LECTURE: 27

Title COMPLEX SEROLOGICAL LABORATORY TECHNIQUES

LEARNING OBJECTIVES:

The student should be able to:

• Define the term "complex serological techniques". • Enumerate some serological applications in the immunology laboratory. • Explain the principle of the complement fixation test. • Explain the immunofluorescence (direct and indirect). • Explain the virus neutralization. • Enumerate procedures involving agglutination, and explaining the principle of each such as:

- Hemagglutination inhibition. - Passive indirect agglutination. - Coombs (antiglobulin) "direct and indirect" tests.

• Explain the principle of direct and indirect enzyme linked immunosorbent assay (ELISA). • Tests for rheumatoid factor such as:

1. Latex agglutination test (Latex coated with IgG). 2. Rose-Waaler test (tannic acid treated-SRBCs coated with rabbit IgG anti- human antibody). 3. Bis-diazotized Benzedine. 4. Test for soluble antigens (A two-step test can be employed to detect soluble antigens that react and neutralize a hemagglutinating antibody). • Explain the principle of direct and indirect radioimmunoassay (RIA).

LECTURE REFRENCE:

1. TEXTBOOK: ROITT, BROSTOFF, MALE IMMUNOLOGY. 6th edition. Chapter 27. pg. 417-434.

2. TEXTBOOK: MARY LOUISE TURGEON. IMMUNOLOGY & SEROLOGY. IN LABORATORY MEDICINE. 2ND EDITION. Chapter 6. pg 111-131.

3. TEXTBOOK: Richard M. Hyde. NATIONAL MEDICAL SERIES FOR INDEPENDENT STUDY. pp 159-169.

1 COMPLEX SEROLOGIC PROCEDRURES

I. Complex Serological Procedures

Antigen – antibody reactions in which the visible manifestation requires the participation of accessory factors, indicator systems, and specialized equipment can be measured by several techniques.

A) Complement, a protein constituent of normal blood serum, is consumed (i.e., fixed) during the interaction of antigens and antibodies. The phenomenon forms the basis for the complement fixation test, which is sensitive test that can be used to detect and quantitate antigens and antibodies.

1. The primary reacting ingredients are antigen, antibody, and complement.

a. Normal guinea pig serum often is used as a primary source of complement because guinea pigs have high levels of complement with efficient lytic properties.

b. Different sources of complement are used in different vitro test (e.g., rabbit complement is used in cytotoxicity tests performed for transplantation).

2. To use the complement fixation test to determine the presence of antibody to a known antigen in a patient's serum, a test system and an indicator system are used.

a. Test system. The serum which is heated to 56oC to inactive native complement, has added to it measured amounts of antigen and complement (e.g., normal guinea pig serum). If antibody specific for the known antigen is present in the serum, antigen-antibody complexes will form that consume (or fix) all the complement. The initial reaction, however, cannot be seen.

b. Indicator system. In a second step, an indicator system consisting of sheep red blood cell (SRBC) plus hemolysin, antibody specific for SRBC, is added to test for the presence of free complement. Interpretation of the test is based on the presence of hemolysis.

1) If all the complement has been fixed, none will be free to lyse the SRBCs, which constitutes a positive complement fixation test.

2) If no antibody is present in the patient's serum, then the complement is not fixed and is free to interact in the indicator system and lyse the SRBCs, which constitutes a negative complement fixation test.

3. Properly conducted complement fixation tests require the incorporation of appropriate controls of ensure that the result are not adversely affected by the presence of anticomplementary ingredients. The antigen or the serum itself may have anti- complementary properties (e.g., denatured or aggregated immunoglobulin, heparin, chelating agents, microbial contaminants), may fix all the complement in the system, or may remove calcium or magnesium ions (both of which are essential for complement- mediated lysis).

B) Fluorescent dyes (e.g., fluorescein isothiocyanate) can be conjugated to antibody molecules to allow visualization of the molecules under ultraviolet light and a fluorescence microscope. Such labeled antibody may then be used to identify antigen. Both direct and indirect techniques are available.

