Vol. 23, No. 1JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1986, p. 109-1130095-1137/86/010109-05$02.00/0Copyright C 1986, American Society for Microbiology

Rapid Dot-Immunobinding Assay on Nitrocellulose forViral Antibodies

R. L. HEBERLING* AND S. S. KALTERDepartment of Virology and Immunology, National Institutes of Health and World Health Organization CollaboratingCenter for Reference and Research in Simian Viruses, Southwest Foundation for Biomedical Research, San Antonio,

Texas 78284

Received 17 July 1985/Accepted 4 October 1985

A procedure is described for the routine laboratory diagnosis of viral serum antibodies. Antigens are dottedon nitrocellulose strips or sheets, and sera are applied on absorbent paper strips. Antigen-antibody complexesare detected with enzyme-conjugated antiglobulin and development of a colored, insoluble substrate product.The test allows processing of multiple sera in one 3- to 5-h operation and is equal to or more sensitive thanserum neutralization, hemagglutination inhibition, and fluorescent antibody assays. Highly infectious virusesinactivated with a psoralen derivative and long-wavelength UV light irradiation can be used as antigens,allowing the study of human pathogens. Although the test detects cross-reacting, group-specific herpesvirusantigens, the intensity of the antibody reaction is greatest with type-specific antigens. Preliminary data suggestthat the technique will be useful for the rapid typing of viruses from clinical specimens.

There is a continuous need for more rapid, sensitive, andspecific procedures for the diagnosis of viral diseases. Thisneed stems from the realization that a definitive diagnosis ishelpful in appropriate patient management and in evaluatingthe use of newer therapeutic regimens now available. Properviral diagnosis is not only important in human medicine butis also necessary in animal colony management, especiallywhere experimental animals are maintained and the controlof infectious diseases is mandatory.Although there are a number of procedures that provide

valuable diagnostic information, many are deficient for oneor another reason. In general, the important factors are timeinvolved in obtaining results and specificity and sensitivity ofthe assay. In addition, reagents which are difficult or costlyto prepare and the need for specialized equipment (fre-quently an important consideration for field studies) oftenmake these procedures the venue of specialty laboratories orimpractical for routine purposes. The use of radioisotopes insome procedures also presents problems of handling wastesand the cost of equipment needed in such assays (3, 15).Another concern is the use of infectious viral antigens inserological assays; tests which permit the use of inactivatedantigens would be more desirable.Enzyme-linked immunosorbent assays have become a

standard for the rapid detection of viral antigens and anti-bodies and, as now performed, can meet most of the criteriadescribed. However, these assays generally use polystyreneor other materials which have a rather low binding capacityfor proteins. The use of nitrocellulose for the detection ofproteins in a polyacrylamide gel following electrophoresis bythe Western blot technique has demonstrated the superiorbinding capacity of this substrate (16). Recently, this prop-erty has been exploited by several laboratories to dot crudeviral antigens directly on nitrocellulose for in situ visualiza-tion of antigen-antibody complexes with enzyme-linkedantiglobulins and a substrate which forms an insoluble,colored end product (1, 6). This approach has been used byus for the routine assay of viral antibodies in human and

* Corresponding author.

nonhuman primate sera, as well as for the identification ofunknown viral antigens in culture fluids.By using viral antigens dotted on nitrocellulose sheets, we

developed a simple enzyme-linked immunoassay (dot-immunobinding assay [DIA]) for the rapid detection of viralantibodies without the need for containment or other specialequipment. Antigens associated with viruses (herpesvirussimiae and herpes simplex virus) considered to be highlypathogenic for humans were inactivated with a psoralenderivative and long-wavelength UV light irradiation.

