JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1989, p. 448-452 Vol. 27, No. 30095-1137/89/030448-05$02.00/0Copyright © 1989, American Society for Microbiology

Evaluation of a Monoclonal Antibody Pool for Rapid Diagnosis ofRespiratory Viral Infections

CAROL STOUT,1* M. DIANNE MURPHY,2 SANDRA LAWRENCE,1 AND SANDRA JULIAN'Department of Pathologyl and Department of Pediatrics,2 University of Tennessee Medical Center at Knoxville,

1924 Alcoa Highway, Knoxville, Tennessee 37920

Received 6 September 1988/Accepted 13 December 1988

A pool of monoclonal antibodies (MAbP) was evaluated both as a method of cell culture confirmation and asa rapid diagnostic screen for viral infection in respiratory secretions. The MAbP was used in a two-stepfluorescent staining procedure on cells harvested from cultures (phase 1) and on exfoliated nasopharyngeal ortracheal cells (phase 2). Antibodies in the MAbP were directed against respiratory syncytial virus, adenovi-ruses, parainfluenza virus types 1, 2, and 3, and influenza viruses A and B. Individual antiviral antibody stainswere used to identify specific viruses from MAbP-positive specimens. In phase 1 (cell culture confirmationonly), 241 respiratory specimens were tested. MAbP sensitivity and specificity were 91 and 94%, respectively.In phase 2, 376 respiratory specimens were evaluated by direct staining of exfoliated cells, and these resultswere compared with results of cell culture isolation. The sensitivity and specificity of the MAbP used in directspecimen testing were 69 and 97%, respectively. These results indicate that the MAbP is highly specific andfairly sensitive for detection of seven different respiratory viruses in one testing procedure. The MAbP is arapid screening technique for respiratory secretions and is potentially a cost-effective approach to viraldetection.

Annually, viral respiratory diseases in adults and childrencause significant morbidity and mortality in addition tomillions of dollars in medical costs. Proper and rapid diag-nosis of these etiological agents is necessary for the appro-priate use of effective antiviral drugs (3, 7, 14) and todecrease unnecessary antibiotic therapy. In addition, be-cause different respiratory viruses are spread via differentmechanisms (fomite, small or large aerosol particles), it ishelpful to identify specific viruses in order to instituteappropriate nosocomial infection precautions (6, 9, 16).Rapid identification of respiratory viruses, therefore, expe-dites favorable patient management and effective cost con-tainment.Although cell culture isolation remains the "gold stan-

dard," enzyme-linked immunosorbent assays (ELISA) orfluorescent-antibody staining can rapidly test for viral anti-gens. Virus isolation can take up to 14 days for somerespiratory viruses, including adenoviruses (AD), parainflu-enza viruses, and respiratory syncytial virus (RSV). ELISAfor RSV performed directly on nasopharyngeal specimens iscommercially available (2, 8, 10, 12, 13, 20), but it is notavailable for other respiratory viruses. Fluorescent-antibodystaining performed directly on nasopharyngeal specimens(DFA) has been applied to all respiratory viruses withvarious degrees of sensitivity and specificity (1, 5, 8, 10, 11,15, 17-19, 21, 22).

Past reluctance on the part of physicians to utilize diag-nostic viral procedures has been related to a variety ofconcepts which are no longer valid. Physicians have fre-quently felt it unnecessary to know which virus was causingthe infection because no specific therapy was available andthe isolation of a specific virus (3 to 14 days) was often toolate to be of benefit to the patient. With the advent of manynew antiviral agents, an increasing population of immuno-compromised individuals, and the ability to provide a spe-cific viral diagnosis in 1 to 72 h, the importance of a specific

* Corresponding author.

viral diagnosis has become obvious. The variety of etiologicagents has made it difficult for physicians to know whichrapid test should be ordered. The ability to rapidly screen forseven respiratory viruses should immensely assist the phy-sician facing these diagnostic problems.We undertook an evaluation of a reagent of pooled mouse

monoclonal antibodies (MAbP) directed against several res-piratory viruses. In phase 1 of the evaluation, the reagentcontained monoclonal antibodies against AD, RSV, influ-enza viruses A and B, parainfluenza virus type 1 (Pi), andparainfluenza virus type 3 (P3) and was evaluated only as acell culture confirmation technique. In phase 2, the reagentcontained monoclonal antibodies to AD, RSV, influenzaviruses A and B, and Pi, P3, and parainfluenza type 2 (P2)and was evaluated on direct respiratory specimens (exfoli-ated nasopharyngeal and tracheal cells) and compared withstandard cell culture isolation.