2 1. Direct immunofluorescence uses antibody that is specific for a particular antigen or parasite labeled with a fluorescent dye, usually fluorescein. This allowed to react with an unknown tissue or organism. If the antibody reacts, it will be visualized as a green stain on the specimen when it is examined under ultraviolet light. To identify Treponema pallidum in exudates from a patient suspected of having syphilis, the following procedure is used.

a. A slide of exudate is prepared and flooded with tagged, specific antibody. If organisms are present in the exudate, they will bind the tagged antibody.

b. Excess antibody is then washed from the slide, and the slide is examined with a fluorescence microscope. T. pallidum will fluoresce against the black background. This is a remarkable rapid, useful test for the identification of unknown microorganisms.

c. This same procedure is used in the identification of other human pathogens such as Legionella pneumophila and Mcoplasma pneumoniae. It also is useful in the detection of vial antigens in tissues. In the latter instance, horseradish peroxidase may be conjugated to the antibody. After the enzyme - antibody complex has reacted with the tissue, the excess reagent is washed away and an appropriate enzyme substrate is added to the tissue section. Bound antibody is detected by the presence of a dark precipitate at the site of antibody binding.

d. Immunoperoxidase tests have advantages over immunofluorescent techniques.

(1) The specimen can be stained with conventional histochemical dyes so structural detail can be used.

(2) The tissue can be examined by standard light microscope procedures.

2. Indirect immunofluorescence procedures use antibody against antibody that has a fluorescent compound covalently coupled to it (e.g., rabbit antihuman gamma globulin antiserum).

a. The "sandwich technique" allows for detection of antibody.

(1) In the serodiagnosis of syphilis by the fluorescent treponemal antibody absorption (FTA- ABS) test, T. pallidum fixed to a slide is flooded with the patient's serum that is to be tested for antibody. If antibodies to the spirochete are present, they will bind to the organisms on the slide.

(2) Excess antibody is removed by washing; to detect bound antibody, the preparation is overlain with fluorescein-tagged antibody to human gamma globulin. If the patient's serum contains antibody to T. pallidum, fluorescing organisms will be seen when the slide is examined with the fluorescence microscope.

b. Indirect immunofluorescence also is used in detecting antinuclear antibodies (e.g., DNA, RNA, and histone). Antinuclear antibodies are present in systemic lupus erythematosus (SLE), and sometimes in rheumatoid arthritis and other autoimmune collagen – vascular diseases. For diagnosing SLE, the procedure is essentially identical to that described above for T. pallidum, except that the antigen is DNA in forms such as animal or human buffy coat cells, rat kidney sections, and beef thymus sections.

C) Hemagglutination inhibition test. Hemagglutination involves the agglutination of red blood cells by antibodies (i.e., hemagglutinations), certain virus particles (e.g., influenza and mumps viruses), or other substances. Although viral hemagglutination inhibition test, which is extremely valuable as a viral diagnostic test (i.e., it demonstrates the presence of serum antibody to hemagglutinating viral substances). 3

1. Similar test can be used to detect soluble antigens that react with and neutralize a hemagglutinating antibody.

2. To examine the serum of a patient suspected of having influenza, the patient's serum is mixed with known influenza virus and red blood cells.

a. If antibody is present, hemagglutination will be inhibited due to the ability of the antibody to bind to the virus and block its ability to Hemagglutinate: Serum antibody + Virus + Red blood cells = no Hemagglutination

b. If no antibody is present, Hemagglutination will occur: Virus + Red blood cells = Hemagglutination

D) Passive agglutination is the conversion of a reaction system from one that precipitates to one that agglutinates, thus yielding a more sensitive indication of antibody.

1. The use of latex particles in the diagnosis of rheumatoid arthritis is an example of passive agglutination. In this disease, the patient produces an antibody (mainly IgM) to his own IgG. The test consists of coating latex particles with IgG and reaching them with the patient's serum. Agglutination indicates the presence of antibodies (i.e., positive test). This antibody is called rheumatoid factor.

2. Bis-diazotized benzidine is a coupling reagent that can be used to conjugate proteins or haptens to red blood cells, thus allowing the detection of specific antibodies to these materials by passive hemagglutination procedures.

3. In the Rose-Waaler test, which detects rheumatoid factor in serum (an anti-IgG antibodies specific for the SRBCs.

E) Coombs' (antiglobulin) test. In certain people, antibodies directed against antigenic determinants are unable to form visible aggregates when subjected to precipitation or agglutination procedures. To demonstrate the presence of antibody in such cases, the Coombs' (antiglobulin) test may be used. Coombs' test involves adding an antibody directed against gamma globulin, which provides a bridge between two antibody-coated cells or particles. The test is performed in two ways. The major use of the Coombs' tests is to direct non-agglutinating anti-red cell antibodies.