MATERIALS AND METHODS

Viral antigens. Cell cultures for virus production weregrown in modified Eagle medium (DM 313; KC Biologicals,Lenexa, Kans.) containing 10% fetal bovine serum and 50 ,ugof gentamicin per ml. Herpes simplex virus type 1 (Mayo)(HSV-1), herpes simplex virus type 2 (MS) (HSV-2),herpesvirus simiae (E2490) (HBV), herpesvirus tamarinus(MV5-4PSL) (HT), and SA8 (B264) were retrieved from theNational Institutes of Health and World Health OrganizationCollaborating Center for Reference and Research in SimianViruses repository (10, 11) and grown in Vero cells in mediumwithout fetal bovine serum. Herpesvirus saimiri was grown inowl monkey kidney cells with medium containing 10% fetalbovine serum. Simian rotavirus SAl1 was grown in MA104cells in medium containing 0.5 ,ug of trypsin IX (SigmaChemical Co., St. Louis, Mo.) per ml without FBS. Infectedcultures were harvested when cytopathology involved theentire cell sheet. The cultures were frozen and thawed threetimes and clarified by low-speed centrifugation (2,000 rpm, 30min). HSV-1, HSV-2, HBV, HT, and SA8 were used withoutfurther purification. Herpesvirus saimiri and SA 1 virus werecentrifuged through a 20%/60% discontinuous sucrosegradient for 2 h at 27,000 rpm in an SW27 rotor (BeckmanInstruments, Inc., Fullerton, Calif.). The virus band wascollected, diluted in phosphate-buffered saline (pH 7.2)(PBS), and pelleted for 2 h at 27,000 rpm. The virus pellet wassuspended in 0.15 M NaCI-0.002M EDTA-0.01 M Tris buffer(pH 9.0) at a 100x concentration. All of these antigens wereused undiluted or diluted 1:2 when inactivated. Uninfectedcell cultures were processed in the same manner as viral

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110 HEBERLING AND KALTER

antigens for controls. Measles (Philadelphia) hemagglutinin,human cytomegalovirus (AD169) (CMV) complement-fixingantigen, and controls were purchased from MA Bioproducts(Walkersville, Md.) and diluted 1:20 and 1:5, respectively, inPBS before use. All antigens were stored at -70°C.

Psoralen inactivation of viral antigens. HSV-1, HSV-2,HBV, HT, and SA8 were inactivated with the psoralenderivative trioxsalen 4'-aminomethyl hydrochloridemonohydrate (10 ,ug/ml; Calbiochem-Behring, La Jolla,Calif.) in the presence of long-wavelength UV light (F15-T8BL; General Electric Co., Schenectady, N.Y.) irradiation asdescribed by Redfield et al. (12) and recently modified by us(R. L. Heberling and S. S. Kalter, Abstr. Annu. Meet. Am.Soc. Microbiol. 1985, S10, p. 21). A 3- to 5-min irradiationwith an intensity of approximately 4,000 jiW/cm2 was ade-quate to destroy 108 infectious doses per ml when the viruswas diluted 1:2 in PBS. Virus preparations to be used fordotting on nitrocellulose were irradiated for 20 min as anadded safety factor and used without further dilution. It wasexperimentally determined that this amount of irradiationdid not affect the antigenicity of these preparations. Al-though HT and SA8 are not known to be human pathogens,they were inactivated to allow comparison of results withherpes simplex virus- and HBV-related herpesviruses.

Sera. Five hundred and sixteen sera (the rhesus monkeysera tested for HBV included those tested for HSV-1)derived from various primate (including human) sourceswere used in this study and were obtained from the reposi-tory of the National Institutes of Health and World HealthOrganization Collaborating Center for Reference and Re-search in Simian Viruses (Southwest Foundation for Bio-medical Research). These sera (see Table 1) had beensubmitted for diagnostic purposes and have been stored at-20°C for reference. The sera were diluted 1:3 as an initialdilution in other serological assays and were used at thatdilution in the DIA.SN, HI, and FA assays. The procedures used for the serum

neutralization (SN), hemagglutination inhibition (HI), andfluorescent antibody (FA) assays have been described pre-viously (2, 7, 9).DIA. The procedure described by Hawkes et al. (6) was

