Fluorescent labeling is another method used to demonstrate the complexing of antigens and antibodies (Fig. 1). Fluorescent molecules are used as substitutes for radioisotope or enzyme labels. The fluorescent antibody technique consists of labeling antibody with fluorescein isothiocyanate (FITC), a fluorescent compound with an affinity for proteins, to form a complex (conjugate). This conjugate is able to react with antibody-specific antigen.

Fig1. Principles of direct and indirect fluorescent techniques. A, Direct fluorescence. B, Indirect fluorescence. 1, Microscopic slide; 2, cell (cytoplasm and nucleus); 3, antiserum (conjugate  in A, unconjugate in B); 4, conjugated antiglobulin serum.

Fluorescent techniques are extremely specific and sensitive. Antibodies may be conjugated to other markers in addition to fluorescent dyes; the use of these markers is called colorimetric immunologic probe detection. The use of enzyme-substrate marker systems has been expanded. HRP, ALP, and avidin biotin conjugated enzyme labels have all been used as visual tags for the presence of antibody. These reagents have the advantage of requiring only a standard light microscope.

Fluorescent conjugates are used in the following basic methods, which are widely used:

 • Direct immunofluorescent assay

• Inhibition immunofluorescent assay

• Indirect immunofluorescent assay

Direct Immunofluorescent Assay

 In the direct fluorescent antibody (DFA) technique, a conjugated antibody is used to detect antigen-antibody reactions at a microscopic level (Fig. 2). DFA can be applied to tissue sections or in smears for microorganisms.

Fig2. Direct fluorescent antibody (DFA) technique. After the  labeling of a specific antibody with FITC, it can be reacted with its  antigen and identified microscopically.

Fluorescein-conjugated antibodies bound to the fluorochrome FITC are used to visualize many bacteria in direct specimens (see later, “Direct Fluorescent Antibody Test for Neisseria gonorrhoeae”). HRP conjugated to antibody, the immunoperoxidase stain, can be used to detect CMV, other viruses, or nucleic acids in cells. In biotin-avidin, enzyme- conjugated methods, single stranded nucleic acid probes, antimicrobial antibodies, or antibiotin antibodies can be bound to the small biotin molecule. These molecules have a strong affinity for the protein avidin, which has four binding sites. Biotin bound to avidin or antibody can be complexed to fluorescent dyes or to color-producing enzymes to form specific detector systems. This system can be applied to the detection of nucleic acids in organisms such as CMV, hepatitis B virus (HBV), Epstein-Barr virus (EBV), and Chlamydia.

The chemical manipulation in labeling antibodies with fluorescent dyes to permit detection by direct microscopic examination does not seriously impair antibody activity, the ability of fluorescent antibody conjugate to react specifically with its homologous antigen. Monoclonal antibodies (MAbs) have also been successfully conjugated to fluorescein for the detection of chlamydiae, rabies virus, and other pathogens in directly stained specimens.

When absorbing light of one wavelength, a fluorescent sub stance emits light of another (longer) wavelength. In fluorescent antibody (FA) microscopy, the incident or exciting light is often blue-green to ultraviolet. The light is provided by a high-pressure mercury arc lamp with a primary (e.g., blue-violet) filter between the lamp and the object that passes only fluorescein- exciting wavelengths. The color of the emitted light depends on the nature of the substance. Fluorescein gives off yellow-green light and the rhodamines fluoresce in the red portion of the spectrum. The color observed in the fluorescence microscope depends on the secondary or barrier filter used in the eyepiece. A yellow filter absorbs the green fluorescence of fluorescein and transmits only yellow. Fluorescein fluoresces an intense apple-green color when excited.

Inhibition Immunofluorescent Assay

The inhibition immunofluorescent assay is a blocking test in which an antigen is first exposed to unlabeled antibody and then to labeled antibody, and is finally washed and examined. If the unlabeled and labeled antibodies are both homologous to the antigen, there should be no fluorescence. This result confirms the specificity of the FA technique. Antibody in an unknown serum can also be detected and identified by the inhibition test.

Indirect Immunofluorescent Assay

The basis for indirect fluorescent assay (IFA) is that anti bodies (immunoglobulins) not only react with homologous antigens, but also can act as antigens and react with antiimmunoglobulins (Box 1). IFA is the serologic method most widely used for the detection of diverse antibodies. Immunofluorescence is used extensively in the detection of autoantibodies and antibodies to tissue and cellular antigens. For example, antinuclear antibodies (ANAs)—a heterogeneous group of circulating immunoglobulins that react with the whole nucleus or nuclear components (e.g., nuclear proteins, DNA, histones) in host tissues—are frequently assayed by indirect fluorescence. By using tissue sections that contain a large number of antigens, it is possible to identify antibodies to several different antigens in a single test. The antigens are differentiated according to their different staining patterns.

Box1. Immunologic Assays Performed by Indirect Fluorescent Antibody Technique

Immunofluorescence can also be used to identify specific antigens on live cells in suspension (flow cytometry). When a live stained cell suspension is put through a fluorescence- activated cell sorter (FACS), which measures its fluorescent intensity, the cells are separated according to their particular fluorescent brightness. This technique permits the isolation of different cell populations with different surface antigens (e.g., CD4+ and CD8+ lymphocytes).

In the IFA, the antigen source (e.g., whole Toxoplasma microorganism, virus in infected tissue culture cells) to the specific antibody being tested is affixed to the surface of a microscope slide. The patient’s serum is diluted and placed on the slide to cover the antigen source. If antibody is present in the serum, it will bind to its specific antigen. Unbound antibody is then removed by washing the slide. In the second phase, antihuman globulin (AHG, directed specifically against IgM or IgG) conjugated to a fluorescent substance that will fluoresce when exposed to ultraviolet light is placed on the slide. This conjugated marker for human antibody will bind to the antibody already bound to the antigen on the slide and will serve as a marker for the antibody when viewed under a fluorescence microscope.

A major problem in interpreting IFA results is background staining. For most IFAs, laboratories must choose a screening dilution because undiluted specimens will show background staining resulting from nonspecific binding or clinically insignificant levels of circulating autoantibodies. The screening dilution plays a critical role; the more dilute the specimen becomes, the less sensitive but more specific the procedure.

An example of a changing clinical situation is that many laboratories have replaced indirect immunofluorescence, once the standard for ANA testing, with the EIA. Less labor and technical experience are cited as reasons for switching from indirect immunofluorescence. However, the trade-off may not be valuable if patients have antibody titers of less than 1:160

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مواضيع ذات صلة

Chemiluminescence

Enzyme Immunoassay

Capillary Electrophoresis

Antibody Structure and Function

B Cell Receptor for Antigen

Antigen Processing and Presentation

Methods of Enhancing Agglutination

Mechanisms of Agglutination

The Major Histocompatibility Complex

Cellular Basis of the Adaptive Immune Response

Mechanisms of Innate Immunity

Overview of Acute-Phase Proteins