JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1989, p. 2096-20990095-1137/89/092096-04$02.00/0Copyright © 1989, American Society for Microbiology

Dot Enzyme Immunoassay for Visual Detection of Peste-des-Petits-Ruminants Virus Antigen from Infected Caprine Tissues

TIMOTHY U. OBI* AND CLEMENT K. OJEH

Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria

Received 27 July 1988/Accepted 20 April 1989

An enzyme-linked immunosorbent microassay using nitrocellulose paper as the solid-phase support wasdeveloped for the detection of peste-des-petits-ruminants virus antigens in infected caprine tissue homogenates.Dots of tissue homogenates were applied to nitrocellulose papers, and any unreacted sites were blocked with 5%skim milk powder in triethanolamine-buffered saline. After incubation of the papers in tissue culturesupernatant monoclonal antibody against the peste-des-petits-ruminants virus, the antigen-antibody reactionwas detected with peroxidase-conjugated anti-mouse immunoglobulin G and the enzyme substrate 4-chloro-1-naphthol. Positive results were visualized as blue dots. Results of the dot enzyme immunoassaycompared favorably with those of the standard enzyme-linked immunosorbent assay. Incorporation of NonidetP-40 in the washing solution did not improve the sensitivity of the dot enzyme immunoassay, and pretreatmentof homogenates with Nonidet P-40 before application to the nitrocellulose paper inhibited the binding of theantigen to the paper and reduced the intensity of the color development.

Peste-des-petits-ruminants (PPR), a highly contagious in-fectious and fatal rinderpestlike disease of small ruminantsand some wildlife species (9), is caused by a virus thatbelongs to the Morbillivirus genus of the Paramyxoviridae(10). Apart from virus isolation and characterization, suchserological tests as the complement fixation test (11), theagar gel precipitation test (10, 15), immuno-electro-osmo-phoresis (13, 15), and the microplate enzyme-linked im-munosorbent assay (ELISA) (T. U. Obi et al., manuscript inpreparation) are available for PPR virus (PPRV) antigendetection.On the basis of the tremendous amount of published work

on the application of the ELISA in the diagnosis of viraldiseases of humans and animals, it is indisputable that theELISA offers obvious advantages of simplicity, sensitivity,and specificity over mosn other serological tests for antigenand antibody detection. Although ELISA results can be readvisually, it is conventional to use a spectrophotometer, a

relatively expensive piece of equipment, for quantification.Another test, the dot enzyme immunoassay (dot EIA),

which can also be visually assessed, was developed for thedetection of antibodies to a number of human and animalpathogens, such as pseudorabies virus (2), herpes simplexvirus type 1, herpes simplex virus type 2, and herpesvirussimiae (12); the agents of visceral leishmaniasis (16) andbovine brucellosis (6); and rinderpest virus (RPV) (1).

In this paper, we describe a rapid dot EIA for thedetection of PPRV antigens in infected postmortem speci-mens.

MATERIALS AND METHODSPPRV antigen. A Nigerian PPRV isolate (NIG.75/1 Vero/

44BK/lSK/lVero/2) was grown in suspension cultures ofVero cells with Eagle medium containing 10% fetal bovineserum. When the cells became confluent, the medium wasreplaced with Eagle medium containing 1% fetal bovineserum. At 5 to 6 days postinoculation, when there was aboutan 80 to 90% cytopathic effect, the cells and the supernatantwere harvested and the harvest was subjected to three cycles

* Corresponding author.

of freezing and thawing and then clarified at 1.84 x g for 30min at 4°C. The supernatant was then centrifuged at 7.37 xg in a Beckman 42.1 rotor for 60 min at 4°C, and the pelletwas suspended to 1/50 of the original volume with phos-phate-buffered saline (PBS; pH 7.4). The negative controlantigen consisted of uninfected Vero cells processed in a

manner similar to that of the PPRV antigen.MAbs. Hybridoma cell lines secreting monoclonal anti-

bodies (MAbs) to structural proteins of PPRV were pro-duced by fusion of splenocytes from BALB/c mice whichhad been immunized with PPRV and SP2/0 myeloma cells(Obi et al., in preparation). One of the MAbs (hybridomasupernatant) to the matrix (M) protein of the virus was usedfor this study.

