JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1987, p. 1207-12120095-1137/87/071207-06$02.00/0Copyright © 1987, American Society for Microbiology

Enzyme Immunoassay Using a Novel Recombinant Polypeptide ToDetect Human Immunodeficiency Virus env Antibody

RICHARD M. THORN,* GERALD A. BELTZ, CHUNG-HO HUNG, BARBARA F. FALLIS, SUSAN WINKLE,KING-LAN CHENG, AND DANTE J. MARCIANI

Cambridge BioScience Corporation, Hopkinton, Massachusetts 01748

Received 8 October 1986/Accepted 25 March 1987

A unique antigen, CBre3, has been synthesized from a genetically engineered clone to detect humanimmunodeficiency virus (HIV) env antibodies with high sensitivity and specificity. The antigen containssequences derived from both envelope proteins of HIV, i.e., gpl20 and gp4l, and was purified free ofEscherichia coli proteins detectable by Coomassie stain or immunoblotting with E. coli antiserum. The purifiedrecombinant polypeptides were used as antigen in an enzyme immunoassay (EIA) to screen serum samples fromhealthy and HIV-infected individuals. The same samples were also tested by radioimmunoprecipitation (RIP)for gp120 and gp160 HIV antibodies. AUl samples containing gpl20 and gp160 antibodies by RIP had CBre3EIA values greater than 0.35 (n, 122; range, 0.37 to 2.1+; median, 1.65). Ail RIP HIV antibody-negativesamples had CBre3 EIA values less than 0.25 (n, 140; mean, 0.052; standard deviation, 0.045; range, 0.00 to0.22). The endpoint titer of a standard positive control serum was 1:10,000 by RIP and by CBre3 EIA. Theassay was 100% accurate in three proficiency panels. It easily detected six samples from individuals whoseinfections were confirmed by culture; these samples were reactive only with p24 by Western blot. The samplesalso were positive for gpl20 and gpl60 antibodies by RIP. These data suggest that the CBre3 EIA can detectenv antibodies as sensitively and specifically as RIP and with more sensitivity than Western blot.

It is generally agreed that the etiologic agent of acquiredimmune deficiency syndrome (AIDS) is a retrovirus (2, 12),which is referred to as human immunodeficiency virus(HIV). Epidemiological data (26), studies of transfusion-associated AIDS (11), and experimental transmission studiesin chimpanzees (16) have established that HIV can betransmitted by blood, blood products, and intimate contactwith infected body fluids. It is therefore essential thatreliable, rapid, and inexpensive screening tests be availablefor detection of contaminated blood and blood products.

Currently screening is done by using HIV in an enzymeimmunoassay (EIA) to detect antibody. The sensitivity andspecificity of these tests are not known exactly since there isno accepted standard. However, there are two assays, HIVimmunoblotting (24) and radioimmunoprecipitation of gpl20and gpl6O (18), that have consistently shown better sensitiv-ity and specificity than the screening assays and are used asconfirmatory tests. Unfortunately, both these confirmatoryassays are impractical for screening.Although there have been many attempts, no one has

reported the development of a screening test with thesensitivity and specificity of the current confirmatory assays(4, 6, 7, 9, 25, 27, 30). We show here that a geneticallyengineered DNA clone produces antigenic peptides, calledCBre3, at reasonably high levels in Escherichia coli whichcan be purified and used in an EIA screening test.

MATERIALS AND METHODS

Recombinant antigen CBre3. (i) Cloning. CBre3 containstwo HIV-derived proteins which are synthesized in E. coliby using a bacteriophage lambda PL expression systemsimilar to that described by Lautenberger et al. (21). A35-kilodalton (kDa) protein is the primary product expected

* Corresponding author.

from the plasmid construction and contains 164 amino acidsderived from the C-terminal end of envelope glycoproteingpl20 and 133 amino acids from the N-terminal end of gp4l(HIV env amino acids 350 to 402, 408 to 523, and 547 to 674[22]). A 19-kDa protein results from initiation of translationat an internal methionine and contains 37 amino acidsderived from the C-terminal end of gpl20 and 133 aminoacids from the N-terminal end of gp4l (HIV env amino acids482 to 523 and 547 to 674 [22]).

