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Testing Immunoassay



  • Testing Immunoassay
  • Immunoassay
  • Scalability, Reliability and Simplicity
  • Understanding the method used for a test provides a broader context for understanding your test results. Immunoassay Overview. A wide range of medical tests are immunoassays, called immunodiagnostics in this context. Many home pregnancy tests are. Enzyme immunoassay (EIA) is now widely used as a diagnostic tool in always accurately reflect the amount of antibody in the test fluid.

    Testing Immunoassay

    The amount of light striking the tube is proportional to antigen concentration. The antigen concentration can be determined within minutes of the reaction. A calibration curve based on a nonlinear model such as a cubic spline plot is used to calculate the antigen concentration from the reaction rate. RIA is a method employing radioactive isotopes to label either the antigen or antibody. Isotopes are atoms that have unstable nuclei and emit radiation in order to transform into stable atoms.

    Most RIA methods employ iodine I as the radiolabel. This isotope emits gamma rays. It has a high specific activity so that a very small mass of isotope is needed, and a short half-life 60 days. These properties result in minimal disposal problems with leftover or spent reagents. Gamma rays emitted by the immune complexes are usually measured following removal of unbound free radiolabel.

    Since background radiation is very low and the counting time can be extended if needed to generate more counts, RIA is the most sensitive of all immunoassay methods. There are two types of RIA, competitive and immunoradiometric sandwich assays. Competitive assays use radiolabeled antigen.

    The labeled antigen "competes" with non-radioactive antigen in the sample for a limited number of binding sites on the reagent antibody. Following incubation, the free radiolabeled antigens are removed by decanting or washing and the radioactivity of the antibody-bound antigens is measured. The radioactivity of the antibody-antigen complexes is inversely proportional to antigen concentration. In the immunoradiometric IRMA or sandwich assay, two antibodies are used and one is radiolabeled.

    In the test system, the sample is incubated with a specific antibody usually attached to a solid phase such as a plastic bead or the wall of a plastic test tube. After washing to remove unbound sample components, a radioactively labeled antibody is added. The second antibody may be directed against a different part of the antigen molecule, or it may be directed against the first antibody e.

    The second antibody binds to the immune complexes making an antibody-antigen-antibody "sandwich. The amount of radioactivity is directly proportional to antigen concentration. As with immunonephelometric assays, the calibration curve for RIA is nonlinear. The reagent antibodies reacting with different parts of the antigen have different binding affinities causing the curve to be hyperbolic.

    Various methods are used to transform the plot so that result can be more accurately determined. Concentration is plotted on the x-axis and radioactivity on the y-axis. This keeps the slope of the curve from changing each day as the amount of radioactivity of the reagent decreases naturally.

    This produces a linear plot from which the concentration of unknown can be easily determined. The major advantages of RIA when compared to other immunoassays are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassay.

    It is still the gold standard to which other immunochemical methods are compared and is still performed in reference laboratories for analytes such as deoxycortisol, which are not available by other methods. Enzyme immunoassay was developed as an alter-native to RIA. These methods use an enzyme to label either the antibody or antigen. Competitive assays use enzyme labeled antigen, and sandwich assays use an enzyme labeled antibody. However, EIA requires an additional step, the addition of substrate which follows the immunological reaction.

    The sensitivity of EIA approaches that for RIA because a single enzyme molecule can catalyze the conversion of many molecules of substrate to produce. Therefore, the enzyme label amplifies the reaction by producing many colored, fluorescent, or chemiluminescent molecules for each antibody-antigen reaction.

    As with RIA, the relationship between enzyme activity and concentration is nonlinear, and curve-fitting methods such as the cubic spline plot or the four-parameter logistic curve are used to calculate concentration.

    Many EIA assays use monoclonal antibodies as reagents to increase the sensitivity and lot to lot reproducibility of the assay. Monoclonal antibodies are made by fusing the immunoglobulin genes from a B lymphocyte, which produces the desired antibody specificity with a malignant plasmacytoma cell line. This results in a malignant cell line called a hybridoma that secretes large quantities of the desired antibody. Since the antibodies are derived from identical cells, the antibody molecules are identical and all have the same binding affinity for the antigen.