1. The direct Coombs' test is used to detect cell-bound antibody. The red blood cells are washed free of serum and unbound antibody, and the antiglobulin reagent is added directly to the cell suspension. The direct Coombs' test is of value in the detection of antibodies to red blood cells associated with syndromes such as hemolytic disease. The antibodies associated with these diseases have the ability to attach to, but not agglutinate, the target red blood cell. The absorbed antibody (i.e., gamma globulin) can be detected, however, by the use of an antibody (i.e., Coombs' serum) to human gamma globulin.

2. The indirect Coombs' test is used to detect the presence of circulating non-agglutinating antibody. A serum sample is incubated with donor red blood cells, the cells are washed, and the antiglobulin reagent is added. At this point, if antibody is absorbed to the red blood cells, it is agglutinated. The indirect Coombs' test is of value in detecting IgG-associated antibody in the serum of a woman who is thought to be sensitized to the Rh antigen and at risk for carrying an erythroblastotic fetus.

F) Viral neutralization is very similar to hemagglutination inhibition, which also is a "neutralization" event. The assays are based on the ability of specific antibodies to interfere

4 with some biologic function of the virus. Usually, the infective property of the virus is blocked.

1. Cytopathic effects (CPE). Certain viruses (e.g., polio virus), when added to appropriate target cells growing in tissue culture (e.g., rabbit kidney cells), produce observable cell destruction referred to as CPE.

2. The phenomenon of CPE is useful in the search for virus-neutralizing antibodies in a serum sample. The serum suspected of containing antibody is added to a virus suspension, and then a susceptible cell culture is inoculated with the mixture.

a. If the cell culture fails to develop CPE, then antibodies were present in the serum sample. Serum antibody + Virus + Target cell = No CPE

b. If CPE develops, then no neutralizing antibodies were present Virus + Target cell = CPE

G) Radioimmunoassay (RIA) is an extremely sensitive method that can be used for the quantitation of any substance that is immunogenic or heptenic and can be labeled with a radioactive isotope (i.e., idone-125). The method is capable of measuring pictogram quantities or less, depending on the substance being assayed.

1. Liquid-phase RIA depends on the competition between labeled (known) and unlabeled (unknown) antigen for the same antibody.

a. A known amount of labeled antigen, a known amount of specific antibody, and an unknown amount of unlabeled antigen are allowed to react together. The antigen-antibody complexes that form are then separated out by either physicochemical or immunologic means (e.g., ammonium sulfate or second antibody precipitation, respectively), and their radioactivity is determined.

b. By measuring the radioactivity still remaining in the supernatant (i.e., unbound, labeled antigen), the percentage of labeled antigen bound to the antibody can be calculated.

c. The concentration of an unknown (unlabeled) antigen can be determined by referring to a standard curve constructed from data obtained by allowing varying amounts of unlabelled antigen to compete.

2. Solid-phase RIA. Recent modifications of the liquid-phase RIA involve absorption or covalent linkage of antibody to a solid matrix (i.e., solid-phase RIA). Unlabeled antigen is then added, followed by labeled antigen. Determination of bound versus free labeled antigen is then made, and the amount of antigen in the unknown sample is calculated by referring to a standard curve.

3. RIA has wide application in the quantitation of a range of biologic substances, including:

a. a number of hormone (e.g., insulin, growth hormone, adrenocorticotropic hormone, triiodothyronine, thyroxine, estrogen) b. Serum proteins (e.g., carcinoembryonic antigen, IgE) c. Metabolites (e.g., adenosine 3':5'-cyclic phosphate, folic acid) d. Drugs (e.g., digoxin, digitoxin, morphine) e. Other microbial agents and antibodies [e.g., hepatitis B surface antigen (HBsAg)]

H) Enzyme-linked immunosorbent assay (ELIS) has virtually the same sensitivity as RIA. The ELISA is both highly sensitive (>99%) and specific (>99% in high-risk populations).

5 1. ELISA can be used to assay either antigens or antibodies. The requisites are as follows.

a. Antigen or antibody can be attached to a solid-phase support (e.g. plastic surfaces, paper disks) and still retain its immunologic activity. b. Either antigen or antibody can be linked to an enzyme (e.g., horseradish peroxidase, alkaline phosphatase) and both immunologic and enzymatic activity is retained by the antigen-enzyme complex.

