Practical tasks

1. Study the results of demonstration of the Bacteriolysis Test. Draw Table 13-1 in your protocol notebook and enter the result in it.

2. Examine and explain the results of the Immune Hemolysis Test (demonstration). Fill in Table 13-2.

3. Determine the titer of complement and its working dose with the help of Complement Titration Test. Write down and fill in Table –13-3. Make a conclusion.

4. Perform the Complement Fixation test to detect the specific antibodies in the patient’s serum. Draw Table 13-4. Make a conclusion.

LESSON 14

SEROLOGICAL REACTIONS with LABELED COMPONENTS. IMMUNOFLUORESCENCE (IF-test). RADIOIMMUNOASSAY (RIA). ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA). IMMUNOBLOTTING

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 which is 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)».