Enzyme Immunoassay for Direct Detection of Influenza Type

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1982, p /82/ $02.00/0 Vol. 15, No.1 Enzyme Immunoassay for Direct Detection of Influenza Type A and Adenovirus Antigens in Clinical Specimens MAURICE W. HARMON* AND KATHY M. PAWLIK Influenza Research Center, Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 7700 Received 4 May 1981/Accepted 10 August 1981 Detection of viral antigens in specimens without prior cultivation in cell culture provides the most rapid method for specific viral diagnosis. A solid-phase, doubleantibody enzyme immunoassay was developed for this purpose and tested with clinical specimens containing influenza type A and adenovirus. Polystyrene microtiter wells were the solid phase and were coated with virus-specific guinea pig immunoglobulins. Specimens were added, and bound viral antigens were detected by addition of virus-specific rabbit immunoglobulins followed by enzyme-labeled goat antirabbit immunoglobulin G. Two methods of labeling goat anti-rabbit immunoglobulin G with horseradish peroxidase were investigated: covalent attachment and a noncovalent, immunological binding of antibody to enzyme, the peroxidase-antiperoxidase method. Both methods of labeling resulted in assays that could detect % tissue culture infectious doses of influenza type A and % tissue culture infectious doses of adenovirus. Equal sensitivity was noted with alkaline phosphatase-labeled goat anti-rabbit immunoglobulin G. An increase in sensitivity of three- to sixfold was achieved when virusspecific rabbit immunoglobulins and conjugate were diluted in 1% gelatin. The solid-phase, double-antibody enzyme immunoassay detected influenza type A and adenovirus in isolation-positive clinical specimens with 5% (21/40) and 62% (1/ 21) efficiency, respectively. The solid-phase, double-antibody enzyme immunoassay has considerable potential as a practical and rapid method for detection of respiratory viral antigens in nasal wash and throat swab specimens. For optimal value, however, greater sensitivity than was provided by the present methods is desirable. Most respiratory virus infections are diagnosed by isolating the agent in a permissive host such as cell culture or embryonated eggs. Although sensitive, isolation involves a delay of several days between inoculation and evidence of virus replication. As advances in antiviral chemotherapy are made, concurrent advances in rapid diagnosis are desirable since antiviral agents are apt to be virus specific, and patients will benefit most by earlier use of an antiviral agent. Detection and identification of viral antigen directly in clinical specimens would accelerate viral diagnosis. One approach toward rapid respiratory virus diagnosis is immunofluorescent staining of cells removed from the site of infection (8, 12, 18). Although this method is rapid, it is also subjective and has not received widespread application. Moreover, some investigators have found that immunofluorescent staining is not as sensitive as isolation and that the use of both techniques results in a significant increase in the number of positive virus identifications (11, 21). 5 Other techniques for direct detection of antigen include radioimmunoassay and enzyme immunoassay (EIA). These methods are necessary to detect viruses that do not readily replicate in cell culture, such as rotavirus (), hepatitis B (1), and hepatitis A (19); however, they may also be applied to viruses that can be isolated in cell culture (4, 26, 6). Relatively little data are available on the performance of EIA when clinical specimens from respiratory disease are used. This report describes several EIA systems; the most satisfactory system was tested with clinical specimens. Antigen could be rapidly detected in 62% of the adenovirus and 5% of the influenza type A virus isolation-positive specimens. MATERIALS AND METHODS Antiserum. Rabbits and guinea pigs were used to prepare antisera to viral antigens. Purification of adenovirus type 5 hexon subunits and immunization of guinea pigs have been described (1, 2). Rabbits were given 5,ug of hexon in complete Freund adjuvant and 5 Fg of hexon in saline after 4 weeks, and they were bled weeks later. Rabbit and guinea pig antisera had