2. ELISA is the screening test to detect antibodies to the human immunodeficiency virus (HIV).

a. The virus is grown in vitro in a human T-cell culture.

b. Purified whole virus is disrupted and viral proteins are immobilized onto plastic beads or multiwell trays.

c. Antibodies to any of these antigens will bind to them and be immobilized.

d. Excess serum proteins are removed by washing the beads (or wells) and an enzyme-linked anti- human gamma globulin antibody is added. The presence of this second antibody can be detected calorimetrically by adding a substrate for the enzyme that will yield a colored end product.

e. The rate of substrate degradation is determined by the amount of enzyme-labeled antibody that is bond, which is directly proportional to the amount of antigen in the solution being tested. A substrate that gives a color change when degraded is chosen. The color change can be measured quantitatively in a spectrophotometer.

3. The application of one variant of ELISA, the double-antibody sandwich for the assay of an antigen (e.g., hepatitis B antigen) uses two antibodies and is performed as follows.

a. The first antibody (e.g., antibody specific for HBsAg is coated on a plastic surface (polystyrene plate). The solution being tested for HBsAg is then applied to the surface. Any unreacted material is removed by washing.

b. The second antibody (i.e., enzyme-labeled anti-HBsAg-specific antibody) is then applied. Any excess conjugate is removed by washing. Finally, the enzyme substrate is added to detect the presence of the enzyme.

II. ASSAYS OF IMMUNE COMPETENCE involves evaluating phagocytic cells, B cells and T cells.

A) Analysis of phagocytic cells involves three different assays.

1. The assay for metabolism and generation of toxic molecules determines whether phagocytic cells are using the hexose monophosphate (HMP) shunt and generating toxic materials to kill microorganisms. It is used in the diagnosis of chronic granulaomaphagocytic function of neutrophils.

a. When neutrophils go through oxidative burst, they reduce the yellow NBT dye to form a dark blue formazan precipitate. Cells that do this and contain the formazan precipitate in their cytoplasm are described as formazan-positive (f+) and can be counted. The assay has two parts.

(1) Resting. Dye is placed on the cell, but nothing is administered to trigger oxidation. A normal resting value is 1% to 2% f+.

(2) Stimulated. Neutrophils are stimulated to phagocytize. In healthy people, 100% of the cells can be stimulated to be f+. 6

b. People with CGD, however, have abnormal cytochrome B protein subunits, and therefore their phagocytes cannot form oxygen radicals and hydrogen peroxide and do not kill intracellulary. None of the cells of people with CGD is f+ in either the resting or stimulated states.

c. The NBT test is particularly useful in genetic counseling (CGD) is usually an X-linked disease). If the mother is carrier, she may have 1% to 2% f+ cells in resting neutrophils and an intermediate value when stimulated (e.g., 50%).

2. Assay for ingestion and killing of microorganisms. Some phagocytic disorders are viewed as a defect in killing, with normal NBT values. In these cases, the microbicidal assay, or intracellular killing assay, is used in testing the functional integrity of phagocytic cells.

a. Phagocytosis. Cells can be incubated with bacteria or other engulfable materials (e.g., latex, polystyrene particles) for 1 to 3 hours, and then stained examined for uptake of the foreign bodies.

b. Microbicidal activity. Intracellular killing by phagocytic cells can be measured by direct plate counting of mixtures of microorganisms and cells.

(1) In this test – the microbicidal (intracellular killing assay – viable bacteria are added to a tube of neutrophils and incubated for 30 to 60 minutes.

(2) Counted the surviving bacteria by culture of the mixture, and comparing the counts to control tube with bacteria but no neutrophils, gives the percentage of kill. A normal population of cells kills 85% to 90% of the bacteria within 30 minutes.

(3) The viability of the engulfed microbes can be determined more easily if acridine orange, a fluorescent dye, is added to the test system. When observed with a fluorescence microscope, live bacteria stain green, whereas dead organisms appear red.

