Prelab conference. Topics for discussion:
1. Mechanism of IF-test (direct and indirect techniques). Application.
2. ELISA. Mechanism, components, application.
3. RIA. Mechanism, components, application.
4. Immunoblotting (The Western Blot). Mechanism, components and application.
¨ Serological Reactions with Labeled Components
Recent years have seen wide application of serological reactions which make use of antibodies or antigens labeled in some way. The «label» may be different but should meet the basic requirement: it should be easily detected by means of definite reactions, under the microscope, or with the help of special technical equipment. Apart from retaining the specificity of immunological reactions, serological reactions with labels make it possible to obtain rapidly the results and are usually characterized by high sensitivity. These reactions, therefore, have found widespread application for the rapid diagnosis of viral and bacterial infections.
The following types of labels are employed most frequently:
1) fluorochromes (for example, fluoresceinisothiocyanate (FITC) and rhodamineisothiocyanate (RITC)), which are capable to glow in ultraviolet rays. Fluorochromes are utilized for performing the immunofluorescence reactions;
2) ferritin, a protein, containing up to 23 per cent of iron, which is readily visible by electron microscopy and thus is well suitable as a label in the immunoelectron microscopy;
3) enzymes, which induce the breakdown of the substrate with the formation of stained products when they get in contact with the substrate. They are used to perform the enzyme immunoassay;
4) radioactive labels are employed in highly sensitive radioimmunoassay.
In serological tests with labeled components both labeled antigens and labeled antibodies can be employed.
NOTE: labeled diagnostic antibodies are also called “conjugate”
The serological tests with labeled reagents vary in their sensitivity and diagnostic value, and some of them (e.g., RIA) require special measures of protection from radiation.
There are two modifications of the immunological reactions with the labeled reagents: a liquid phase modification (when all necessary reagents are mixed in the tube) and a solid phase modification whichis most often employed in microbiology. This modification is characterized by the fact that reagents (antigens or antibodies) are sorbed on a solid material and only after that the remaining ingredients of serological reaction are added. Plastic plates, beads, films or tubes made of various synthetic inert materials are usually used as a solid phase carrier of antigens or antibodies. Being absorbed on the surface of such materials, antibodies or antigens, even in a dry state, retain their immunological specificity and ability to participate in serological reactions for a long time.
NOTE: the performance of serological tests with labeled components requires the careful washing to eliminate unreacted reagents.
There are numerous methodological variants of performance immunological tests with the labeled components. When the antigen to be tested attaches to the labeled antibody and then the results are registered, this is a direct variant. In some cases the antigen is caught by antibodies bound to the solid phase. Following incubation with the material, the antigen tested attaches to the antibody and thus to the solid phase. Then the «linked» antigen is demonstrated by means of labeled antibodies against this antigen. This variant is termed «sandwich» test, it is considered as modification of direct variant. In a indirect variant antiglobulin antisera labeled with some mark are used.
NOTE: For competitive variant see point «Radioimmunoassay (RIA)».
¨ Immunofluorescence (IF-test)
The IF-test is commonly used to identify the unknown antigen in the specimen.
NOTE: IF-test is also known as Coons’ reaction.
The special diagnostic antibodies labeled with some fluorochromes are employed in this test. Fluorescent dyes (e.g., fluorescein, rhodamine) can be covalently attached to antibody molecules and made visible by ultraviolet light in the fluorescence microscope. The antibodies labeled by FITC (or RITC) retain the immunological specificity and interact with strictly definite antigens. Such labeled antibody can be used to identify antigens both on the surface of bacteria and in the cells in histologic section or other specimens. Labeled antibody-antigen complexes are readily recognized by intense yellow-green fluorescence (for FITC) during examination with the fluorescent microscopy. The antibodies labeled by RITC in specific antigen-antibody complexes induce the orange-red fluorescence under the fluorescent microscope. There are several variants of IF-test:
1. A direct immunofluorescence reaction occurs when known labeled antibody interacts directly with unknown antigen. If the antigen is present in the specimen, the antibody will react, and the antibody-coated microbes will glow when observed with the fluorescent microscope.
2. An indirect immunofluorescence reaction occurs when a 2-stages process is used. This variant of IF-test can be performed for detection of antibody or antigen.
3. An anti-complement immunofluorescence reaction is used in cases where the antigen-antibody complex can fix complement. The microbe is fixed to a slide and mixed with unlabeled specific antiserum and guinea pig complement. After washing anti-complement fluorescent-labeled antibodies are added. The labeled antibodies will bind only if the first reaction produced antigen-antibody-complement complexes, which happens only if the antibody in the antiserum is specific for the unknown microbe on the slide.
