Vol. 3, No. 2

CLINICAL MICROBIOLOGY REVIEWS, Apr. 1990, p. 132-152 0893-8512/90/020132-21$02.00/0 Copyright © 1990, American Society for Microbiology

Immunoserology of Infectious Diseases KAREN JAMES

Central DuPage Hospital, Winfield, Illinois 60190,t and Loyola University Medical Center, Maywood, Illinois 60153 IMMUNE RESPONSE TO MICROORGANISMS ........................................................... 133 133 Immunity to Bacteria ........................................................... 133 Immunity to Viruses ........................................................... 133 Immunity to Fungi ........................................................... TEST SYSTEMS FOR IMMUNODIAGNOSIS OF INFECTIOUS DISEASES ................................... 133 133 Nonspecific Indicators of Infectious Diseases ........................................................... CRP ............................................................ 133 Endotoxin ........................................................... 134 134 TNF ........................................................... 134 Antibody Production and Purification ........................................................... 134 Polyclonal antibodies ........................................................... 135 Affinity-purified antibodies ........................................................... Fractionated antibodies ........................................................... 135 135 MAbs ........................................................... 135 Soluble Antigen-Antibody Reactions ........................................................... 135 Double diffusion in agar (Ouchterlony reactions) ........................................................... CIE ........................................................... 135 135 Particulate Antigen-Antibody Reactions ........................................................... 135 Hemagglutination assays ........................................................... 136 HI assays ........................................................... Latex agglutination (LA) ............................................................ 136 136 Coagglutination ........................................................... 136 Lytic Assays ........................................................... 136 CF ........................................................... Neutralization assays ........................................................... 136 Immunohistochemical Techniques ........................................................... 136 Direct IFAs ........................................................... 137 Indirect IFAs for total antibody ........................................................... 137 Indirect IFAs for IgM antibody ........................................................... 138 False-positive and false-negative IFAs for IgM antibody .......................................................... 138 138 Amplification IFAs ........................................................... 139 Immunoassay Techniques ............................................................ 139 Rapid EIAs to detect bacterial antigens ........................................................... 139 Solid-phase methods for detection of antibodies ............................................................ 140 IgM and IgG separation methods ........................................................... 140 Capture assays ........................................................... SELECTION OF METHODS FOR CLINICAL LABORATORY USE ............................................. 141 Detection of Antibody to Verify Immunity ........................................................... 141

Preemployment screening ........................................................... Prenatal screening ........................................................... Pretransplant screening ........................................................... Detection of Antibody to Diagnose Disease ........................................................... Acute and convalescent specimens ........................................................... IgM-specific assays ........................................................... Method comparisons ........................................................... Congenital infections ........................................................... Transplantation and immunosuppression ........................................................... Serologic tests for syphilis ............................................................ Streptococcal antibodies ........................................................... EBV antibodies ...........................................................

Legionella antibodies ...........................................................

Rickettsia antibodies ............................................................ Lyme disease serology ........................................................... Antibodies to other microorganisms ............................................................ t Address for corrrespondence. 132

141 141

143 143 143 143

144 144 144

145 145 145

146 146 146

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Other viral antibodies ............................................. 147 Detection of Soluble Antigen Correlates with Active Disease ............................................. 148 Neonatal bacterial infections .............................................148 Neonatal viral infections ............................................. 148 Group A streptococcal infections in children .............................................148 Other microbial antigens ............................................. 148 Other viruses ............................................. 149 CONCLUSIONS ............................................. 149 ACKNOWLEDGMENTS ............................................. 149 LITERATURE CITED ............................................. 149 IMMUNE RESPONSE TO MICROORGANISMS

Immunity to Bacteria The immune response to extracellular bacteria must counteract all of the mechanisms of invasion elicited by these organisms (22, 26). The immune response includes antibodies to capsular polysaccharides, to exotoxins (e.g., antistreptolysin 0 [ASO]), and to extracellular enzymes (antihyaluronidase). Antibodies to tetanus toxin or diphtheria toxin can neutralize the effects of these toxins and prevent host tissue destruction (16). Complement activation promotes effective opsonization with and without antibody (67). The membrane attack complex of the terminal complement components are required to lyse and eliminate certain gram-negative organisms (Neisseria spp.). For other gram-negative bacteria, a synergistic destruction by complement in conjunction with lysozyme is necessary (21). Complement activation is also necessary to release chemotactic factors to attract phagocytic cells to the site of infection. Endotoxins elicited from certain gram-negative bacteria can initiate the activation of the complement alternative pathway in the absence of antibody. Endotoxin can also degranulate neutrophils, enhance cytotoxicity, and prompt a variety of other severe metabolic and potentially lethal effects if gram-negative bacterial infections are not efficiently treated (8). Immunity to intracellular pathogens is primarily cellular immunity, i.e., delayed T-cell hypersensitivity involving lymphocytes, cytokines, and macrophages (48). There are only two methods available to detect delayed T-cell hypersensitivity: in vivo cutaneous injection of purified antigens (skin or anergy testing) or in vitro lymphocyte transformation studies with purified antigens. Neither method is highly reproducible and may be falsely negative due to the immunosuppression experienced by individuals suffering from invasion by intracellular pathogens. In many situations, antibody is produced, but serves no demonstrable protective mechanism. If antibody production is stimulated by the intracellular pathogen, detection of that antibody and its class specificity can be useful in diagnosing the invading organism(s). Immunity to Viruses Antibody (immunoglobulin G [IgG] and IgM) capable of binding directly to extracellular viruses may prevent viruses from infecting other cells. If the virus has a viremic phase, neutralizing antibodies may be produced. Two types of neutralizing antibodies that can be demonstrated are complement independent and complement facilitated. Antibodies of the G, M, and A classes have been shown to neutralize the infectivity of virtually all known viruses (21). Intercellular or vertically transmitted viruses or both would not be subject to the neutralizing effects of antibodies.

