Methods Of Antibody Detection

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Direct Whole Pathogen Agglutination Assays

The most basic tests for antibody detection are those that measure the antibody produced by a host to determinants on the surface of a bacterial agent in response to infection with that agent. Specific antibodies bind to surface antigens of the bacteria in a thick suspension and cause the bacteria to clump together in visible aggregates. Such antibodies are called agglutinins, and the test is called bacterial agglutination. Electrostatic and other forces influence the formation of aggregates in solutions so that certain conditions are usually necessary for good results. Because most bacterial surfaces exhibit a negative charge, they tend to repel each other. Performance of agglutination tests in sterile physiologic saline (0.85% sodium chloride in distilled water), which contains free positive ions, enhances the ability of antibody to cause aggregation of bacteria. Although bacterial agglutination tests can be performed on the surface of both glass slides and in test tubes, tube agglutination tests are often more sensitive, because a longer incubation period (allowing more antigen and antibody to interact) can be used. The small volume of liquid used for slide tests requires a rather rapid reading of the result, before the liquid can evaporate.

Examples of bacterial agglutination tests are the tests for antibodies to Francisella tularensis and Brucella spp., which are part of a panel called febrile agglutinin tests. Bacterial agglutination tests are often used to diagnose diseases in which the bacterial agent is difficult to cultivate in vitro. Diseases that can be diagnosed by this technique include tetanus, yersiniosis, leptospirosis, brucellosis, and tularemia. The reagents necessary to perform many of these tests are commercially available, singly or as complete systems. Because most laboratories are able to culture and identify the causative agent agglutination tests for certain diseases, such as typhoid fever, are seldom used today. Furthermore, the typhoid febrile agglutinin test (called the Widal test) is often positive in patients with infections caused by other bacteria because of cross-reacting antibodies or a previous immunization against typhoid. Appropriate specimens from patients suspected of having typhoid fever should be cultured for the presence of salmonellae instead.

Whole cells of parasites, including Plasmodium, Leishmania, or Toxoplasma gondii, have also been used for direct detection of antibody by agglutination. In addition to using the actual infecting bacteria or parasites as the agglutinating particles for the detection of antibodies, certain bacteria may be agglutinated by antibodies produced against another infectious agent. Many patients infected with one of the rickettsiae produce antibodies that can nonspedfically agglutinate bacteria of the genus Proteus. The Weil-Felix test detects these cross-reacting antibodies. As newer, more specific serologic methods of diagnosing rickettsial disease become more widely available, however, the use of the Proteus agglutinating test is being discontinued

Particle Agglutination Tests

Numerous serologic procedures have been developed to detect antibody via the agglutination of an artificial carrier particle with antigen bound to its surface. As noted in Chapter 9, similar systems employing artificial carriers coated with antibodies are commonly used for detection of microbial antigens. Either artificial carriers, such as latex particles or treated red blood cells, or biologic carriers, such as whole bacterial cells, can carry an antigen on their surface that will bind with antibody produced in response to that antigen when it was introduced to the host. The size of the carrier enhances the visibility of the agglutination reaction, and the artificial nature of the system allows the antigen bound to the surface to be extremely specific.

Results of {article agglutination tests are dependent on several factors, including the amount and avidity of antigen conjugated to the carrier, the time of incubation together with the patient's serum (or other source of antibody), and the microenvironment of the interaction (including pH and protein concentration). Commercial tests have been developed as systems, complete with their own diluents, controls, and containers. For accurate results, they should be used as units without modifications. If tests have been developed for use with cerebrospinal fluid, for example, they should not be used with serum unless the package insert or the technical representative has certified such usage.

Treated animal red blood cells have also been used as carriers of antigen for agglutination tests; these tests are called indirect hemagglutination, or passive hemagglutination tests, because it is not the antigens of the blood cells themselves, but rather the passively attached antigens, that are being bound by antibody. The most widely used of these tests are the microhemagglutination test for antibody to T pallidum (MBA-TP, so called because it is performed in a microliter plate), the hemagglutination treponemal test for syphilis (HATTS), the passive hemagglutination tests for antibody to extracellular antigens of streptococci and the rubella indirect hemagglutination tests, all of which are available commercially. Certain reference laboratories, such as the Centers for Disease Control and Prevention (CDC), also perform indirect hemagglutination tests for antibodies to some Clostridia, Burkhol-deria pseudomallei, Bacillus anthracis, Corynebacterium diphtheriae, Leptospira, and the agents of several viral and parasitic diseases.

Complete systems for the use of latex or other particle agglutination tests are available commercially for the accurate and sensitive detection of antibody to cytomegalovirus, rubella virus, varicella-zoster virus, the heterophile antibody of infectious mononucleosis, teidioic acid antibodies of staphylococci, antistrepto-coccal antibodies, mycoplasma antibodies, and others. Latex tests for antibodies to Coccidioides, Sporothrix, Echinococcus, and Trichinella are available, although they are not widely used because of the uncommon occurrence of the corresponding infection or its limited geographic distribution. Use of tests for Candida antibodies has not yet shown results reliable enough for accurate diagnosis of disease.

