JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1987, p. 199-2020095-1137/87/020199-04$02.00/0Copyright © 1987, American Society for Microbiology

Vol. 25, No. 2

Indirect Immunofluorescence Assay for Antibodies toHuman Immunodeficiency Virus

EVELYNE T. LENNETTE,1* SIMON KARPATKIN,2 AND JAY A. LEVY3Virolab, Inc., Berkeley, California 947101; New York University Medical Center, New York, New York 100162; and

Cancer Research Institute, University of California School of Medicine, San Francisco, California 941433Received 30 June 1986/Accepted 3 October 1986

We compared an indirect immunofluorescence assay (IFA) with a commercial enzyme-linked immunosor-bent assay (ELISA) screening procedure for the detection of antibodies to the human immunodeficiency virus(HIV). The IFA assay was as sensitive and specific as the ELISA. No false results were observed with IFA onsingle testing of 181 sera, while the ELISA produced five false-positive results which required retesting forresolution. In addition, the IFA was suitable for quantitation of anti-HIV responses. The IFA was superior tothe ELISA for its reliability, simplicity, and rapidity in the diagnostic laboratory.

Serologic testing for human immunodeficiency virus(HIV) is currently performed primarily in blood banks forthe screening of potentially infected blood donors. Whileuseful diagnostic information such as the clinical spectrumand pathogenesis ofHIV infections is still being sought after,practical virologic and serologic testing procedures are beingdeveloped by various research laboratories. The serologicassays for anti-HIV antibodies are the enzyme-linked immu-nosorbent assay (ELISA) and immunoblot assay (2, 3). Theformer (ELISA) is used as a screening test, while the latteris used mainly to confirm the ELISA results. Both proce-dures have drawbacks for the reference diagnostic labora-tory. ELISA require lengthy incubations and multiple con-trols per run; they also produce erroneous results, necessi-tating that positive results be confirmed by a second proce-dure. The immunoblot assay is cumbersome and poorlystandardized. For these reasons, we evaluated the indirectimmunofluorescence assay (IFA) as a potentially usefulquantitative procedure suitable for the diagnostic virologylaboratory.

MATERIALS AND METHODS

Sera were collected from various patients at high risk forboth the acquired immunodeficiency syndrome (AIDS) andAIDS-related complex as defined by the case definitioncriteria of the Centers of Disease Control (1). They include:(i) homosexuals with and without lymphadenopathy andidiotypic thrombocytopenia (ITP), (ii) intravenous drugabusers with ITP, (iii) AIDS patients who are either homo-sexuals or heterosexual drug users, and (iv) hemophiliacs.The patients were from both the San Francisco and NewYork City metropolitan areas. Sera from laboratory person-nel were used as seronegative controls.The uninfected human T-lymphoid cell line HUT-78 and

its HIV-positive derivative E-2B were described previouslyby Kaminsky et al. (5). E-2B has 80 to 95% of the cellsinfected with HIV SF2. A few minor modifications ofmethods were made to provide a simplified procedure suit-able for the diagnostic laboratory. Both HUT-78 and theinfected E-2B cultures were propagated in antibiotic-freeRPMI 1640 medium with 7.5% bovine serum and 20 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic

* Corresponding author.

acid); the cultures were subdivided weekly by diluting them1:5 in fresh medium.To prepare IFA smears, we centrifuged actively growing

HUT-78 and E-2B cultures (less than 5 days old) at 400 x gfor 10 min. The pelleted cells were suspended gently inphosphate-buffered saline containing 0.25% bovine serumalbumin to yield a final density of approximately 1 x 107 cellsper ml. The HUT-78 cell suspension was then mixed withE-2B suspension in a 3:1 ratio to provide negative controland positive cells on the same smear. The mixture (3 to 5 pil)was spread on glass slides and air dried rapidly. The driedsmears were then fixed in acetone at room temperature for 1min and then stored at 5°C for up to 2 weeks, or frozen at-20°C for longer storage.For the IFAs, the sera were diluted at 1:10 in phosphate-

buffered saline and heated at 56°C for 30 min to inactivateHIV. The diluted specimens were incubated with the cellsmears for 30 min, rinsed with two changes of phosphate-buffered saline, and stained with fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin G (IgG;Hyclone) or IgM or IgA (Tago) for 30 min.ELISAs for HIV antibodies were done with the Abbott

