Diagnostic Assays : Other assays

Diagnostic Assays Portfolios


Porous Silicon Nanomembranes for the Rapid Separation of Macromolecules by Size and Shape

Researchers at the University of Wisconsin-Platteville have developed a unique nanomembrane for the separation of biomolecules based on their three-dimensional geometries. These “macromolecular sieves” are produced from laser-etched silicon nanomembranes. The pores in these membranes have openings in the sub-micron range but are designed to significantly reduce the flow impedance of the filtered solution. This design feature allows for faster filtration time when compared with traditional membranes. Nanomembranes with square and rectangular geometries have been produced. Desirable characteristics of the square opening membrane include a high open area of 45% and low standard deviation in opening size (less than 5%). Additionally, the fabricated membranes have been tested with vacuum pumps and show no signs of damage after repeated filtrations with 15 psi of applied pressure differential. Currently, reducing opening size below 100 nm and introducing openings of varying geometries is under development. Further efforts are also underway to decrease the manufacture time and increase the overall scalability of the membrane patterning process.

Noninvasive Assay for Bovine Embryo Quality

UW–Madison researchers have identified 11 microRNAs (miRNAs) and 18 mRNAs as indicators of healthy IVF embryo development. They discovered that the miRNAs are differentially expressed between bovine blastocyst-stage embryos and those that fail to develop (‘degenerates’). This is the first report that miRNA levels in the culture medium differ among embryos of different developmental fate and can be used as indicators of embryo viability.

Predicting Glucoregulatory Dysfunction

UW–Madison researchers have developed a method based on blood lipid chemistry to identify a subject at risk for glucoregulatory dysfunction. The method involves obtaining a biosample from the subject, separating the diacylglycerol fatty acids and determining if the concentration is above or below a control range.

One-Step DNA Extraction from Dried Blood Spots for Newborn Screening

A Wisconsin researcher has developed a one-step method for eluting DNA from a blood sample. The method involves using a DNA elution solution and agitating the blood sample in the solution with heat. The purified DNA is suitable for use in techniques such as enzymatic DNA amplification and real-time PCR.

Measuring the Activity of a Specific Fraction of Albumin for Early Diagnosis of COPD or Sepsis

UW-Madison researchers have developed a sensitive and rapid high throughput assay that can be used for early detection of COPD or sepsis. This assay measures the functional activity of a specific fraction of albumin (SFA) in serum or plasma.

The assay uses a liposome that contains a fluorescently labeled fatty acid and a negatively charged phospholipid as a substrate. The liposome is mixed with phospholipase A2 (PLA2), an enzyme that plays a key role in inflammation, and a biological sample from a patient. The SFA in the sample then acts to remove the labeled fatty acid from the liposome, causing a detectable change in fluorescence intensity. This change can be used to determine SFA activity.

The measured SFA activity then can be compared to a normal range of SFA activity or to a baseline measurement from the patient to determine if it is decreasing, abnormally low or normal. A decrease or low measurement of SFA activity can indicate inflammation. If the patient is a tobacco smoker, a decrease in activity as compared to an earlier measurement from the patient suggests the patient has developed or will soon develop COPD. If a patient has recently undergone surgery, a decrease in SFA activity can indicate infection and the possibility of sepsis. On the other hand, a return of SFA activity to normal levels may indicate that the patient has recovered.

Non-Invasive Diagnosis and Evaluation of Disease

UW-Madison researchers have developed a non-invasive method for rapidly measuring early onset or progression of disease by identifying specific biomarkers in a biological sample. This method allows for “fingerprinting” the dynamic changes of disease progression and aids in evaluating the disease process.

A biological sample, such as blood or plasma, is taken from a subject undergoing testing for a disease, and one or more biomarkers are measured. The measurements then are compared to a standard biomarker profile that describes how the biomarkers change as a result of the specific disease. Because the profile changes as the disease progresses, measuring a biological sample at a single point in time provides information on whether or not an individual has the disease, the type (e.g., bacterial or viral) and the stage of the disease (e.g., early or late). 

Synthetic Cofactor Analogs of S-Adenosylmethionine as Ligatable Probes of Biological Methylation

UW-Madison researchers have developed compounds and methods for specifically labeling the substrates of SAM-dependent methyltransferases. The methods use SAM analogs that have been modified at the C5` position so the analog is transferred by the methyltransferase to a methylation site in a substrate, such as a peptide or nucleic acid. Once anchored to the substrate, these cofactor analogs allow for the addition of a detectable and/or isolable label. The label may contain various moieties that aid in determining the methylation state of the substrate. The SAM analogs can also be used with nucleic acid methyltransferases to allow for the rapid identification of specific DNA or RNA residues that are typically methylated.