Samples for antigen detection. Tissues used for this studywere from goats which died of natural PPRV infection.Diagnosis of the disease was based on clinical signs andgross and histopathological findings (14), as well as results ofthe agar gel precipitation test done by previously describedmethods (15). During postmortem examination, tissues werecollected from the lungs, trachea, mediastinal and mesen-teric lymph nodes, tonsils, spleen, small intestine, colon,cecum, and rectum. These tissues were extracted as 20%(wt/vol) homogenates in PBS (pH 7.4), and the homogenateswere stored for between 6 months and 2 years at -20°Cbefore they were tested. Since the homogenates were pre-pared essentially for agar gel precipitation tests, aseptictechniques were not used.Dot EIA procedure. Plain nitrocellulose paper (Transblot

no. 162-0115; Bio-Rad Laboratories, Richmond, Calif.) wascut into portions (6 by 5 cm) with a clean, greaseless scalpelblade, and 30 1-cm squares were marked out on each paperwith a pencil. During these procedures, the nitrocellulosepaper was handIed with clean fine forceps and surgicalgloves to avoid any alterations on the paper surface. Foroptimization, a checkerboard titration of a twofold dilutionof the positive PPRV antigen from 1:5 to 1:160 and the MAb(1:5 to 1:80) was performed.For the actual test, 2 il of the optimal dilution (1:10) of the

PPRV antigen in PBS (pH 7.4) and the same dilution of thehomogenates and the negative control antigen were dotted at

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the centers of the squares on the nitrocellulose paper byusing a Finpipette (Labsystem, Helsinki, Finland). Thepapers were then dried at 37°C for 15 min, immersed intriethanolamine-buffered saline (TBS; pH 7.5) containing0.01% Tween 20 and 5% skim milk powder (SMP; Marvel)(TBSTSMP), and incubated at 37°C for 15 min to block anyunreacted binding sites. After a 5-min wash in PBS, thepapers were immersed in a predetermined (1:20) dilution ofthe MAb in TBSTSMP and incubated at 37°C for 60 min. Thenitrocellulose papers were then washed in three changes ofPBS for 15 min and incubated in the optimal dilution (1:200)of horseradish peroxidase-conjugated rabbit anti-mouse im-munoglobulin G (Dakopatts, Denmark) in TBSTSMP at 37°Cfor 30 min. After another 15-min wash in PBS, the paperswere immersed in freshly prepared enzyme substrate, 4-chloro-1-naphthol, prepared as a 3-mg/ml stock solution inanhydrous methanol. Before use, 10 ml of TBS and 4 FLl of30% hydrogen peroxide were added to 2 ml of the stocksolution. The enzyme reaction was stopped after 10 min bywashing the paper in tap water. The papers were then airdried and visually assessed for blue color development. In anattempt to determine the sensitivity of this test, tissue-culture-grown PPRV with a titer of 104-5 50% tissue infectivedoses per ml was titrated by doubling dilution in the dot EIAas described above.Comparison of dot EIA and standard indirect EIA. Thirty-

eight randomly selected tissue homogenates were screenedfor PPRV antigen by the dot EIA and by an indirectmicrotiter plate EIA. The method of the indirect ELISA willbe described elsewhere (Obi et al., in preparation). Briefly,microtiter EIA plates (NUNC Immunoplate 1; GIBCO-Europe, Ltd.) were sensitized in duplicate with a 1:10dilution of homogenates in carbonate-bicarbonate buffer (pH9.6); and following washing in PBS and drying on papertowels, hybridoma supernatant containing MAbs to thePPRV was added and the plate was incubated at 37°C for 60min. The antigen-antibody reaction was detected with horse-radish peroxidase-conjugated rabbit anti-mouse immuno-globulin G (1:2,000) in PBS containing 5% SMP and 0.01%Tween 20, followed by the enzyme substrate o-phenylene-diamine. The enzyme reaction was stopped with 1 M sulfuricacid, and the plate was read spectrophotometrically (Ti-tertek Multiscan; Flow Laboratories) at a wavelength of 492nm. Samples giving optical densities twice the mean of thatof the negative control antigen were regarded as positive.