MATERIALS AND METHODSClinical specimens. (i) Phase 1. From September 1986 to

March 1987, 241 respiratory specimens were obtained fromhospitalized patients (80% pediatric) at the University ofTennessee Medical Center, Knoxville, and East TennesseeChildren's Hospital, Knoxville. Of the specimens, 65% werenasal washes (NW), 21% were throat/nasopharyngeal spec-imens, and 14% were other respiratory specimens.

(ii) Phase 2. From November 1987 to April 1988, 376respiratory specimens were collected from hospitalized pa-tients who presented with respiratory symptoms. The ma-jority (299 specimens) of the specimens were NW; all others(77 specimens) were tracheal aspirates.Specimen collection. NW were obtained by using a syringe

and butterfly technique. A butterfly was attached to a sterilesyringe, and 1 to 5 ml of sterile water or saline was drawninto the syringe. The needle and all but 3 to 4 cm of thetubing were cut away. The remaining tubing was insertedinto the nasopharynx, and 1 to 2 ml was injected andimmediately aspirated.

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EVALUATION OF A MONOCLONAL ANTIBODY POOL FOR VIRUSES

The specimens arrived in the laboratory on wet ice,usually within 0.25 to 2 h of collection, and were processedimmediately. If the specimen was not received in viraltransport medium, approximately half of each NW or tra-cheal aspirate was placed in viral transport medium (vealinfusion broth with gelatin and antibiotics) before inocula-tion into cell culture tubes.

Reagents. The MAbP and individual fluorescent stains toeach of the seven viruses were manufactured by BartelsImmunodiagnostic Supplies, Bellevue, Wash. These re-agents are under investigation for U.S. Food and DrugAdministration approval; we evaluated lots A, B, and C. Thereference fluorescent stains used for comparison in phase 1were obtained from Whittaker Bioproducts, Walkersville,Md. (cytomegalovirus, AD, and influenza viruses A and B),Bartels Immunodiagnostics (RSV), Wellcome Diagnostics,Research Triangle Park, N.C. (Pi and P3), and Syva Co.,Palo Alto, Calif. (herpes simplex virus). In phase 2, thereference fluorescent strains (AD Pi, P2, P3, and influenzaviruses A and B) used for comparison were obtained fromthe Centers for Disease Control (CDC), Atlanta, Ga. Rabbitanti-mouse fluorescein isothiocyanate (FITC) conjugate wassupplied by Bartels to be used with the Bartels and CDCantiviral stains. FITC-conjugated monoclonal anti-RSV an-tiserum was purchased from Bartels and was used as areference fluorescent stain for RSV.

Sensitivity and specificity of all fluorescent stains wereevaluated upon receipt of the reagents. The MAbP showedno cross-reactivity with other viruses (herpesviruses, en-teroviruses, and cytomegalovirus) and exhibited a 3 to 4+reaction against each of the seven respiratory viruses oncontrol slides. The individual monoclonal antibodies to eachof the seven viruses also exhibited a 3 to 4+ reaction againsttheir homologous antigens on control slides. All other fluo-rescent antisera tested positive against their homologousantigens.The MAbP and individual monoclonal antibodies devel-

oped by Bartels are affinity-purified antibodies collectedfroni mouse ascites fluid. The antibodies are currently beingcharacterized as to their antigenicity against particular viralproteins. The anti-mouse FITC conjugate used with theMAbP and CDC stains was supplied by Bartels. Both apurified anti-mouse conjugate and F(ab')2 fragment conju-gate were used in phase 2.

Cells. Rhesus monkey kidney (RMK) and HEp-2 cellculture tubes were purchased weekly from Whittaker. Hu-man newborn fibroblasts (HNF) and A549 cells were pur-chased weekly from Bartels.

