J Clin Pathol 1988;41:57-61

Comparison of immunoperoxidase staining withindirect immunofluorescence, ELISA, and Westernblotting assays for detecting anti-HTLV-I antibodiesin systemic lupus erythematosusK YAMAGUCHI, ESTELA MATUTES, T KIYOKAWA,* YOKO NISHIMURA,*T ISHII,* K TAKATSUKI,* D CATOVSKY

From the Medical Research Council Leukaemia Unit, Royal Postgraduate Medical School, London, and the*Blood Transfusion Service and Department ofInternal Medicine, Kumamoto University Medical School,Kumamoto, Japan

ABSTRACT Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were

investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immun-ofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based onthe Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five variousautoimmune diseases with one or more circulating autoantibodies. Serum samples from threepatients were found to be HTLV-I-positive by the ELISA assay and sera from five patients showed anon-specific reaction by indirect immunofluorescence. These sera were negative when tested byindirect immunoperoxidase staining and Western blotting assay. All four methods gave positiveresults when tested with samples from 19 HTLV-I carriers and 16 patients with adult T cell leukaemia.Indirect immunoperoxidase staining and Western blotting assay are probably useful and more

specific assays for the detection of anti-HTLV-I antibodies in samples from patients withautoimmune diseases.

Several methods to detect antibodies against HTLV-I-namely, indirect immunofluorescence using fixedMT-I cells,' membrane immunofluorescence, solidphase immunocompetition assay,23 enzyme linkedimmunosorbent assay (ELISA),4 gelatin particleagglutination,5 and strip radioimmunoassay, based onthe Western blotting assay,6 have been reported. Thereare still difficulties, however, in detecting specific anti-HTLV-I antibodies in patients with autoimmunedisease with circulating autoantibodies. Indeed, withindirect immunofluorescence and ELISA assays it isnot always easy to exclude a non-specific reaction. Inthis study we investigated the presence ofantibodies toHTLV-I in the sera from patients with systemic lupuserythematosus and other autoimmune diseases byseveral of the above methods and a new immuno-peroxidase technique.

Accepted for publication 4 August 1987

Material and methods

PATIENTS WITH AUTOIMMUNE DISEASESera from seven patients with systemic lupus eryth-ematosus and five patients with progressive systemicsclerosis (n = 1), idiopathic thrombocytopenic pur-pura (n = 1), Sj6gren's syndrome (n = 1),polymyositis, (n = 1), and rheumatoid arthritis (n =1), treated in Kumamoto (an area where the incidenceof HTLV-I infection is high) were studied. All werewomen aged between 28 and 67 years who had one or.more circulating autoantibodies (anti-DNA, anti-mitochondria, thyroid autoantibodies etc). As positivecontrols, sera from 16 patients with adult T cellleukaemia (ATL) and 19 HTLV-I carriers were testedby indirect immunofluorescence, and sera from 26healthy subjects were used as negative controls.

SCREENING OF SERA FOR ANTI-HTLV-IANTIBODIESSerum samples were surveyed for anti-HTLV-Iantibodies by indirect immunofluorescence, ELISA,

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Yamaguchi, Matutes, Kiyokawa, Nishimura, Ishii, Takatsuki, CatovskyWestern blotting assay and indirect immunoperox-idase staining. The procedure for indirect immuno-fluorescence was as follows.' MT-1 cells fixed inacetone7 were reacted with diluted sera and thenfluorescein isothiocyanate conjugated goat antihumanIgG was added and incubated at 37C for 30 minutes.The MT- I cell line was derived from peripheral bloodleukaemic cells from a patient with ATL. As describedpreviously,' cells bearing HTLV-I associated antigens(ATLA) in MT-1 cells are few (1-2%), and almost allMT-1 cells do not express ATLA. Anti-HTLV-Iantibodies, positive, and negative reference sera wereincluded in each test as parallel controls. Sera showingbright fluorescence at a 1/10 dilution were regarded aspositive. The fluorescence was somewhat granular,often appearing as large aggregates in the cytoplasmbut not in the nucleus of the cells. On the other hand,reactions showing diffuse stain in both cytoplasm andnucleus were regarded as non-specific.

The procedure for ELISA (Eisai Kit, Tokyo, Japan)was as follows.4 Coated with antigen solution, extrac-ted MT-2 cells' were reacted with diluted sera in plasticcups and then with alkaline phosphate labelled mouseantihuman IgG monoclonal antibody at 37°C for 60minutes. The MT-2 cell line was established from cordblood lymphocytes cocultivated with leukaemic cellsfrom a patient with ATL. Most MT-2 cells release a

large number of virus particles. After adding a p-nitrophenyl phosphate solution at 37°C for 60 minutesthe reaction was stopped by the addition of4 N NaOH. The absorbence at 405 nm was measuredusing a spectrophotometer. The cut off value ofELISA was the mean value for serum samplesobtained from 104 healthy subjects determined to beantibody negative by the indirect immunofluorescence

assay plus three times the standard deviation. Absor-bence readings of more than 0 400 were considered tobe positive.