4. To detect the unknown antibodies in the patient’s serum can be used «sandwich» technique. To do this, the specific known antigen is attached to a slide. Then patient’s serum with “unknown antibodies” is added, and the preparation is washed. If the unknown serum antibody matches the antigen, it will remain fixed to it on the slide and can be detected by adding a fluorescent-labeled antibody and examining the slide by ultraviolet (fluorescent) microscopy.
An indirect immunofluorescence reaction is most commonly used in microbiology. This method is based on the using of two types of antisera. To identify the unknown antigen the unlabeled antibodies against the antigen to be assayed are applied on the slide with the specimen (containing the “unknown” suspected antigen). The preparation is thoroughly washed. At the second stage, the formed antigen-antibody complex is treated with the labeled antiserum containing antibodies against immunoglobulins in the first (unlabeled) antiserum. In this case, antiglobulin antiserum, labeled with fluorochromes coats the unlabeled antibody-unknown antigen complex. And as a result, the antigen becomes visible under the fluorescent microscope. The indirect test is often more sensitive than a direct test, because more labeled antibody adheres to antigenic site. Furthermore, the labeled antiglobulin becomes a «universal reagent» (independent of the nature of the antigen used) reactive with all Ig G of that species (e.g., human Ig G antibodies).
¨ Enzyme-Linked Immunosorbent Assay (ELISA)
The enzyme-linked immunosorbent assay (ELISA), also known as the enzyme immunoassay (EIA), is an immunologic test that uses an enzyme-mediated reaction as an indicator of antigen-antibody complex production. ELISA is an extremely sensitive technique for rapidly detecting specific antibodies and antigens. This method, which has many variations, depends on the conjugation of an enzyme to either an antigen or an antibody. The enzyme is detected by assaying for enzyme activity with its substrate. Virtually any enzyme that converts its substrate to a detectable product can be used as the indicator. Peroxidase and alkaline phosphatase are commonly utilized as enzymes while 5-aminosalicylic acid, orthophenylendiamine, and other substances are used as the substrate for peroxidase.
There are two basic methods of EIA. The direct ELISA commonly detects antigens, and the indirect ELISA detects antibodies (Fig. 10). A microtiter plates with numerous shallow wells are used in both procedures. The test has become highly automated in many applications, such as testing for the AIDS antibodies. To measure antibody, known antigen is fixed on a solid phase (e.g., plastic microdilution plate), incubated with test material (e.g., patient’s serum dilutions), washed, and reincubated with an anti-immunoglobulin (antiglobulin) antiserum labeled with an enzyme (e.g., horseradish peroxidase). Enzyme activity, measured by adding the specific substrate and estimating the color reaction, is a direct function of the amount of antibody bound. The number of formed enzyme-antigen-antibody complexes corresponds to the intensity of substrate staining. Therefore, the activity of the enzyme can be determined by the intensity of post-incubation staining with the appropriate substrate by means of the automatic device. The results are registered by a spectrophotometer.
NOTE: For the mechanism and the procedure of ELISA see «PRACTICAL WORK».
¨ Radioimmunoassay (RIA)
Standard antigen preparations labeled with radioactive iodine (125 I) provide another highly sensitive method for detecting minute amounts of antigen. The common technique (liquid phase modification) of competitive radioimmunoassay (RIA) employs the following procedure:
1. A known antibody preparation is mixed with the sample for assay. Any specific antigen in this sample will complex with the antibody. This antibody contains no radioactive label, so these complexes will not be radioactive.
2. 125I-labeled antigen is then added to this mixture to react with any remaining antibody that wasn’t bound by the antigen in the sample. The number of radioactive antigen-antibody complexes is thus inversely proportional to the amount of antigen in the sample. If the sample contains none of the antigen, for example, all the antibody will be available to combine with the radioactive antigen indicator.
3. All antigen- antibody complexes are precipitated and separated from solution, and the radioactivity of the precipitate is determined. (Unreacted 125I antigen remains in solution and is discarded.) The radioactivity of precipitate is inversely proportional to the amount of antigen in the test specimen. If this sample had large amount of antigen, it would react with most of the antibody, leaving little or no immunoglobulin to react with labeled antigen; very few radioactive antigen-antibody complexes would form, and the precipitate would have little radioactivity. Conversely, if the original sample had small amounts of antigen, the binding sites of the antibodies would still be available to react with the labeled antigen, yielding a highly radioactive precipitate.