(ii) Production of antigen. E. coli transfected with recom-binant clone CBre3 was grown in LB broth supplementedwith 1% glucose and 0.1% Casamino Acids (Difco Labora-tories) at 32°C to an optical density (560 nm) of 0.4 to 0.6.The culture was then shifted to 42°C and incubated for anadditional 1 or 2 h to induce the expression of recombinantprotein. At the end of induction, cells were harvested bycentrifugation at 4,000 x g for 30 min, washed with 50 mMTris hydrochloride (pH 7.5) containing 0.15 M NaCI, andstored at -70°C until use.

The HIV-specific antigen is produced in E. coli host cellsat levels accounting for about 5 to 10% of total cellularproteins. To purify the antigen, about 4 g of E. coli cell pastewas thawed at room temperature and suspended in 40 ml of50 mM Tris hydrochloride (pH 7.5) containing 1 mMphenylmenthylsulfonyl fluoride and 1 mg of aprotinin. Thecells were then digested with lysozyme (0.5 mg/ml) at roomtemperature for 30 min and sonicated on ice (four 30-sintervals, with intermittent cooling) by using the intermedi-ate titanium tip of a Fisher Sonic Dismembrator set at 45%maximum energy output. The lysate was then made up to0.5% Triton X-100, 5 mM dithiothreitol, and 10 mM EDTA.

After being stirred at room temperature for 30 min, thelysate was sedimented for 30 min at 5,000 x g. The pelletwas suspended in 40 ml of 50 mM Tris hydrochloride (pH8.0)0.5% Triton X-100-10 mM EDTA-1 mM dithiothreitol,sonicated for 2 min by using the same procedures described

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above, and stirred at room temperature for 1 h. The insolublepellets were sedimented by centrifugation at 5,000 x g for 30min and extracted once more with the same buffer asdescribed in the preceding step. The pellet fraction waswashed once with 1 M NaCI, twice with 0.2% Zwittergent3-14 (Calbiochem-Behring), once with 6 M urea, and oncewith 8 M urea in 50 mM Tris hydrochloride (pH 8.5). Theresulting pellet contained the HIV-specific antigen at about80 to 85% purity. The pellet was suspended in 6 M guanidinehydrochloride in 50 mM Tris hydrochloride (pH 8.0)-S5 mMdithiothreitol and sedimented at 100,000 x g for 30 min, andthe supernatant was applied to a Sephacryl S-300 columnequilibrated and eluted with the same buffer. Fractionscontaining HIV-specific antigen were pooled and rechrom-atographed on the same column under identical conditions toobtain purified antigen.

Protein determinations. Protein determinations were madeby using BCA (Pierce Chemical Co.) with bovine albumin asa standard.

Rabbit anti-E. coli serum. E. coli was pelleted and soni-cated, and insoluble material was collected by centrifuga-tion. New Zealand White rabbits were given one subcutane-ous injection of the insoluble material approximately every 3weeks for 9 weeks. The rabbits were then given three dosesof insoluble material from E. coli transfected with thelambda PL vector containing coding sequences for an unre-lated peptide. After the immunizations, the rabbits were bledperiodically and tested by Western blot for reactivity with E.coli proteins.Immunoblots with rabbit and human sera. E. coli or

purified proteins were analyzed by the sodium dodecylsulfate-polyacrylamide system described by Laemmli (19).Electrophoretic transfer to ntirocellulose was performed asdescribed by Towbin et al. (28). After transfer, the nitrocel-lulose was soaked in 0.5 M NaCI-50 mM Tris hydrochloride(pH 7.5) (TBS) plus 0.1% Brij 58 for more than 1 h. Sera(rabbit or human) were diluted with the same solution andincubated for 1 h at 22°C with the nitrocellulose. After beingwashed three times for 5 min each with TBS, affinity-purified, peroxidase-labeled anti-immunoglobulin in TBSplus 0.1% Brij 58 was incubated for 30 to 60 min at 22°C.After another three 5-min washes with TBS, substrate (60mg of 4-chloronaphthol, 20 ml of methanol, 100 ml of TBS,60 ,ul of 30% H202) was added and incubated for 5 to 10 minat 220C.CBre3 (Env) EIA. Purified deltaG71A in 6 M guanidine