Effects of NP-40 on dot EIA. In an attempt to evaluate theeffect of Nonidet P-40 (NP-40) (BDH Chemicals Ltd., Poole,England) on the detectability of PPRV antigen, 120 homoge-nates were tested by the dot EIA with the washing stepsdone with PBS and then with PBS to which 1% NP-40 hadbeen added. In addition, the homogenates were treated with1% NP-40 for 60 min at room temperature and then centri-fuged at 1.84 x g for 15 min; the supernatant was dotted ata 1:10 dilution in PBS onto the nitrocellulose papers, and thedot EIA was performed as described above.

RESULTS

Antigen detection by dot EIA. Of the 124 tissue homoge-nates tested by the dot EIA, 103 (83.1%) were positive forPPRV antigens. Positive homogenates gave distinct bluedots within 10 min (Fig. 1), while the negative ones and thenegative control antigen failed to give any distinct colorreaction. Results were assessed semiquantitatively fromintense blue (4+) to no color or barely perceptible (-) (Fig.2). The detectability of PPRV antigen from the various

12 3 4 5 6

B

FIG. 1. Dot EIA reactions with six PPRV-positive tissue ho-mogenates (row A, 1 to 6), one tissue culture-negative controlantigen (row B, 1), and five negative homogenates (row B, 2 to 6).

organs is summarized in Table 1. It can be seen that highpercentages of all the tissues except the trachea were posi-tive. Semiquantitative analysis of the results showed that 39(37.9%) of the positive samples were scored 1+, 34 (33%)were scored 2+, 26 (25.2%) were scored 3+, and only 4(3.9%) were scored 4+.

It was also observed that 63.2% of the lung samples,16.7% of the tracheal samples, 42.9% of the mediastinallymph node samples, 66.7% of the rectal samples, 42.9% ofthe splenic samples, 42.3% of the mesenteric lymph nodesamples, 66.7% of the tonsilar samples, 30% of the small-intestinal samples, 66.7% of the colonic samples, and 63.6%of the cecal samples were assessed as 2+ and 3+. Whentissue culture PPRV was titrated, clearly visible color reac-tions were seen at up to a 1:20,480 dilution.

Detection of PPRV antigen by dot EIA and standard indi-rect EIA. PPRV antigen was detected in 33 (86.8%) of the 38randomly selected tissue homogenates by the dot EIA and in31 (81.6%) of these homogenates by the standard indirectmicroplate EIA. These results are summarized in Table 2.

Effect of NP-40 on PPRV antigen detection. Of a total of 120tissue homogenates tested, 105 (87.5%) were positive for

1 2 3 4

A *_ ou&

R

c'- ,. oz e* o

E !FIG. 2. Semiquantitative evaluation of dot EIA reactions for

PPRV antigen detection. Reactions: 4+, Ai (lungs), A2 (mesentericlymph nodes), and D2 (small intestine); 3+, B3 (lungs), C4 (rectum),Dl (spleen), D4 (cecum), El (spleen), and E6 (lungs); 2+, A3(lungs), A4 (colon), A5 (tonsils), A6 (trachea), Bi (lungs), B5(cecum), B6 (mesenteric lymph node), C2 (spleen), C3 (mesentericlymph node), C5 (mediastinal lymph node), C6 (mesenteric lymphnode), D3 (lungs), D6 (small intestine), E3 (colon), E4 (mesentericlymph node), and E5 (rectum); 1+, B2 (lungs), B4 (lungs), D5(trachea), and E2 (spleen); -, Cl (spleen). Reactions are explainedin the text.