2 6 HARMON AND PAWLIK J. CLIN. MICROBIOL. neutralization titers of 512 and -,125, respectively, against adenovirus type 5. Influenza type A (A/USSR/90/77) from the Centers for Disease Control was grown in 11-day-old chicken embryos. Allantoic fluid was clarified by low-speed centrifugation, and viruses were pelleted twice by ultracentrifugation, (80,000 x g for 0 min). The second pellet was suspended in 2 ml of phosphatebuffered saline (PBS) and centrifuged (65,000 x g for 45 min) through a discontinuous sucrose gradient (5, 20, and 60% sucrose). The virus band at the 20/60% sucrose interface was removed, sonicated, titered by hemagglutination, and stored at -70 C until used for immunization. Rabbits were immunized with 20,000 hemagglutination units of influenza type A in complete Freund adjuvant and reimmunized with the same amount of antigen in incomplete adjuvant 4 weeks later. Antiserum was obtained 4 weeks after reimmunization. Guinea pigs were immunized in a similar manner with 10,000 hemagglutination units per injection. Periodatetreated rabbit and guinea pig antisera both had hemagglutination inhibition titers of 10,240. Antiserum to peroxidase was produced by immunization of rabbits with 15 mg of peroxidase in complete Freund adjuvant followed by 10 mg in incomplete adjuvant 4 weeks later. Animals were bled 8 weeks after the second injection. Conjugates. Three enzyme-antibody conjugates were prepared, one with alkaline phosphatase and two with horseradish peroxidase. All three systems used the same goat anti-rabbit immunoglobulin G (IgG) (Immuno-Reagents, Inc., Seguin, Tex.). For covalent linkage, immunoglobulins were precipitated with ammonium sulfate as outlined by Cherry (5) and partially purified by DEAE-cellulose (DE-52; Whatman, Clifton, N.J.) chromatography. Conditions for conjugating alkaline phosphatase to IgG with glutaraldehyde were described by Engvall and Perlmann (9). The source of alkaline phosphatase was calf intestine (Sigma type VII; Sigma Chemical Co., St. Louis, Mo.), and the specific activity was 1,100 U per mg of protein. Two methods of labeling antibody with peroxidase were investigated. Goat anti-rabbit IgG was covalently attached to the carbohydrate moiety of horseradish peroxidase (Sigma type VI) by the method of Nakane and Kawaoi (22). Specifically, we used 0.04 M periodate and reacted 0.5 mg of peroxidase per mg of IgG. The second method of labeling antibody with peroxidase was a noncovalent, immunological method called the peroxidase-antiperoxidase (PAP) method (27, 28). This technique involves two antibodies, one against peroxidase and the other against the viral antigen, both of which are produced in the same species (in this case, rabbits). The two rabbit antibodies are bonded together immunologically by goat antirabbit IgG; therefore, this method avoids loss of enzyme activity or antibody specificity due to the labeling process itself. In immunoperoxidase microscopy, the addition of peroxidase and antiperoxidase as a soluble complex is more sensitive than the sequential addition of antiperoxidase (as antiserum or purified antibody) followed by peroxidase (24). Details for production of PAP complexes are outlined by Stemberger (27). EIA. The method used was a solid-phase, doubleantibody assay. Immunoreagents were immunoglobulin fractions of antisera obtained by ammonium sulfate precipitation (5). The optimal concentrations were determined by checkerboard titration. Diluent for immunoreagents (and antigen, when diluted) consisted of PBS containing 0.05% Tween 20 (PBS-T). The addition of 1.0% gelatin (USP powder; W. H. Curtin and Co., Houston, Tex.) to the diluent for certain immunoreagents increased sensitivity three- to sixfold (see Results) and was used in all tests with clinical specimens. Virus-specific guinea pig immunoglobulins were diluted in carbonate buffer (ph 9.6) (0) and added to the inner 60 wells (0.2 ml) of Microelisa substrate plates (Dynatech Laboratories, Inc., Alexandria, Va.), which served as the solid phase. Plates were incubated at 7 C for 5 h. After two washes with PBS, wells were postcoated with 1% gelatin in PBS for 1 h at 7 C. After this and each successive incubation period, plates were washed four times with PBS-T. Test samples (0.2 ml) were added in duplicate and incubated at 7 C for 18 h. Virus-specific rabbit immunoglobulins and conjugates were each incubated for 2 h at 7 C. In the PAP system, unconjugated goat antirabbit IgG (immunoglobulin fraction) and rabbit PAP complexes were each incubated for 2 h at 7 C. Substrate for peroxidase consisted of 0.01% hydrogen peroxide and 0.01% 2,2'-azino-di-[-ethylbenzthiazoline sulfonate (6)] (Boehringer-Mannheim Biochemicals, Indianapolis, Ind.) as chromagen in 0.1 M sodium citrate buffer (ph 4.0). Peroxidase reactions were not stopped for reading; however, test runs were small, and all wells were read within minutes. Optical density was measured at 417 nm in a spectrophotometer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio). Substrate for alkaline phosphatase was p-nitrophenylphosphate (Sigma 104 phosphatase substrate tablets) diluted to 1 mg/ml in 10% diethanolamine buffer (ph 9.8). Reactions were stopped by