hydrochloride-50 mM Tris (pH 8.0) was diluted to 3 ,ug/mlwith 2 M guanidine hydrochloride-50 mM Tris (pH 8.0), and100 pul was added to polystyrene microwells. The antigenwas allowed to absorb at 37°C for 16 h. The antigen solutionwas removed from the wells, and 100 pil per well of 0.15 MNaCl-10 mM phosphate (pH 7.3) plus 5% normal goat serumwas added. The wells were then incubated at 37°C for 2 h,following which the phosphate-buffered saline-normal goatserum solution was removed and 100 ,uI per well of TBS wasadded. Serum samples (5 pil) were added and incubated at37°C for 1 h. The wells were emptied and washed four timeswith 0.05% Tween 20 in deionized water. To each well, 100pul of mouse monoclonal anti-human immunoglobulin Gconjugated to horseradish peroxidase (Ortho Diagnostics,Inc.) was added and incubated for 30 min at 37°C. The wellswere emptied and washed five times with 0.05% Tween 20 indeionized water. To each well, 100 ,ul of o-phenyl-enediamine-2 hydrochloride and H202 substrate in citrate-phosphate buffer (Ortho) was added and reacted for 30 minat 20 to 220C. To each well, 25 ,ul of 4 N H2SO4 was added,

and the optical density at 490 nm was read on a DynatechMR600 microplate reader.

Radioimmunoprecipitation. Radioimmunoprecipitation as-says were performed as previously described (18) by usingextracts from [35S]cysteine-labeled human T-cell lympho-tropic virus type IIIB-infected cells. Exposures were stan-dardized by including a known positive sample at a dilution(1:10,000) which was just discernibly different from a stan-dard negative serum sample. A sample was consideredpositive if the HIV env gene-encoded polypeptides gpl20and gpl6O were visible.Serum specimens. Serum specimens were generously pro-

vided by S. Cunningham-Rundles, W. Parks, M. Ascher, T.Spira, T. H. Lee, M. Essex, F. Polk, and D. Bing. All serawere received coded, and no information was obtained fromthe supplier until radioimmunoprecipitation and EIA testingwith recombinant antigen were done. The sera were notrandomly selected. We requested a mix of difficult positivesand controls. The clinical signs of the blood donors are givenin Table 1. Three proficiency panels were also tested in blindfashion. They were from the College of American Patholo-gists (panel WM), D. S. Burke and R. Redfield at WalterReed Hospital, and T. Spira at the Centers for DiseaseControl.

RESULTSAs described in Materials and Methods, CBre3 is made in

E. coli by using a bacteriophage lambda PL vector. With thisvector, the recombinant genes are repressed at 32°C andinduced at 42°C. A Coomassie blue-stained polyacrylamidegel of an induced E. coli culture is shown in Fig. 1, lane 1.New protein bands at approximately 19 and 35 kDa wereseen after the induction of transfected E. coli. A Coomassieblue stain of the purified antigen, which shows strong bandsat 19 and 35 kDa and a weak diffuse band at 30 kDa, is shownin lane 2. N-terminal sequencing of the 19-kDa peptideshowed that it was produced by initiation of translation of aninternal methionine (env amino acid number 482 with theRatner et al. [22] numbering system). The 30-kDa peptideswere reactive with HIV antisera (see below), but they havenot been further characterized.An AIDS serum sample apparently free of E. coli antibody

showed reactivity in Western blots with all the bands in thepurified antigen preparation (Fig. 1, lane 4) but no reactivitywith induced E. coli extracts which did not have recombi-nant DNA inserts (lane 3). Blots incubated with HIV anti-body-negative sera did not react with the CBre3 proteins(data not shown). There were also no reactions with thepurified peptide when HIV antibody-negative human serawhich had extensive reactivity to E. coli proteins were used(data not shown). In contrast, rabbit E. coli antiserum didnot show reactivity with the purified antigen (lane 5) but didreact with the E. coli extracts (lane 6). Prebleed rabbit serumdid not react with any proteins (data not shown). Thus, weconcluded that the recombinant antigen peptides were HIVencoded, immunoreactive, and free of apparent E. coliprotein contamination.The purified antigen was subsequently used to develop an

EIA to test sera for HIV env antibodies. To determine thesensitivity of the CBre3 EIA, a saturating amount of antigen(2.5 ,ug/ml) was absorbed to the plastic and a positive controlserum was titrated and assayed by both radioimmunoprecip-itation and EIA. This serum had an endpoint titer between10-4 and 10-5 by either method (Fig. 2). Thus, by titrationthe two assays seemed to have comparable sensitivities forenv antibodies.

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RECOMBINANT HIV env EIA 1209

A 1 2

98K -

68K --

43K -

30K --

18KK-

B3 4

35K -.

19K -.

