208 MICROBIOLOGY

SECTION 3 LABORATORY WEEK 13

- SEROLOGICAL IDENTIFICATION OF MICROORGANISMS, DIRECT SEROLOGICAL TESTING - EXERCISE 40. LATEX AGGLUTINATION - ENZYME IMMUNO ASSAY - SEROLOGICAL IDENTIFICATION OF PATIENT ANTIBODIES, INDIRECT SEROLOGICAL TESTING - QUALITATIVE DETECTION OF INFECTIOUS MONONUCLEOSIS, HUMAN IgM ANTIBODIES TO HETEROPHILE ANTIGEN - ANTIBODY TITER

SEROLOGICAL IDENTIFICATION OF MICROORGANISMS, DIRECT SEROLOGICAL TESTING

Highly specific identification of microorganisms can be obtained by serological techniques. In vitro (that is, outside the body and in an artificial environment, such as a test tube), antigens and antibodies react together in certain visible ways. The chemical composition of antigens differ, and therefore, the reactions are highly specific; that is, each antigen provokes an antibody response with that antibody only. When it provokes an antibody response, the antigen is known as an immunogen. In gram-negative bacilli, the carbohydrate antigens within the wall of the organism are called somatic (associated with the soma, that is, the body of the cell) or O antigens. Each species has a different array of O antigens that can detected in serological tests. In like manner, those bacilli that are motile also contain characteristic flagellar protein components called H antigens (H is from the German word hauch , which refers to motility). In streptococci, the carbohydrate wall antigens are used to group the organisms by alphabetic designations A through V. Many bacteria also contain antigenic carbohydrate capsules that can be used for identification, the primary example being the pneumococci, whose capsules permit them to be differentiated into more than 80 different types. Exotoxins and other protein metabolites of bacterial cells are also antigenic. The interaction of antibody with antigen may be demonstrated in several ways. Examples of these are latex agglutination, coagglutination, and Enzyme-linked assays. These tests depend on linking antibody to a particle or enzyme in order for a positive reaction to be observed. The fluorescent antibody test is similar to the enzyme immunoassay except that the antibody is linked to a dye that fluoresces when it is reviewed microscopically under an ultraviolet light source. Fluorescent antibody tests can provide rapid diagnosis of infections caused by pathogens that are difficult to grow in culture, or that grow slowly. Thus they have become popular for detecting such organisms as Legionella pneumophilia (the agent of Legionnaires disease), Bordetella pertussis , Chlamydia trachomatis and several viruses, directly in patient specimens. A portion of the specimen dried on a microscope slide is treated with the fluorescent antibody reagent, rinsed to remove unbound antibody, and then viewed under a fluorescence microscope with an ultraviolet light source.

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In a positive test, bacteria or viral inclusions fluoresce apple green. This test is used in a similar way to identify microorganisms isolated on culture plates or in cell cultures. A simpler test, which detects O and H antigens of gram-negative enteric bacilli (usually Salmonella and Shigella species and Escherichia coli ), is the bacterial agglutination test. When the unknown organism isolated in culture us mixed with an antiserum (prepared in animals) that contains antibodies specific for its antigenic makeup, agglutination (clumping) of the bacteria occurs. If the antiserum does not contain specific antibodies, no clumping is seen. A control test in which saline is substituted for the antiserum must always be included to be certain that the organism does not clump in the absence of the antibodies. In this exercise, you will note how a microorganism can be identified by an interaction of its surface antigens with a known agglutinin that produces a visible agglutination of the bacterial cells. The test is referred to as a Staph Latex Test.

Exercise 40. CULTURE

LATEX AGGLUTINATION

You are given two unknown gram positive, catalase positive cocci on Blood Agar labeled A and B. review laboratory 7

PROCEDURE
Step 1. Each desk is provided with a STAPH LATEX TEST. Step 2. You will perform a Latex agglutination assay according to the instructions provided with you test kit. The instructor will demonstrate the Latex agglutination assay. Step 3. Record results below. Color plate 13.1

LATEX AGGLUTINATION

ASSAY

FOR THE IDENTIFICATION

OF S. aureus

unknown A

unknown B

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ENZYME IMMUNO ASSAY

The instructor will demonstrate and discuss the Abbott TESTPACK STREP A Enzyme Immunoassay for the rapid detection and confirmation of S. pyogenes (Group A Streptococci). Color plate 13.2