addition of 50,ul of N NaOH and read on a Multiskan microplate reader with a 40-nm filter. Background reactions were determined by substituting PBS-T, normal nasal washes in Ringer lactate, or normal throat swabs in veal infusion broth with 0.5% bovine serum albumin (2), where appropriate. Six to 10 controls were included in each assay. A sample was considered positive if the optical density was greater than standard deviations above the mean optical density of the appropriate controls. Confirmatory test. In cases where enough clinical specimens were available, a confirmatory test was conducted to test for reaction specificity. The test consisted of adding the specimen, in duplicate, to wells coated with postimmunization immunoglobulins and to wells coated with preimmunization immunoglobulins at the same dilution from the same guinea pig. The EIA was then carried out as described. The optical density of the postimmunization wells was divided by the optical density of the preimmunization wells, and a ratio (post/pre) was obtained. Background ratios were determined on nine normal throat swabs tested on five separate plates (45 determinations). The mean ratio of optical densities (post/pre) was 0.95, with a standard deviation of We considered a ratio that was standard deviations greater than the mean (i.e., a ratio of 1.1) to be evidence of specific binding of the virus in question. A ratio of less than

3 VOL. 15, 1982 this in a reactive specimen was considered a nonspecific reaction. Immunoreagent absoption. Since cross-reactions between immunoreagents were detected when we used appropriate controls, immunoreagents were absorbed against normal globulins of the appropriate species. Normal globulins were obtained by ammonium sulfate precipitation (5) and covalently attached to cyanogen bromide-activated Sepharose 4-B (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) by the method of Cuatrecasas (7). This involved coupling 8 mg of protein per ml of packed Sepharose in 0.2 M sodium citrate buffer (ph 6.5). After coupling, beads were washed with five cycles of alternating buffers (0.1 M sodium acetate with 1 M NaCl, ph 4.0; 0.1 M sodium borate with 1 M NaCl, ph 8.0). Finally, beads were washed and stored in PBS. Equal volumes of the immunoreagent to be absorbed and Sepharose 4-B with covalently coupled normal globulins were incubated at 7 C for 1 h followed by overnight incubation at 4 C. Horseradish peroxidase and alkaline phosphatase conjugates and virus-specific rabbit immunoglobulins were absorbed against normal guinea pig globulins. In addition, guinea pig immunoglobulins against influenza type A were absorbed against normal rabbit and goat globulins. Overall background reactions were reduced by 60 to 90% after such absorptions. Test specimens. Tissue culture harvests of adenovirus type 5 and influenza type A were used to determine the sensitivity and specificity of the EIA methods. Adenovirus was grown in HEp-2 cells and assayed in primary human embryonic kidney cells. Growth medium for cells consisted of Eagle minimal essential medium supplemented with heat-inactivated (56 C, 0 min) fetal bovine serum (10%), penicillin (100 UIml), and streptomycin (100,ug/ml). For growth and assay of adenovirus type 5, the concentration of fetal bovine serum was reduced to 2%. Influenza type A was prepared and assayed in MDCK cells. For growth and assay of virus, serum-free minimal essential medium supplemented with 2,g of trypsin per ml (Worthington Biochemicals Corp., Freehold, N.J.) was used (29). Uninfected cell controls were prepared in parallel. Each cell culture system was the most sensitive for detection of the respective virus (10, 17, 20). Titers were calculated by the Karber method (15). The clinical specimens tested were specimens routinely submitted to the Viral Diagnostic Laboratory at The Methodist Hospital and to the community surveillance program of the Influenza Research Center, Houston, Tex. The nasal wash specimens were collected in Ringer lactate. Throat swabs were placed in veal infusion broth containing 0.5% bovine serum albumin (2). Specimens were stored at -70 C until tested by EIA. Adenoviruses were isolated in human embryonic kidney or HEp-2 cells. Viruses were detected by cytopathic effects and identified by immunofluorescence as previously described (1). Adenoviruses were not typed. Influenza type A (HiN1) viruses were isolated in primary rhesus monkey kidney or MDCK cells and detected by hemadsorption of guinea pig erythrocytes. Isolates were identified by immunofluorescence, using influenza type A (HlN1)-specific antiserum. EIA FOR DETECTION OF VIRAL ANTIGENS 7 RESULTS Preliminary experiments were concerned with determining whether the method of labeling