simplified and modified to allow the simultaneous assay ofmultiple serum samples for antibody to one or more antigensin 3 to 5 h. Plain white nitrocellulose (NC) sheets (BA85,0.45 jim [pore size]; Schleicher & Schuell, Inc., Keene,N.H.) were cut to the desired size and marked with ink toidentify antigen-dotting sites, or the NC was purchased withan imprinted 3-mm grid (BA85/21 SD). Generally, sheets (8by 11 cm) with 96 antigen dots 1 cm apart or gridded sheets(8.8 by 8.8 cm) with dots every third grid (81 per sheet) wereused. The sheets with grids proved to be more convenient touse. One-microliter samples of virus and control antigenswere spotted on the NC with a syringe (The Hamilton Co.,Reno, Nev.). After 0.5 h at room temperature to allow theantigen to dry, the NC sheet was submerged in 5% nonfatdry milk in PBS containing 0.01% Antifoam A (Sigma) and0.0001% merthiolate to block the unreacted protein-bindingsites. The blocking procedure could be extended overnight.Other blocking agents such as horse, goat, or bovine serumhave been used, but this milk preparation (BLOTTO) wasfound to be most effective (3). After 1 h at room temperature,the BLOTTO was rinsed off with PBS-Tween 20 (0.1%;Sigma). The NC sheet may be used at this point or dried andstored at 4°C for future use.For use, the NC sheet was placed on blotting paper (no.

470; Schleicher & Schuell) saturated with PBS-Tween 20 to

keep it moist. Test sera previously diluted in PBS for SN orother comparative tests were applied by saturating absor-bent paper strips or disks 3 mm in width (lintless paperstrips; Gelman Sciences, Inc., Ann Arbor, Mich.) and plac-ing them on the NC sheet over the test antigen dots. (Contactbetween strip ends is to be avoided.) The sheet was thenplaced in a humidified incubator for 1 h at 37°C, washedthree times with shaking for 5 min with PBS-Tween 20, andsubmerged in goat anti-human immunoglobulin G (IgG)conjugated with alkaline phosphatase (Kirkegaard and PerryLaboratories, Gaithersburg, Md.) diluted 1:1,000 in PBS.This preparation may be used for both human and nonhuman(Old World) primates because of the close antigenic relation-ship of their immunoglobulins (14). In testing New Worldmonkey and rabbit sera, the less species-specific staphylo-coccal protein A-enzyme conjugate (horseradish peroxidase[Kirkegaard and Perry] diluted 1:1,000 in PBS [pH 8.1]) wasused, because it reacts with IgG from a variety of mamma-lian species. This allowed the testing of primate immuno-globulin species which are not closely related to human IgG(13). Following 1 h of incubation at 37°C, the sheet waswashed three times for 5 min in PBS-Tween 20 and sub-merged in substrate. The substrate used for the alkalinephosphatase conjugate was naphthol AS-MX phosphate withfast red (1). This produced a red spot at the site of antigen-antibody reaction. The substrate for horseradish peroxidasewas 4-chloro-1-naphthol-hydrogen peroxide, yielding a bluespot (6). In both instances, the intensity of color was directlyrelated to the amount of conjugate fixed to the antigen-antibody complex. The avidin-biotin-glucose oxidase system(Vectastain ABC kit; Vector Laboratories, Inc., Burl-ingame, Calif.) was also used successfully in the DIA. Thesubstrate was allowed to react until color development onpositive controls was adequate (5 to 15 min at room temper-ature). The substrate was then washed off with tap water,and the NC was allowed to dry before visual interpretation.Washing had to be thorough; otherwise, the enzyme-substrate reaction continued during drying, and nonspecificstaining occurred.

Controls consisted of uninfected cell culture antigens andknown positive and negative sera. A positive reaction was adetectable colored spot with an intensity greater than thoseof the cell control and negative sera. Control dots weregenerally negative or barely perceptible.