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TABLE 1. Results of detection by dot EIA of PPRV antigen in different organs of goats naturally infected with PPRV

Sample No. (%) positive No. (%) of positive samples assessed visually asa:(no. tested by dot EIA) by dot EIA 1+ 2+ 3+ 4+

Lung (19) 18 (94.7) 5 (27.8) 5 (27.8) 7 (38.9) 1 (5.6)Trachea (12) 8 (58.3) 5 (71.4) 1 (14.3) 1 (14.3) 0 (0)Mediastinal lymph node (7) 7 (100) 4 (57.1) 3 (42.9) 0 (0) 0 (0)Rectum (9) 8 (88.9) 2 (25.0) 2 (25.0) 4 (50.0) 0 (0)Spleen (14) 10 (71.4) 4 (40.0) 3 (30.0) 3 (30.0) 0 (0)Mesenteric lymph node (26) 21 (80.8) 8 (38.1) 7 (33.3) 4 (19.1) 2 (9.5)Tonsil (6) 5 (83.3) 1 (20.0) 3 (60.0) 1 (20.0) 0 (0)Small intestine (10) 8 (80.0) 4 (50.0) 3 (37.5) 0 (0) 1 (12.5)Colon (9) 8 (88.9) 2 (25.0) 3 (37.5) 3 (37.5) 0 (0)Cecum (12) 11 (91.7) 4 (36.4) 4 (36.4) 3 (27.3) 0 (0)

Total (124) 103 (83.1) 39 (37.9) 34 (33.0) 26 (25.2) 4 (3.9)a Reactions are explained in the text.

PPRV antigen when PBS alone was used in the washingsteps, while 93 (77.5%) were positive when NP-40 was addedto the washing solution. The addition of NP-40 did notimprove the intensity of color development. In addition,when the homogenates were treated with NP-40 to 1%(vol/vol) before being dotted on the nitrocellulose paper,only 85 (70.8%) were positive for PPRV antigen by the dotEIA. It was further observed that NP-40 treatment pre-vented the formation of distinct dots, thus resulting invarious degrees of dispersion of homogenate dots from thepoint of application, as well as decreased intensity of colordevelopment (Fig. 3).

DISCUSSION

The isolation of PPRV in such countries as Oman (19),Sudan (8), and the United Arab Emirates (9), hithertothought to be free of the infection, has raised the question ofthe precise geographical spread of the infection (19). For aneffective PPRV diagnosis, as well as for epidemiologicalsurveillance, a cheap, sensitive, and specific diagnostic testsuch as the ELISA or the dot EIA is therefore imperative.The results obtained in this study showed that PPRV

antigen was detectable in 83.1% of 124 infected tissuehomogenates which were examined by the dot EIA. This testdetected PPRV antigen in a very high proportion of all theorgans from which the tissues were taken, despite the factthat some of the homogenates had been stored for up to 24months at -20°C with occasional power interruptions. Be-cause these tissues were from animals which died at various

TABLE 2. Comparison of dot EIA and standard indirectmicroplate ELISA for detecting PPRV antigen in

infected caprine tissue homogenates

Sample No. (%) positive by:(no. tested) Dot EIA Indirect EIA

Lung (8) 8 (100) 7 (87.5)Spleen (10) 6 (60.0) 8 (80.0)Esophagus (3) 3 (100) 3 (100)Cecum (3) 3 (100) 2 (66.7)Mesenteric lymph nodes (7) 6 (85.7) 4 (57.1)Colon (3) 3 (100) 3 (100)Trachea (4) 4 (100) 4 (100)

Total (38) 33 (86.8) 31 (81.6)

stages of disease, the severity of which also varied, it is notpossible to unequivocally suggest preferred specimens fordiagnosis based on this study. However, it has been sug-gested on the basis of results of the agar gel precipitation testwith tissues from experimentally infected goats that thepreferred specimens for PPRV antigen detection are thecolon, cecum, rectum, spleen, small intestine, and mesen-teric lymph nodes, in that order (15). When one considersthe number of samples that gave definitively distinct colorreactions (2+ or 3+) in this study, it seems that the preferredsamples are the colon, rectum, tonsils, cecum, and lungs.

