Tecnicas Laboratorias Abbas

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  • 513

    LABORATORY METHODS USING ANTIBODIES

    The exquisite specifi city of antibodies for particular anti-gens makes antibodies valuable reagents for detecting, purifying, and quantitating antigens. Because antibodies can be produced against virtually any type of macromol-ecule and small chemical, antibody-based techniques may be used to study virtually any type of molecule in solution or in cells. The method for producing monoclo-nal antibodies (see Chapter 5 ) has greatly increased our ability to generate antibodies of almost any desired speci-fi city. Historically, many of the uses of antibody depended on the ability of antibody and specifi c antigen to form large immune complexes, either in solution or in gels, that could be detected by various optical methods. These methods were of great importance in early studies but have now been replaced almost entirely by simpler methods based on immobilized antibodies or antigens.

    Quantitation of Antigen by Immunoassays

    Immunologic methods of quantifying antigen concentra-tion provide exquisite sensitivity and specifi city and have become standard techniques for both research and clini-cal applications. All modern immunochemical methods of quantitation are based on having a pure antigen or antibody whose quantity can be measured by an indica-tor molecule (or a label). When the antigen or antibody is labeled with a radioisotope, as fi rst introduced by Rosalyn Yalow and colleagues, it may be quantifi ed by instruments that detect radioactive decay events; the assay is called a radioimmunoassay (RIA) . When the antigen or antibody is covalently coupled to an enzyme, it may be quantifi ed by determining with a spectropho-tometer the rate at which the enzyme converts a clear substrate to a colored product; the assay is called an enzyme-linked immunosorbent assay (ELISA) . Several variations of RIA and ELISA exist, but the most commonly used version is the sandwich assay ( Fig. A-1 ). The sandwich assay uses two different antibodies reactive with different epitopes on the antigen whose concentra-tion needs to be determined. A fi xed quantity of one

    LABORATORY TECHNIQUES COMMONLY USED IN IMMUNOLOGY

    LABORATORY METHODS USING ANTIBODIES, 513

    Quantitation of Antigen by Immunoassays, 513

    Identifi cation and Purifi cation of Proteins, 514

    Labeling and Detection of Antigens in Cells and Tissues, 516

    Purifi cation of Cells, 519

    Measurement of Antigen-Antibody Interactions, 519

    TRANSGENIC MICE AND GENE TARGETING, 520

    METHODS FOR STUDYING T LYMPHOCYTE RESPONSES, 523

    Polyclonal Activation of T Cells, 523

    Antigen-Induced Activation of Polyclonal T Cell Populations, 523

    Antigen-Induced Activation of T Cell Populations with a Single Antigen Specifi city, 523

    Methods to Enumerate and Study Functional Reponses of T Cells, 524

    METHODS FOR STUDYING B LYMPHOCYTE RESPONSES, 525

    Activation of Polyclonal B Cell Populations, 525

    Antigen-Induced Activation of B Cell Populations with a Single Antigen Specifi city, 525

    Assays to Measure B Cell Proliferation and Antibody Production, 525

    Many laboratory techniques that are routine in research and clinical settings are based on the use of antibodies. In addition, many of the techniques of modern molecular biology have provided invaluable information about the immune system. We have mentioned these techniques often throughout the book. In this appendix, we describe the principles underlying some of the most commonly used laboratory methods in immunology. In addition, we summarize how B and T lymphocyte responses are studied with use of laboratory techniques. Details of how to carry out various assays may be found in laboratory manuals.

    Appendix

    IV

  • Appendix IV Laboratory Techniques Commonly Used in Immunology514

    presence of antibodies that are specifi c for a microbial antigen (e.g., antibodies reactive with proteins from human immunodefi ciency virus [HIV] or hepatitis B virus) as indicators of infection. In this case, a saturating quantity of antigen is added to replicate wells containing plate-bound antibody or the antigen is attached directly to the plate, and serial dilutions of the patient s serum are then allowed to bind. The amount of the patient s antibody bound to the immobilized antigen is determined by use of an enzyme-linked or radiolabeled second anti-human immunoglobulin (Ig) antibody.