the goat anti-rabbit antibody with horseradish peroxidase influenced the sensitivity of the assay. Both covalent and immunological attachment of label to antibody resulted in assays with detection limits of % tissue culture infectious doses (TCID50) for a stock influenza type A pool (Fig. 1). Since the immunological (PAP) method required one extra incubation period and one more antiserum (rabbit antiperoxidase), subsequent testing used covalently attached label. In a similar manner, the covalently linked horseradish peroxidase and alkaline phospha- I E 1.5 I 1.0 n rl_ U..' J._ ' 1.5 c t- Covalent I I I attachment Conjugate Dilution 0 1:200 *-* 1:400 I mmunologic attachment PAP Dilution o0-1: : o l.5 I nfl uenza A/US SR (TC I D50/0.2ml) FIG. 1. Comparison of covalent and immunological methods of labeling goat anti-rabbit antibody for detection of influenza type A. Equal portions of the same virus dilutions were used for both comparisons. Both assays detected influenza type A at a concentration of 10-5 TCID5O/0.2 ml. Control values for conjugate dilutions 1:200 and 1:400 were 0.20 ± and , respectively. Control values for PAP dilutions 1:00 and 1:600 were and , respectively. Standard deviations for the experimental values were comparable.

4 8 HARMON AND PAWLIK tase conjugates were compared for sensitivity. Each had a detection limit of 10 8 TCID50 for a stock adenovirus pool (data not shown). The alkaline phosphatase conjugate was selected for further use. While testing methods to reduce nonspecific binding, we observed that dilution of virusspecific rabbit immunoglobulins and conjugate in PBS-T containing 1% gelatin (compared with PBS-T alone) increased the test sensitivity (Table 1). Although a 10-fold increase in sensitivity was not always achieved, the mean increase in sensitivity in seven experiments using adenovirus as antigen was sixfold. The mean increase in sensitivity using influenza type A as antigen was.2-fold in five experiments. Table 2 shows the specificity of the adenovirus and influenza type A EIA systems. As expected, the adenovirus system cross-reacted with all of the adenovirus antigens tested. It did not react with any of the other respiratory virus antigens. The influenza type A (HlNi) EIA cross-reacted with influenza type A (HN2) (A/ Bangkok/l/79) at a low level but not with any of the other viral antigens tested. Table shows our experience with the adenovirus EIA, using clinical specimens. Sixty-two percent of isolation-positive specimens were positive by EIA. Eight percent of the clinical specimens that did not yield an adenovirus were reactive by EIA. The confirmatory test indicated that these were nonspecific reactions. Five of the 1 EIA-positive, isolation-positive specimens were available for confirmatory testing, and all indicated adenovirus-specific binding. Table 4 shows a comparison of isolation and TABLE 1. Enhancement of EIA sensitivity by dilution of immunoreagents in PBS containing 1% gelatin Adenovirus type 5 Optical density (405 nm)' dilution' PBS-T PBS-T with 1% gelatin ± ± ± ± ± ± ± ± ± ± PBS ± a Equal portions of the same virus dilutions were added to both sets of wells. b Appropriate dilutions of rabbit anti-adenovirus type 5 immunoglobulin and goat anti-rabbit IgG alkaline phosphatase conjugate were prepared in PBS-T or PBS-T containing 1% gelatin. All other manipulations were identical. c, Positive reactions, which were standard deviations greater than the mean of 10 PBS controls. TABLE 2. virus antigena Adenovirus type Rhinovirus type 1A Coxsackievirus A type 21 Respiratory syncytial virus Parainfluenza virus type 1 2 Influenza virus type B Influenza virus type A HlNl HN2 None (PBS-T, n = 8) Specificity of adenovirus and influenza type A EIA systems Optical density (405 nm) Adenovirus Influenza virus EIA EIA ± J. CLIN. MICROBIOL a The amount of viral antigen added ranged between 104 and 106. TCID50/0.2 ml. EIA for detection of influenza type A (HIN1). Four of eight nasal washes were positive by EIA. These four specimens were also positive when tested with the horseradish peroxidase conjugate (data not shown). EIA testing of throat swab specimens from the 1978 to 1979 season resulted in 6 positive reactions out of the 11 (55%) isolation-positive specimens tested. Influenza type A virus (HiN1) isolates from the 1980 to 1981 season tested by EIA were shown by monoclonal antibody tests to be a mixture of 1 A/Brazil/79-like and others more distantly related (H. R. Six, personal communication). Five isolates tested by EIA were not tested by monoclonal antibodies. Even though some of the virus strains encountered exhibited increased antigenic drift of the hemagglutinin antigen relative to the A/USSR/90/77 prototype to which the antisera were raised, 11 of 21 specimens (52%) were EIA positive. Seven positive specimens were A/Brazil/79-like, two were more distantly related, and two were not typed with monoclonal antibodies. All positive specimens

