for molecular diagnosis of cystic fibrosis

Cystic fibrosis (CF) is one of the most common recessive genetic diseases in North America. So far, 1200 mutations causing CF have been identified. Several techniques such as allele specific oligonucleotide (ASO) dot-blot, reverse dot-blot, amplification refractory mutation (ARMS), and an oligo-ligation assay, are available to detect the most common mutations. However, detecting compound heterozygotes between DeltaF508, the most common disease causing mutation, and other mutations which are rare is difficult as some mutations are common only to particular ethnic groups. Therefore, new diagnostic tests such as restriction enzyme assays and single stranded conformational polymorphism (SSCP) have been designed to recognize rare and population-specific mutations. This review will describe the most commonly used CF mutation detecting diagnostic techniques, as well as novel assays and techniques currently in development that might be employed in future.     

Development of two real-time quantitative TaqMan PCR assays to detect circulating Aspergillus fumigatus DNA in serum

Several PCR assays have been developed for detecting Aspergillus fumigatus DNA in blood of patients with invasive aspergillosis. However, the best blood fraction to be assayed has not been defined and the multicopy genes used as the DNA targets for amplification not characterized. Firstly, we developed a real-time PCR assays based on the TaqMan technology targeted to a single copy gene. To compare serum, white cell pellet, and plasma for effectiveness as blood assay fractions, we spiked whole blood with A. fumigatus DNA and processed these fractions similarly. The difference between white cell pellet and serum was not significant. In contrast, the yield from plasma was 10 times lower than from serum. Then, we compared serum processed immediately or after 24 h at room temperature and observed a lower yield after 24 h. Secondly, a real-time PCR assay targeted to a mitochondrial gene was also developed. The copy number was estimated between 9 and 10 mitochondrial genes per single copy gene. Therefore, we recommend serum, stored and frozen as soon as possible, to be used for detecting circulating A. fumigatus DNA for diagnosis. Moreover, the mitochondrial multicopy gene was characterized in order to compare results from different patients.     

Competition between Serum IgG,IgM, and IgA Anti-Glycan Antibodies

Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies (IgA, IgG, and IgM) compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies.     

A method to detect ras point mutations in small subpopulations of cells.

Biochemical assays for ras mutations are capable of detecting a mutant allele only if it is present in at least 5% of cells tested. Further, ras mutation assays which utilize the polymerase chain reaction (PCR) are unable to distinguish a ras mutation in a small population of cells from mutations resulting from Taq DNA polymerase base misincorporation. We used a standard restriction fragment length polymorphism assay of PCR-amplified c-Ki-ras to detect codon 12 mutations in tumor cells and found a cumulative error frequency for Taq DNA polymerase of one codon 12 mutation per 2 X 10(4) molecules of total amplification product. The Taq polymerase-induced mutations were found to be multiple base transitions and represented a constant proportion of the amplification product at each step of the PCR. The ability to detect the in vitro generated mutation was dependent on the number of thermal cycles and the sensitivity of the detection assay. With these considerations in mind, we developed a two-step RFLP assay in which the thermal cycle number was kept low and molecules containing mutations at codon 12 were selectively amplified in the second step. We were able to detect a ras mutation occurring in 1 per 1000 cells (a two log improvement over standard RFLP methods) without detecting mutations resulting from Taq DNA polymerase infidelity.     

A quantitative lateral flow assay to detect complement activation in blood

Complement is a major effector arm of the innate immune system that responds rapidly to pathogens or altered self. The central protein of the system, C3, participates in an amplification loop that can lead to rapid complement deposition on a target and, if excessive, can result in host tissue damage. Currently, complement activation is routinely monitored by assessing total C3 levels, which is an indirect and relatively insensitive method. An alternative approach would be to measure downstream C3 activation products such as C3a and iC3b. However, in vitro activation can produce falsely elevated levels of these biomarkers. To circumvent this issue, a lateral flow immunoassay system was developed that measures iC3b in whole blood, plasma, and serum and avoids in vitro activation by minimizing sample handling. This assay system returns results within 15 min and specifically measures iC3b while having minimal cross-reactivity to other C3 split products. While evaluating the potential of this assay, it was observed that circulating iC3b levels can distinguish healthy individuals from those with complement activation-associated diseases. This tool is engineered to provide an improved method to assess complement activation at point of care and could facilitate studies to monitor disease progression in a variety of inflammatory conditions.     