Virus isolation. Maintenance medium in cell culture tubeswas removed, and the cell layer was washed with 1 ml ofserum-free minimum essential medium. Duplicate tubes ofRMK, HEp-2, and HNF cells were inoculated with 0.2 ml ofspecimen in viral transport medium and allowed to adsorbfor 90 min at room temperature. If volumes were sufficient,the specimens were also inoculated onto duplicate A549 cells(phase 1 only). Tubes were then fed 1 ml of 2% agamma calfserum in minimum essential medium. Only RMK cells werefed with serum-free minimum essential medium. RMK cellswere incubated at 33°C on stationary racks; HEp-2 and HNFtubes were incubated at 35°C. Tubes were examined at 2-dayintervals for viral cytopathic effect (CPE), and hemadsorp-tion of guinea pig erythrocytes was performed biweekly.DFA. NW and tracheal aspirate suspensions were washed

and centrifuged two times at 1,500 rpm for 10 min. Super-natant was decanted, and 0.1 to 0.2 ml of phosphate bufferedsaline (PBS) was added to the cell pellet; a suspension was

made by pipetting up and down, and a drop was deposited ontwo or three microscope slides. Slides were allowed to airdry and then placed in cold acetone for 10 min.The MAbP and individual fluorescent stains obtained from

Bartels and CDC are two-step stains (excluding the refer-ence RSV stain). Briefly, the slides were incubated for 30min at 35°C with the MAbP or individual antiviral antibody,washed once in PBS, and then incubated for 30 min at 35°Cwith anti-mouse FITC conjugate. The slides were againwashed in PBS for 10 min, quickly immersed in distilledwater, and air dried. All other reference stains were usedaccording to the instructions of the manufacturer.The presence of at least one cell exhibiting characteristic

fluorescent viral inclusion bodies per specimen was consid-ered a positive test result. Any slides containing no cells orfew cells (<6 cells per spot) were recorded as such. All slideswere read blindly.

Cell culture FA confirmation (phase 1 only). Cell layersfrom each pair of tubes were scraped, washed once in PBS,and stained with the MAbP when any of the followingoccurred: (i) positive CPE, (ii) positive hemadsorption, or(iii) negative CPE at 14 days postinoculation. A cell pelletwas obtained and suspended in 0.2 ml of PBS and spottedonto two slides. The slides were allowed to air dry and thenfixed in cold acetone for 10 min.For the cell layers negative at 14 days, one slide only was

stained with the MAbP. For the cell layers positive for CPEor hemadsorption, one slide was stained with the MAbP anda reference stain corresponding to the type of CPE orhemadsorption. If the MAbP slide was found to be positive,slides were stained with Bartels individual monoclonal fluo-rescent antibodies to each of the six respiratory viruses.During phase 2, all cell culture isolates were confirmed in

a manner standard for the laboratory.

RESULTS

Phase 1. Of the 241 respiratory specimens tested, 61 (25%)were positive for a viral agent. The positive specimensconsisted of 35 RSV, 10 enteroviruses, 8 AD, 4 cytomega-lovirus, 2 herpes simplex virus, and 2 P3. Only 45 ofthese 61isolates were considered respiratory viral agents (i.e., RSV,Pi, P2, P3, AD, and influenza viruses). Because influenzavirus activity was low in this winter season and population,influenza virus was not isolated from the specimens tested.The average time for isolation of RSV was 5.6 days, with arange of 2 to 13 days, based on syncytia formation in HEp-2cells. The average isolation times were 9.4 days for AD and10 days for P3, based on CPE or hemadsorption in cellculture, respectively.The reference procedures, including CPE and confirma-

tory FA on cell cultures, detected 50 of the 61 total positiveisolates, which included 34 respiratory isolates. All viruseswere presumptively identified in cell cultures by their char-acteristic CPE or hemadsorption. Further identification wasaccomplished by FA staining of cell layers. Enterovirusisolates were sent to the Tennessee state lab for identifica-tion by using pools of neutralizing antibodies.The MAbP detected 44 of 61 isolates, including 42 of the

45 respiratory isolates. The MAbP showed a weak cross-reactivity on one enterovirus isolate and one herpes simplexvirus type 1 isolate. Both were considered nonspecific whentested with the individual monoclonal antisera. The MAbPdid not detect two RSV isolates. Both RSV isolates missedby the MAbP had syncytia upon initial isolation; one hadsyncytia upon reisolation, and one was DFA positive (Table

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TABLE 1. Discrepant specimens and technique comparison for phase 1

Results with:Virus

isolated MAbP/individual Reference CPE (celi line)a Reference FA Reisolation. DFA for RSVantibody (cell line)a of cell culture