Antibody can also diminish infectivity of viruses by preventing attachment to the specific receptor or by introducing conformational changes in the viral structure that promote aggregation. Aggregation facilitates more effective elimination by antibody-mediated mechanisms such as opsonization or complement activation or both. Arboviruses and hepatitis B virus are examples of viruses that can be eliminated by antibody-mediated events during their release into the bloodstream or lymphatics. In certain situations, antibody to viral proteins can be detrimental to the host. For example, serum antibody to respiratory syncytial virus (RSV), which is not protective but was passively acquired across the placenta from the mother, may produce an Arthus (immune complex) type of hypersensitivity reaction in the lungs of infants (20). Similar damaging effects of antiviral antibody have been described with measles infections in infants. The immune response to intercellularly or vertically transmitted viruses involves cell-mediated cytotoxicity. Cytotoxic effector cells recognize the alterations to the membrane antigens that are perturbed by viruses and either require specific (T-cell) or nonspecific (natural killer cells or macrophages) cytolytic effector cells (88). Antibody-dependent cell-mediated cytotoxicity has also been shown to be an effector mechanism of antiviral cytotoxicity (85). Immunity to Fungi Immunity to fungi is primarily cell mediated. Detection of specific IgM and IgG antibodies to certain fungi by immunoprecitin reactions can be helpful in establishing the diagnosis and following the course of the disease. However, the antibodies do not play a protective role. TEST SYSTEMS FOR IMMUNODIAGNOSIS OF INFECTIOUS DISEASES

Nonspecific Indicators of Infectious Diseases The local response to infection or tissue injury or both is acute inflammation, resulting in vascular changes and attracting leukocytes. During the first few days following insult, systemic and metabolic changes occur which comprise the acute-phase response (57). Acute-phase proteins include those that usually increase by 50% (complement components and ferritin), alpha-1-antitrypsin, fibrinogen, and haptoglobin which increase 200 to 400%, and C-reactive protein (CRP) which can increase up to 1,000% in severe tissue injury. CRP and complement components are the only acute-phase proteins that have been shown to be directly involved in the elimination of microorganisms. CRP. CRP is the prototype acute-phase protein. CRP was originally recognized for its ability to precipitate with the C-polysaccharide fraction of pneumococcus (95). CRP did

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50

(including the production of TNF) referred to as septic or shock (65). Endotoxins are phagocytized and gram-negative 300 detoxified by the liver; consequently, the concentration of 40 detectable LPS is high in portal blood, but often not detectable in the peripheral blood circulation (94). The Limulus lysate assay is currently the only available ~~~~ 200 ~~ ~ ~ ~ ~ ~ 2 method for detecting trace quantities of endotoxin. LPS 250 7 causes gelation of an extract from the lysate of Limulus polyphemus, the horseshoe crab (88). The Limulus assay is O ~~~~~~~~~~~~~~~~~~O not specific for a particular microorganism, but detects LPS from all gram-negative bacteria including Eschericia coli, Neisseria meningitidis, and Haemophilus influenzae. Since endotoxin is so rapidly cleared from peripheral blood, deDays post antigen stimdation tecting LPS in serum is unreliable, but detecting endotoxin ----- IgM in cerebrospinal fluid (CSF) is a sensitive indicator of the ECRP ESR IgG of gram-negative bacterial meningitis (88). presence FIG. 1. Nonspecific and specific immune responses in relation to TNF. TNF is a cytotoxin which participates in the immune time after antigenic stimulation. ESR, Erythrocyte sedimentation response to microorganisms as well as effects the antitumor rate. activity for which it was named. TNF was first characterized as an activity which appeared in murine serum after injection with Mycobacterium bovis BCG (bacillus Calmette-Gudrin) not appear to be an antibody to pneumococcus since its level and LPS. When sera containing TNF were injected into decreased when patients recovered from the pneumonia. tumor-bearing mice, necrosis of the tumor was induced and CRP was present in sera from patients with other bacterial the tumor regressed (78). TNF can induce interleukin-1 illnesses, but not detectable in normal human sera. production and can induce a factor that is directly cytotoxic CRP binds to phosphocholine and galactose residues, to malaria and other parasites (8). ligands which are widely distributed among microbial prodCachectin was first described when investigators were ucts including fungi, parasites, lactobacilli, and streptococci searching for the mediators responsible for cachexia (wast(67). When CRP binds to these surfaces, it activates coming) associated with parasitic infections (8). Subsequently, plement in much the same manner as antigen-antibody cachectin and TNF were shown to have strong DNA seactivation of Clq (10). When CRP and C3 are bound to an quence homologies and are now considered to be identical organism, phagocytosis is promoted by this opsonization cytotoxins. (23). Human CRP provides protection in vivo from a lethal When recombinant TNF is administered to mice, several dose of Streptococcus pneumoniae in mice (99). CRP is distinctive among human acute-phase proteins pathologic events occur, including severe metabolic acidobecause it is usually present in nanogram-per-milliliter consis, marked hemoconcentration, biphasic changes in blood glucose concentrations, severe pulmonary leukostasis and centrations, but can increase dramatically and rapidly to hundreds of micrograms per milliliter within 3 days (9, 28). edema, hemorrhagic necrosis of adrenals and pancreas, and The highest CRP levels are found in patients with bacterial tubular necrosis of the kidney (8). When TNF was neutralinfections (>100 ,ug/ml), while moderate CRP elevations (10 ized with (actively or passively acquired) antibodies, these to 100 ,ug/ml) are commonly found in chronic inflammatory pathologic changes were prevented. conditions such as autoimmune diseases, malignancies, alRecently, methods have become available to measure coholic hepatitis, congestive heart failure, and pregnancy TNF levels in human sera. Waage et al. (96) noted a (68). When elevated CRP levels are found in patients with correlation between TNF levels and the degree of septic chronic inflammation, superimposed bacterial infections shock and subsequent death in a series of patients with have been confirmed (68). meningococcal septicemia. Although TNF is a nonspecifiCRP elevates more rapidly and decreases sooner with cally induced cytotoxin, detection of TNF may provide an resolution of the infectious process than does the erythocyte indicator of the extent of damage or the nature of the sedimentation rate, the classic nonspecific indicator of ininfectious process or both. flammation (Fig. 1). Measurement of spinal fluid CRP levels has been shown to be sensitive and specific for differentiAntibody Production and Purification ating bacterial from viral meningitis, enabling efficient therapeutic intervention (13, 72). Serum CRP levels have been Polyclonal antibodies. Antisera used in many of the assay used to differentiate patients with bacteremia from those systems to be described are prepared by hyperimmunizing with contaminated blood cultures (63), monitor treatment of animals (rabbits or goats) with purified antigens emulsified in infective endocarditis (64), monitor spinal cord-injured paadjuvants. After 6 to 8 weeks of injections, the animals are tients for earlier detection of urinary tract infections (59), bled and serum samples are removed and tested to ensure differentiate pyelonephritis from cystitis in children (45) and the monospecificity of the resulting antisera for the antigen adults (68), differentiate bacterial pneumonia from acute injected. If contaminating antibodies or antibodies with bronchitis (68), and monitor for postoperative infections (28) other specificities are present, they are removed by absorpand for other noninfectious disease applications (68, 73). tion with their corresponding antigens. The immunoglobulin Endotoxin. Endotoxins are the lipopolysaccharide (LPS) fraction of the animal sera is isolated by selective salt components of the outer membrane of gram-negative bacteprecipitation (NH4SO4) or by ion-exchange chromatograria. When released in vivo, endotoxin has toxic and pyrophy. Even a high-titered antibody represents only 10 to 15% genic properties (65), including release of interleukin-1 and of the total IgG fraction of a polyclonal antibody. The tumor necrosis factor (TNF). Administration of endotoxin remaining IgG molecules are of undefined specificity, recan prompt severe metabolic and physiologic disturbances flecting the myriad of antigens to which the animal has been