This variant of RIA is based on the competition for specific antibody between the labeled (known) and the unlabeled (unknown) concentration of material (e.g., antigen). Therefore, this method is termed competitive RIA. The concentration of the unknown (unlabeled) antigen or hapten is determined by comparing the results with those obtained using several concentrations of a predetermined standard antigen.
In the competitive RIA in solid phase modification specific antibodies to the tested antigen are sorbed on the surface of shallow wells of commercial microtiter plates. Then, the antigen-containing material to be tested is placed into the wells and incubated. If the antigen reacts specifically with the antibody adsorbed to the well, the antigen will be retained there when the well is washed free of unbound antigen. The 125I-labeled antigen is then added. If the material contained the antigen specific to immobilized antibodies, some of the active centers would be engaged. In this case the labeled antigen could react only with remaining antibody that wasn’t bound by the antigen in the sample. The number of radioactive antigen-antibody complexes is thus inversely proportional to the amount of antigen in the known sample.
The indirect RIA can be also employed for detecting both “unknown” antibodies (serological diagnosis) and unknown antigens. In both cases an anti-globulin labeled antisera are used. To carry out the serological diagnosis by indirect RIA, the known antigen is adsorbed to the well surface and then the patient’s serum is added. If it contains specific antibodies, the antigen-antibody complex is formed on the well surface. After washing the anti-globulin antiserum labeled by 125I is inoculated into the wells. The greater the concentration of antibodies to the antigen in the patient’s serum, the larger the level of the radioactive label linked to the well surface.
RIA is highly sensitive method applied to the assay of hormones or drugs in serum. A specialized RIA, the radioallergosorbent test (RAST), is used to measure the amount of serum Ig E antibody that reacts with a known allergen (antigen).
¨ Immunoblotting (Western Analysis)
Immunoblotting (is also called «Western blotting») is a method for identifying a particular antigen in a complex mixture of proteins. The complex mixture of proteins is subject to sodium dodecyl sulfate (SDS) - polyacrylamide gel electrophoresis (PAGE). This separates the proteins according to molecular size. The gel is then covered with a membrane (usually a sheet of nitrocellulose), and the proteins are «transferred» by electrophoresis to the membrane. The nitrocellulose membrane (blot) acquires a replica of the proteins separated by SDS-PAGE. During the transfer, the SDS is largely removed from the proteins and, at least for some proteins, there is refolding into a conformation such that antibodies can react with the proteins on the membrane.
The nitrocellulose is then incubated with either a radioactive or enzyme-labeled antibody in a direct test or in an indirect test with antibody followed by a labeled antiglobulin. The protein antigen then becomes visible as a band on the membrane. None of the other proteins in the mixture are detected.
PRACTICAL WORK
1. The Mechanism of ELISA for Detecting Hbs Ag in the Patient’s Serum. Specific antibodies against Hbs Ag are fixed to a solid surface (commercially prepared plastic microtiter plate), and then the patient’s serum is added.
NOTE: Patient’s serum is usually examined to detect the antibodies. But hepatitis B is also known as parenteral hepatitis. Therefore viral antigen Hbs Ag can be detected in the serum and blood.
Therefore, the direct ELISA («sandwich» modification) is performed.
If Hbs Ag is present in the tested serum, it will bind to antibody adsorbed to a well and be fixed to the container. The serum and all unbound components are washed away before the next step. Enzyme conjugate (enzyme-linked specific antibodies against Hbs Ag) is then added. The conjugate is prepared by chemically linking the indicator enzyme (in this case, peroxidase) with specific anti-Hbs antibodies. This conjugate will bind to the fixed antigen-antibody complex. If both the antibody adsorbed to the well and the antibody known to be specific for Hbs Ag have reacted with the antigen, a «sandwich» will have formed, with the antigen between two antibody molecules. This in turn fixes peroxidase to the microtiter plate. Unbound enzyme conjugate is washed away before the next step. The substrate for enzyme action, (hydrogen peroxide), and a colorless chemical indicator (chromogen) are then added. Peroxidase converts H2O2 to H2O and O2 . In the presence of O2 , the indicator changes color, indicating a positive reaction. Serum of the health person does not contain Hbs Ag and consequently fail to provide bridges that link the enzyme conjugate to the fixed antibodies. All peroxidase is therefore removed during the second wash, and no O2 is generated. Negative reaction therefore shows no color (see Fig. 10).
The Procedure of Detecting Hbs Ag in the Patient’s Serum by ELISA. The ELISA is performed according to Table 14-1.
Table 14-1