Liquid competition radioimmunoassay for the detection and quantitation of the HIV p24

Journal of Virological Methods, Elsevier

17 (1987) 199-210

199

JVM 00610

Liquid competition radioimmunoassay for the detection and quantitation of the HIV p24 Arnaldo Caruso, Luigina Terlenghi, Roberto Ceccarelli, Rosanna Verardi, Ida Foresti, Gemma Scura, Nino Manta, Carlo Bonfanti and Adolf0 Turano Institute of Microbiology,

University of Brescia, Brescia, Ital)

(Accepted

7 April

1987)

Summary

Productive infection of permissive cell cultures by HIV has been detected by different assays of which the measurement of reverse transcriptase (RT) activity has been considered highly specific and sensitive. Here we describe the production and characterization of a mouse hybridoma cell line, MB12, secreting monoclonal antibodies to HIV ~24, the major core protein, and the use of this monoclonal antibody to develop a type specific indirect liquid competitive radioimmunoassay (RIA) capable of providing earlier detection of the replicating virus than the RT assay. This assay also provides a quantitative analysis of HIV ~24, which can be used to study the viral replication in permissive cell cultures. The ease of methodology and the adaptability of the competitive RIA to various assay conditions make this immunoassay suitable for the study of HIV expression in infected cell cultures. Monoclonal

antibody;

Liquid

competitive

RIA;

HIV

Introduction Human immunodeficiency virus (HIV) is the etiologic agent of AIDS (BarreSinoussi et al., 1983; Gallo et al., 1984; Klatzmann et al., 1984; Levy et al., 1984; Popovic et al., 1984; Sarngadharan et al., 1984; Schupbach et al., 1984), a disease

Correspondence to: Arnaldo Caruso. Civili 1. 25100 Brescia, Italy.

Institute

016609341871$03.50

Science

0

1987 Elsevier

of Microbiology,

Publishers

University

B.V. (Biomedical

of Brescia,

Division)

Piazza

Spedali

which causes depletion of the OKT4+ subset of T-lymphocytes (CDC, 1982; Gottlieb et al., 1981) and is accompanied by opportunistic infections and malignancies (Feorino et al., 1984). Isolation of HIV (the AIDS agent) and the development of cell lines resistant to HIV cytopathic effect and yet permissive for virus replication (Popovic et al., 1984; Casareale et al., 1985), enabled the continuous production of large amounts of virus, its characterization and the development of specific assays and reagents. Evidence of HIV infection in man has obvious important clinical implications. Large scale screening for antibody in high risk individuals can be based on ELISA tests, immunofluorescence test and an antibody capture assay (Parry, 1986) as suggested by Mortimer et al. (1985), or by the use of Western blot analysis as a confirmatory test for sera found positive in ELISA tests (Sarngadharan et al., 1984). Virus isolation, where possible, remains the most specific test for infectivity. HIV infection of permissive cell cultures is usually monitored by detecting replicating virus with the electron microscope, by immunofluorescence, by measuring virionassociated reverse transcriptase (RT) activity in culture supernatants and, recently by Sandwich-enzyme-linked-immunosorbent assays (McDougal et al., 1985; Higgins et al., 1986). Here we describe the production and characterization of a mouse hybridoma, MB12, secreting monoclonal antibodies to HIV ~24, the viral major core protein and its use for the development of a highly sensitive and specific liquid competitive radioimmunoassay (LICRIA) for the detection and quantitative analysis of HIV ~24. This LICRIA test has advantages with respect to the other tests available to detect replicating HIV in permissive cell lines, such as a more rapid diagnosis of infection and the possibility of a truly quantitative analysis of HIV ~24, which can be used as a specific marker of virus replication.