Flocculation Tests

In contrast to the aggregates formed when particulate antigens bind to specific antibody, the interaction of soluble antigen with antibody may result in the formation of a precipitate, a concentration of fine particles, usually visible only because the precipitated product is forced to remain in a defined space within a matrix. Two variations of the precipitin test, flocculation and counterimmunoelectrophoresis, are widely used for serologic studies.

In flocculation tests the precipitin end product forms macroscopically or microscopically visible clumps. The Venereal Disease Research Laboratory test, known as the VDRL, is the most widely used flocculation test. Patients infected with pathogenic treponemes, most commonly T, pallidum, the agent of syphilis, form an antibody-like protein called reagin that binds to the test antigen, cardiolipin-Iecithin-coated cholesterol particles, causing die particles to flocculate. Because reagin is not a specific antihody directed against T. pallidum antigens, the test is not highly specific but it is a good screening test, detecting more than 99% of cases of secondary syphilis.

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Widal Test Slide Method

Figure 10-4 MACRO-VUE RPR card test. R, Reactive (positive) test; N, nonreactive (negative) test. (Courtesy Becton Dickinson Diagnostic Systems, Sparks, Md.)

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Figure 10-4 MACRO-VUE RPR card test. R, Reactive (positive) test; N, nonreactive (negative) test. (Courtesy Becton Dickinson Diagnostic Systems, Sparks, Md.)

The VDRL is the single most useful test available for testing cerebrospinal fluid in cases of suspected neurosyphilis, although it may be falsely positive in the absence of this disease. The performance of the VDRL test requires scrupulously clean glassware and exacting attention to detail, including numerous daily quality control checks. In addition, the reagents must be prepared fresh each time the test is performed, patients' sera must be inactivated by heating for 30 minutes at 56° C before testing, and the reaction must be read microscopically. For all these reasons, it has been supplanted in many laboratories by a qualitatively comparable test, the rapid plasma reagin, or RPR, test.

The RPR test is commercially available as a complete system containing positive and negative controls, the reaction card, and the prepared antigen suspension. Hie antigen, cardiofipin-ledthin-coated cholesterol with choline chloride, also contains charcoal partides to allow for macroscopically visible flocculation. Sera are tested without heating, and the reaction takes place on the surface of a specially treated cardboard card, which is then discarded (Figure 10-4). The RPR test is not recommended for testing cerebrospinal fluid. All procedures are standardized and dearly described in product inserts, and these procedures should be adhered to strictly. Overall, the RPR appears to be a more specific screening test for syphilis than the VDRL, and it is certainly easier to perform. Several modifications have been made, such as the use of dyes to enhance visualization of results.

Conditions and infections other than syphilis can cause a patient's serum to yidd a positive result in the VDRL or RPR tests; these are called biologic false-positive tests. Autoimmune diseases, such as systemic iupus erythematosus and rheumatic fever; infectious mononudeosis; hepatitis; pregnancy; and old age have caused false-positive tests, so results of screening tests should be considered presumptive until confirmed with a spedfic treponemal test.


Another variation of the classic predpitin test has been widely used to detect small amounts of antibody. This test takes advantage of the net electric charge of the antigens and antibodies being tested in a particular test buffer. Because the antigen and antibody being sought migrate toward each other in a semisolid matrix under the influence of an electrical current, the method is called counterimmunoelectrophoresis (CIE). The prindple of this test is outlined in Chapter 9; the same methodology is used to identify spedfic antigen or antibody. When antigen and antibody meet in optimal proportions, a line of precipitation appears. Because all variables, such as buffer pH, type of gel or agarose matrix, amount of electrical current, amount and concentration of antigen and antibody, size of antigen and antibody inocula, and placement of these inocula, must be carefully controlled for maximum reactivity, CIE tests are difficult to develop and perform. Other methods for detection of antibody to infectious agents are more commonly used in most diagnostic laboratories.

Immunodiffusion Assays

Closdy resembling the predpitin test is the Ouchterlony double immunodiffusion assay, which is widely used for detecting antibodies directed against fungal cell components. This test is described in Chapter 9. Whole-cell extracts or other antigens of the suspected fungus are placed in wells in an agarose plate, and the patient's serum and a control positive serum are placed in adjoining wells. If the patient has produced specific antibody against the fungus, predpitin lines will be visible within the agarose between the homologous (identical) antigen and antibody wells; their identity with similar lines from the control serum hdps confirm the results. The type and thickness of the predpitin bands may have prognostic, as well as diagnostic, value. Antibodies against the pathogenic fungi Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, and some opportunistic fungi are routinely detected by immunodiffusion. The test usually requires at least 48 hours, but additional time may be required to develop the bands.