5 VOL. 15, 1982 TABLE. Detection of adenovirus in clinical specimens by virus isolation and EIA EIA Cell culture result No. tested positive' No. % Adenovirus isolatedb Adenovirus not isolated 21 9c d a Specimens were considered positive if the optical density was greater than standard deviations above the mean optical density of six to eight normal throat swab specimens. b Adenoviruses were isolated in human embryonic kidney or HEp-2 cells and identified by immunofluorescent staining. c Includes eight specimens with other virus isolates: three herpes simplex, two influenza type A, one influenza type B, one picornavirus, and one enterovirus ḋ The confirmatory test indicated that these were false-positive reactions. were influenza specific by the confirmatory test. One specimen (collected on 18 July 1980) was positive by EIA, and no influenza or heterologous virus was isolated. The confirmatory test indicated influenza-specific binding (post/pre ratio =.2). Paired sera were not available for serological testing to confirm an influenza infection in this patient. DISCUSSION EIA tests for a variety of substances are being applied with increasing frequency in clinical laboratories; included is the use of EIA for viral diagnosis (2). Initially used to detect viruses that do not replicate in cell culture, such as rotavirus (), hepatitis B (1), and hepatitis A (19), EIA is now being used for more rapid detection and identification of viruses that can be isolated in cell culture. Herrmann et al. (16) and Yolken and Torsch (6) have used EIA to identify tissue culture isolates of enteroviruses. We reported using EIA to rapidly detect adenovirus in tissue culture cell extracts before development of cytopathic effects (1). That assay was a singleantibody EIA, and sensitivity was too low for detection of viral antigen in clinical specimens (2% positive). The same specimens, nasal washes from an artificial adenovirus challenge (6), were tested by the double-antibody EIA described in this report, and 29% were positive (data not shown). Nearly 40% were positive at the peak of virus shedding (day 6 postinoculation). Thus, the double-antibody EIA is more sensitive than the single-antibody assay (5). The reason for a lower number of positive reactions here (i.e., 29 to 40%) compared with EIA FOR DETECTION OF VIRAL ANTIGENS 9 the results in Table is unknown. It may be related to the fact that the previous specimens were nasal washes from artificially challenged volunteers, whereas results in Table were from throat swab specimens from naturally acquired disease. In the present study, viral antigens in specimens from naturally acquired adenovirus and influenza type A infections were detected by EIA with 62 and 5% efficiency, respectively. These positive reaction rates are lower but comparable to those reported for other virus systems. Pronovost et al. (25) found 67% of herpes simplex-containing specimens positive by standard EIA; measurement of the enzyme reaction product by chemiluminescence detected viral antigen in three more specimens (8%). Yolken and Torsch (6) detected coxsackievirus B antigen in seven of nine rectal swabs (78%). Chao et al. (4) reported 2 of 29 (79%) isolation-positive respiratory syncytial virus specimens positive TABLE 4. Detection of influenza type A in clinical specimens by virus isolation and EIA Cell culture No. EIA result Specimen tested positive' Influenza virus Nasal wash 8 4 isolated' ( season) Throat swab 11 6 ( season) Throat swab 21 lc ( season) Total (5) Influenza virus Nasal wash 5 0 not isolated Throat swab 2d 1 Total 7 1 () a Specimens were considered positive if the optical density was standard deviations greater than the mean optical density of 7 normal nasal washes or 10 normal throat swabs. Percentages are given in parentheses. b Influenza viruses were isolated in rhesus monkey kidney or MDCK cells and identified as influenza type A by immunofluorescent staining. c Viruses isolated in the 1980 to 1981 season were a mixture of A/Brazil/79-like and others that were more distantly related. Included as EIA positive were three specimens in which the optical density was only 2 standard deviations greater than the mean of nine normal throat swabs. The remaining eight positive specimens were greater than standard deviations above the mean control value. All positive reactions were influenza specific by the confirmatory test. d Includes five specimens with other virus isolates: two herpes simplex, one picornavirus, one enterovirus, and one influenza type B.