SEROLOGICAL IDENTIFICATION OF PATIENT ANTIBODIES, INDIRECT SEROLOGICAL TESTING

The serology laboratory tests patients sera to detect specific antibodies. The results of such tests may provide a serological diagnosis of an infectious disease in which antibody was produced in specific response to the microbial antigens of the infecting microorganism. Demonstrating increasing quantities of antibody in the serum during the course of disease form its onset (when they may be little or no antibody) and acute stages through convalescence (when large amounts of antibody have been produced) constitutes evidences of current active infection and indicates the natures of the etiological agent. The interaction of a patients antibody with a specific antigen may be demonstrated in one of several ways. Descriptive terms for antibodies refer to the type of visible reaction produced. AGGLUTININS are antibodies that produce agglutination, a reaction that occurs when the bacterial cells or other insoluble particles are visibly clumped by antibody combined with antigens on the cell surfaces. PRECIPITINS are antibodies that produce precipitation of soluble antigens (free in solution and unassociated with cells). When antibodies combine with such antigens, the large complexes that result simply precipitate out of solution in visible aggregates. COMPLEMENT-FIXING ANTIBODIES are those that, in combination with their antigens, bind or fix complement, a normal component of human or animal serum. ANTITOXINS are antibodies produced in response to antigenic toxins. Since toxins are soluble antigens, in vitro interactions with antitoxins are seen as precipitation. OPSONINS are antibodies that coat the surfaces of microorganisms by combining with their surface antigens. This coating on the bacterial cell makes them highly susceptible to phagocytosis by white blood cells. (The word opsonin has a Greek root that means to relish food) Serological tests may be performed in vitro (in the test tube) or in vivo (in the body of an animal or human). In in vitro tests, such as those just described, quantitative methods are often employed. An in vivo test may employ experimental animals or cell cultures to demonstrate neutralization of an antigen by its antibody. Depending on the nature of the antigen injected intradermally, a humoral or cellular (delayed hypersensitivity) immune response may occur. Patients immune to diptheria experience no reaction at the site of injected diptheria toxin (Schick test) because their circulating antitoxin neutralize this antigen. Conversely, when persons who are (or have been) infected by tubercle bacilli are injected with a purified protein derivative of this microorganisms, the response is a reddened area of induration at the injection site. This Delayed Hypersensitivity reaction results from vasodilation and infiltration of lymphocytes.

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HUMAN IgM ANTIBODIES TO HETEROPHILE

ANTIGEN

Infectious mononucleosis (often called simply "mono") is a common viral infection that causes fever, sore throat, and enlarged lymph nodes. The most common complaint is a sore throat. It is commonly caused by Epstein-Barr virus but can be caused by other viruses such as cytomegalovirus (CMV). It is diagnosed most frequently among teenagers and young adults. The illness may last weeks to months. Treatment mainly is to releive symptoms and can nearly always be done at home with plenty of rest. By adulthood, 90-95% of men and women have already been infected. Mono usually occurs between the ages of 15-25 and is highly contagious; 1-3% of college students contract mono each year. Infection is spread through exposure to body fluids containing the virus. It is most often transmitted via saliva (hence the name "kissing disease"). However, mono can also be spread through blood and genital secretions. A blood test that detects the presence of Heterophile antibodies (antibodies that nonspecifically react against different proteins and are useful in the diagnosis of infectious mononucleosis) may be employed about 1 week after the onset of the disease. The antibodies peak at weeks 2 to 5 and may persist for several months to 1 year.

QUALITATIVE DETECTION OF INFECTIOUS MONONUCLEOSIS,

The instructor will demonstrate and discuss the COLOR SLIDE II Mononucleosis Test for the rapid detection of Heterophile antibodies of Infectious Mononucleosis.

ANTIBODY TITER
To quantitate antibody in serum, serial dilutions of the serum are made by setting up a row of test tubes, each containing the same measured volume of saline diluent. A measured quantity of serum is added to the first tune and mixed well. The dilution in this tube is noted (1:2, 1:4, 1:10). A measured aliquot of this first dilution is then removed and placed in the second tube, containing measured saline. Material in the second tube is mixed, and an aliquot is removed and placed in the third tube. The procedure is repeated down the line of tubes, so that a graded series of serum dilutions is obtained. (This procedure is analogous to preparing antimicrobial dilutions). The antigen is then added in a constant volume per tube. After allowing time (at the right temperature) for antigen-antibody combination to occur, the tubes are examined for visible evidence of such combination. The reciprocal of the last (highest) dilution of serum that produces a visible reaction is reported as the titer of the serum because it indicates the relative quantity of the antibody present. If two sera are compared for reactivity with the same antigen, the one that can be diluted furthest and still show reactivity is said to have the highest titer, that is, the most antibody.

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In serological diagnosis of infectious disease, it is almost always necessary to test two samples of the patients serum: one drawn soon after the onset of symptoms during the acute stage, and another taken 10 to 14 days later(convalescent sera). The reason for this is that the antibody production takes time to begin and to build up to detectable concentrations during the course of active infection. The first sample may show no antibody, or a low titer that could either reflect past infection or previous vaccination with the microbial antigen in question. If the second sample shows at least a fourfold or greater increase in titer as compared with the first, it is evident that current active infection had induced a rising production of antibody. Such laboratory information is of great value both in diagnosis and in evaluation of the immunologic status of the patient with respect to any antigen tested. The instructor will demonstrate and discuss procedures involved in pooling blood serum, saline-serum dilution and agglutination assays to measure an antibody titer.

TERMS AND QUESTIONS FOR STUDY

ANTIGEN (Ag)

O ANTIGEN H ANTIGEN

ANTIBODY (Ab)

HOMOSPECIFIC REACTION

AGGLUTINATION

BLOOD SERUM

ANTISERUM

ANTIBODY TITER

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1. Name the components of microbial cells that are antigenic (immunogenic).

2. What is the purpose of the control test run in parallel with the bacterial agglutination?

3. What is the principle of serological identification of microorganisms?

4. What is the value of serological identification of a microorganism as compared with culture identification?

5. If two separate species of bacteria share the same antigenic chemical group, what would be the result when each is mixed with antibody prepared against one of them?

6. What is a fluorescent antibody?

7. Describe a doubling serial dilution of six tubes, beginning with a serum dilution of 1:2 in the first tube.

8. Define serum titer.

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9. What are acute and convalescent sera? Why must they both be tested in making a serological diagnosis of infectious disease?

10. Define toxin, antitoxin, and toxoid.

11. What is the difference between an agglutination and a precipitation test?

12. How do immunological tests for detecting microorganisms or their antigens in patient specimens differ from serological tests to detect antibodies in patient sera? Describe direct and indirect testing.

13. Why is immunity to tuberculosis detected by a skin test rather than by a test for the patients serum antibodies?

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