Materials and Methods Cells and viruses The virus producer cell line H9/HTLV-III, the HIV purified on gradient density and the affinity purified HIV p24 were provided by Dr. M.G. Sarngadharan. The virus producer cell line CEMiLAV 1 was provided by Dr. F. Barre-Sinoussi. Production of monoclonal

antibody (MAb)

BALBic mice were immunized with three intraperitoneal (100 pg) and one intravenous (10 kg) inoculations of HIV purified on density gradient and lysed by detergent treatment. The disrupted virus was emulsified in complete Freund’s adjuvant for the first intraperitoneal inoculation and in incomplete adjuvant for the following two doses given 1 wk apart. Three days after the final intravenous booster of the lysate virus in sterile phosphate buffered saline (PBS), splenic lymphocytes were fused with the NS-1 mouse myeloma line. The fusion procedure, cell culturing, screening of monoclonal antibodies, cloning of hybridoma lines, mouse ascitic

201

fluid preparation and determination of immunoglobulin essentially as described (Galfre et al., 1977; Reading, et al., 1985). Preparation

and culture of virus-infected

subclass secretion were 1982; Di Marzo Veronese

cells

H9 cell line was cultured in RPMI-1640 (Flow Laboratories Inc., Rockville, MD) containing 20% fetal calf serum and prepared for infection as described (Popovic et al., 1984). Briefly, 1 X 10’ cells were incubated with DEAE-dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) at a concentration of 25 pgiml for 30 min at 37°C and then washed with RPMI-1640. Supernatant (5 ml) from HB/HTLV-III cultures, an immortalized and HIV chronically infected cell line (Popovic et al., 1984), was used to infect 5 X lo6 cells. Uninfected H9 cells (5 X 106) were cultured as negative control. On days 2, 4, 6, 9, 13 and 16, 10 ml of cell culture medium from either infected or uninfected cells was harvested, and cultures were refed with 10 ml of fresh growth medium. The harvest was clarified by low speed centrifugation, and the resulting cell pellet and supernatant were stored separately at -70°C. Each supernatant was thawed, divided in two aliquots of 5 ml each, diluted 1:1.5 with a 30% polyethyleneglycol-6000 (Serva Feinbiochemica, Heidelberg, F.R.G.) solution (w/v) and incubated overnight at 4°C. The suspensions were then centrifuged at 1600 x g for 90 min at 4°C and the supernatants discarded. The pellets derived from a series of aliquots were assayed for the presence of a specific RT activity as described (Gallo et al., 1978). The pellets from the second series of aliquots were resuspended in PBS containing 1% Triton X-100 (pH 7.2), boiled for 2 min and tested by LICRIA for HIV p24 detection, after determination of protein concentration (Markwell et al., 1978). Protein

extract of cells

Both infected and uninfected cell pellets were resuspended in PBS containing 1% Triton X-100 (w/v), pH 7.2, and then frozen and thawed three times. Nuclei and debris were pelleted at 1200 x g for 10 min and the clarified supernatants were boiled for 2 min. Following a centrifugation at 3500 X g for 10 min, the supernatants were assayed for the presence of HIV p24 by LICRIA and Western blot technique. Protein concentration was determined (Markwell et al., 1978). Western

blotting

analysis

The affinity purified HIV ~24, the HIV lysate and extracts from H9, H9/HTLVIII, and CEM/LAV 1 cell lines were fractionated by electrophoresis on a 12% polyacrylamide slab gel in the presence of sodium dodecylsulphate (Laemmli, 1970). The proteins were electrophoretically transferred to a nitrocellulose sheet as described (Towbin et al., 1979). The sheet was then incubated with PBS containing 5% BSA (w/v) for 2 h at room temperature (r.t.), washed with PBST and cut into strips. Each strip was then incubated for 2 h at r.t. with 1.6 ug of affinity purified