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exposed during its lifetime. It is necessary to characterize painstakingly each polyclonal antibody to ensure that it is detecting only the desired antigen. Still, the high percentage (85 to 90%) of undesired antibodies increases the occurrence of nonspecific binding of conjugated (but irrelevant) antibodies in test systems. Affinity-purified antibodies. To eliminate the antibodies of undesired specificity, the IgG fraction of a polyclonal antibody can be purified by binding to and eluting from an insolubilized form of the antigen. This is performed by column chromatography, using a matrix to which the immunizing antigen is coupled with a spacer molecule (an inert molecule that serves as a carrier). The spacer is covalently coupled to the antigen and to the insoluble matrix to preserve optimally the relevant antigenic determinants. The antibody is then applied to the column, using conditions favorable for antigen-antibody reactions to occur (pH 7.5 to 8.5, physiologic ionic strength). IgG molecules of unrelated specificity would pass through the column with the wash buffer. Antigen-specific IgG molecules are then eluted off the antigen matrix, using an elution buffer with a lower pH and ionic strength. In this way, the functional integrity of the IgG molecules is preserved, while the antibodies are gently dissociated from the insolubilized antigen. The resulting affinity-purified antibody is significantly higher (85 to 95%) in specific antibody activity. This process decreases or entirely eliminates any nonspecific binding due to extraneous antibodies, resulting in a much better reagent for use with immunohistochemical assays. Polyclonal antibodies that have been affinity purified have many uses in the clinical laboratory. Most antibodies to human immunoglobulins, conjugated with fluorescent or enzyme labels, are affinity-purified polyclonal antibodies. Polyclonal antibodies bind to several antigenic determinants, increasing the ability to detect their respective antigens. Fractionated antibodies. Antibodies in antisera used to detect antigens in cultured cell lines (especially herpesviruses) bind nonspecifically to the IgG Fc receptors of the cells. One effective approach to decrease that nonspecific binding significantly is to fractionate the IgG antisera to eliminate the Fc regions of the IgG antibody molecules. Under controlled conditions, the enzyme pepsin sequentially cleaves the C-terminal end of the IgG molecule (the Fc region), leaving the antibody-combining N terminals [the F(ab')2 region]. The F(ab')2 fragments can still be effectively conjugated, e.g., with fluorochromes or enzymes. Affinitypurified antibodies can be pepsin digested, resulting in affinity-purified F(ab')2 antisera. MAbs. The production of monoclonal antibodies (MAbs) was first described in 1975 by Kohler and Milstein (53). The potential applications for clinical laboratory assays and in immunotherapy were immediately recognized. MAbs are prepared by hybridizing antibody-forming cells to continuously replicating cell lines. Each antibody-forming cell, programmed to produce antibody of a single (mono-) specificity with a single heavy-chain and single light-chain class, can be cloned to replicate itself almost indefinitely. These antibody-producing clones of cells (hybridomas) are used to prepare virtually unlimited quantities of MAbs that are chemically, physically, and immunologically completely homogeneous and definitively characterizable (93). Hybridoma cells can be stored indefinitely, frozen in liquid nitrogen. As needed, these hybridomas can be grown in large quantities in tissue culture or propagated in syngeneic mice to form ascites from which MAbs can be isolated. The advantages of MAbs are obvious: the minimal purifi-