2D gel blood serum biomarkers reveal differential clinical proteomics of the neurodegenerative diseases

This review addresses the challenges of neuroproteomics and recent progress in biomarkers and tests for neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. The review will discuss how the application of quantitative 2D gel electrophoresis, combined with appropriate single-variable and multivariate biostatistics, allows for selection of disease-specific serum biomarkers. It will also address how the use of large cohorts of specifically targeted patient blood serum samples and complimentary age-matched controls, in parallel with the use of selected panels of these biomarkers, are being applied to the development of blood tests to specifically address unmet pressing needs in the differential diagnosis of these diseases, and to provide potential avenues for mechanism-based drug targeting and treatment monitoring. While exploring recent findings in this area, the review discusses differences in critical pathways of immune/inflammation and amyloid formation between Parkinson’s disease and amyotrophic lateral sclerosis, as well as discernable synergistic relationships between these pathways that are revealed by this approach. The potential for pathway measurement in blood tests for differential diagnosis, disease burden and therapeutic monitoring is also outlined.     

Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases

Recent developments in DNA and RNA amplification technology are enabling the design of ultra-sensitive diagnostic assays for infectious diseases. The leading amplification technology is the polymerase chain reaction (PCR). An alternative approach, Q-beta amplification, also promises remarkable speed and precise quantification of assay results.     

Polymerase chain reaction diagnosis of fungal disease: Finally coming of age

Definitive diagnosis of invasive fungal disease (IFD) remains limited, and nonculture diagnostic tests could improve this. Serologic testing for biomarkers is standardized through commercial manufacture and is gaining widespread application and incorporation into disease-defining guidelines, whereas most molecular assays remain “in-house” with few commercial tests and limited clinical validation. Standardization of molecular testing will need to be genus-specific because different diseases present and progress in various ways, influencing the optimal specimen choice. Methodologic standardization is almost complete for Aspergillus polymerase chain reaction and will be used to determine clinical performance. The new generation of broad-range systems capable of detecting and differentiating fungi may provide the best route to covering the spectrum of IFD, but evaluation is required and will be difficult to achieve for some of the less prevalent IFDs.     

A real-time PCR assay for DNA-methylation using methylation-specific blockers

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.     

A new method for high speed, sensitive detection of minimal residual disease

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.     

肝细胞癌循环肿瘤细胞标志物及检测方法研究进展

肝细胞癌(hepatocellular carcinoma, HCC)在中国是一种高发病率和高死亡率的恶性肿瘤。肿瘤切除、肝移植是治疗该病最有效的手段,但术后的高复发率和高转移率是影响患者预后的重要因素。外周血循环肿瘤细胞(circulating tumor cells, CTCs)是导致肝细胞癌术后复发和转移的必要因子。综述了CTCs的标记物——磷脂酰肌醇蛋白聚糖-3、转铁蛋白受体、甲胎蛋白、α-L岩藻糖苷酶、上皮细胞粘附因子、高尔基蛋白73和异常凝血酶原等,以及利用这些标记物检测CTCs的特异性和灵敏度,以期为肝细胞癌转移的早期检测、术后的复发、预后评估和选择治疗方案等提供依据。     

Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.     

Maladies à prions humaines

Human prions diseases are rare, fatal, transmissible neurodegenerative diseases, presumably caused by new infectious protein-only agents or prions. The diagnosis is established post mortem by neuropathological examination of the brain. During the clinical course of the disease, cerebrospinal fluid biomarkers are valuable diagnostic tests of the disease in patients with rapidly progressive dementia. The tests accuracy varies among the subtypes of the disease. Researches are in progress for new biomarkers discovery.     

Physical characterization of the "immunosignaturing effect"

Identifying new, effective biomarkers for diseases is proving to be a challenging problem. We have proposed that antibodies may offer a solution to this problem. The physical features and abundance of antibodies make them ideal biomarkers. Additionally, antibodies are often elicited early in the ontogeny of different chronic and infectious diseases. We previously reported that antibodies from patients with infectious disease and separately those with Alzheimer's disease display a characteristic and reproducible "immunosignature" on a microarray of 10,000 random sequence peptides. Here we investigate the physical and chemical parameters underlying how immunosignaturing works. We first show that a variety of monoclonal and polyclonal antibodies raised against different classes of antigens produce distinct profiles on this microarray and the relative affinities are determined. A proposal for how antibodies bind the random sequences is tested. Sera from vaccinated mice and people suffering from a fugal infection are individually assayed to determine the complexity of signals that can be distinguished. Based on these results, we propose that this simple, general and inexpensive system could be optimized to generate a new class of antibody biomarkers for a wide variety of diseases.     