35K

19K -

C 6

'ik

a-

.,

FIG. 1. (A) Coomassie blue-stained 12.5% polyacrylamide gelsof transfected E. coli and purified deltaG71A peptides. Lanes: 1,approximately 80 F±g of induced E. coli transfected with lambda PL

containing recombinant DNA sequences; 2, 5 p.g of purified CBre3.(B) Immunoblots with HIV antibody. Lanes: 3, approximately 80 ,gof induced E. coli transfected with lambda PL without recombinantsequences; 4, 5 ,ug of purified CBre3. The blots were reacted with an

HIV-positive serum at a 1:100 dilution as described in Materials andMethods. (C) Immunoblots with E. coli antibody. Lanes: 5, 5 FLg ofpurified CBre3; 6, approximately 80 ptg of induced E. colitransfected with lambda PL without recombinant sequences. Thetransferred proteins were reacted with E. coli antiserum at a 1:100dilution as described in Materials and Methods.

To characterize further the CBre3 EIA sensitivity andspecificity, serum samples were obtained from several col-laborators. The distribution of the CBre3 EIA data is shownin Fig. 3. The CBre3 EIA range of radioimmunoprecipita-tion-negative samples (n, 140) was 0.00 to 0.22, with a mean

and standard deviation of 0.052 ± 0.045. Samples with gpl6Oand gp120 antibodies (n. 122) had CBre3 EIA values rangingfrom 0.37 to 2.1 +, with a median value of 1.65. There was

no overlap in CBre3 EIA values between gp120 and gpl6Oantibody-positive and -negative samples. The positive sam-

ple with the lowest CBre3 EIA value was from a multiple-transfusion patient without AIDS and may represent pas-sively acquired antibody or very recent seroconversion. Nofurther information could be obtained about this individual,nor were follow-up samples available. Ail the samples were

tested in separate assays three or more times, and the valueswere within 30% of one another. From these data, it appearsthat the minimum A490 value which can be judged as positiveis 0.25. Relative to radioimmunoprecipitation, the CBre3EIA was 100% sensitive and 100% specific when 0.25 was

used as the minimum value for a positive specimen.

We also extended these results with a CBre3 antigenprepared by using a different purification method (data notshown). Serum samples from approximately 250 more indi-viduals were analyzed, and the results showed 100% corre-

lation with radioimmunoprecipitation. Included were

seroconversions occurring in hemophiliacs in which theCBre3 EIA was positive at the earliest sample positive byWestern blot or radioimmunoprecipitation.The correlations between the assay and the clinical char-

acteristics of the samples are given in Table 1. The AIDScases were from pediatric and adult groups and from theeast, south, and west coasts of the United States. As can beseen, there was complete agreement between CBre3 EIAand clinical AIDS or AIDS-related complex (ARC) symp-

toms. The CBre3 EIA-positive samples in people withoutAIDS or ARC were confirmed by radioimmunoprecipitation.Thus, the CBre3 EIA was 100% sensitive relative to expertclinical diagnoses and to the radioimmunoprecipitation ref-erence assay.The results of blind testing of three independent profi-

ciency panels are summarized in Table 2. In each case theCBre3 EIA easily discriminated between positive and nega-tive samples and was 100% accurate. Included in the panelfrom the Centers for Disease Control were six sampleswhich reacted in Western blots only in the 24-kDa area. Allthe p24-only samples positive by CBre3 EIA had gpl20 andgpl60 antibodies detectable by radioimmunoassay and were

from culture-positive samples. Conversely, the radioimmu-noassay-negative samples were unreactive in the EIA.

DISCUSSION

The results show that the CBre3 antigen can be used in a

screening-type EIA which is apparently as sensitive andspecific as radioimmunoprecipitation is for detecting HIVenv antibody. We attribute this performance to the followingfactors. (i) CBre3 antigen contains many conserved epitopes(9, 13, 31). (ii) Relatively large amounts of pure antigen werepresent in the assay. (iii) The antigen was free of immuno-logically detectable amounts of contaminants.By comparison, other recombinant proteins have not been

used as successfully or developed as fully. Chang et al. (5)analyzed 20 AIDS sera by using a small recombinant-derived, clone 121 peptide in a blot assay and observed 1false-negative serum. Although 1 in 20 is a small fraction, itis not acceptable for HIV screening. Subsequently, the samepeptide was expressed in a different vector system (7). Byusing purified antigen in a solid-phase radioimmunoassay, 2