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cation required ensures optimal functional activity; characterization of the specificity needs to be done only once instead of each time an animal is immunized; MAbs generally do not bind to human IgG Fc receptors; and they generally are unencumbered by nonspecificity or crossreactivity (93). The disadvantages of MAbs are more subtle: a single specificity does not enable effective cross-linking of antigens with antibodies so one MAb cannot be used in agglutination or precipitation reactions, although "cocktails" of several MAbs can overcome that limitation; murine MAbs do not efficiently bind Clq to activate complement so are not useful in assays that rely on complement activation; the single antigenic determinant to which a MAb binds may not be expressed under certain conditions of antigen presentation (e.g., viable versus fixed organisms). Nevertheless, an entirely new field of study is available to use these reagents effectively for clinically applicable testing systems. Soluble Antigen-Antibody Reactions Double diffusion in agar (Ouchterlony reactions). The classic method for detecting antibody and evaluating its specificity (identity, partial identity, or nonidentity) is Ouchterlony double diffusion. Antigens can also be characterized by Ouchterlony diffusion when antibodies of known specificity are available. Since the procedure requires 18 to 24 h of diffusion for the reactions to occur, this method is not as helpful in the rapid diagnosis of acute infections as other methods discussed below. The Ouchterlony reaction is primarily used to detect antibodies in patients with suspected histoplasmosis, coccidiomycosis, or aspergillosis and to detect other fungal antigens associated with hypersensitivity pneumonitis. CIE. Counterimmunoelectrophoresis (CIE), one-dimensional double electrophoresis, specifically directs the movement of antigen and antibody toward each other in an electric field. The buffer pH is selected to optimize the electroendosmotic effects of antibody toward the cathode (negative pole) while the antigen moves toward the anode (positive pole). This electrophic movement rapidly (30 min) concentrates the antigen and antibody in the zone between the adjacent wells. CIE is approximately 10 times more sensitive than double diffusion. This was the original method used to detect hepatitis B surface antigen and antibody known then as Australian antigen and antibody. CIE has also been useful for rapid identification of antigens from bacteria associated with meningitis, septicemia, disseminated intravascular coagulation, pneumonia, and septic arthritis (88). Depending on the sensitivity and specificity of the antibody used, minimal detectable concentrations of bacterial antigen range from 50 to 10 ng/ml (30). CIE has largely been replaced with particulate antigen-antibody reactions for detecting many bacterial antigens. Particulate Antigen-Antibody Reactions Hemagglutination assays. A variety of antigens can be coupled to erythrocytes (RBCs) to provide the indicator system to detect antibodies. Target antigens such as polysaccharides readily adhere to RBCs, including antigens from E. coli, N. meningitidis, and Toxoplasma sp. as well as purified protein derivative from M. tuberculosis. Carbohydrate antigens readily adhere to RBCs, but protein antigens require pretreatment with tannic acid (producing "tanned" RBCs) or with chromium chloride. Tanning the RBCs facilitates a high-density coating which increases the sensitivity of the test system. Subsequent Formalin or glutaraldehyde treatment of tanned RBCs coated with either protein or carbo-

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hydrate allows long-term storage. Treated tanned RBCs coated with protein antigens have been used for detection of antibodies to toxins, e.g., diphtheria toxin or tetanus toxin (16). Treponemal antibodies are detected by using treponemal antigens adsorbed to tanned RBCs in the micro-hemagglutination assay for Treponema pallidum (MHA-TP) (98). HI assays. Hemagglutination inhibition (HI) assays used in infectious disease serology are based on the capacity of certain viral antigens to agglutinate RBCs of selected species spontaneously. Antibodies present in patient sera prevent the spontaneous agglutination of the RBCs, thus resulting in inhibition of agglutination, indicating a positive test for the presence of antibody. HI was the original method used to detect antibodies to rubella virus (76). HI tests cannot distinguish between IgM and IgG classes of antibodies. Immunoassay techniques which have replaced HI for detection of rubella virus antibodies will be reviewed below. Antibodies to other less prevalent hemagglutinating viruses are still detected by the HI method. These include influenza viruses, arboviruses, reoviruses, and certain enteroviruses (49). Latex agglutination (LA). Latex particles are spheres of polystyrene which readily bind IgG molecules by the Fc region at pH 9.0 when a low-ionic-strength buffer is used. When antibodies are bound by their Fc region, the antibodycombining sites [F(ab) regions] remain exposed and are capable of binding antigens. When the target antigens have repetitive antigenic structures (e.g., polysaccharides), multivalent antibodies coupled to multiple latex particles can bind antigen molecules and cross-link the latex particles, resulting in agglutination. The latex particles serve as the indicator system to detect the antigen-antibody reaction. Theoretically, any antigen (but not hapten) to which antibodies can be produced should be detectable by LA. LA is particularly useful to detect bacterial polysaccharide antigens in CSF or urine or both. LA has essentially replaced CIE for the detection in CSF of capsular antigens of H. influenzae type b, several N. meningiditis groups, and S. pneumoniae for diagnosing bacterial meningitis and for detection of Cryptococcus neoformans capsular antigens in immunosuppressed patients. Group B streptococcal antigens are detected by LA in urine or CSF from newborns.

Coagglutination. Staphylococcus

aureus

(Cowan strain)

contains protein A distributed evenly on the outermost layer of the cell wall. Protein A binds the Fc region of IgG subclasses 1, 2, and 4 (which constitute 95% of the total IgG), analogous to the binding of IgG to latex particles. The

antibody-coated Staphylococcus becomes the indicator reagent to detect the presence of antigens corresponding to the specificity of the coupled antibody. Coagglutination has been used to detect the presence of bacterial antigens in CSF or urine. Coagglutination is useful in the immunologic identification of bacteria from culture, with commercially available reagents for grouping streptococci and detecting Staphylococcus aureus coagulase production (30). Since protein A is an effective cross-linking reagent for most IgG subclasses, any other preformed complexes containing IgG and antigen would also cause agglutination of the sensitized Staphylococcus reagent. Nonspecific agglutination is prevented by treating the body fluid to be tested with soluble protein A to block binding of preformed complexes or by heating the body fluid to 100'C to denature patient IgG molecules before testing. Coagglutination must be well controlled to detect nonspecific agglutination in the body fluid, using unsensitized Staphylococcus particles and particles sensitized with an

irrelevant, but species-specific antibody. Coagglutination reagents have a much shorter shelf life than latex reagents due to deterioration of the bacteria upon storage. Many of the clinical laboratory applications of coagglutination have been essentially replaced by LA or immunoassay techniques.