3. Two assays for chemotaxis are available for testing the ability of neutrophils to move in a directed migratory pattern toward a chemotactic stimulus such as endotoxin or the complement split product C5a.

a. The neutrophils and the stimulus can be separated by a nitrocellulose membrane with a pore size of 3 to 5 µm. The cells that migrate through the small pores can be counted by staining and observation of the opposite side of the membrane.

b. The migration may occur through an agar medium, with the cells migrating from a well punched in the agar toward a chemoattractant placed in a second well.

d. The assay must be done in concurrence with a normal blood specimen to provide a frame of reference.

B) Analysis of B and pre-B cells. In all evaluations of immunity, it is important to establish both the number of immune cells that are present and whether these cells are functional.

1. Membrane marker assays are used to enumerate B cells.

a. For B cells, the marker can be a surface immunoglobulin molecule or a cluster of differentiation (CD) molecules such as CD19 or CD20; these molecules are all integral membrane components (not just absorbed by a receptor).

b. The procedure using immunoglobulin is as follows.

7 (1) Because the immunoglobulin is a protein, it can serve as an antigen. The B cells are mixed with an antibody to gamma globulin that has been labeled with fluorescein dye.

(2) The antibody recognizes the antigen, so that after incubation, the cell has fluorescent antibody all around its membrane. Under a fluorescence microscope, B cells can be identified by their bright green halo.

(3) The cells are commonly enumerated by flow cytometric techniques (see IV C 1 b; VII A 1 a.).

2. Enumeration of pre-B cells

a. The pre-B cell has µ heavy (H) chains in its cytoplasm but none on its membrane. Antibody does not normally enter the cell cytoplasm, and it can label the membrane only if the membrane contains IgM molecules.

b. Pre-B cells are incubated with fluorescein-labeled anti-Human µ chain antiserum.

c. The cells are then washed and fixed on a slide. At this point, the membrane is permeable, and rhodamine (red)-labeled anti-human µ serum is added to label any µ chains in the cytoplasm.

d. Pre-B cells show red fluorescence in the cytoplasm and no fluorescence on the membrane. Mature B cells have a red cytoplasm and a green membrane.

3. Evaluation of function a. The quantitation of serum immunoglobulin levels is accomplished by radial immunodiffusion.

b. Some patients have normal serum immunoglobulin levels but lack specific antibodies. In this case, it must be determined whether the patient can respond to antigens by synthesizing antibodies.

(1) Possible test antigens include poliovirus, tetanus toxoid, pneumococcal polysaccharide, and keyhole-limpet hemocyanin (KLH).

(2) The patient's immunization history is examined before deciding which antigen to use.

(a) Because most people have had immunization, they should respond to poliovirus, tetanus toxoid, and diphtheria toxoid antigens.

(b) If the patient has not been immunized, vaccination is necessary. A killed vaccine is used because attenuated vaccines can cause infection and death in immunodeficient people.

(c) If an antibody assay of an immunized patient is negative, a booster injection (using a killed vaccine) can be given.

c. Isohemagglutinin levels. People in blood group A, B, or O should have anti-B, anti-A, or anti-A and anti-B antibodies, respectively, in their serum by the age of 2 years. If these antibodies are absent, a defect in B-cell function is indicated.

C) Analysis of T cells and their subsets

1. Enumeration of T-cell subset

a. Fluorescent monoclonal antibodies can be used to assay specific T-cell subsets, because different monoclonal antibodies interact with different subsets of cells (Table -1). 8

b. The fluorescence-activated cell sorter (FACS) uses flow cytometric techniques to analyze and separate cells according to their fluorescence and light-scattering properties (Figure-1).

(1) A cell suspension is stained with a fluorescent dye conjugated to a particular monoclonal antibody.

TABLE-1 Immunologic Specificity of Selected Ant-T-Cell Serums

Antibody Normal Blood Against Cells Detected Values (%) CD2* T cells and natural killer (NK) cells 85+ CD3+ T cells 75 CD4 Helper and inducer T cells 50 CD8 Suppressor and cytotoxic T cells 25

*CD = cluster of differentiation molecules, which are membrane components of various, cells of the body that aid in identification of cellular function. +Called the pan-T reagent because it reacts with all peripheral blood cells; CD3 antigen is complexed with the T cell antigen receptor in the T-cell membrane.