HTLV-III EIA kit, according to their instructions. For thecomparison of ELISA and IFA, any discrepant results wereretested by both methods.

RESULTSHIV antigen expression. We found several factors which

affected the level of antigen production in E-2B cells. Asdescribed previously (5), the highest level of HIV antigenwas expressed during the growth phase of the cells. How-ever, log phase could be maintained at 37°C with media with10% bovine serum for only about 3 days, after which thecells rapidly lost viability. If the cells were not subcultivatedpromptly, the HIV-antigen expression would gradually belost. Hence, subcultivation was needed twice weekly. Wefound the following alternative procedures to be both sim-pler and less labor intensive. Log-phase growth of the cellline was maintained for a longer period (5 to 6 days) bylowering: (i) the seeding density to 1 x 105 to 2 x 105 cellsper ml, (ii) the temperature from 37 to 35°C, and (iii) thebovine serum concentration from 10 to 7.5%. We alsoadopted the following routine to maintain HIV antigenexpression of the culture. After 1-week-old E-2B andHUT-78 cultures were diluted 1:5 in fresh medium, they

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FIG. 1. Indirect immunfluorescence staining patterns for HIV antibodies in sera from a negative control patient (A) and HIV-positivepatients (B to F). (D) Large IFA-positive cell fusing with negative cell. (E) Cytoplasmic focal pattern. (F) Diffuse and membrane staining.

were mixed at a 2:1 ratio. With weekly addition of uninfectedcells, the E-2B line was maintained in a continuously repli-cating viral cycle. E-2B has been in continuous culture in ourlaboratory for over 8 months. The level of infectivity of thissubline was similar to those previously described for theparent line E; it varied from 80 to 100%, while the cultureexhibited a moderate-to-large number of vacuolating giantcells with a striking ballooned appearance. While the

HUT-78 fine also has a few large cells, they are not nearly aslarge nor are they as striking in appearance as those seen inE-2B.We noted some lot-to-lot variation in our smear prepara-

tions. With the high-titered sera, it became apparent thatsome lots of smears showed prozones at a serum dilution of1:10, but stained brilliantly at higher serum dilutions. Thiseffect may be due to limiting antigen concentration in the

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INDIRECT IMMUNOFLUORESCENCE ASSAY FOR HIV ANTIBODIES 201

TABLE 1. Prevalence of anti-HIV antibodies

Clinical group No. of sera % Sero-(total) IFA + IFA - positive

AIDS (27) 27 0 100Symptomatic homosexuals (ITP) (33) 33 0 100Drug users with ITP (14) 12 2 86Asymptomatic homosexuals (12) 4 8 33Asymptomatic hemophiliacs (65) 15 50 23Controls (laboratory workers) (30) 0 30 0

smears. The prozone effect can cause false-negative resultsif sera are only screened at a 1:10 dilution. To avoid thispotential problem, we tested each lot of smears for prozonesusing a 1:10 dilution of reference high-titered sera.