Western blotting assay was performed as follows.Cell lysates were obtained by incubating 10 millionMT-2 cells in I ml of lysing buffer (10 mM Tris-hydrochloric acid, pH 7-2, containing 0 9% sodiumchloride, 0 5% NP-40 and 2 mM phenylmethylsul-fonyl fluoride) for 20 minutes on ice. After removingcell debris at 150 000 rpm for 10 minutes recoveredproteins were diluted 1/1 with Laemmli sample bufferand electrophoresed on 12% polyacrylamide in thepresence of sodium dodecyl sulphate (SDS). Theseparated protein was electroblotted on nitrocellulosesheets by the Western blotting technique. The virusstrip radioimmunoassay was done by the methodsdescribed previously,9 using non-fat dried mild insteadof bovine serum albumin to prevent non-specificbinding of proteins to the nitrocellulose.

Indirect immunoperoxidase was performed as foll-ows.'0 C91/PL cells fixed in acetone were reacted withdiluted sera for 30 minutes at 37°C in a moist chamberand then with peroxidase conjugated rabbit anti-human immunoglobulin (Dako, Denmark) at 37°C for30 minutes. The C91 /PL cell line was established fromcord blood lymphocytes cocultivated with HTLV-Ipositive neoplastic T cells." The C91/PL cell line isalso a high producer of HTLV-I viral particles. Thecytochemical reaction was developed for five minuteswith 30 mg of diaminobenzidine tetrahydrochloride(DAB, Sigma) in 50 ml of PBS and 20 p1 of hydrogenperoxide (30 vol), and then washed in PBS and distilledwater and counterstained with Harris's haematoxylin.Sera showing peroxidase activity in a multiple gran-ular pattern were considered to be positive.

Table HTL V-I antibodies in autoantibody positive sera

Indirect Indirectimmuno- Western immuno-fluores- blotting peroxidase

Case No Age/Sex Diagnosis ELISA cence assay staining

I 35/F Systemic lupus erythematous - - - -2 28/F Systemic lupus erythematous -

3 41/F Systemic lupus erythematous - NS4 36/F Systemic lupus erythematous - - - -5 39/F Systemic lupus erythematous -

6 45/F Systemic lupus erythematous - NS7 38/F Systemic lupus erythematous 0-676 NS - -8 52/F Progressive systemic sclerosis - NS9 37/F Idiopathic thrombocytopenic purpura -

10 62/F Sj6gren's syndrome 0-566II 46/F Polymyositis 0-623 NS12 67/F Rheumatoid arthritis -

ATL (n = 16) 0 870-overscale* + + +HTLV-I carriers (n = 19) 1 043-overscale + + +Healthy subjects (n = 26) <0400 - - -

*range; NS, non-specific staining.

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HTLV-I antibodies in systemic lupus erythematosus

Fig 1 Photomicrographs ofdetection ofanti-HTLV-I antibodies by indirect immunoperoxidase staining: (a) positive (patientwith A TL); (b) negative (healthy subject); (c) negative (patient with systemic lupus erythematosus).

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Yamaguchi, Matutes, Kiyokawa, Nishimura, Ishii, Takatsuki, CatovskyResults

*Sj-_

Fig 2 Detection ofanti-HTL V-I antibodies by Western

blotting method. lane a) sera ofhealthy subject; lane b) sera

ofpatient with A TL, lane c) sera ofpatient with systemic

lupus erythematosus.

Fig 3 Scheme ofanti-HTL V-I anlibody positive patterns byWestern blotting method: (a) 90% ofpositive sera show bothanti-gag and env gene products ofHTL V-I; (b) 5% ofpositive sera show only anti-gag gene products; (c) <5% ofpositive sera show only anti-env gene products.