(2) The suspension is forced through a narrow aperture so that the cells are lined up single file. (3) The stream passes through a laser beam, and photocells measure fluorescence emission and light scatter, giving an index of cell size and granularity. (4) Vibrations in the flow chamber disperse the particles into droplets. (5) The failing droplets are next given an electrical charge, the nature of which depends on the photocell measurements. (6) The droplets then pass between a pair of charged plates, where they are deflected into new paths according to their electrical charge. Thus, the cells can be separated and sorted. (7) The viability of the cells is not affected by this procedure; therefore, they can be used in subsequent experiments such as in vivo reconstitution of immunodeficient animals.

2. Evaluation of function

a. Delayed-type hypersensitivity skin testing. Skin tests for delayed hypersensitivity are useful in overall evaluations of immunocompetence and as part of the diagnostic workup in certain infectious disease (e.g., tuberculosis).

(1) The patient undergoes a battery of skin tests intended to evoke a response to one or more antigens. Multiple antigens may be tested simultaneously.

(a) Available antigens include mumps virus, trichophytin (a fungal antigen), candidin (a yeast antigen to which most humans have been exposed), streptokinase-streptodornase (SKSD); a product of group A β-hemolytic streptococci), purified protein derivative (PPD; a skin test reagent for Mycobacterium tuberculosis infection), and diphtheria toxoid (inactivated toxin from the diphtheria bacillus).

(b) As with serum antibody assay, the occurrence of a response depends on what antigens the patient has encountered during his or her lifetime.

9

Figure-1 Principle of the fluorescence-activated cell sorter (FACS)

10

(2) For each test, the patient is given an intradermal injection of the material, and the injection site is observed for the next 3 days. (3) A positive test result typically is indicated by an erythematosus, indurated lesion that peaks after 48 hours.

(a) This reaction is due to the infiltration of cells (lymphocytes and macrophages) into the area. (b) Usually the erythema is larger than the induration, but it is the indurated area that is measured.

b. Lymphocyte proliferation assays

(1) These assays test T-cell function in terms of response to a mitogen by determining the amount of radioactive tritium incorporated into the DNA of the cell.

(2) Specific antigens, such as PPD, also can be used in people known to be sensitive to a particular substance.

c. Mixed lymphocyte culture

(1) These assays depend on the fact that the patient's T cells have different histocompatibility antigens than do target cells from a nonrelated donor.

(2) The target cells are treated with a drug (e.g., mitomycin C) or with irradiation to prevent DNA synthesis and proliferation. The patient's T cells are then mixed with the treated target cells.

(3) The patient's cell should recognize the target cells as foreign and being proliferation, which can be measured by the addition of tritiated thymidine.

(4) The degree of incompatibility between donor and recipient can be estimated by the amount of radioactivity taken up by the lymphocytes. d. Cell-mediated cytotoxicity assays are used to determine the functional integrity of cytotoxic T lymphocytes and also of natural killer (NK) cells.

(1) The effector cells are incubated with the 'target' which can be another lymphocyte, a tumor cell, or other human cells in culture. After a few hours of incubation, a dye such as eosin or trypan blue is added to the cell mixture.

(2) The end product of the test is a dead target cell with a porous membrane. The pores, which are created by perform molecules released by the cytotoxic cells, allow the dye to penetrate into the cytoplasm so that dead cells can be microscopically observed and counted.

(3) Another index of cell death that is commonly use is the release of chromium-51 by dead cells. This has the advantage of greater sensitivity and objectivity because the result is 'read' by a machine.

11

III. IDENTIFICATION OF SPECIFIC ALLERGENS IN IMMEDIATE (TYPE 1) HYPERSENSITIVITY REACTIONS

A) General considerations

1. A thorough patient history can be extremely valuable for the identification of allergens, particularly if environmental agents are involved.

2. A trial exposure may confirm the cause of an immediate hypersensitivity reaction; however, more precise techniques often must be used.

B) In vivo tests

1. The potential allergens may be administered either via a scratch test or intradermally. Multiple allergens may be tested simultaneously. Antihistamines must be discontinued 24 hours or more before skin testing.

2. A positive test is characterized by the appearance, where seconds or minutes, of a typical wheal (edema) and flare (erythema) reaction in the skin at the site of allergen administration, caused by the local release of mediators.

3. False-positive and false-negative results are possible. Histamine or a compound that can induce histamine release from mast cells may be administered as a positive control.

C) In vitro IgE assays. Either total IgE or specific IgE levels may be measured.