RSV +/+ (R, H, A5) Questionable on day 13 (H) RSV+, AD+ - -+/+ (A5) No CPE NDc + -+/+ (H) Questionable on day 13 (H) ND _ ++/+ (R, A5) Contaminated (H) ND ++/+ (R) Contaminated (H), questionable (R, A5) ND+/- (R, H) Syncytia on day 6 (H) ND +-/ND (R, H, A5) Syncytia on day 12 (H) ND _ +

P3 +/+ (R, H) Weak hemadsorption on days 8 and 13 (R) ND - ND+/+ (R) No hemadsorption ND - ND

AD +/+ (H) CPE on day 13 (H) AD- + ND+/+ (H, A5) Round on days 10 and 12 (H, A5) AD- + ND+/+ (H, A5) Round on days 9 and 11 (HI, A5) AD- ++/+ (R, H) Round on day 4 (R, H) AD- + ND-/+ (R, H) CPE on days 11 and 14 (H, A5) AD+ + ND

a H, HEp-2; R, RMK; A5, A549.b Reinoculation of specimen frozen at -70°C.c ND, Not done.

1). Of the five RSV isolates missed by routine referencetechniques, one showed no CPE, twd had questionable CPE,and two were contaminated. The two with questionable CPEwere confirmed by either a DFA-positive specimen or apositive reference FA performed from the culture. Uponreisolation, two of these five reference-negative specimensproduced late CPE (days 12 and 15), which was confirmed asRSV by reference FA. Only one specimen positive by theMAbP was not confirmed as having RSV by either referencetechniques or reisolation. Two P3 isolates were identified byusing the MAbP. One of these was negative for hemadsorp-tion of guinea pig erythrocytes. The other was very weaklyhemadsorption positive at days 8 and 13, which was con-strued as nonspecific. Of the eight AD isolated, the MAbPdetected seven; the reference techniques detected only four.Of the five discrepant specimens, all had AD-like CPEinitially; five had CPE upon reisolation. The discrepancydelineated was that the reference FA antiserum used forconfirmatory testing of cell cultures was not sensitive to allAD types, as was the MAbP. The MAbP compared favor-ably with cell culture and confirmatory FA, with a sensitivityof 91% and a specificity of 94% for respiratory specimens inour testing. The MAbP gave consistent patterns of stainingfor each virus, and positive viral staining had a 4+ apple-green fluorescence, while negative cells appeared red. Vir-tually no nonspecific staining of cells or debris was ob-served. The MAbP served as a good screening procedure forpositive cultures, negative cultures, and contaminated cul-tures of both RMK and HEp-2 cells.

Phase 2. Of the 376 specimens tested in phase 2, 191 (51%)were positive for 194 viral isolates, while 185 yielded no viralagent by culture, the MAbP, or DFA. Three of the positivespecimens yielded two respiratory viruses (two dual RSVand influenza virus A and one dual RSV and Pi). Thenumber and types of viruses found positive by culture,MAbP, or both are listed in Table 2. Three of the eight ADculture-positive and MAbP-negative specimens and two ofthe twelve influenza virus A culture-positive and MAbP-negative specimens had no cells for interpretation of theMAbP DFA. Overall, only 6% of the specimens tested withthe MAbP had either no cells or too few cells present on theslide. Influenza virus B was isolated but not from the

specimens tested in this study. The influenza virus A isolateswere not delineated as to H and N types.Of the 23 culture-negative and MAbP-positive RSV spec-

imens, the DFA (using reference anti-RSV antiserum) waspositive for 18, negative for 2, and not performed for 3. Oneof the five patients without a confirmatory DFA had hadanother specimen positive for RSV by culture 2 days earlier.For the one culture-negative and MAbP-positive influenzavirus A specimen, no reference DFA test was done. All but5 of 24 culture-negative and MAbP-positive specimens wereconfirmed by another method, and therefore only 5 areconsidered false-positives.

In phase 2, the sensitivity and specificity of the MAbP forrapid identification of respiratory viruses were 69 and 97%,respectively. Positive and negative predictive values were 96and 79%, respectively. The efficiencies of the MAbP withrespect to specific respiratory viruses are given in Table 3.Both P2 and P3 had a small number of positive specimens.Therefore, MAbP efficiency for these viruses is still underquestion. The low sensitivity for AD (based also on a smallnumber of positive samples) could be due to collection ofNW instead of throat swabs; the latter may yield higher titersof AD.

