Immunoassays in Coagulation Testing
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Fibrinolysis Volume 4, Issue 3 , July , Pages Author links open overlay panel C. Legnani 1 M. Maccaferri 1 G. Clarke, W.
Harris, N. Chapter 10, Immunoassays.
To detect or measure an antibody in a person's blood, a known antigen is attached to a solid surface. A solution containing the patient sample is added. If the patient's sample contains antibody, it will bind to the antigen. If the enzyme-linked antibody binds to human antibodies, the enzyme will create a detectable change that indicates the presence and amount of the antibody in the patient sample.
Millipore Corporation [On-line information]. Gerostamoulos, J. It combines an electrophoresis step with a step that transfers blots the separated proteins onto a membrane. To perform a western blot test, a sample containing the protein is applied to a spot along one end of a layer of gel. Multiple samples and a control may be placed side by side along one end of the gel in separate "lanes.
These sample and control ladders are then "blotted" transferred onto a thin membrane that is put in contact with the gel. Labelled or tagged antibodies are then used in a one or two step process to detect the proteins bound to the membrane. For example, to confirm HIV or Lyme antibody tests, the proteins separated are those of the causative organism. The presence of the certain proteins is interpreted by comparison with known negative or positive control samples in the other lanes. Sources Khalsa, G. Western blotting. Louis: Elsevier Saunders; Humans normally have 23 pairs of chromosomes: 22 pairs of non-sex-determining chromosomes autosomes and 1 pair of sex chromosomes XX for females and XY for males.
Chromosomes are made up of DNA, repeating sequences of four bases that form the thousands of genes that direct protein production in the body and determine our physical characteristics. DNA consists of two strands bound together in a double helix structure like a spiral staircase.
Each half of the helix is a complement of the other. Fluorescent probes are then added to the sample. Fluorescent probes are sections of single-stranded DNA that are complementary to the specific portions of DNA of interest. The probe, which is labeled with a fluorescent dye, attaches to the specific piece of DNA. When the slides are examined using a special microscope, the genes that match the probe can be seen as areas of fluorescence, which will appear as bright spots on a dark background. This technique can be used to show the presence of extra gene copies duplicated or amplified genes , and genetic sequences that are missing gene deletions or have been moved translocated genes.
Increased numbers of chromosomes, as seen in certain genetic disorders, are also diagnosed using FISH technologies trisomy 21 or Down syndrome, for example. The targeted area s or sequences of DNA are determined by the probes that are used. Multiple targeted areas in the DNA can be assessed at the same time using FISH probes labeled with a number of different fluorescent dyes.
Application of enzyme immunoassays to coagulation testing.
The following photographs show cells that have been evaluated using the FISH methodology. These are just a few examples of the use of FISH technique. Down syndrome In Figure 1, FISH testing is applied to cells in amniotic fluid, obtained from a pregnant woman carrying a baby suspected of having Down syndrome trisomy Binding of cations present in serum, e. Surfactants are commonly used to decrease non-specific adsorption, but they must be carefully selected and optimized for immunoassays since, at high concentrations, they may cause the loss of antibodies passively adsorbed onto the solid support beads used in immunoassays.
Silicone surfactants interact with ion-specific electrode membranes to increase the measured voltage during magnesium and lithium determinations. In addition, water-soluble silicone polymer coatings in separator tubes can physically mask antibodies and alter avidin-biotin binding reactions in immune-radiometric assays.
Interference in immunoassay is one factor that contributes to the uncertainty of medical testing. The recognition of such aberrant test results requires constant surveillance by both laboratory and doctor.
Immunoassays in Coagulation Testing [electronic resource]
One potential source of chemical interferences, the purified water, can be eliminated by choice of a purpose-designed system to produce high-purity water. Let's talk about lab water. Categories Analytical Chemistry. Type your search. Immunoassay — Examples of Sources of Interference and their Effects. Components from blood collection tubes, such as stoppers, lubricants, surfactants and separator gels, can leach into specimens or adsorb analytes. Special additives may also affect analyte stability. These may arise from the sample itself, from earlier contamination as listed above or from the purified water and other reagents used.
Analytical Interferences Biologically active species A wide range of interferences can arise from biologically active species that are present in the sample. Chemical and Other interferences Other interferences can arise from the effects of reagents added to stabilise samples, to enhance other tests or to mask interferences. Citrate Citrate is also added as an anticoagulant by binding calcium ions.
S urfactants Surfactants are commonly used to decrease non-specific adsorption, but they must be carefully selected and optimized for immunoassays since, at high concentrations, they may cause the loss of antibodies passively adsorbed onto the solid support beads used in immunoassays. Importance of Interferences in Immunoassay.
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