TABLE 1. Clinical analysis of sera testedNo. of samples positive or negative

Clinical signs n by CBre3 EIA (range of OD values)'RIP negative RIP positive

AIDS/KS/OI 83 (14 female) 0 83 (0.49-2.1+)ARC/LAD 13 0 13 (0.83-2.00)Healthy 68 68 (0.00-0.22) 0Unhealthy 39 37 (0.00-0.16) 2 (0.37-1.10)Unknown 59 35 (0.01-0.19) 24 (1.02-2.02)

a KS, Kaposi's sarcoma; QI, opportunistic infections; LAD, lymphadeno-pathy disease. The specimens designated unhealthy were from individualswith the following clinical signs or diseases: 4 multiple transfusions, 5rheumatoid factor, 6 antinuclear antibody, 1 rectal cancer, 3 multiple sclero-sis, 3 primary immunodeficiency, 10 breast cancer, 3 adult lymphocyticsarcoma, 1 melanoma, 1 carcinoma, 1 classical Kaposi's sarcoma, and 1human T-cell lymphotropic virus type I adult T-cell leukemia.

b Negative and positive, Absence and presence, respectively, of gpl20 andgpl60 antibodies by radioimmunoprecipitation (RIP); OD, optical density.

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Dilution-1.1~~~~~~~~~~~~~~~~~~~~~~I, 1 0(J 10 101 1.O

Neg r-- Positive----

2.0 -i

1.8

16

14

160K120K

1.2 --

1 .0 ---:o

CD

0.8 -

0.6

24K 04'\

0.2

Dilution"FIG. 2. Titration of HIV-positive serum in radioimmunoprecipitation and CBre3 EIA tests. Serial dilutions of a positive serum were made

in a negative serum and tested in the two assays as described in Materials and Methods. Serially diluted positive ( ) or negative ---)

sera were used.

of 132 AIDS samples reacted either negatively or apparentlynot very significantly. They also noted that in preliminaryresults with an EIA format not all AIDS and ARC specimenswere positive. It appears that the clone 121 peptide is notsufficiently sensitive for use in a screening assay.

In another study, a recombinant protein from clone O1R6,representing approximately half of HIV p31, was found toreact with 20 of 20 AIDS sera in Western blots (6). However,since it is now known that p3l antibody is not always foundin HIV-infected people at detectable levels (27), this peptidewould not be useful for a screening test. Crowl et al. (9)tested a large portion of the env gene which was expressed in

E. coli and found it reactive with 50 of 50 AIDS sera byWestern blot analysis. To our knowledge, use of this proteinin an EIA format has not yet been described.

Cabradilla et al. (4) developed an EIA with a 102-amino-acid recombinant protein from the HIV gp4l region whichwas fused with a bacterial protein. They reported 11 appar-ent false-negative reactions.

Recently, Schultz et al. (25) described the results of usinga recombinant peptide which included all of the gp4l andpart of the gpl20 amino acids. The recombinant material wasexpressed in E. coli, and the whole preparation was usedonly in blots. They found 100% correlations between recom-binant blots and virus blots or radioimmunoprecipitation,

20-

18-

à. 16-

.0 14-

E 12-

Z 10-

8-

6-

4-

2

0 .2 .4

O RIP Negative (N=140)* env Antibody Positive (N=122)

* a _unm

;~~~~~~~~~.8 .0 1.2 1.4 1.6 1.8 2.0

A490

FIG. 3. Distribution of CBre3 EIA values for 262 radioimmuno-precipitation (RIP)-analyzed sera. Radioimmunoprecipitation expo-sures were standardized with a diluted positive control as describedin Materials and Methods. The maximum optical density of theMR600 microplate reader was 2.10.

TABLE 2. Proficiency panel resultsNo. of samples % Correlation with

Panel CBr3 EIA radioimmuno- Range of Range ofpositive/total precipitation

CAP 2/4 100 0.80-0.95 0.04-0.06CDC 22/40C 100 1.02-2.10 0.02-0.15WR-I, II, III 32184d 100 0.63-1.94 0.00-0.16WR-RR 27/52 10oe 0.63-1.90 0.01-0.05

a CAP, College of American Pathologists; CDC, Centers for DiseaseControl; WR I, II, and III, Walter Reed Hospital, panels I, II, and III; WR-RR, Walter Reed Hospital, RR panel, individual samples only.

b Radioimmunoprecipitation was scored positive if both gpl60 and gp120bands were present.