RESULTSFour members of the Alphaherpesvirinae subfamily (11), a

CMV (Betaherpesvirinae), herpesvirus saimiri (Gam-maherpesvirinae), measles virus, and simian rotavirus SAllwere used in the DIA in a comparative study of relatedviruses and to indicate the versatility of the test for use withvarious types of virus. Table 1 lists the results of testing 680human and nonhuman primate sera by the DIA and SN, HI,or FA assays. The overall sensitivity (SN, HI, or FA andDIA positive) of the DIA was 97.8% (317 of 324). All of the231 Alphaherpesvirinae SN-positive sera were also positivefor antibody by DIA. Of 46 herpesvirus saimiri SN-positivesera, 1 was not detected by DIA (sensitivity, 97.8%) and 6 of37 rotavirus SN-positive sera were negative by DIA (sensi-tivity, 83%). Sera positive for CMV (FA) or measles (HI)antibody were all positive by DIA.A fairly large number of rhesus monkey sera were positive

by DIA and negative by SN when tested against HBV orHSV-1 antigens (specificity, 88.8 and 89.16%, respectively).Three chimpanzee sera were positive for CMV by DIA butnegative by FA.

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DOT-IMMUNOBINDING ASSAY 111

When individual sera, including human and rhesus, Afri-can green, and squirrel monkey sera, were tested by DIAagainst all four of the Alphaherpesvirinae, the positivehuman and Old World monkey sera reacted to all of theantigens. However, positive human and rhesus and Africangreen monkey sera reacted most strongly with antigens ofthe virus common to each species, i.e., human with HSV-1,rhesus monkey with HBV, and African green monkey withSA8 (Fig. 1). Negative sera reacted with none of the anti-gens. Positive New World squirrel monkey sera reacted onlywith HIT antigens. Therefore, the Old World species, includ-ing humans, showed a group-specific reaction with a degreeof specificity indicated by the intensity of the reaction. NewWorld squirrel monkey sera failed to react with the group-

specific antigens common to herpesviruses of Old Worldprimates.

Sensitivity of the DIA. When rhesus monkey sera weretitrated to an endpoint for SN and DIA antibody againstHBV and HSV-1, several points were noted (Table 2). Acomparison of DIA serum titers with SN or FA indicatedthat the DIA was at least 10 times more sensitive. Thereseemed to be no direct correlation between SN and DIAtiters, except that high SN titers tended to give higher DIAtiters. Although the HSV-1 SN titers were higher than theHBV titers in five of the sera titrated, the DIA HBV titerswere uniformly higher than those obtained with HSV-1antigen. Therefore, the DIA is probably a more accuratereflection of a response to HBV infection with a heterolo-gous cross-reacting HSV-1 antibody response than is the SNtest. Further indication that the DIA is a highly sensitive testwas obtained in a comparison of two sera for CMV antibodywith FA titers of 1:10 and 1:40, which showed DIA titers of>1:500.

Identification of viral antigens. Preliminary studies on theuse of hyperimmune polyclonal rabbit sera for the detectionand identification of viral antigens were done with cellculture antigens of HSV-1, HSV-2, HBV, SA8, and HTdotted on NC. The protein A-horseradish peroxidase conju-gate was used to detect antigen-rabbit antibody complexes.As with the primate test sera, cross-reactions were notedamong the HSV-1, HSV-2, HBV, and SA8 viruses, buthomologous reactions were most intense. The HT rabbitserum reacted only with its homologous viral antigens.These results are similar to those obtained with the SN test.

TABLE 1. Comparison of SN and DIA for detection ofviral antibody

Virus

No.

Serum source ofsera

tested

No. of No. ofSN-positive DIA-positivesera (%)a sera (%)'

HBV Rhesus monkey 240 115 (47.9) 129 (53.8)HSV-1 Rhesus monkey 164 67 (40.9) 77 (47.0)HSV-1 Human 27 23 (85.2) 23 (85.2)SA8 African green 48 22 (45.8) 22 (45.8)

monkeyHT Squirrel monkey 40 4 (10.0) 4 (10.0)Herpesvirus Squirrel monkey 98 46 (46.9) 45 (45.9)

saimiriCMV Chimpanzee 8 2b (25) 5 (62.5)Measles Rhesus monkey 16 8' (50) 10 (62.5)SA1l Baboon 39 37 (94.9) 31 (79.5)

a There was 93.5% concordance between SN and DIA results.b FA positive.r HI positive.