    Identifi cation and Purifi cation of Proteins

    Antibodies can be used to identify and characterize pro-teins and to purify specifi c proteins from mixtures. Two commonly used methods to identify and purify proteins are immunoprecipitation and immuno affi nity chroma-tography. Western blotting is a widely used technique to

    antibody is attached to a series of replicate solid supports, such as plastic microtiter wells. Test solutions containing antigen at an unknown concentration or a series of stan-dard solutions with known concentrations of antigen are added to the wells and allowed to bind. Unbound antigen is removed by washing, and the second antibody, which is enzyme linked or radiolabeled, is allowed to bind. The antigen serves as a bridge, so the more antigen in the test or standard solutions, the more enzyme-linked or radio-labeled second antibody will bind. The results from the standard solutions are used to construct a binding curve for the second antibody as a function of antigen concen-tration, from which the quantities of antigen in the test solutions may be inferred. When this test is performed with two monoclonal antibodies, it is essential that these antibodies see nonoverlapping determinants on the antigen; otherwise, the second antibody cannot bind.

    In an important clinical variant of immunobinding assays, samples from patients may be tested for the

    FIGURE A 1 Sandwich enzyme-linked immunosorbent assay or radioimmu-noassay. A fi xed amount of one immobilized antibody is used to capture an antigen. The binding of a second, labeled antibody that recog-nizes a nonoverlapping determinant on the antigen will increase as the concentration of antigen increases and thus allow quantifi cation of the antigen.

    ** ***

    ***

    Bind first antibody to well of microtiter plate

    Remove unbound antigen by washing

    Add labeled second antibody specific for nonoverlapping epitopes of antigen

    Remove unbound labeled second antibody by washing; measure amount of secondantibody bound

    Determine amount of bound second antibody as a function of the concentration of antigen added (construction of a standard curve)B

    ound

    labe

    l

    Concentration of antigen

    2

    3

    4

    5

    6

    1

    Add varying amount of antigen ( )

  • 515LABORATORY METHODS USING ANTIBODIES

    washing the beads (by repeated detergent addition and centrifugation). The specifi c protein that is recognized by and now bound to the antibody may be eluted from the beads and dissociated from the antibody by use of a harsh denaturant (such as sodium dodecyl sulfate), and the proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins may be detected after electrophoresis by staining the polyacrylamide gel with a protein stain or by a Western blot analysis (described later). If the original mixture contained radioactively labeled proteins, specifi c proteins immunoprecipitated by the antibody may be revealed by autofl uorography or autoradiography, protein bands being captured on x-ray fi lm placed on the dried SDS polyacrylamide gel containing separated proteins.

    determine the presence and size of a protein in a biologic sample.

    Immunoprecipitation and Immuno Affi nity Chromatography Immunoprecipitation is a technique in which an anti-body specifi c for one protein antigen in a mixture of proteins is used to identify this specifi c antigen ( Fig. A-2A ). The antibody is typically added to a protein mixture (usually a detergent lysate of specifi c cells), and staphylococcal protein A (or protein G) covalently attached to agarose beads is added to the mixture. The Fab portions of the antibody bind to the target protein, and the Fc portion of the antibody is captured by the protein A or protein G on the beads. Unwanted proteins that do not bind to the antibody are then removed by

    FIGURE A 2 Isolation of an antigen by immunoprecipitation or affi nity chromatography. A, A particular antigen can be purifi ed from a mixture of antigens in serum or other solutions by adding antibodies specifi c to the antigen that are bound to insoluble beads. Unbound antigens are then washed away, and the desired antigen is recovered by changing the pH or ionic strength of the solution so that the affi nity of antibody-antigen binding is lowered. Immunoprecipitation can be used as a means of purifi cation, as a means of quantifi cation, or as a means of identifi cation of an antigen. Antigens purifi ed by immunoprecipitation are often analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. B, Affi nity chromatography is based on the same principle as immunoprecipitation, except that the antibody is fi xed to an insoluble matrix or beads, usually in a column. The method is often used to isolate soluble antigens (shown) or antibodies specifi c for an immobilized antigen.

    Mixture of antigen of interest( ) with other antigens

    Collectimmobilizedantibody by

    centrifugation

    Wash withfresh solution

    to removeunboundantigens

    Denatureantibodyto eluteantigen

    Add excessimmobilized