C Six of the samples were p24 only by Western blot. All six were positive byCBre3 EIA and radioimmunoprecipitation.

d These included 52 normal controls and 16 ARC and 16 AIDS samples.One ARC serum had no detectable gp4l by Western blot. All ARC and AIDSsamples were positive by all assays. Among the normals were 22 specimenswhich were false-positives (0.33 to 1.2) by the Abbott Laboratories screeningassay.

e Correlated with Western blot done by Walter Reed Hospital. Blots wereconsidered positive if gp4l or p24 plus p55 were seen.

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RECOMBINANT HIV env EIA 1211

but to our knowledge this recombinant peptide has not beenpurified and used in a screening test.Wang et al. (30) have described an EIA using a 21-amino-

acid env peptide, SM284, which did not react with 4 of 228AIDS and 9 of 145 ARC sera. Unfortunately, the unreactivesera were not tested by radioimmunoprecipitation to deter-mine whether they had env antibody. The apparent differ-ences in sensitivity between the various recombinant pep-tides may be due to the number of conserved epitopes. Wehave also found that combining peptides with large fusionproteins can diminish immunoreactivity (unpublished re-sults), implying that the method of expression may alsoinfluence the reactivity.

It is significant that our assay is able to detect envantibodies even when they are not observed in Westernblots. This is important, since it has been reported that gagantibodies are sometimes the first ones observed in Westernblot analysis of seroconversions (10, 20). In neither study,however, were the sera analyzed by radioimmunoprecipita-tion for env antibodies. Furthermore, using Western blots inwhich gpl20 and gpl6O antibodies were detectable, Ulstrupet al. (29) found that gpl20 and gpl6O antibodies are detect-able prior to or coincident with p24 antibody in seroconver-sions. In our study, we found six sera which reacted onlywith p24 by Western blots but had gpl20 and gpl6O antibod-ies by radioimmunoprecipitation. These six sera were easilydetected in the CBre3 EIA (Table 2). In addition, titrationsof positive sera by Western blots and radioimmunoprecipi-tation also show that immunoprecipitation is a more sensi-tive assay for env antibody (R. M. Thorn, unpublished data),which is consistent with studies by others (17). These resultssuggest that the Western blot as currently utilized (23) isrelatively insensitive for env antibody detection.Western blot and radioimmunoprecipitation assays can

determine whether an EIA-positive reaction is due to antigenreactivity but cannot necessarily determine whether thereaction is HIV specific. A serum showing gp4l and otherbands on a blot is good evidence of HIV exposure, but seranot showing gp4l reactivity are not conclusive, particularlysera which react with p24 only. Some sera reactive with p24only may be blot artifacts (3) or may be due to antibodiescross-reactive with other related viruses (8, 14, 15). Simi-larly, a serum with both gpl20 and gpl6O reactivities inradioimmunoprecipitation is evidence of HIV exposure, butsamples with reactivity to p24 only or gpl6O only may beartifacts or examples of cross-reactions. Perhaps the easiestmethod of identifying HIV-infected true positives is torebleed the individual at a later date and reanalyze theantibody reactivities against pure env, gag, and pol recom-binant antigens. Analyses of seroconversions show thatinfected individuals develop antibodies to more HIV pro-teins with time.The results described in this paper indicate that the CBre3

antigen can be used in EIAs which are as sensitive andspecific as radioimmunoprecipitation for env antibody. Weare, therefore, developing improved EIA methods and test-ing larger numbers of samples to determine if CBre3 canreplace virus antigen in screening or reference immunoas-says.

ACKNOWLEDGMENTS

We thank Donald S. Burke, Jim Dahlberg, Max Essex, and MarkI. Greene for their helpful comments in the writing of this paper.

LITERATURE CITED1. Barnes, D. M. 1986. Keeping the AIDS virus out of the blood

supply. Science 233:514-515.2. Barre-Sinoussi, F., J.-C. Chermann, F. Rey, M. T. Nugeyre, S.

Charmaret, J. Gruest, C. Dauguet, C. Axler-Blin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum, and L. Montagnier. 1983.Isolation of a T-lymphotropic retrovirus from a patient at risk ofacquired immune deficiency syndrome (AIDS). Science 220:868-871.

3. Burke, S. D., and R. R. Redfield. 1986. False-positive Westernblot tests for antibodies to HTLV-III. J. Am. Med. Assoc. 256:347.

4. Cabradilla, C. D., J. E. Groopman, J. Lanigan, M. Renz, L. A.Lasky, and D. J. Capon. 1986. Serodiagnosis of antibodies to thehuman AIDS retrovirus with a bilaterally synthesized envpolypeptide. Biotechnology 4:128-133.