ANTi'IGENS

00

'-4 C'1l

(- := :r cncr

>: > > 00u )r. nP < H

EH = En=a:

1

2

3

4

5

6

7

8

AgC

A

Human

A

Rhesus

A

African

Green

FIG. 1. DIA of herpesvirus antibodies in eight different humanand rhesus and African green monkey sera with an alkaline phos-phatase-goat anti-human IgG conjugate. All three no. 1 sera and theno. 5 rhesus and African green monkey sera were negative by DIAand SN. The no. 5 human serum was DIA positive but SN negative.Arrows indicate homologous antibody reactions.

No attempt was made to determine the minimum numberof viral particles required for detection, but as little as 103infectious doses of virus was detected in this system whenvirus dilutions were dotted. Although preliminary in nature,these studies suggest that the DIA can be used as a sensitiveassay for identifying unknown viral isolates and possibly fordetecting specific viral antigens in clinical specimens. Theuse of monoclonal antibodies should improve the specificityof this test, if not the sensitivity.Other parameters of the DIA. The procedure described by

Hawkes et al. (6) differs from that reported here principally

TABLE 2. Comparison of SN and DIA antibody titers for HBVand HSV-1 in rhesus monkey sera

Serum HBV HSV-1 HBV HSV-1no.

1 1:24 1:48 1:800 1:2002 1:24 21:96 1:800 1:8003 1:24 .1:96 1:800 1:4004 1:3 1:12 1:200 1:1005 1:3 1:24 1:800 1:4006 <1:3 <1:3 1:200 1:1007 21:96 1:24 1:400 1:4008 1:48 1:6 .1:1,600 1:8009 1:24 1:12 1:400 1:20010 1:24 1:12 1:800 1:200

- ... I[ I I I

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112 HEBERLING AND KALTER

in our use of paper disks or strips to apply test sera to antigendots. This allows the testing of multiple sera on a single pieceof NC. A major concern in using this technique was thepotential for running or diffusing of sera from one serum toanother, causing false-positive results. Although this wasobserved when sera were dropped directly onto antigens, aspread beyond the edge of the paper strips of more than 2mm was not detected when antigen dots were spaced 3 mmapart. Therefore, with a distance of 6 mm between strips, nomixing of sera occurs.As observed by Hawkes et al. (6), binding of antigen to

NC appears to be instantaneous. This is indicated by the sizeof the antigen dot, which never exceeds the limit of the 3-mmgrid, even though a 1-,ul drop wets an area larger than this.For this reason, increasing the volume of antigen applied ina dot does not proportionately increase the intensity of theobserved color reaction. Instead a larger dot of similarintensity occurs. It is better to apply multiple small drops ofantigen with drying between applications.Once the antigen was dried on the NC sheet, it was quite

stable. Dry sheets stored in sealed plastic bags at roomtemperature showed very little loss in activity after a month.Storage at refrigerator or freezer temperatures increased thisshelf life. NC sheets should be thoroughly dried beforerefrigerator or room temperature storage to discouragegrowth of molds, This occjirred despite the presence ofmerthiolate in the BLOTTO.

DISCUSSIONThe diagnosis of viral infections requires the use of rapid,

sensitive assays if they are to be of value in the detection ortreatment of disease. Ideally, the test should be useful in thesmallest laboratory, where sophisticated equipment andhighly trained technical support may not be available, or forfield conditions. In addition, it should be useful for antibodydeterminations as well as for the early detection of viralantigens in clinical specimens. The DIA described here withNC as a substrate for antigen adsorption can be completed in3 to 5 h and allows the testing of one or many sera at a timeby simply cutting the NC sheet to accommodate the desirednumber of antigen dots. This can be done before or afterantigen application.For antibody detection, the antigens used have been

relatively crude virus preparations for the most part, con-sisting of clarified culture media or commercially preparedHI and complement fixation antigens. However, in thisstudy herpesvirus saimiri and SAl antigens were partiallypurified and concentrated. Although crude preparations ofthese viruses were used successfully in preliminary trials,concentration enhanced the sensitivity of the results withthese viruses and may be required in the preparation of otherantigens. Use of subvirion proteins would provide morespecificity but would limit the availability of the test. Inaddition, sensitivity might be lost through failure to detectother pertinent antibodies.Our results indicate that the DIA is a highly sensitive test.