Point-of-care molecular diagnostic systems--past, present and future

The field of molecular diagnostics has greatly decreased the time it takes to identify infectious agents and to test their antimicrobial resistance. Portable devices are currently in development that can easily identify a variety of nucleic acid targets (either DNA or RNA) from multiple sample types in under an hour. This is done by a variety of methods including real-time polymerase chain reaction, probe-based assays, bioluminescence real-time amplification, and microarray or micro-pump technologies. These self-contained systems require only minimal training and perform all steps of the assay from extraction through detection with little or no operator intervention.     

Differential interferometry in the analytical ultracentrifuge. I. Design, construction, and alignment of differential interferometer

The application of an enzyme-linked immunosorbent assay to the study of histone antigens is described. This method is much more sensitive than microcomplement fixation tests and allows the interaction between histone antibodies and nucleosomes to be measured directly on the solid phase under a variety of ionic conditions. The results indicate that the enzyme-linked immunosorbent assay is a sensitive assay for investigating different conformational states of chromatin and for detecting low levels of histone antibodies in antisera.     

Polymerase chain reaction-based methods of DNA methylation analysis

DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.     

Tumor antigen NB/70K and CA 125 levels in the blood of preoperative ovarian cancer patients and controls: a preliminary report of the use of the NB12123 and CA 125 radioimmunoassays alone and in combination

The NB12123 and CA125 radioimmunoassays, murine monoclonal antibody assays for measuring circulating levels of human ovarian tumor associated antigens NB/70K and CA 125, respectively, have been previously described. In the present study, preoperative serum samples were obtained from patients undergoing laparotomy for benign neoplastic ovarian tumors (N = 16), cancer of the cervix (N = 22), cancer of the uterus (N = 20), and cancer of the ovary (N = 47). Controls (N = 50) were obtained from healthy blood bank donors. No correlation was observed between the levels of NB/70K and CA 125 in these samples (r2 = .079, linear regression analysis). In general, increasing levels of both antigens were present with increasing tumor burden and higher histological grade. In addition, both markers were most elevated in the serum of ovarian cancer patients with serous and unclassified adenocarcinomas. Using 40 AU and 35 unit cut-offs for the NB/70K and CA 125 assay, respectively, overall specificity for healthy controls and patients with benign diseases approaches 100%. The combined sensitivity of the assays for ovarian cancer patient sera in this study indicates that the assays may be helpful in establishing a pre operative diagnosis of ovarian cancer. Complementarity of the NB/70K and CA 125 assays has been demonstrated, indicating that one or both assays may be used to monitor as many as 85% of ovarian cancer patients.     

The use of post‐translationally modified peptides for detection of biomarkers of immune‐mediated diseases

Biomarkers are decision‐making tools at the basis of clinical diagnostics and essential for guiding therapeutic treatments. In this context, autoimmune diseases represent a class of disorders that need early diagnosis and steady monitoring. These diseases are usually associated with humoral or cell‐mediated immune reactions against one or more of the body's own constituents. Autoantibodies fluctuating in biological fluids can be used as disease biomarkers and they can be, thus, detected by diagnostic immunoassays using native autoantigens. However, it is now accepted that post‐translational modifications may affect the immunogenicity of self‐protein antigens, triggering an autoimmune response and creating neo‐antigens. In this case, post‐translationally modified peptides represent a more valuable tool with respect to isolated or recombinant proteins. In fact, synthetic peptides can be specifically modified to mimic neo‐antigens and to selectively detect autoantibodies as disease biomarkers. A ‘chemical reverse approach’ to select synthetic peptides, bearing specific post‐translational modifications, able to fishing out autoantibodies from patients' biological fluids, can be successfully applied for the development of specific in vitro diagnostic/prognostic assays of autoimmune diseases. Herein, we report the successful application of this approach to the identification of biomarkers in different autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.