5. Chang, N. T., P. K. Chanda, A. D. Barone, S. McKinney, D. P.Rhodes, S. H. Tam, C. W. Sherman, J. Huang, T. W. Chang,R. C. Gallo, and F. Wong-Staal. 1985. Expression in Esche-richia coli of open reading frame gene segments of HTLV-III.Science 228:93-96.

6. Chang, N. T., J. Huang, J. Ghrayeh, S. McKinney, P. K.Chanda, T. W. Chang, S. Putney, M. G. Sarngadharan, F.Wong-Staal, and R. C. Gallo. 1985. An HTLV-III peptideproduced by recombinant DNA is immunoreactive with serafrom patients with AIDS. Nature (London) 315:151-154.

7. Chang, T. W., I. Kato, S. McKinney, P. Chanda, A. D. Barone,F. Wong-Staal, R. C. Gallo, and N. T. Chang. 1985. Detection ofantibodies to human T-cell lymphotropic virus-Ill (HTLV-III)with an immunoassay enmploying a recombinant Escherichiacoli-derived viral antigenic peptide. Biotechnology 3:905-909.

8. Clavel, F., D. Guetard, F. Brun-Vezinet, S. Chamaret, M. A.Rey, M. O. Santos-Ferreira, A. G. Laurent, C. Dauguet, C.Katlama, C. Rouzioux, D. Klatzmann, J. L. Champalimand, andL. Montaginer. 1986. Isolation of a new human retrovirus fromWest African patients with AIDS. Science 233:343-346.

9. Crowl, R., K. Ganguly, M. Gordon, R. Conroy, M. Schaber, R.Kramer, G. Shaw, F. Wong-Staal, and E. P. Reddy. 1985.HTLV-III env gene products synthesized in E. coli are recog-nized by antibodies present in the sera of AIDS patients. Cell41:979-986.

10. Esteban, J. I., C.-C. Tai, J. W. D. Kay, J. W.-K. Shih, A. J.Bodner, and H. J. Alter. 1985. Importance of Western blotanalysis in predicting infectivity of HTLV-III/LAV positiveblood. Lancet ii:1083-1086.

11. Feorino, P. M., V. S. Kalyanaraman, H. W. Haverkos, C. D.Cabradilla, D. T. Warfield, H. W. Jaffe, A. K. Harrison, M. S.Gottlieb, D. Goldfinger, J. C. Chermann, F. Barre-Simonssi,T. T. Spira, J. S. McDougal, J. W. Curran, L. Montagnier, F. A.Murphy, and D. P. Francis. 1984. Lymphadenopathy associatedvirus infection of a blood donor-recipient pair with acquiredimmunodeficiency syndrome. Science 225:69-72.

12. Gallo, R. C., S. Z. Salahuddin, M. Popovic, G. M. Shearer, N.Kaplan, B. F. Haynes, T. J. Palker, R. Redfield, J. Oleske, B.Safai, G. White, P. Foster, and P. D. Markham. 1984. Frequentdetection and isolation of cytopathic retroviruses (HTLV-III)from patients with AIDS and at risk for AIDS. Science224:500-503.

13. Hahn, B. H., G. M. Shaw, M. E. Taylor, R. R. Redfield, P. D.Markham, S. Z. Salhuddin, F. Wong-Staal, R. C. Gallo, E. S.Parks, and W. P. Parks. 1986. Genetic variation in HTLV-III/LAV over time in patients with AIDS or at risk for AIDS.Science 232:1548-1553.

14. Kanki, P. J., F. Barin, S. M'Boup, J. S. Allan, J. L. Romet-Lemonne, R. Marlink, M. F. McLane, T. H. Lee, B. Arbeille, F.Denis, and M. Essex. 1986. New human T-lymphotropic retro-virus related to simian T-lymphotropic virus type III (STLV-III/AGM). Science 232:239-243.

15. Kanki, P. J., M. F. McLane, N. W. King, Jr., N. L. Letvin, R. D.Hunt, P. Sehgal, M. D. Daniel, R. C. Desrosiers, and M. Essex.1985. Serologic identification and characterization of a macaqueT-lymphotropic retrovirus closely related to HTLV-III. Science228:1199-1201.