Lytic Assays CF. Complement fixation (CF) is a two-step procedure which uses complement to lyse indicator RBCs. The first step involves reacting antigen with antibody in the presence of complement in fluid phase. The complement used must be from a standard source, e.g., rabbit serum which has been appropriately collected and stored to preserve the hemolytic activity. If corresponding antigens and antibodies are present, the complement cascade will be activated through the classical pathway. RBCs coated with RBC antibody (the indicator system) are added to the first reaction mixture. If complement had been activated (fixed) during the first incubation, the indicator particles are not lysed. If the first step did not contain either the specific antigen or antibody, complement will bind to the antigen-antibody complex present on the indicator particles, and lysis of the indicator RBCs will occur. CF is a semiquantitative method that can be used to detect either antigens or antibodies if the corresponding specific antibody or antigen is available. CF does not distinguish IgM from IgG antibodies since both can fix complement. This method is extremely sensitive and has broad applications for infectious disease serology, but is rarely used outside of reference laboratory situations because it is cumbersome and complex (93). CF may be the only method available for detecting antibody to less prevalent viruses (e.g., coxsackieviruses). An advantage of the CF method is that, by keeping all other test parameters unchanged, many antigens can be tested to determine population exposure to rare organisms. Neutralization assays. Beta-hemolytic group A streptococci produce a number of extracellular toxins that stimulate the production of antibodies by infected patients (54). Several of these toxins also function as hemolysins and lyse RBCs. Specific antibodies neutralize the hemolysins and inhibit RBC lysis. Analogous to the CF test, a positive test is negative for hemolysis, and in a negative test hemolysis is detectable. In the first step of the reaction, patient serum (antibody) is incubated with the specific hemolysin being assayed (antigen). In the second step, group 0 RBCs are added. If the hemolysin has been neutralized during the first incubation, the indicator particles will not lyse. If specific antibody was not present to neutralize the hemolysin, the RBCs will lyse. ASO is the most commonly used neutralization test to detect immunologic evidence of exposure to streptococci. Immunohistochemical Techniques The most widely used immunohistochemical techniques are immunofluorescence assays (IFAs). IFA uses tissue or bacterial cells as the substrate (source of antigen) affixed to a glass slide and a fluorochrome-conjugated (antibody) detection system. IFA remains the "gold standard" for many infectious disease serology test systems. The advantages of IFA are that (i) the substrate can be visualized, ensuring specificity of the reaction; (ii) it is significantly less cumbersome than CF; (iii) it is highly reproducible when performed

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DOUBLE INDIRECT IFA

INDIRECT IFA labeled rabbit

anti-goat Ig

Labeled goat antibody

2Goat antibody

Patient antibody

Patient antibody

Antigens .*.,.D....

ANTI-COMPLEMENT IFA

AVIDIN-BIOTIN COMPLEX

C MATRIX ~ D ~ MATRIX: FIG. 2. Amplification immunoassay systems. (A) Indirect IFA (shown for comparison) uses 'labeled goat anti-human immunoglobulin as the indicator molecule to detect bound patient antibody. (B) Double-indirect IFA uses 'labeled rabbit anti-goat immunoglobubin to detect unlabeled goat anti-human immunoglobulin which has bound to patient antibody. Fewer patient antibody molecules can be detected with amplification techniques, thereby increasing sensitivity. (C) Anti-complement IFA uses 'labeled F(ab')2 goat anti-human immunoglobulin as the indicator molecule to detect guinea pig 'complement bound to patient antibody. (D) Avidin-biotin complex reactions use 6biotinylated goat anti-human immunoglobulin to bind to patient antibody. The indicator system is the 'labeled avidin which binds to the biotin.

by well-trained technologists; and (iv) in indirect IFA, the same conjugate and dilution of patient sera can be used to detect antibody to many different organisms or antigens. The disadvantages of IFA are that it: (i) requires fresh or frozen tissue or cells (processed tissue or smears fixed for Grams stain are not usable); (ii) requires special equipment and conditions (a fluorescence microscope and a dark room for reading); (iii) is labor intensive, even when the substrate for indirect IFAs can be purchased (reagent dilution, multiple incubation and wash steps, and cover slips are required); (iv) is subjective, requiring extensive training to read the reactions and multiple controls to ensure test specificity and sensitivity; and (v) has not been successfully automated. Immunohistochemical assays have been developed that use enzyme-conjugated antibodies (e.g., horseradish peroxidase) with correponding substrates that yield different colors when hydrolyzed to contrast with the colors of typical histochemical strains. Immunoperoxidase techniques may avoid the first two disadvantages of IFA, but the subjective interpretation and labor intensiveness of immunoperoxidase methods are even greater than with IFA. The systems described below for direct and indirect IFA also apply to immunoperoxidase techniques which have generally not been used for microbiology applications. Direct IFAs. Direct IFA is used to detect antigens or organisms present in cells or tissues, using fluorochromeconjugated antisera (conjugate) specific for the antigen(s) in question. Microbiologic applications of direct IFA include detection of Chlamydia trachomatis elementary bodies in columnar epithelial cells from the cervical canal, urethra, eye, or rectum (2). Direct IFA is useful not only to detect the

microorganism, but also to evaluate whether the correct and/or appropriate specimen was collected. Treponema pallidum can be detected by direct IFA during early stages of the disease when the organisms are concentrated in the chancre (primary) or in the mucocutaneous lesions (secondary). Direct IFA, using conjugate to T. pallidum which has been absorbed with Reiter treponemes, has essentially replaced dark-field examination as a diagnostic test for early stages of syphilis before reaginic antibodies are produced (4). Direct IFA has been used successfully for detection of Legionnella spp. (22) and for many viruses, including herpes simplex virus (HSV) types 1 and 2, cytomegalovirus (CMV), RSV, influenza virus types A and B, parainfluenza virus 1, 2, and 3, varicella-zoster virus (VZV), and adenovirus (D. Scholes, J. R. Daling, A. S. Stergachis, S. P. Wang, and J. T. Grayston, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1171, 1988). Enzyme-conjugated antibodies have also been used to detect these microorganisms in histologic tissue sections. Indirect IFAs for total antibody. Indirect IFA is used to detect antibodies in patient sera (Fig. 2A). Standardized antigens (organisms or virus-infected cell cultures) are fixed to glass slides. Patient serum is diluted, layered over the substrate, and incubated to allow the antigen-antibody complex to form. Unbound antibody is washed away, leaving only bound antibody, which is then incubated with the fluorescent conjugate. When antibodies are present in the patient's serum, a second antigen-antibody reaction will take place, with the conjugate becoming the third layer on the slide.