Identification of Disease Characteristics Using Isotope Ratios in Breath

The researchers now have developed a method of using isotope changes in breath to distinguish bacterial infections from viral infections in humans and other animals. A cavity ringdown spectrometer is used to collect and analyze the isotopes in breath samples taken from a subject over time. A change in the ratio of one stable isotope to another within several hours of exposure to an infectious agent indicates a bacterial infection. On the other hand, a delayed change in isotope ratios, followed by periodic alterations in the ratios, indicates a viral infection.

Method to Diagnose and Treat Degenerative Joint Disease

UW-Madison researchers have developed methods for detecting and treating joint disease, particularly in joints containing a functionally important intra-articular ligament or tendon. To diagnose joint or ligament degeneration, a sample is taken from a joint or ligament and tested for the expression of cathepsin K, cathepsin S, or tartrate-resistant acid phosphatase (TRAP). Because these enzymes are up-regulated during the development of inflammatory arthritis or ligament degeneration, increased levels indicate the presence of disease. Treatment of joint or ligament disease would involve inhibiting the activity of cathepsin K, cathepsin S, or TRAP.

White Blood Cell Assay for Determining Risk of Sepsis and Other Inflammatory Disorders

UW-Madison researchers have developed a method of rapidly assaying P2X7 pore activity in white blood cells within a sample of whole blood. The white blood cells are labeled and then depolarized in an isotonic depolarizing solution. Next, the cells are mixed with a dye and a P2X7 agonist to activate the pore activity of P2X7. Finally, the cells are mixed with a divalent cation to deactivate P2X7’s pore activity. To quantify P2X7 pore activity, the amount of dye taken up by the labeled cells is compared to the dye uptake in control cells without the P2X7 agonist. The amount of activity may help indicate prognosis and suggest a treatment plan for patients at risk for sepsis or other inflammatory disorders.

Phospholipase Activity Provides a Simple Test for Systemic Inflammation in Acute and Chronic Disease

UW-Madison researchers have developed a fluorescence assay for measuring phospholipase activity, including secretory PLA2 (sPLA2) activity.  This assay provides a simple, rapid and highly reproducible blood test that can be used to monitor systemic inflammation over an extended period and evaluate the effectiveness of anti-inflammatory therapies.

The assay uses a unique, fluorescently labeled liposome.  When a sample containing a phospholipase is added to the liposome, the phospholipase hydrolyzes the phospholipid components of the liposome, causing a detectable change in fluorescence intensity.  The degree of change indicates the activity of the phospholipase.

The sample can be compared to a control to determine if an individual has elevated PLA2 activity, which may indicate a disorder associated with systemic inflammation, such as sepsis, heart disease, cystic fibrosis or chronic obstructive pulmonary disease.  The assay also can be used to identify agents capable of altering phospholipase activity. 

Because the activity of sPLA2 in serum is a critical marker for monitoring the onset and severity of systemic inflammation in patients with sepsis or heart disease, this assay also could be used to create a portable, bedside device for early detection of hyper-inflammation.  Such a device could provide accurate, real-time monitoring of changes in fluorescence intensity resulting from changes in sPLA2 activity and may enable early interventions for the prevention of multi-organ failure.

DNA Encoding a DNA Repair Protein

UW-Madison researchers had developed an isolated and purified DNA molecule encoding p95, a DNA repair protein. p95 is a product of the NBS1 gene, the defective gene in the disorder NBS. It also is an integral member of the double strand break repair complex, the function of which is impaired in cells from NBS patients.

Among other uses, this discovery provides an assay in which agents that increase or decrease the activity of p95 can be identified by first expressing p95 in a cell line. Some of the cells are then exposed to the agent, and the amount of p95 is measured relative to that of the control cells.

Novel Assay for Measuring Stress-Induced CREB Phosphorylation

UW-Madison researchers have developed a phospho-specific antibody that can be used to monitor stress-induced phosphorylation of the CREB transcription factor. CREB is phosphorylated in response to a wide variety of cell stresses that result in oxidative or radiation damage to cellular DNA. The researchers identified three sites on CREB that are phosphorylated by the ATM kinase. This affinity-purified, rabbit polyclonal antibody specifically recognizes one of these sites after phosphorylation has occurred. The antibody can be used to determine if ATM has phosphorylated CREB.