VOL. 25, 1987

on Septem

ber 29, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

1212 THORN ET AL.

16. King, N. W. 1986. Simian models of acquired immunodeficiencysyndrome (AIDS): a review. Vet. Pathol. 23:345-353.

17. Kitchen, L., G. Malone, S. Orgad, F. Barin, R. Zaizov, B.Ramot, E. Gazit, J. Kreiss, M. Leal, I. Wichmann, U.Martinowitz, and M. Essex. 1986. Viral envelope protein ofHTLV-III is the major target antigen for antibodies in hemo-philiac patients. J. Infect. Dis. 153:788-790.

18. Kitchen, L. W., F. Barin, J. L. Sullivan, M. F. McLane, D. B.Brettler, P. H. Levine, and M. Essex. 1984. Aetiotogy of AIDS-antibodies to human T-cell leukemia virus (type III) in hemo-philiacs. Nature (London) 312:367-369.

19. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

20. Lange, J. M. A., R. A. Coutinho, W. J. A. Krone, L. F.Verdonck, S. A. Danner, J. V. D. Noordaa, and J. Goudsmit.1986. Distinct IgG recognition patterns during progression ofsubclinical and clinical infection with lymphadenopathy associ-ated virus/human T lymphotropic virus. Br. Med. J. 292:228-230.

21. Lautenberger, J. A., A. Seth, C. Jorcyk, and T. S. Papas. 1984.Useful modifications of the Escherichia coli expression plasmidpJL6. Gene Anal. Tech. 1:63-66.

22. Ratner, L., W. Haseltine, R. Patarca, K. J. Kivak, B. Starcich,S. F. Josephs, E. R. Doran, J. A. Rafalski, E. A. Whitehorn, K.Bairmeister, L. Ivanoff, S. R. Petteway, Jr., M. L. Pearson, J. A.Lautenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C.Gallo, and F. Wong-Staal. 1985. Complete nucleotide sequenceof the AIDS virus, HTLV-III. Nature (London) 313:277-284.

23. Sarngadharen, M. G., M. Papovic, L. Bruch, J. Schupbach, andR. C. Gallo. 1985. Antibodies reactive with human T-lympho-tropic retroviruses (HTLV-III) in the serum of patients withAIDS. Science 224:506-508.

24. Schubach, J., M. Popovic, R. V. Gilden, M. A. Gonda, M. G.

Sarnadharan, and R. C. Gallo. 1984. Serological analysis of asubgroup of human T-lymphotropic retroviruses (HTLV-III)associated with AIDS. Science 224:503-505.

25. Schultz, T. F., J. M. Aschauer, P. Hengster, C. Larcher, H.Wachter, B. Fleckenstein, and M. P. Dierich. 1986. Envelopegene-derived recombinant peptide in the serodiagnosis of hu-man immunodeficiency virus infection. Lancet ii:111-112.

26. Shaw, G. M., S. Broder, M. Essex, and R. C. Gallo. 1984.Human T-cell leukemia virus: its discovery and role in leuke-mogenesis and immunosuppression. Adv. Intern. Med. 30;1-51.

27. Steimer, K. S., K. W. Higgins, M. A. Powers, J. C. Stephans, A.Gyenes, C. George-Nascimento, P. A. Luciw, P. J. Barr, R. A.Hallewell, and R. Sanchez-Pescador. 1986. Recombinant poly-peptide from the endonuclease region of the acquired immunedeficiency syndrome retrovirus polymerase (pot) gene detectsserum antibodies in most infected individuals. J. Virol. 58:9-16.

28. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nicrocellulosesheets: procedure and some applications. Proç. Natl. Acad. Sci.USA 76:4350-4354.

29. Ulstrup, J. C., K. Skaug, K. J. Figenscham, I. Orstavik, J. N.Brunn, and G. Petersen. 1986. Sensitivity of Western blotting(compared with ELISA and immunofluorescence) duringseroconversion after HTLV-III infection. Lancet ii:1151-1152.

30. Wang, J. J. G., S. Steel, R. Wisniewolski, and C. Y. Wang. 1986.Detection of antibodies to human T-lymphotropic virus type IIIby using a synthetic peptide of 21 amino acid residues corre-sponding to a highly antigenic segment of gp41 envelope pro-tein. Proc. Natl. Acad. Sci. USA 83:6159-6163.

31. Willey, R. L., R. A. Rutledge, S. Dias, T. Folks, T. Theodore,C. E. Buckler, and M. A. Martin. 1986. Identification of con-served and divergent domains within the envelope gene of theacquired immunodeficiency syndrome retrovirus. Proc. Natl.Acad. Sci. USA 83:5038-5042.

J. CLIN. MICROBIOL.

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nloaded from