TABLE 2. Results of culture and MAbP in phase 2"

No. of resultsVirus Culture+, Culture-, Culture+, Total

MAbP- MAbP+ MAbP+ positive

RSV 25 23 (18 DFA+) 95 143AD 8 (3)b 0 1 9Pl 4 0 3 7P2 3 0 1 4P3 3 0 0 3Influenza virus A 12 (2)b 1 12 25Herpes simplex 3C 0 3

virus type 1All 58 24 112 194

a MAbP (sensitivity, 69%; specificity, 97%; positive predictive value, 96%;negative predictive value, 79%) versus cell culture isolation.

b Inadequate or poor specimen for DFA.C HSV would not be detected by the MAbP.

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TABLE 3. Efficiency of MAbP versus culture in phase 2

Results with MAbPVirus Sensitivity Specificity ppVa NPV' Sensitivityb

(%) (%) (%) (%) (%)

RSV 79 98 96 90 87AD 17 100 NCC NC 50Pl 43 100 NC NC 57P2 25 100 NC NC 25P3 0 100 NC NC 33Influenza virus A 45 99.7 91 97 77

a PpV, Positive predictive value; NPV, negative predictive value.b Sensitivity defined as MAbP negative, individual antibody positive, and

culture positive versus all culture positives.c NC, Not calculated.

Of the 50 culture-positive and MAbP-negative specimenswith adequate cells for interpretation (3 herpes simplexvirus-positive specimens excluded), 46 were tested with theindividual antiviral stains even though the MAbP was nega-tive. This was done after observation of culture positivity orwhen the MAbP slide had few cells. The individual slideswere still read in a blinded manner. Even though the MAbPslide was negative, 20 of 46 individual slides were positivefor a viral agent which corresponded to the virus found byculture.The non-affinity-purified rabbit anti-mouse conjugate used

on approximately the first 45 specimens tested with theMAbP exhibited substantial nonspecificity and staining ofdebris. This was eliminated when the F(ab')2 fragmentconjugate was used. Use of this conjugate gave a consis-tently red background. It was also observed that differentviruses (RSV, AD, and Pi) showed different patterns andsizes of inclusion bodies (speckled, globular, cytoplasmic,etc.), most noticeable on the individual slides but also seenwith the MAbP.Average times in days for CPE detection in cell cultures

were 6.6 for RSV, 7 for AD, 9 for influenza virus A, 10 forP3, 10.6 for Pi, and 12 for P2.

DISCUSSION

DFA of respiratory specimens for viral antigens is a rapidalternative and/or adjunct to virus isolation. Reported DFAsensitivities and specificities are, respectively, 79 to 96% and70 to 100% for RSV, 33 to 88 and 91 to 100% for influenzaviruses A and B, 31 to 94 and 93 to 100% for parainfluenzaviruses, and 28 to 69 and 100% for AD (1, 5, 8, 10, 11, 15,17-22). The differences seen in sensitivities between report-ing laboratories for the same virus can be due to theinconsistency in availability and quality of reagents and tothe important parameters of specimen collection, specimentransportation, and interpretation of DFA by the microsco-pist. Clearly, not all viral antigens can, at this point in time,be detected by DFA with the same sensitivity. Our MAbPDFA results correlate with the reported efficiencies for eachvirus, although more evaluation is needed for influenza virusB and Pi, P2, and P3. The reported specificities observedbetween 91 and 100%, except for RSV, are acceptable. TheMAbP showed very good specificity for all viruses (98 to100%), including RSV, most likely due to its monoclonalantibody composition. Neither DFA nor culture can detectevery positive viral specimen, owing to mistreatment ofspecimens rendering infectious virus nonviable or to insuf-ficient cellular material. It has been shown by Gardner et al.(4) that infectious RSV may not be recovered by culture,

whereas the DFA may remain positive late in RSV infection.This could explain the wide ranges in sensitivity (79 to 100%)and specificity (70 to 100%) seen for RSV DFA.DFA has many advantages for the clinical virology/micro-

biology laboratory. It is certainly considerably more rapidthan either cell culture isolation (45 to 90 min for DFAcompared with 3 to 14 days for isolation) or ELISA (45 to 90min for DFA versus 2 to 4 h for ELISA). It is also, at thispoint, more useful for a wide array of viruses than is ELISA,since a commercial ELISA is only available for RSV directspecimen testing. With DFA there is no need to batchspecimens, as is sometimes the case with ELISA. Also,DFA can be used for viral diagnosis in laboratories in whichcell culture isolation is not done. In addition, DFA canimpart cost savings, especially compared with labor-con-suming isolation techniques.Excluding influenza virus B, the MAbP to seven respira-