The somewhat lower correlation of SN and DIA results withrotavirus antigen cannot be explained, except that 5 of 6SN-positive, DIA-negative sera had SN titers of 1:6 orlower. However, in detecting other antibodies, the DIAprocedure generally indicated more positive results than didthe SN test.Although the sensitivity in herpesvirus antibody assay was

almost 100%, specificity for HBV and HSV-1 in rhesusmonkey sera was somewhat lower (89%). The higher anti-body titers obtained by DIA suggest that false-positive

results may be a reflection of this increased sensitivity. Itmay be, however, that the DIA detects antibody to a broaderspectrum of virion antigens, some of which are not associ-ated with neutralization of viral infectivity. The presence ofthese nonneutralizing antibodies would also explain thefalse-positive results. Similar reasoning can be applied to themeasles and CMV results.The results of DIA testing of rhesus monkey and human

sera for HBV and HSV-1 antibody are interesting in com-parison with SN results. In a previous study, it was shownthat monkey sera with SN titers of >1:6 for B virus generallyhad HSV-1 titers of equal or greater magnitude (8). Humansera with IISV-1 antibody show little or no HBV antibody.In contrast, the DIA results always showed higher titers toHBV in monkey sera, and HSV-1-positive human seraalways showed a reaction with HBV antigen, but the titerswere lower than the homologous HSV-1 titers.Whereas the specificity of the herpesvirus antibody assays

was good in relation to detecting positive and negative sera,group-specific h,erpesvirus antigens were detected. How-ever, subjective evaluation of the intensity of the coloredenzyme produAt §uggests that the type-specific reaction isalways the most intense. In this regard, DIA appears to bemore specific than the SN test; however, HSV-1 and HSV-2reactions have not been evaluated in human sera fro,mpatients with authenticated infections. Diagnosis of mixedinfections would require the use of type-specific antigens.Although the detection of herpesvirus antibody in nonhu-

man primate sera is emphasized herein, preliminary studieswith a number of other viral antigens (influenza, parainfluen-za, adenovirus, rubella) indicate the utility of the DIA for therapid screening of sera against any number of viral antigens.In these studies, we purchased commercial complementfixation and HI antigens, when possible, to determine theusefulness of these readily available antigens. Nonspecificreactions were sometimes observed with tissue controls, butthese could be diluted out and still retain specific reactivity.Hawkes et al. also used commercially prepared antigens intheir studies (6). The successful use of these relatively crudepreparations indicates the potential for the use of this test inmost laboratories as a general serological diagnostic proce-dure.Of practical importance is the stability of the viral antigens

after drying on the NC membrane. The ability to maintainthese sheets for long periods at ambient temperatures with-out significant lo'ss of activity indicates their utility for fieldstudies. Dotting of clinical specimens on NC sheets or stripsin the field may provide a method for in situ studies ortransporting to the laboratory without loss of viral antigenic-ity.The psoralen derivative trioxsalen 4'-aminomethyl hydro-

chloride monohydrate was selected for virus inactivationwith long-wavelength UV, because previous studies haveshown that there is little or no effect on the antigenicity ofviruses treated in this manner (5, 12).At this point, the usefulness of DIA is primarily in the

detection of viral antibodies. Preliminary studies indicatethat unknown virus isolates can be identified with thisprocedure. In addition, the sensitivity of the procedure mayallow the use of NC for recovery of viral antigens fromclinical specimens. Concentration of the antigen might beaccomplished by repeated application and drying or filtrationof the specimen through NC membranes. This would befollowed by application of known antisera for detection ofspecific antigens. A similar approach was suggested byFuruya et al. (4) for detection of influenza viruses. The

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DOT-IMMUNOBINDING ASSAY 113

effects of b6dy fluids (immunoglobulins) and proteins thatmay be ill clinical specimens were not evaluated in this studyor in that of Furaya et al., however, and would have to beconsidered.