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FALSE POSITIVE IgM ASSAY

FALSE NEGATIVE IgM ASSAY Labeled Anti-IgM

Patient antibodies

AnBige

M

| B 10 t ; -; "if: 't' - . . -'. . '-"'.-' " .'-"'""-'...'.'..........

FIG. 3. Sources of error in specific IgM assays. (A) False-positive IgM assay due to patient RF binding to patient specific IgG. (B) False-negative (or decreased intensity resulting in a borderline result) due to specific IgG competing with specific IgM for antigen-binding sites.

For general testing, fluorescein-conjugated anti-human immunoglobulin is usually anti-IgG with reactivity to both kappa and lambda light chains. This light chain reactivity also detects antibodies of the IgA or IgM class or both, which would be advantageous to detect all classes of antibody reactive with microorganisms. The classic indirect IFA is the fluorescent treponemal antibody absorption (FTA-ABS) test. The antigen, T. pallidum Nichols, is fixed to glass slides. Prior to incubation with the antigen, the patient's serum is absorbed with the nonpathogenic T. pallidum Reiter. This absorption step significantly increases the specificity of the test (18); however, false-positive reactions can still occur in patients with autoimmune diseases (55) and hypergammaglobulinemia and in those with Lyme disease due to immunological cross-reactivity with antigenic sites on the spirochetes (60). Other commonly performed indirect IFAs include TORCH titers for evaluating the immune status of pregnant women. TORCH tests include toxoplasma (TO), rubella virus (R), CMV (C), and HSV (H). These organisms cause significant congenital infections resulting in stillbirths or a spectrum of congenital diseases, although infections in adults or children frequently are mild or even subclinical. Although once ordered as a panel of tests, current preferred practice is to perform only the specific test based on the mother's clinical and exposure history. Indirect IFAs for IgM antibody. If the test system is designed to detect antibodies produced during acute infection, the conjugate must be specific for IgM heavy chains with no light-chain or other heavy-chain reactivity. When congenital infections are suspected as the cause of stillbirth or abnormalities of a newborn, indirect IFAs for IgM antibody should be used to detect IgM antibodies produced by neonates. Unlike maternal IgG, IgM does not cross the placenta, so any specific IgM antibodies detected would have been produced by the fetus in response to a congenital infection. Healthy newborns have 5 to 15% of the adult level of total IgM since the in utero environment is essentially sterile. If an organism is transmitted to the fetus from the mother during gestation, the fetus will develop its own IgM response to the organism, which would be detectable in an IgM-specific indirect IFA. False-positive and false-negative IFAs for IgM antibody. Indirect IFAs for IgM antibody are subject to false-positive reactions attributable to the presence of rheumatoid factor (RF) activity in the serum being tested (Fig. 3A). RF binds to IgG when IgG is bound to antigen. The process of binding to an antigen causes a confirmational change in IgG which exposes new antigens on the Fc region of the IgG molecule.

The IgM RF binds to these newly exposed IgG determinants. Unless separation methods are used, IgM RF cannot be distinguished from organism-specific IgM. Distinguishing RF from organism-specific IgM is particularly important in neonatal sera since a high percentage of congenitally infected neonates have detectable RF (31). False-positive IgM assays due to heterotypic antibody responses between herpesviruses may also be detected; i.e., patients infected with Epstein-Barr virus (EBV) or VZV may demonstrate CMV IgM antibodies without evidence of CMV infection (56). IgM assays can also be subject to false-negative results if IgG antibody inhibits or competes with IgM for binding sites on the antigen (Fig. 3B). To avoid any possibility of falsepositive or false-negative reactions in most methods, IgG should be separated from IgM before the assays are performed. Separation methods will be discussed below. The demand for assays that detect IgM antibodies to CMV and HSV has recently increased due to infections in organ transplant patients. The immunosuppressed state of transplant patients increases their susceptibility to these opportunistic viruses since both humoral and cell-mediated immunity are required for optimal viral immunity. Although specific IgG antibody may be present, it may not be as effective in controlling viral infections in patients whose cell-mediated immunity has been abrogated to prevent transplant rejection. The production of IgM antibodies is T independent, i.e., does not require the presence or cooperation of T cells (77). IgM antibodies are produced in immunosuppressed patients and can be useful indicators of acute infection. Amplification IFAs. The sensitivity of IFAs is limited by the level of fluorescence detectable by the human eye. The optimal fluorescein/protein ratio of a polyclonal antibody conjugate is 2.5 (34), i.e., two to three fluorescein molecules for each immunoglobulin molecule, which is satisfactory for detecting high-density antigens such as bacterial or protozoan cell surface antigens. Directly conjugating antibodies at a higher fluorescein/protein ratio results in significantly increased nonspecific staining. Detection of low-density antigens or using MAbs or both may require a higher fluorescein/protein ratio to achieve desired sensitivity. Indirect or double-indirect IFA has been used to amplify the fluorescent signal and improve sensitivity. If the antigen can be detected by direct IFA, indirect IFA increases the test sensitivity. If the test antibody is difficult to detect by indirect IFA, a double-indirect IFA would increase the sensitivity of detection (Fig. 2B). For example, if organisms were not visible in the T. pallidum direct IFA, the test could be repeated with unconjugated rabbit antibody to the