tory viruses evaluated in this study showed very goodspecificity (97%) and fairly good sensitivity (69%). The lackof sensitivity of the MAbP may have been influenced byseveral factors. As with all DFA procedures, it is essential toobtain a quality specimen with an adequate number of cells.Even though 80% of our respiratory specimens were NW,which yield a higher percentage of positive cultures thannasal or throat swabs (W. H. Greene, R. F. Betts, and M. A.Menegus, Program Abstr. 28th Intersci. Conf. Antimicrob.Agents Chemother., abstr. no. 205, 1988), it is implicit that alow number of viral particles in culture may replicate andyield a positive culture, while antigen detection approachesare limited to the number of viral particles initially obtained.Also, since recollection of specimens is often not done orpossible, we used a low cutofffor the number of cells presentto interpret each specimen; thus, only 6% of our specimensfor DFA were rejected. Although all specimens are trans-ported to our virology laboratory on ice, delays are inevita-ble, and long periods of delay may result in proteolyticenzymatic breakdown of cells and loss of viral antigen.Strain differences within virus types will certainly affect thesensitivity of an antiviral reagent. The property of monoclo-nal antibodies being immunogenic to only one epitope of asingle virus inherently makes them more specific but possi-bly less sensitive when many strains and/or types are exhib-ited by one virus. This is particularly true with AD, with >35types, and the influenza A viruses, with different strainscirculating each year. Because of a low number of samples(<10) for the parainfluenza viruses and AD, the sensitivity ofthe MAbP for these viruses may be understated. In anystatistical analysis, a small number of samples may decreaseaccuracy because each variant is such a large proportion ofthe whole. The sensitivity increased when the individualmonoclonal antibodies were used in designated circum-stances even after the MAbP was observed to be negative.This may suggest that the MAbP contains too many compet-ing antibodies or that a larger sample is required for stainingwith the MAbP.Ray and Minnich (17) have suggested that a broader

routine battery of rapid viral antigen detection tests shouldbe considered in certain clinical circumstances. We heartilyagree. In the months surrounding peak RSV season, aroutine viral respiratory battery antigen detection test wouldbe quite useful for rapid diagnosis.The MAbP contains all monoclonal antibodies and is

supplied at working dilution. The FITC conjugate is alsosupplied at working dilution and is used with the MAbP andall individual stains, which aids in convenience and consis-tency. In summary, the MAbP is an excellent cell culture

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confirmation reagent, and a rapid adjunct to cell culture fortesting of direct specimens for more than one respiratoryvirus. Several possibilities remain regarding the MAbP. (i)Its sensitivity as a rapid screen of direct specimens mayincrease when a larger sampling of AD- and parainfluenzavirus-positive specimens are tested. (ii) Its sensitivity mayincrease if antibody composition or quantity is altered. (iii)Its use should be evaluated on cell cultures at 2 to 5 days forviral identification before CPE is evident but viral antigen ispresent.

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13. Masters, H. B., B. J. Bate, C. Wren, and B. A. Lauer. 1988.Detection of respiratory syncytial virus antigen in nasopharyn-geal secretions by Abbott Diagnostics enzyme immunoassay. J.Clin. Microbiol. 26:1103-1105.

14. McClung, H. W., V. Knight, B. E. Gilbert, S. Z. Wilson, J. M.Quarles, and G. W. Divine. 1983. Ribavirin aerosol treatment ofinfluenza B virus infection. J. Am. Med. Assoc. 249:2671-2674.

15. Minnich, L., and C. G. Ray. 1980. Comparison of directimmunofluorescent staining of clinical specimens for respiratoryvirus antigens with conventional isolation techniques. J. Clin.Microbiol. 12:391-394.

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17. Ray, G. C., and L. L. Minnich. 1987. Efficiency of immunoflu-orescence for rapid detection of common respiratory viruses. J.Clin. Microbiol. 25:355-357.

18. Shalit, I., P. A. McKee, H. Beauchamp, and J. L. Waner. 1985.Comparison of polyclonal antiserum versus monoclonal anti-bodies for the rapid diagnosis of influenza A virus infections byimmunofluorescence in clinical specimens. J. Clin. Microbiol.22:877-879.

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