1. Radioimmunosorbent test (RIST) is the technique used to measure total IgE concentration. In this case, the IgE serves as antigen.

a. Sheep or rabbit antibody to human IgE binds to a solid phase, such as a paper disk, and the serum to be examined is then added. The IgE in the serum reacts specifically with the coated disk.

b. Unbound serum proteins are removed by washing, after which radiolabeled antibody to human IgE is added; the labeled antibody combines with the IgE that is bound to the disk.

c. After rewashing, the radioactivity of the complex on the disk is measured. The bound radioactive material is directly proportional to the amount of IgE in the serum sample.

2. Radioallergosorbent test (RAST) is the technique used to measure specific IgE levels.

a. A specific allergen (e.g., ragweed pollen antigen) is bound to a solid phase, such as a cellulose particle or paper disk. The serum to be examined is then reacted with the solid phase-allergen complex. If IgE specific for ragweed is presented, it binds to the solid phase.

b. Unbound IgE is removed by washing, after which radiolabeled antibody to human IgE is reacted with the solid phase.

c. After rewashing, the radioactivity of the particles is measured to give the serum level of IgE specific for ragweed antigen.

IV. DETECTION OF IMMUNE COMPLEXES. Several types of assays are available for detecting immune complexes in serum and other biologic fluid. 12

A) C1q binding assay. Complement component C1q has an affinity for human complexes.

1. C1q solid-phase binding assay

a.Serum to be tested is incubated in a polystyrene tube that has been coated with C1q. After incubation, the sample is washed out of the tube.

b. The amount of immune complex bound to C1q is measured by using radiolabeled antibody to immunoglobulin.

2. C1q liquid-phase binding assay

a. Radiolabeled C1q is added to the serum being tested.

b. Approximately 1 hour later, the bound C1q is removed from the free C1q by precipitation with polyethylene glycol.

c. The radioactivity level of the bound C1q corresponds to the C1q-binding capacity and indicates the amount of immune complexes.

B) Raji cell binding assay

1. Raji cells (a human lymphoblastoid cell line) are well suited for the detection of immune complexes because;

a. They can bind immune complexes through C3 receptors.

b. They lack surface immunoglobulin.

2. Immune complexes bound to the surface of the Raji cells can be assayed by the addition of radiolabeled anti-IgG antibody.

V. PRODUCTON AND USE OF MONOCLONAL ANTIBODIES

A) Present and potential uses of monoclonal antibodies

1. Monoclonal antibodies are becoming increasingly useful in laboratory procedures.

a. For example, fluorescent monoclonal antibodies are being used to assay specific T cell subsets by flow cytometric techniques.

b. A constant and uniform source of antibody with a single specificity (instead of the usual mixture produced by the immune system) not only affords a powerful research tool but also can be expected to provide quicker and more accurate identification of viruses, bacteria, and cancer cells.

2. The long-range benefit of monoclonal antibodies is that they have been shown to be therapeutically useful.

a. Monoclonal antibodies are being used in the treatment of cancer, primarily as carriers of anticancer agents such as radioisotopes or cytotoxic agents.

b. In addition, monoclonal antibodies may represent an alternative source of immunogens for human anti-idiotype antibody vaccines. Monoclonal antibodies are homogeneous proteins, and if a monoclonal antibody carried an idiotope that resembled the epitope of the original immunogen (i.e., an anti-idiotypic antibody), it could substitute for the original immunogen in a vaccine.

13 B) Production of monoclonal antibodies by the murine hybridoma technique. Hybridomas are artificially created cell that produce pure or monoclonal antibodies.

1. A mouse is injected with antigen, and plasma cells from its spleen are fused with cancerous mouse cells (i.e., plasmacytoma or myeloma cells)

a. The cells are cultured at high dilutions to allow only one fused cell per culture well.

b. Only hybrid (fused) cells replicate because plasma cells are fully differentiated cells (which do not multiply), and the plasmacytoma cells used are deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT), which makes them susceptible to metabolic poisoning by appropriate alternation in the media.

2. Each hybrid cell formed by this technique has two important properties:

a. It produces the single type of antibody molecule of its spleen cell parent.

b. It continually grows and divides, like its plasmacytoma cell parent.

3. The clone of hybrid cell that produces the desired antibody is identified and is then grown as a continuous cell line, from which large amounts of the homogeneous or monoclonal antibody can be harvested.

Dr. MUSTAFA HASAN LINJAWI

14