Tech Primer
Testing Technologies and Methodologies Athena Diagnostics is committed to providing high quality and highly reliable assays. To do this, and to continue on the leading edge of neurological diagnosis, our laboratory uses many sophisticated technologies and methodologies. Below is a brief description of some of the procedures used to perform our assays. Fluorometry
Fluorometry is based on a molecule’s ability to absorb light energy and subsequently emit the energy at a different wavelength. This highly sensitive assay enables monitoring of an analyte’s interaction with patient specimens by the presence or absence of fluorescence. Gas Chromatography
Gas Chromatography is a process by which a biological fluid becomes volatile and is separated into its constituent components by moving a mobile gas phase over a liquid phase. This process is used to detect, identify, and quantitate volatile fatty acids in clinical assays. Mass Spectroscopy
Mass Spectroscopy (MS) provides information about the elemental composition and structure of organic compounds. By manipulation through electrostatic and magnetic fields, ionized compounds are positively identified. In clinical assays, MS can identify compounds, such as very long chain fatty acids, in body fluids. Isotope Substitution
Isotope Substitution is a procedure that substitutes radioactive labeled antibodies or analytes into a biological reaction in conjunction with a clinical specimen. Highly sensitive radioactive counters monitor the uptake of radioactivity and reveal both qualitative and quantitative information about specific, targeted patient specimen components. Spectrophotometry
Many determinations in clinical assays are based upon analyte detection by the measure of absorbed radiant energy through specific wavelength filters. Spectrophotometers can detect minute levels of patient antibodies or analytes when reacted with antibodies bound to a chemical tag that the instrument is optimized to detect. Nanogen SNP Analysis
The Nanogen NC400 is an open platform single nucleotide polymorphism (SNP) technology that uses a re-usable Chip platform to assay the genotype of single or multiple SNPs in a single reaction. This technology combines PCR methodology with fluorescence probe hybridization to accuracy detect SNP genotypes. Enzyme-Linked Immunosorbent Assay (ELISA)
The ELISA method is a highly efficient, quantitative analysis that generally involves linking a specific antibody or antigen to a 96-well microtiter plate. This attachment allows the introduction of sample or standard to distinct locations. An incubation period for antigen/antibody binding is followed by washing away the excess or unreacted specimen. A second enzyme-labeled antibody, which will specifically bind to the antigen-antibody complex linked to the microtiter plate, is added.
A reaction, in the form of oxidation of the bound enzyme-labeled antibody, produces a color formation that is proportional to the amount of antigen or antibody present in the sample. Spectrophotometry is utilized to detect and to quantify the enzyme-linked antibody complex. Radioimmune Assay (RIA)
The RIA method is very similar to the ELISA method, except that it uses radioisotopes instead of enzyme-labeled antibody to detect the antigen/antibody complex. Western Blot
Western blot analysis is a method that is used diagnostically to determine the presence or absence of a specific antibody or antigen in a patient specimen. The test antigen is isolated on a polyacrylamide gel through the process of electrophoresis. It is then transferred and covalently linked to nitrocellulose. Specimens or control standards are introduced to discrete areas of the nitrocellulose that contain the linked antigen. Specific antibodies to the antigen in the specimen will complex with the nitrocellulose-bound antigen. An additional enzyme-linked antibody is introduced and allowed to bind with the antigen-antibody complex.
Reaction of the bound enzyme-labeled antibody produces a visible colored band in the discrete location, or molecular weight, of the originally-transferred antigen, indicating the presence of complexing antibodies in the specimen. Southern Blot
The Southern blot technique is used to detect specific DNA fragments of varying size. It relies upon the digestion of genomic DNA by restriction enzymes. Double-stranded DNA is split into single strands, cut, and electrophoretically separated on an agarose gel.
The gel is transferred to a nitrocellulose membrane filter and hybridized to a radioactively labeled cDNA probe that binds only with its complementary genomic DNA fragment. The membrane is exposed to X-ray film, creating an autoradiogram that permits visual detection of the hybridized cDNA. For each test, the presence of a specific pattern is used to detect or rule out a mutation in the individual’s gene. Polymerase Chain Reaction (PCR)
The PCR process is an elegant means to greatly amplify a DNA sequence. Initially, a DNA helix is denatured by exposure to heat. A pair of customized strands of oligonucleotides, or primers, are annealed to their complementary genomic DNA sequence. A heat-resistant bacterial enzyme known as Taq polymerase allows free-floating nucleotides to attach to the primers. This extends the strands across the region of interest and forms two new double helices from the original one. When utilizing the PCR method only the critical DNA fragment is copied and amplified. This process is repeated multiple times, exponentially increasing the amount of newly copied DNA with each cycle. PCR methodology is used either as an independent diagnostic tool or in addition to other molecular genetic techniques, such as Southern blot. Pulsed Field Gel Electrophoresis (PFGE)
Most DNA fragments used in clinical diagnosis are small and may be resolved using simple gel electrophoresis. However, larger fragments require greater care to resolve into discrete bands. Pulsed field gel electrophoresis uses electric current in several directions to “sift” large DNA fragments through a gel. The technique allows for resolution of these fragments by altering the electric field between spatially distinct pairs of electrodes. Once the fragments are separated, they are transferred to a nitrocellulose membrane filter and hybridized in a process similar to that of Southern blotting. Covalent Technology
Covalent technology is an improved ELISA technique that increases assay sensitivity and accuracy. This improvement is due to three principle differences between covalent technology and standard ELISAs. First, covalent plates have chemical groups that are capable of forming strong covalent bonds grafted to the surface of the plate’s plastic wells. By contrast, standard ELISA plates attach substances by physical and electrical forces of attraction. Second, covalent plates possess spacer arms that extend the attached antigens up off the bottom of the plate into the solution phase where they have a better chance of binding to antibodies. Third, the binding sites of covalent linked antigens tend to have a uniform specific orientation, whereas on standard plates the antigen binding sites are oriented in a random fashion. This means that, compared to typical ELISA technology, more of the covalent plate antigen is properly aligned and held in the liquid phase, exposing the antigen to the epitopes of the antibody. Viral Cytopathic Assay
The viral cytopathic assay is a technique used to detect neutralizing antibodies (NAbs) to interferon (IFN) by directly evaluating cell survival in the presence of IFN. This is accomplished by a viral cytopathic effect reduction bioassay in human lung carcinoma cells. These cells are plated in 96-well plates and exposed to a mixture of previously incubated interferon and the patient’s serum (which may or may not contain NAbs). When EMC virus is added, it kills cells not protected by IFN. The number of cells killed by the virus indicates cells unprotected by IFN due to neutralization by NAbs. Cells are stained and quantitated with a spectrophotometric plate reader. Automated DNA Sequencing
Automated sequencing technology detects fluorescence from four different dyes that are used to identify the four nucleotide bases -- A, G, C, and T -- and their sequence in a given gene. Each dye emits light at a different wavelength when excited by a laser. When the DNA fragments are separated by size through polyacrylamide gel electrophoresis, all four colors can be detected simultaneously. The fluorescent light is ultimately converted to an electrical signal and the final sequence data is derived using computer software.
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