Although interpretation of this test was visual and, there-fore, subjective, we had little difficulty in detecting positiveand hegative reactions. The test might be automated with arecording densitometer as shown by Furuya et al. (4), but wefeel that the utility of this test for routine diagnosis lies in itssimplicity and rapidity in obtaining a yes-no-type answer.Endpoint titrations can be performed but may not be neces-sary in a clinical laboratory setting. No attempt was made torelatV the intensity of the dot color to the history of thepertinent infection, since the necessary sera are not availableat this time.

ACKNOWLEDGMENTS

This study was supported by Public Health Service grantRR00361-17 from the National Institutes of Health and World HealthOrganization grant V4/181/38.The able technical assistance of Steven L. Woolley and Lydia C.

North is appreciated.

LITERATURE CITED1. Bode, L., L. Beutin, and H. Kohler. 1984. Nitrocellulose-

enzyme-linked immunosorbent assay (NC-ELISA)-a sensitivetechnique for the rapid visual detection of both V'iral antigensand antibodies. J. Virol. Methods 8:111-121.

2. Boulter, E. A., S. S. Kalter, R. L. Heberling, J. E. Guajardo,and T. L. Lester. 1982. A comparison of neutralization tests forthe detection of antibodies to Herpesvirus simiae (monkey Bvirus). Lab. Anim. Sci. 32:150-152.

3. Elder, J. H., and M. Munson. 1984. Modification of immuno-blotting technique for detection and quantitation of infectiousvirus in cell monolayers. Biotechniques 2:170-172.

4. Furuya, K., S. Noro, T. Yamagishi, and N. Sakurada. 1984.Adsorption of influenza viruses to nitrocellulose membranefilters by filtration and their quantitative densitometric determi-nation. J. Virol. Methods 9:193-199.

5. Hanson, C. V., J. L. Riggs, and E. H. Lennette. 1978. Photo-chemical inactivation of DNA and RNA viruses by psoralenderivatives. J. Gen. Virol. 40:345-358.

6. Hawkes, R., E. Niday, and J. Gordon. 1982. A dot-immunobinding assay for monoclonal and other antibodies.Anal. Biochem. 119:142-147.

7. Hierholzer, J. C., M. T. Suggs, and E. C. Hall. 1969. Standard-ized viral hemagglutination and hemagglutination-inhibitiontests. II. Description and statistical evaluation. Appl. Microbiol.18:824-833.

8. Hutt, R., J. E. Guajardo, and S. S. Kalter. 1981. Detection ofantibodies to Herpesi'irus sithiae and Herpesvirus hmoninis innonhuman primnates. Lab. Anim. Sci. 31:184-189.

9. Kalter, S. S. 1972. Identification arid study of viruses, p.382-468. In R. N. T.-W. Fiennes (ed.), Pathology of simianprimates, part 2. Infectious and parasitic diseases. S. Karger,Basel.

10. Kalter, S. S., and R. L. Heberling. 1974. Activities of theNIH/WHO Regional Reference Center for Simian Viruses. Lab.Anim. Sci. 24:150-158.

11. Matthews, R. E. F. 1982. Classification and nomenclature ofviruses. International Committee on Taxonomy of Viruses, 4threport. Intervirology 17:1-199.

12. Redfield, D. C., D. D. Richman, M. N. Oxman4 atid L. H.Kronenberg. 1981. Psoralen inactivation of influenza and herpessimplex viruses and of virus-infected cells. Infect. Immun.32:1216-1226.

13. Richman, D. D., P. H. Cleveland, M. N. Oxman, and K. M.Johnson. 1982. The binding of staphylococcal protein A by thesera of different animal species. J. Immunol. 128:2300-2305.

14. Shuster, J., N. L. Warner, and H. H. Fudenberg. 1969. Cross-reactivity of primate immunoglobulins. Ann. N.Y. Acad. Sci.162:195-201.

15. Talbot, P. J., R. L. Knobler, and M. J. Buchmeir. 1984. Westernand dot immunoblotting analysis of viral antigens and antibod-ies: application to murine hepatitis Virus. J. Immunol. Methods73:177-188.

16. Towbin, A., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.USA 76:4350-4354.

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