The table shows the incidence of anti-HTLV-Iantibodies in the sera from the 12 patients withautoimmune disease by ELISA, indirect immuno-fluorescence, Western blotting assay, and indirectimmunoperoxidase staining. Three of the 12 sera werepositive in the ELISA assay and five showed a non-specific reaction by indirect immunofluorescence, butnone was found to be positive by Western blottingassay or indirect immunoperoxidase staining. All serafrom patients with ATL and HTLV-I carriers shownto be positive by ELISA and indirect immunofluores-cence assays were also positive by Western blottingassay and indirect immunoperoxidase staining. Serafrom healthy subjects, however, were negative by allassays. Fig la illustrates the reactivity ofa patient withATL (HTLV-I antibody positive serum); fig lb, apatient with systemic lupus erythematosus (HTLV-Iantibody non-specific reaction by ELISA and indirectimmunofluorescence assays); and fig Ic, HTLV-1antibody negative serum from a healthy subject byindirect immunoperoxidase-staining. Fig 2 shows theprofiles of recognised virus related proteins obtainedby Western blotting assay. Usually, positive serareacted with all or some of the HTLV-I glycoproteingp46, p28, p24, and p19'2; sera from patients withautoantibodies did not show antibodies to HTLV-Ispecific proteins-instead many non-specific bandswere observed. Fig 3 shows the characteristic profilesobtained by Western blotting assay in HTLV-Ipositive patients, which are easily distinguishable fromthe non-specific bands shown in patients with auto-antibodies.

Discussion

It has been shown previously that sera from patientswith systemic lupus erythematosus or other autoim-mune diseases and malaria may react with HTLV-I-positive cell lines when studied by indirect immuno-fluorescence and ELISA methods.'3 14 This is likely tobe caused by a false positive reaction due to therecognition ofunknown cellular proteins on HTLV-I-positive cells rather than the detection of virus specificantigens, as the findings have not been confirmed whenapplying more specific assays such as Western blottingassay (fig 2). Therefore, although indirect immuno-fluorescence, ELISA, and particle agglutinationassays are relatively simple first line techniques for thescreening of blood donors, there is always the need toconfirm the positive results by Western blotting assay.

In this study we have shown that a simple indirectimmunoperoxidase staining method, which is com-monly used to detect cytoplasmic antigens and thenuclear enzyme terminal deoxynucleotidyl transferase

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HTL V-I antibodies in systemic lupus erythematosus 61(TdT), may be useful and more specific than indirectimmunofluorescence and ELISA to detect antibodiesto HTLV-I. Thus sera from all six patients whichshowed a non-specific reaction for HTLV-I antibodieswhen assessed by indirect immunofluorescence orELISA, or both, were shown to be unreactive with theindirect immunoperoxidase staining method. Theindirect immunoperoxidase staining method alwaysshowed clearly granular positive patterns in sera frompatients with ATL and in HTLV-I carriers. Theseresults were further confirmed by the Western blottingassay.A similar indirect immunoperoxidase staining

method has previously been reported to be useful indetecting the presence of antibody to HTLV-III,'5 andit has been shown that results obtained by indirectimmunoperoxidase staining are comparable withthose ofthe indirect immunofluorescence method. Theindirect immunoperoxidase staining method,however, has not been applied systematically toinvestigate sera from patients with autoantibodies,which may show a non-specific pattern of reactivity byindirect immunofluorescence and the ELISA meth-ods. In this study we have shown that the indirectimmunoperoxidase staining method may be morespecific than the indirect immunofluorescence andELISA methods as, unlike indirect immunofluores-cence and ELISA, a non-specific reaction has not beendetected by indirect immunoperoxidase staining. Inrelation to the ELISA assay, we performed thestandard ELISA test by an antiglobulin technique; it ispossible, however, that a competitive ELISA tech-nique might avoid some of the difficulties (non-specificstaining) derived from the standard ELISA test andtherefore might be as specific as the indirectimmunoperoxidase staining method. We have alsoshown that indirect immunoperoxidase staining isequally sensitive for testing samples of HTLV-I-positive subjects (healthy carriers and ATL patients).In fact, the indirect immunoperoxidase stainingmethod described here seems to be reliable, relativelysimple, and reproducible, and can be used as a first linescreening method for HTLV-I antibody.Although a role for viruses in the aetiology of

systemic lupus erythematosus has been regarded asfeasible,'7 the role of HTLV-I has not been confir-med.'8 Our data further suggest that HTLV-I does notplay a part in the pathogenesis of systemic lupuserythematosus.

KY was supported by the Yamagiwa-YoshidaMemorial International Study Grant awarded by theInternational Union Against Cancer. EM was suppor-ted by the Leukaemia Research Fund ofGreat Britain.

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9 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer ofproteins from polyacrylamide gels to nitrocellulose sheets:procedure and some applications. Proc Natl Acad Sci USA1979;76:4350-4.

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Requests for reprints to: Dr D Catovsky, MRC LeukaemiaUnit, Royal Postgraduate Medical School, Du Cane Road,London W12 OHS, England.

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