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treponemes, any unbound antibody could be washed off, and the bound rabbit antibody could be detected by using a fluorochrome-conjugated goat anti-rabbit IgG as the conjugate. Each rabbit IgG molecule contains multiple antigenic sites which would be recognized by the goat anti-rabbit IgG. If four goat anti-rabbit IgG molecules bind to the rabbit IgG, the fluorescent intensity would be magnified four times. Additional controls would need to be included in each assay to ensure that the extra step did not decrease the specificity while increasing the sensitivity. Complement-amplified IFA, also called anticomplement IFA, has been used primarily for detection of herpesviruses (74). Herpesvirus-infected tissue culture cells have an enhanced expression of IgG Fc receptors that nonspecifically bind IgG antibody molecules. Anticomplement IFA uses complement as the third of four layers (Fig. 2C). Antigen is the substrate on a slide; patient's serum is added analogously to performing an indirect IFA. A source of active complement (e.g., guinea pig) is added. While IgG molecules bound to Fc receptors cannot bind Clq, IgG or IgM bound to antigen by the Fab region does bind Clq and activates the classical complement pathway. After unbound complement and other proteins are washed away, an F(ab')2, fluorochrome-conjugated, anti-guinea pig C3 is added (74). Anticomplement IFA eliminates the need to control for nonspecific IgG binding since a complement component is detected instead of IgG. Although not efficiently performed in a clinical laboratory setting, this fourstep procedure may be the only method available for detecting antibodies to certain herpesvirus antigens (e.g., the nuclear antigen of EBV [EBNA]). A very versatile amplification technique is the avidinbiotin complex. Biotin covalently coupled to antibody is used for the primary reagent. Conjugated avidin is the second reagent (Fig. 2D). Avidin has a very high binding affinity (1015 Kin) for biotin, and each biotin molecule will bind four avidin molecules (3). Avidin can be saturated with fluorescein molecules without loss of its ability to bind to biotin. This results in very bright specific staining with minimal nonspecific staining. Controls are few compared with other amplification methods and include conjugated avidin alone and an unrelated biotinylated antibody. A distinct advantage of the avidin-biotin complex system is that the same biotinylated antibody can be used with several different conjugated avidins, i.e., fluorescein (green fluorescence), phycoerythrin (red fluorescence), peroxidase (light microscopy), or ferritin (for electron microscopy). The avidin-biotin complex technique works well with MAbs because biotinylation is fast (1 h), gentle (reaction at pH 7.0 to 8.5 at 22°C), and efficient (biotinylation of 95% of the amino groups of an antibody molecule does not alter the antigen-binding capacity of the antibody) (35). Biotinylation reagents can be purchased commercially (Vector Laboratories, Burlingame, Calif.), mixed with the desired antibody, and used the same day. Avidin conjugated to any indicator system feasible to use is commercially available. Avidinbiotin complex has not realized its full potential in the field of microbiology. Immunoassay Techniques Rapid EIAs to detect bacterial antigens. A plethora of commercially available products has been released in the past few years which use enzyme immunoassay (EIA) technology for detection of proteins and bacterial antigens. Most of the early methods were qualitative assessments of the

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presence or absence of an antigen by a color change of liquid in a test tube, of a matrix on a stick or a paddle, or of a matrix on a plastic reaction vial (e.g., ICON; Hybritech Inc., San Diego, Calif.). Most of these methods were developed to meet the demands for more rapid test results. The techniques were reported to be simple enough to be performed by nontechnical employees such as those in a doctor's office setting. Although the price per test was high, the EIA technology represented a cost savings to doctors' offices because the results were available before the patient left the office. This was particularly beneficial for rapid testing for streptococcal pharyngitis since the physician could prescribe or withhold antimicrobial agents based on the test results rather than providing empirical treatment or telephone follow-up with the patient or both. Clinical microbiology laboratories have resisted accepting and applying this new technology because of a high percentage of false-negative results (25). The compromise acceptable to many laboratories is to take two throat swabs: if the rapid test is positive, discard the second swab; if the test is negative, use the second swab for culture. A recently introduced technique uses liposomes, artificial lipid spheres, to detect group A streptococcal antigens (BBL Microbiology Systems, Cockeysville, Md.). In this test, specific antibody is adsorbed to a porous matrix. The patient specimen is added and any antigen present binds to the antibodies. Liposomes containing a colored dye inside concentric lamillar layers and coated with antibodies to the same antigen are then added. If no antigen was bound by the first antibody, the liposomes flow through the porous matrix. If liposomes are bound, the dye is released by a wash solution which lyses the liposomes, depositing the dye on the matrix. A recent entry into the solid-phase EIA bacterial antigen marketplace is Chlamydia antigen detection (P. Coleman, V. Varitek, T. Grier, J. Hansen, G. Kurpiewski, J. Safford, B. Marchlewicz, and I. K. Mushahwar, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 1184, 1988). The IFA method done in many clinical microbiology laboratories depends on receiving a satisfactory specimen. With the solid-phase EIA technology, the results are either positive or negative (Eastman Kodak Clinical Products, Rochester, N.Y.; Abbott Laboratories Diagnostics Div., Abbott Park, Tll.). A negative EIA could mean either "no chlamydia" or "unsatisfactory specimen." Reports of negative results should clearly state both possibilities and be confirmed by the IFA method, similar to performing a streptococcal culture when the rapid antigen test is negative. Rapid detection EIA methods for other sexually transmitted diseases may be available to doctors' offices and even to the general public in the very near future. Under development are tests for gonococcus, trichomonas, and human immunodeficiency virus. Concerns of clinical microbiologists include the loss of epidemiologic tracking. Rapid antigen EIA methods simply detect gonococcal antigens and do not evaluate antimicrobial susceptibility. Therefore, they fail to detect antimicrobial agent-resistant strains of N. gonorrhoeae. To respond to resulting problems, clinical microbiologists should be aware of the availability of these test systems, their level of sensitivity, and their degree of specificity. Solid-phase methods for detection of antibodies. Enzymelinked immunosorbent assays (ELISAs) are a variation on the coated-tube assay described above, but the roles of the antibody and the antigen are reversed. Instead of an antibody being the solid-phase component, antigen is coupled to

