Lecture 9 objective To understanding of

• Reactions between antibodies and antigens that can be used in diagnosis of disease and identification of pathogens.

• Serology testing of a patient’s blood serum that can indicate a current or past infection and the degree of immunity.

• Tests that produce visible interactions of antibodies and antigens include agglutination, precipitation, and complement fixation.

Serological and immune tests: measuring the immune response in vitroCharacteristics of antibodies (such as their quantity or specificity) can reveal the history of a patient’s contact with microorganisms or other antigens. This is the underlying basis of serological testing. Serology is the branch of immunology that traditionally deals with in vitro diagnostic testing of serum. Serological testing is based on the familiar concept that antibodies have extreme specificity for antigens, so when a particular antigen is exposed to its specific antibody, it will fit like a hand in a glove. The ability to visualize this interaction by some means provides a powerful tool for detecting, identifying, and quantifying antibodies or for that matter, antigens.One can detect or identify an unknown antibody using a known antigen, or one can use an antibody of known specificity to help detect or identify an unknown antigen (figure 16.3).

Modern serological testing has grown into a field that tests more than just serum. Urine, cerebrospinal fluid, whole tissues, and saliva can also be used to determine the immunologic status of patients. These and other immune tests are helpful in confirming a suspected diagnosis or in screening a certain population for disease.

General Features of Immune Testing

1

University of Thi-QarCollege of Medicine

Dept. of Microbiology

3rd stageSubject : Immunology

The strategies of immunologic tests are diverse, when antibodies and antigens can be used as tools. We will summarize them under the headings of:

1. Agglutination. 2. Precipitation.3. Immunodiffusion. 4. Complement fixation. 5. Fluorescent antibody tests. 6. Immunoassay tests.

The most effective serological tests have a high degree of specificity and sensitivity. Specificity: is the property of a test to focus upon only a certain antibody or antigen and not to react with unrelated or distantly related ones. Sensitivity: means that the test can detect even very small amounts of antibodies or antigens that are the targets of the test. New systems using monoclonal antibodies have greatly improved specificity, and those using radioactivity, enzymes, and electronics have improved sensitivity.

Visualizing Antigen-Antibody Interactions In the case of large antigens such as cells, Ab binds to Ag and creates large clumps or aggregates that are visible macroscopically or microscopically. Smaller Ag-Ab complexes that do not result in readily observable changes will require special indicators in order to be visualized. Endpoints are often revealed by dyes or fluorescent reagents that can tag molecules of interest. Similarly, radioactive isotopes incorporated into antigens or antibodies constitute sensitive tracers that are detectable with photographic film.An antigen-antibody reaction can be used to read a titer, or the quantity of antibodies in the serum. Titer is determined by serially diluting a sample in tubes or in a multiple-welled microtiter plate and mixing it with antigen. It is expressed as the highest dilution of serum that produces a visible reaction with an antigen. The more a sample can be diluted and yet still react with antigen, the greater is the concentration of antibodies in that sample and the higher is its titer

Agglutination and Precipitation ReactionsThe essential differences between agglutination and precipitation are in size, solubility, and location of the antigen. In agglutination, the antigens are whole cells such as red blood cells or bacteria with determinant groups on the surface. In precipitation, the antigen is a soluble molecule. In both instances, when Ag and Ab are optimally combined so that neither is in excess, one antigen is interlinked by several antibodies to form an insoluble, three-dimensional aggregate so large that it cannot remain suspended and it settles out . Agglutination testing examples:

ABO and Rh (Rhesus) blood types in preparation for transfusions. In this type of test, antisera containing antibodies against the blood group

2

antigens on red blood cells are mixed with a small sample of blood and read for the presence or absence of clumping.

The Widal test is an example of a tube agglutination test for diagnosing salmonelloses and undulant fever. In addition to detecting specific antibody, it also gives the serum titer (figure 16.5b).