Hemagglutination Inhibition Assays

Many human viruses can bind to surface structures on red blood cells from different spedes. For example, rubella virus particles can bind to human type O, goose, or chicken erythrocytes and cause agglutination of the red blood cells. Influenza and parainfluenza viruses agglutinate guinea pig, chicken, or human 0 erythrocytes; many arboviruses agglutinate goose red blood cells; adenoviruses agglutinate rat or rhesus monkey cells; mumps virus binds red blood cells of monkeys; and herpesvirus and cytomegalovirus agglutinate sheep red blood cells. Serologic tests for the presence of antibodies to these viruses exploit the agglutinating properties of the virus particles. Patients' sera that have been treated with kaolin or heparin-magnesium chloride (to remove nonspecific inhibitors of red cell agglutination and nonspecific agglutinins of the red cells) are added to a system that contains the virus suspected of causing disease. If antibodies to the virus are present, they will form complexes and block the binding sites on the viral surfaces. When the proper red cells are added to the solution, all of the virus particles will be bound by antibody, preventing the virus from agglutinating the red cells. Thus, the patient's serum is positive for hemagghitination-inbibiting antibodies. As for most serologic procedures, a fourfold increase in Such titers is considered diagnostic. The hemagglutination inhibition tests for most agents are performed only at reference laboratories. Rubella antibodies, however, are often detected with this method in routine diagnostic laboratories. Several commercial rubella hemagglutination inhibition test systems are available.

Neutralization Assays

Antibody that inhibits the infectivity of a virus by blocking its host cell receptor site is called a neutralizing antibody. The serum to be tested is mixed with a suspension of infectious viral particles of the same type as those with which the patient is suspected of being infected. A control suspension of viruses is mixed with normal serum. The viral suspendons are then inoculated into a cell culture system that supports growth of the virus. The control cells will display evidence of viral infection. If the patient's serum contains antibody to the virus, that antibody will bind the viral particles and prevent them from invading the cells in culture. The antibody has neutralized the "infectivity'' of the virus. These tests are technically demanding and time-consuming and are performed only in reference laboratories.

Antibodies to bacterial toxins and other extracellular products that display measurable activities can be tested in the same way. The ability of a patient's serum to neutralize the erythrocyte-lysing capability of streptolysin 0, an extracellular enzyme produced by S. pyogenes during infection, has been used for many years as a test for previous streptococcal infection. After pharyngitis with streptolysin O-producing strains, most patients show a high titer of the antibody to streptolysin 0, that is, antistreptolysin 0 (ASO) antibody Streptococci also produce the enzyme deoxyribonuclease B

(DNase B) during infections of the throat, skin, or other tissue. A neutralization test that prevents activity of this enzyme, the anti-DNase B test, has also been used extensively as an indicator of recent or previous streptococcal disease. However, the use of particle agglutination (latex or indirect hemagglutination) tests for the presence of antibody to many of the streptococcal enzymes has replaced the use of these neutralization tests in many laboratories.

Complement Fixation Assays One of the classic methods for demonstrating the presence of antibody in a patient's serum has been the complement fixation test (CF). This test consists of two separate systems. The first (the test system) consists of the antigen suspected of causing the patient's disease and the patient's serum. The second (the indicator system) consists of a combination of sheep red blood cells, complement-fixing antibody (IgG) raised against the sheep red blood cells in another animal, and an exogenous source of complement (usually guinea pig serum). When these three components are mixed together in optimum concentrations, the antisheep erythrocyte antibody will bind to the surface of the red blood cells and the complement will then bind to the antigen-antibody complex, ultimately causing lysis (bursting) of the red blood cells. For this reason the antisheep red blood cell antibody is also called hemolysin. For the CF test these two systems are tested in sequence (Figure 10-5). The patient's serum is first added to the putative antigen; then the limiting amount of complement is added to the solution. If the patient's serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement added. In the next step the sheep red blood cells and the hemolysin (indicator system) are added. Only if the complement has not been bound by a complex formed with antibody from the patient's serum will the complement be available to bind to the sheep oell-hemolysin complexes and cause lysis. A positive result, meaning the patient does possess complement-fixing antibodies, is revealed by failure of the red blood cells to lyse in the final test system. Lysis of the indicator cells indicates lack of antibody and a negative CF test.

Although requiring many manipulations, at least 48 hours for both stages of the test to be completed, and often yielding nonspecific results, this test has been used over the years to detect many types of antibodies, particularly antiviral and antifungal. Many new systems provide for improved recovery of pathogens or their products and for more sensitive and less demanding procedures for detection of antibodies, including particle agglutination, indirect fluorescent antibody tests, and enzyme-linked immunosorbent assay (EUSA) procedures, and have gradually been introduced to replace

Chapter 10 Serologic Diagnosis of Infectious Diseases 167


Hemolysin Complement

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