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a surface. The second layer of the sandwich is antibody from the patient serum. The third layer is an enzyme-conjugated anti-human IgG or IgM. The indicator system is the color change resulting from cleavage of the substrate by the conjugated enzyme. The intensity of the color is directly proportional to the amount of patient antibody bound to the antigen. Another interesting approach to EIA systems is FAST ELISA (Falcon Assay Screening Test ELISA; Becton Dickinson Labware, Oxnard, Calif.), which uses coated polystyrene beads attached by a tine to the lid of a microdilution tray (39). The advantage of the FAST system is that the same dilution of patient sera, conjugated antibody, and enzyme substrate could be used for multiple tests by simply using beads coated with different antigens. A manufacturer of automated microbiology equipment (Vitek Systems, Hazelwood, Mo.) has developed an automated immunodiagnostic assay system (VIDAS) which uses the solid-phase ELISA format to detect antigens and antibodies directly from patient specimens in 1 to 2 h. The solid-phase receptacle is a pipette tip-shaped device made of polystyrene or polypropylene. Reagents are predispensed into a cuvette strip which also includes containers of predispensed wash solutions. A computer-controlled instrument performs the tests in batch mode or by random access. Antigen detection tests undergoing field trials include Chlamydia trachomatis, RSV, HSV, and Clostridium difficile toxin. Antibody detection tests for human immunodeficiency virus are also being tested (W. M. Janda, M. H. Graves, K. Hoffman, L. M. Wilcoski, J. M. Stevens, and L. M. Gorniak, Abstr. Annu. Meet. Am. Soc. Microbiol. 1989, C-45, p. 401). The company has developed assays for tests that are currently labor intensive or lengthy, have diagnostic utility, and do not need antibiotic susceptibility testing. Plans for future development include direct antigen detection of Mycobacterium spp., Mycoplasma pneumoniae, and antibodies to the other organisms of the TORCH panel. IgM and IgG separation methods. To avoid false-positive and false-negative results in specific IgM and IgG assays, several methods are available to separate IgG from IgM in serum. Physical separation based on the differential size of these immunoglobulins can be achieved by molecularsieving column chromatography, but this is not practical for clinical laboratory test systems. Sucrose gradient ultracentrifugation would also separate IgM from IgG based on size; however, IgG-containing immune complexes sediment with IgM (31). Most clinical laboratories do not have access to ultracentrifugation equipment. An efficient technique commonly used for IFA assays uses miniature ion-exchange columns (46) which are commercially available from at least two sources. IgG passes through the column and IgM is retained. IgM is eluted from the column by a buffer at a lower pH and higher ionic strength than the application buffer. The volume of serum used and the volume of eluting buffer are carefully controlled to ensure that the final eluate is equivalent to a known dilution of serum; therefore, the final results are semiquantitative as a titer. In some methods, serum is absorbed with staphylococcal protein A either by using the actual bacteria or by binding protein A to an insoluble matrix used as an absorption reagent. Protein A absorption removes IgG subclasses 1, 2, and 4 (but not 3) from the serum. There is increasing evidence that protein A also removes significant virusspecific IgM activity (44). To prevent interference by IgG, commercially available

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EIAs for IgM use either absorption with anti-human IgG (44) or aggregated human gamma globulin (AHGG) (47). AntiIgG binds to IgG in the sample, removing the IgG reactivity to the antigen; if no IgG is available to bind to the antigen, RF would not be bound to IgG. AHGG neutralizes RF, but does not eliminate the potential for false-negative results by IgG competing with IgM for binding sites on the antigen. One study that compared various methods of removing IgG from patient sera found anti-IgG treatment superior to AHGG for eliminating nonspecific IgM activities without impairing specific activities (31), but this finding needs confirmation. The simultaneous dilution of serum and absorption with either anti-IgG or AHGG, as used in several commercial ETAs, is more efficient than ion-exchange separation of IgM from IgG. Our experience with IgM EIAs from three sources suggests that the quantity of anti-IgG used by some manufacturers may not be adequate to remove the IgG antibody from patients with hypergammaglobulinemia (K. James, R. Van Enk, and K. Thompson, manuscript in preparation). Capture assays. Capture assays are another variation of solid-phase technology that can be used to detect antibodies or antigens. The capture assay for antigen detection uses either polyclonal antibodies or MAbs attached to the solid phase to bind (capture) the antigen being detected. The enzyme-labeled antibody for detection can be either monoclonal or polyclonal. Polyclonal antibodies more reliably detect different forms of the antigen, in contrast to MAbs which bind to only a single epitope. There is some limited evidence that capture assays are more sensitive when polyclonal antibodies that bind multiple antigenic epitopes are used (M. D. Tolpin and M. A. Collins, Clin. Microbiol. Newsl. 10:109-111, 1988). In the capture assay for detecting IgM antibodies to specific viral antigens, an animal antibody to the Fc region of human IgM is attached to a solid-phase matrix (69). Potentially, all of the IgM molecules in the patients' sera can be bound to the anti-IgM antibody, regardless of their antigenic specificity. The antigen to be bound by the IgM is conjugated with an enzyme (or biotin when an amplification technique is necessary) and incubated with the "captured" IgM. If IgM specific for the enzyme-conjugated antigen is present, the antigen is captured by the patient IgM, and enzyme substrate will be cleaved. The color reaction is directly proportional to the level of specific IgM antibody in the patients' sera. The capture assay has wide potential for detecting IgM antibodies to microorganisms. Microdilution trays can be coated with anti-IgM and used for detecting antibodies with many different antigenic specificities. A single dilution of patient serum can be applied to the coated wells. Different antigens can be enzyme conjugated (or biotinylated and used with a single enzyme-conjugated avidin) and a single substrate can be used to develop the color reaction. The capture assay would be an efficient method to screen sera to detect the presence or absence of several antibodies (e.g., evaluating congenital infections). Recently, a variation of the capture assay technique has been automated for high-volume hepatitis tests (Abbott Laboratories Diagnostics Div.), including hepatitis B surface antigen and antibody, hepatitis B core antigen, and hepatitis A virus antibody (27). The capture assay works well for detecting IgM antibodies, but is not useful for specific IgG antibodies. During the acute phase of infections, especially congenital viral infections, the majority of the IgM present is virus specific. In contrast, virus-specific IgG would be