Staining patterns. The IFA staining patterns in the contin-uously maintained cells were of two main types: (i) a diffusecytoplasmic pattern (Fig. 1F) and (ii) a cytoplasmic focalpattern (Fig. 1E). The majority of the sera we tested exhib-ited all these staining patterns, although an occasional sam-ple showed only one of the patterns. Other patterns, such ascapping and membrane staining (Fig. 1B and C, respec-tively), were also observed, but were not dominant. Nonuclear staining was observed in these cells. The infectedcells stained intensely and were very easily recognized. Afew sera (less than 1%) contained antilymphocytic antibod-ies and could be easily detected when all cells, and not theexpected 25%, were staining. These nonspecific reactionswere confirmed when the sera were retested with uninfectedHUT-78 smears. Preabsorption with HUT-78 cells (1:1, cellto serum volume) permitted an evaluation of these sera foranti-HIV antibodies.We tested 181 sera by both IFA and ELISA to compare

assay sensitivity and specificity. We found nearly perfectcorrelation between the two procedures. Sera were consid-ered positive if they were tested positive by both assays. Oninitial testing, there were 91 positive and 85 negative sera.

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An additional five sera were positive by ELISA only; all fivewere shown to be negative on retesting by either procedures.These five results were scored as false ELISA positives.The distribution of HIV seropositivity is summarized in

Table 1. It ranged from 0% among the laboratory workers,23% in hemophiliacs (from 1981 to 1983), 33% in asympto-matic homosexuals, and 86% in drug users to 100% inhomosexuals with ITP and AIDS.Absorbance readings with ELISA and titers by IFA do not

show close correlation for individual sera. However, therelative geometric mean titers and absorbance values amongthe various clinical groups were similar for each of the twotest procedures. IFA data from Fig. 2 show intravenous drugabusers to have the highest geometric mean antibody titers,followed in decreasing order by homosexual ITP, AIDS, andhemophiliac patients. The geometric mean ELISA absor-bance values for the four groups were in an identicaldecreasing order, 1.078, 0.848, 0.502, and 0.101, respec-tively.We tested all the seropositive hemophiliacs (15 patients)

and 10 of the AIDS patients for anti-HIV IgM. In addition,32 patients with AIDS-related complex were tested for thepresence of anti-HIV IgA. All the assays for anti-HIV IgMand IgA were negative.

DISCUSSIONCurrent testing for HIV antibodies is based primarily on

ELISA and immunoblot (2, 3). For the reference diagnosticlaboratory in which the number of specimens to be testedwould be small and in which a reliably accurate test isneeded, both of these assays have drawbacks. The ELISAprocedure of most commercial kits requires several longincubations and also requires a large number of controls perrun. Of most concern, however, are reports of both false-positive and false-negative results with the ELISA, necessi-tating the use of a second confirmatory test (4, 7). Thepresent kits have not been evaluated for quantitative use anddo not provide for immunoglobulin class-specific testing.

CONTROL ASYMPTOM. ASYMPTOM. HOM0SEXUAL I.V. DRUG USER AIDSHEMÔPHILIAC HOOEXUAL with ITP with ITP

FIG. 2. Distribution of IFA anti-HIV antibodies in various categories of patients and controls. The number and line in each panel indicatethe geometric mean HIV titer for the corresponding patient group. I.V., Intravenous.

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The immunoblot assay in its current format is very cumber-some and labor intensive. It is more suitable as an analyticresearch tool than as a diagnostic procedure. It has not beenstandardized sufficiently for clinical use, and it is also notfree from false results (6, 7). Currently, there are no com-mercially Food and Drug Adminstration-approved diagnos-tic reagents for the immunoblot assay.The potential utility of the IFA for the detection and

quantitation of HIV antibodies in the diagnostic laboratorywas recognized after the publication of results on a largenumber of sera (5). The IFA has been used in diagnosticlaboratories successfully for 2 decades. Its specificity andsensitivity as an assay for antibodies against a wide spectrumof microbes have been well documented. Therefore, it wasnot surprising that our evaluation of the IFA assay forantibodies to the HIV shows it to be a suitable procedure forthe diagnostic laboratory. The IFA procedure is rapid,requiring 1 h of actual incubation time, and it is simpler thanELISA or immunoblot techniques. The IFA antigen reagentsare simple to prepare, within the capability of most referencelaboratories. The reagents are quite stable with shelf life ofmany months, compared with 6 weeks for the Abbott kit. Asthe IFA procedure is in wide use for most diagnosticlaboratories, it would require minimum effort to add HIV tothe inventory and repertory lists.We found the IFA to be as specific and sensitive as the