The Rapid Plasma Reagin (RPR) to test for antibodies to syphilis. The cold agglutinin test, (4°–20°C), to diagnose Mycoplasma pneumonia. The Weil-Felix reaction for diagnosing rickettsial infections.

In latex agglutination tests, the inert particles of tiny latex beads are coated by specific ab.

Pregnancy hormone in the urine, Identifying Candida yeasts and bacteria (staphylococci, streptococci, and

gonococci), Diagnosing rheumatoid arthritis.

In viral hemagglutination testing, agglutinogen is a red blood cell that reacts naturally with certain viral antigens. The RBCs are mixed with a known virus and a patient’s serum of unknown content.

The test is interpreted differently than other agglutination tests because it is based on a competition between the A sample of patient’s serum is serially diluted with saline. The dilution is made in a way that halves the number of antibodies in each subsequent tube. An equal amount of the antigen (here, blue bacterial cells) is added to each tube. The control tube has antigen, but no serum. After incubation and centrifugation, each tube is examined for agglutination clumps as compared with the control, which will be cloudy and clump-free. The titer is defined as the dilution of the last tube in the series that shows agglutination. If the patient’s serum does not contain antibodies specific to the virus, the virus reacts with the RBCs instead and agglutinates them. Thus, agglutination here indicates no antibodies and a seronegative result. On the other hand, if the specific antibodies for the virus are present, they attach to the virus particles, the RBCs remain free, and there is no agglutination. As performed in the wells of microtiter plates, the difference is apparent to the naked eye (figure 16.6). Several viral diseases (measles, rubella, mumps, mononucleosis, and influenza) can be diagnosed with this test.

3

Precipitation Tests In it; the soluble antigen is precipitated (made insoluble) by an antibody. This reaction is observable in a test tube in which antiserum has been carefully laid over an antigen solution. At the point of contact, a cloudy or opaque zone forms. Example of this technique is the VDRL (Veneral Disease Research Lab) test to detect antibodies to syphilis. Although precipitation is a useful detection tool, the precipitates are so easily disrupted in liquid media that most precipitation reactions are carried out in agar gels. These substrates are sufficiently soft to allow the reactants (Ab and Ag) to freely diffuse, yet firm enough to hold the Ag-Ab precipitate in place. One technique with applications in microbial identification and diagnosis of disease is the double diffusion (Ouchterlony) method. It is called double diffusion because it involves diffusion of both antigens and antibodies. The test is performed by punching a pattern of small wells into an agar medium and filling them with test antigens and antibodies. A band forming between two wells indicates that antibodies from one well have met and reacted with antigens from the other well. Variations on this technique provide several results.

Immunoelectrophoresis constitutes yet another refinement of diffusion and precipitation in agar. With this method, a serum sample is first electrophoresed to separate the serum proteins as previously shown. Antibodies that react with specific serum proteins are placed in a trough parallel to the direction of migration, forming reaction arcs specific for each protein. This test is widely used to detect disorders in the production of antibodies. Counter immunoelectrophoresis, which uses an electrical current to speed up the migration of antibody and antigen, is a newer technique for identifying bacterial and viral antigens in blood.

The Western Blot for Detecting ProteinsThe Western blot test involves the electrophoretic separation of proteins, followed by an immunoassay to detect these proteins. This test is a counterpart of the Southern blot test for identifying DNA. It is a highly specific and sensitive way to identify or verify a

4

particular protein (antibody or antigen) in a sample. First, the test material is electrophoresed in a gel to separate out particular bands. The gel is then transferred to a special blotter that binds the reactants in place. The blot is developed by incubating it with a solution of antibody or antigen that has been labeled with radioactive, fluorescent, or luminescent labels. Sites of specific binding will appear as a pattern of bands that can be compared with known positive and negative samples. This is currently the verification test for people who are antibody-positive for HIV in theELISA test, because it tests more types of antibodies and is less subject to misinterpretation than are other antibody tests. The technique has significant applications for detecting microbes and their antigens in specimens.

5