6 10 HARMON AND PAWLIK by EIA. Recently, Sarkkinen et al. (26) found radioimmunoassay and EIA to be as effective as immunofluorescent staining for detecting respiratory syncytial virus, parainfluenza virus type 2, and adenovirus in nasopharyngeal aspirates. Isolation data were not available. Clearly, each of these immunoassays is capable of detecting viral antigen in the majority of positive specimens. However, to be useful in the clinical laboratory, some increase in sensitivity is needed Ẇe attempted to increase EIA sensitivity by improving the quality of the conjugate. Covalent attachment of horseradish peroxidase to antibody with glutaraldehyde results in low yields (2 to 10%) of active enzyme and a heterogeneous conjugate (1). We used the periodate method, which covalently couples the carbohydrate moiety of horseradish peroxidase to antibody with yields as high as 70% (22). We compared this method of labeling with a noncovalent, immunological method (PAP) which avoids loss of enzyme activity or antibody specificity that can occur during covalent linkage. Both methods, however, produced conjugates of equivalent sensitivity. Yolken and Stopa (5) recently demonstrated that attachment of PAP complexes to antibody with staphylococcal protein A also did not improve sensitivity compared with conventional methods of attaching label. Their data and evidence reported here suggest that simple attachment of alkaline phosphatase to antibody by glutaraldehyde provides as sensitive an assay as do more efficient methods of conjugation. One successful method of increasing sensitivity is to use an enzyme substrate in which the reaction product is detectable at a lower concentration, such as fluorescence (4) or radioactivity (14). By performing two inhibition EIA tests on each specimen, one with a fluorogenic substrate and one with a radioactive substrate, Berg et al. () detected influenza type A (HN2) antigen in more nasal wash specimens than were detected by isolation, particularly in the second half (days 5 to 8) of the illness. EIA and isolation are nearly equivalent in the first 4 days of illness when both tests are applied. Interestingly, when only a single EIA test is applied to specimens during the first 4 days of illness, the detection rate ranges between 50 and 91% of those detected by isolation. The observation that dilution of virus-specific rabbit immunoglobulins and conjugate in 1% gelatin increased sensitivity was unexpected. Gelatin was originally added to reduce background reactions; however, background as well as specific reactions were actually increased. The mechanism of this enhancement is unknown. In the described EIA, we observed that a J. CLIN. MICROBIOL. similar percentage of influenza type A viruses were detectable even though the hemagglutinin antigen had undergone antigenic drift. Animals were immunized with whole virus (A/USSR/90/ 77) and most likely responded to other viral antigens present in addition to the hemagglutinin. Evidence that antibodies to the nucleocapsid or matrix antigen were involved is provided by the fact that this EIA cross-reacts with HN2 virus (A/Bangkok/1/79) but not with influenza type B. In a previous report (1) and in Table 2, evidence is presented that an EIA using antibody to adenovirus type 5 hexon antigen is reactive with a number of (theoretically all) adenovirus serotypes. Clinical samples reported in this study were randomly selected from those submitted over a -year period. Although isolates were not typed, we presumably encountered more than one serotype. The use of the confirmatory test with clinical specimens is clearly indicated, particularly considering the limited amount of information available on the use of EIA for respiratory virus detection. In this report, three reactions with the adenovirus EIA were shown to be false by the confirmatory test. Viral respiratory disease constitutes one of the most common human afflictions. However, the majority of serious viral respiratory infections are caused by a relatively few agents, primarily influenza virus types A and B, parainfluenza virus types 1, 2, and, respiratory syncytial virus, and adenoviruses. Data presented and reviewed in this report indicate that progress has been made in the rapid diagnosis of this group of agents. Increased sensitivity through the use of alternative enzyme-substrate systems and some attention to the somewhat neglected area of specimen collection and processing should make rapid diagnosis of this group of agents a reality in the foreseeable future. ACKNOWLEDGMENTS This investigation was supported by Puol Iealth service grant Al from the National Institute of Allergy and Infectious Diseases. 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