ELISA. We experienced some false results with the ELISA,primarily irreproducibly positive results. While we recognizethese false-positives to be due to technical problems and notinherent in the reagents as provided by the manufacturer,they are nevertheless widely encountered (2, 4). With theELISA procedure, the washing steps are both exacting andcritical. In our experience, the most common sources oferrors are residual or unintentionally splashed conjugatedreagent which would yield false-positive results. These ir-regular events, unfortunately, would not be noticed, hencethe necessity of the confirmatory test. This requirement isboth time-consuming and costly. With the IFA neitherfalse-positive nor false-negative results occurred in our trial.This is due in part to the less demanding steps in the test andto the skill of the microscopist. Moreover, the specific HIVantigens are easily recognized in the stained cells. We feelthat the ability of a trained technologist to discriminatespecific reactions from false reactions is a necessary advan-tage for the reference laboratory. This efficiency was re-flected in the lack of erroneous results encountered with theIFA. Since we found the IFA to be more rapid and reliable

than the Abbott ELISA, we feel that it is well suited for usein the reference laboratory as the primary test for antibodiesto HIV, one which would not require a confirmatory test.Even from our limited survey, it appears that the humoral

response in the patients is not an all-or-none effect. Whilethere were overlaps in HIV antibody titers among thevarious groups of patients, similar trends in the geometricmean titers were observed among the various clinical groupsby two independent serologic procedures. These quantita-tive differences have been noted by others (5). The signifi-cance of these observed patterns is not known and should beinvestigated in longitudinal studies. In addition, followingshifts in the antibody titers may be useful for monitoring thecourse of disease or therapy.

ACKNOWLEDGMENT

We are indebted to Mary M. Herrmann for her skillful technicalassistance.

LITERATURE CITED1. Centers for Disease Control. 1985. Revision of the case definition

of acquired-immunodeficiency syndrome for national reporting-United States. Morbid. Mortal. Weekly Rep. 34:373-375.

2. Centers for Disease Control. 1985. Update: Public Health Serviceworkshop on human T-lymphotropic virus III antibody testing-United States. Morbid. Mortal. Weekly Rep. 34:477-478.

3. Centers for Disease Control. 1985. Provisional Public HealthService interagency recommendations for screening donatedblood and plasma for antibody to the virus causing acquiredimmunodeficiency syndrome. Morbid. Mortal. Weekly Rep.35:1-5.

4. Gallo, D., J. L. Diggs, G. R. Shell, P. J. Dailey, M. N. Hoffman,and J. L. Riggs. 1986. Comparison of detection of antibody to theacquired immune deficiency syndrome virus by enzyme im-munoassay, immunofluorescence, and Western blot methods. J.Clin. Microbiol. 23:1049-1051.

5. Kaminsky, L. S., T. McHugh, D. Stites, P. Volberding, G. Henle,W. Henle, and J. Levy. 1985. High prevalence of antibodies toacquired immune deficiency syndrome (AIDS)-associated retro-virus (ARV) in AIDS and related conditions but not in otherdisease states. Proc. Natl. Acad. Sci. USA 82:5535-5539.

6. Saag, M. S., and J. Britz. 1986. Asymptomatic blood donor witha false positive HTLV III Western blot. N. Engl. J. Med.314:118.

7. Weiss, S. H., J. J. Goedert, G. Sarngadharan, A. J. Bodner, TheAIDS Seroepidemiology Collaborative Working Group, R. C.Gallo, and W. A. Blattner. 1985. Screening test for HTLV-III(AIDS agent) antibodies. J. Am. Med. Assoc. 253:221-225.

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