In addition, because the homogenates were not sterile andbecause of long storage at -20°C with occasional powerinterruptions it was not possible to titrate the samples andcorrelate the dot EIA results with infectious titers. Never-theless, this test detected PPRV with a titer of 104-5 tissueculture infective doses per ml at up to a 1:20,480 dilution.The finding that the negative uninfected control antigen

failed to give a color reaction suggests that the dot EIA is notonly sensitive but also specific. However, it was not clearfrom this study whether the dot EIA could differentiatePPRV from the closely related RPV. Further studies usingthe battery ofMAbs to PPRV at our disposal to elucidate thisaspect are under way; our previous results (9) indicated that

rr

FIG. 3. Effects on detectability of PPRV antigen by dot EIA oftreatment of homogenates with NP-40 to 1% (vol/vol) before beingdotted on nitrocellulose paper. Note the dispersion of the homoge-nate dots from the points of application and the decreased intensity.

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the only MAb which differentiated PPRV from RPV was ananti-RPV MAb.

It has been reported that the relative sensitivities andspecificities of the dot EIA and the microplate EIA were ofthe same magnitudes in the detection of antibodies to pseu-dorabies virus (2). In the present study, the dot EIA detectedPPRV antigen in 33 of 38 homogenates while the indirectmicroplate ELISA detected PPRV antigen in 31 samples.The advantages of the dot EIA over the microplate EIAinclude the more rapid and efficient antigen-binding capacityof nitrocellulose paper over polystyrene plates (2, 4, 5), thebetter signal-to-noise ratios resulting in fewer false-positiveresults, and the shorter incubation times (18), as well as thefact that the dot EIA is inexpensive and reagent conservative(16). In addition, the color development in the dot EIA isstable and allows for permanent record keeping of the testresult (17).

It has been shown that nitrocellulose paper dotted withviral antigen can be stored for long periods at room temper-ature without loss of activity (12). In developing countrieswhere transportation networks and cold-storage facilitiesmay be limited, it is suggested that clinical specimens andtissue homogenates could be dotted on nitrocellulose paper,air dried, and then dispatched to even the most basiclaboratory for the dot EIA.The finding in this study that nonionic detergent NP-40

decreased the sensitivity of the dot EIA is noteworthy.Nonionic detergents such as NP-40, Triton X-100, andZwittergent have been used in the processing of viral anti-gens for the standard EIA (3) and in the renaturing steps ofsodium dodecyl sulfate gels before transfer of the proteins tonitrocellulose paper (7). Such detergents have been reportedto decrease the binding capacity of proteins to nitrocellulosepaper (7). Preliminary results (T. U. Obi, unpublished)similarly have shown that NP-40 at high concentrationsdecreased the ability of PPRV and RPV antigens to bind topolystyrene plates. However at antigen dilutions above 1:50,this effect was eliminated. The decreased sensitivity of thedot EIA with the NP-40-treated homogenates may haveresulted from the fact that the homogenates were screened ata 1:10 dilution.The dot EIA is a good example of the adaptation of

sophisticated laboratory-based technology to field situationsor for use in basic laboratories in which the purchase ofexpensive equipment such as a spectrophotometer may notbe feasible.

ACKNOWLEDGMENTS

The technical and secretarial assistance of E. Akhenemhen, T.Emiola, and J. Efrenie is gratefully acknowledged.

LITERATURE CITED1. Afshar, A., and D. J. Myers. 1986. Simple and rapid DOT-

enzyme immunoassay for visual detection of rinderpest antibod-ies in bovine and caprine sera. Trop. Anim. Health Prod.18:209-216.

2. Afshar, A., P. F. Wright, and G. C. Dulac. 1986. Dot-enzyme

immunoassay for visual detection of antibodies to pseudorabiesvirus in swine serum. J. Clin. Microbiol. 23:563-567.