    In addition to safety, another advantage of EIA is that light versus radiation is measured. This obviates the need for a scintillation counter that is more expensive than a light measuring instrument.

    In addition, some competitive EIA methods do not require the separation of antibody-bound and free antigen. These methods are called homogenous assays. The ELISA method is a heterogenous sandwich immunoassay, which means that separation of bound and free enzyme label is required. The term ELISA refers to the use of a solid phase to which the antibody or antigen is bound in order to facilitate the separation.

    ELISA methods are usually performed using a microtiter plate containing 96 wells rather than in test tubes.

    Incubation, washing, and signal reading steps are performed as with EIA sandwich assays described above. FIA refers to immunoassays that utilize a fluorescent label or an enzyme label that acts on the substrate to form a fluorescent product. In fluorescence measurements short wavelength light usually near ultraviolet light is used to excite the molecules.

    Fluorescent molecules stabilize by losing part of the absorbed light energy as heat and part as longer wavelength visible light. Fluorescent measurements are inherently more sensitive than colorimetric spectrophotometric measurements. Therefore, FIA methods have greater analytical sensitivity that EIA methods which employ absorbance optical density measurement.

    Chemiluminescent immunoassays utilize a chemiluminescent label. Chemiluminescent molecules produce light when they are excited by chemical energy. The energy usually comes from an oxidation-reduction reaction. These molecules can be conjugated directly to antigens, or they can be used as substrates for enzyme labels. The most commonly used chemiluminescent labels are acrodinium, luminol, and dioxetane.

    Acrodinium and luminol are excited by peroxidase enzyme reactions and can be used with EIAs that employ a horseradish peroxidase label. Dioxetane-phosphate can be excited by hydrolysis of the phosphate bond using the enzyme alkaline phosphatase as the label.

    Consider this example of a competitive binding assay for thyroxine T4 based on chemiluminescence. The sample is mixed with T4 labeled with alkaline phosphatase ALP in a plastic tube containing anti-T4 conjugated to the tube wall. T4 in the sample competes with the ALP-labeled T4 for the antibody.

    After the reaction, the tube is washed to remove any unbound T4 and dioxetane-phosphate is added. The enzyme hydrolyzes the phosphate ester bond exciting the dioxetane which releases flashes of light.

    These emissions are measured by a light detector and are inversely proportional to the T4 concentration of the sample. Blood samples are collected by venipuncture using standard precautions for reducing exposure to blood-borne pathogens. Risks of venipuncture include bruising of the skin or bleeding into the skin.

    Special safety precautions must be observed when performing radioimmunoassay RIA methods. RIA tests use radioactive isotopes to label antigens or antibodies. Pregnant females should not work in the area where RIA tests are being performed.

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    Fast results and better patient flow with cardiac testing at the point of care. Run several samples in parallel. Joining the fight against sepsis Learn more about the importance of early detection of sepsis and Procalcitonin PCT testing.

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    From an ethical perspective, confirmation testing should be performed on all inconsistent immunoassays, as the repercussions of a false-positive screen can be dramatic. Importantly, the validity for extreme accuracy of both of these types of testing has been established 2,7. Next month, I will discuss the challenges involved in accurately interpreting the results of UDT, the role of the laboratory that you use in assisting you, and how to discuss these results with your patients in a meaningful and productive manner.

    In the meantime, keep your patients comfortable and safe, and keep the faith! Protocol for accuracy of point of care POC or in-office urine drug testing immunoassay in chronic pain patients: J Pain Palliat Care Pharmacother.


    An immunoassay is a test that relies on biochemistry to measure the presence and/or concentration of an analyte. The analyte can be large proteins, antibodies . Immunoassays are used to quantify molecules of biological interest based of the immunoassay should be tested initially for both linearity and. With immunoassay testing at the point of care, you get high-quality results and rapid selection of patients who need urgent treatment e.g. suspected infection.

    Scalability, Reliability and Simplicity



    An immunoassay is a test that relies on biochemistry to measure the presence and/or concentration of an analyte. The analyte can be large proteins, antibodies .


    Immunoassays are used to quantify molecules of biological interest based of the immunoassay should be tested initially for both linearity and.


    With immunoassay testing at the point of care, you get high-quality results and rapid selection of patients who need urgent treatment e.g. suspected infection.

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