3. Anderson, J. 1984. Use of monoclonal antibody in a blockingELISA to detect group specific antibodies to bluetongue virus.J. Immunol. Methods 74:139-149.

4. Beutin, L., L. Bode, T. Richter, G. Peltre, and R. Stephan. 1984.Rapid visual detection of Escherichia coli and Vibrio choleraeheat-labile enterotoxins by nitrocellulose enzyme-linked im-munosorbent assay. J. Clin. Microbiol. 19:371-375.

5. Beyer, C. F. 1984. A "dot-immunobinding assay" on nitrocel-lulose membrane for the determination of immunoglobulin classof mouse monoclonal antibodies. J. Immunol. Methods 67:79-87.

6. Chand, P., H. V. Batra, and J. R. Sadana. 1988. Detection ofbrucella specific protein-A reactive antibodies in buffaloes byDOT-enzyme-linked immunosorbent assay. Vet. Rec. 122:162-163.

7. Dunn, S. D. 1986. Effects of the modifications of transfer buffercomposition and the renaturation of proteins in gels on therecognition of proteins on Western blots by monoclonal anti-bodies. Anal. Biochem. 157:144-147.

8. El Hag Ali, B., and W. P. Taylor. 1984. The isolation of pestedes petits ruminants virus (PPRV) from the Sudan. Res. Vet.Sci. 36:1-4.

9. Furley, C. W., W. P. Taylor, and T. U. Obi. 1987. An outbreakof peste des petits ruminants in a zoological collection. Vet.Rec. 121:443-447.

10. Gibbs, E. P. J., W. P. Taylor, M. J. P. Lawman, and J. Bryant.1979. Classification of peste des petits ruminants virus as thefourth member of the genus Morbillivirus. Intervirology 2:268-274.

11. Hamdy, F. M., A. H. Dardiri, O. Nduaka, S. S. Breese, andE. C. Ihemelandu. 1976. Etiology of stomatitis pneumoenteritiscomplex in Nigerian dwarf goats. Can. J. Comp. Med. 40:276-284.

12. Heberling, R. L., and S. S. Kalter. 1986. Rapid dot-immunobind-ing assay on nitrocellulose for viral antibodies. J. Clin. Micro-biol. 23:109-113.

13. Majiyagbe, K. A., D. R. Nawathe, and A. Abegunde. 1984.Diagnosis of PPR infection using the immuno-electro-osmo-phoresis (IEOP) technique. Rev. Elev. Med. Vet. Pays Trop.37:11-15.

14. Obi, T. U., M. O. Ojo, O. A. Durojaiye, O. B. Kasali, S.Akpavie, and D. B. Opasina. 1983. Peste des petits ruminants(PPR) in goats in Nigeria: clinical, microbiological and patho-logical features. Zentralbl. Veterinaermed. Reihe B 30:751-761.

15. Obi, T. U., and D. Patrick. 1984. The detection of peste despetits ruminants (PPR) virus antigen by agar gel precipitationtest and counter-immunoelectrophoresis. J. Hyg. 93:577-586.

16. Pappas, M. G., R. Hajkowski, and W. T. Hockmeyer. 1983.Dot-enzyme-linked immunosorbent assay (DOT-ELISA): a mi-cro technique for the rapid diagnosis of visceral leishmaniasis. J.Immunol. Methods 40:276-284.

17. Porter, D. D., and H. G. Porter. 1984. A glucose oxidaseimmunoenzyme stain for the detection of viral antigens orantibody on nitrocellulose transfer blots. J. Immunol. Methods72:1-9.

18. Suresh, M. R., and C. Milstein. 1985. A direct antigen-bindingassay to screen hybridoma supernatants. Anal. Biochem. 151:192-195.

19. Taylor, W. P. 1984. The distribution and epidemiology of pestedes petits ruminants. Prev. Vet. Med. 2:157-166.

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