202

MAb MB12 in 10 ml of PBS containing 1% BSA (w/v), washed three times and allowed to react for 1 h at r.t. with a 1:300 dilution of rabbit anti-mouse IgG (H+L chain) (Cappel, Cochranville, PA). Following three washes with PBST, the strips were incubated for 1 h at r.t. with [iz51]protein A (Amersham International plc, Amersham, U.K.), at 5X10” counts per minute (cpm)/ml washed overnight with PBST, briefly dried and exposed to X-ray film (Hyperfilm-MP, Amersham International). Solid phase RIA The reactivity of MAb MB12 was assayed using a solid phase RIA as described (Horan Hand et al., 1984) with minor modifications. Briefly, a lysate of densitybanded HIV, at concentrations ranging from 0.1-0.4 pg (per 50 l.~l of PBS containing calcium and magnesium, pH 7.2) was added to each well of 96-well polyvinyl chloride (PVC) microtiter plates and allowed to dry overnight at 37°C. Microtiter wells were treated with 200 ~1 of PBS containing 5% BSA (w/v) (pH 7.2). After incubation for 1 h at 37”C, the plates were washed with PBS containing 1% BSA (w/v) and 0.5% Tween-20 (v/v) (assay buffer). Dilutions of MAb MB12 tissue culture supernatant ranging from 1:5 to 1:lOOOO (in 50 ~1 of assay buffer) were added to each well. Following a 1 h incubation, the plates were washed with assay buffer and rabbit anti-mouse IgG (H+L chain) at 1:2000 dilution (in 50 ~1 assay buffer) was added to each well. After incubation for 1 h at 37”C, 50000 cpm of [1251]protein A, diluted in 25 l~_lof assay buffer, were added. to each well. Following an additional incubation for 1 h at 37”C, the plates were washed four times with assay buffer and the bound cpm were detected by cutting the wells from plates and counting them in a Gamma Counter. Indirect binding competition RIA for HIV p24 The assay was performed as described previously with some modifications (Caruso et al., 1986). Briefly, lysates of HIV (0.2 pg), diluted in 50 l.~l of PBS containing calcium and magnesium (pH 7.2), were allowed to dry overnight at 37°C on individual wells of 96-well PVC microtiter plates (detection plates). Volumes of 200 ~1 of PBS containing 5% BSA (w/v) were added to each well of the detection plates and to each well of a separate set of 96-well PVC microtiter plates containing no antigen adsorbed to the wells (reaction plates), in order to minimize non-specific protein adsorption. After incubation for 1 h at 37°C the plates were washed once with PBS containing 1% BSA (w/v). To each well of the reaction plate, 75 l_1,1 of the appropriate dilution of MAb MB12 tissue culture supernatant (1:lOO) in assay buffer, and either 75 l.i.1of competitor antigen (diluted in assay buffer containing 1% Triton X-100) or assay buffer containing 1% Triton X-100 were added. The final detergent concentration in each well was 0.05% Tween 20 and 0.5% Triton X-100. Reaction plates were incubated overnight at 4°C. Fifty ~1 aliquots were then transferred from each well of the reaction plates to duplicate wells of the detection plates. The detection plates were then incubated for 1 h at

203

37°C and washed twice with assay buffer. Fifty $ of a 1:2000 dilution (in assay buffer) of rabbit anti-mouse IgG (H+L chain) were then added to each well. The detection plates were incubated for 1 h at 37”C, the wells were then washed twice and 25 ~1 of assay buffer containing 50000 cpm of [‘251]protein A was added to each well. Following an additional 1 h incubation at 37”C, the wells were washed four times with assay buffer, cut from the detection plates and counted in a Gamma Counter. The percent of bound counts was determined by dividing the average of cpm bound to wells of the detection plate in the presence of competitor antigen by the average of the cpm obtained in the absence of competitor antigen, multiplied by 100.

Results Characterization

of MAb MB12

Several hundred hybridoma tissue culture supernatants were tested for their reactivity to a density gradient purified HIV preparation. One of these, designated MB12, was found to react with the HIV major core protein with a MW of 24 kDa

a

MWx103 2000

b

c

d

e

f

92.5‘d” 69 - *a 46-

MWx103 -200 -92.5 e e-69

a,~~_*

iay,. w-46

A

B

Fig. 1. Detection of HIV p24 by the MB 12 hybridoma in the immunoblot assay. Strips were prepared with affinity purified HIV p24 (2 )~g per lane), HIV lysate (5 kg per lane), H9, H9IHTLVIII and CEMiLAV 1 extracts (50 pg per lane) and treated with 1.6 kg of affinity purified MAb MB12 in 10 ml of PBS containing 1% BSA (w/v) as described in Materials and Methods. (A) Lanes: a = H9 cell line; b = H9/HTLV-III producer cell line; c = purified HIV; d = affinity purified HIV ~24. (B) Lanes: e = CEMiLAV 1; f = H9/HTLV-III.

204

loo0 5 10 Kx) l/ANTIBODY DILUTkXl Fig. 2. Reactivity of MAb MB12 with a purified HIV lysate. Decreasing amounts of MAb MB12 (from I:5 to 1:lOOO dilution of cell culture supernatant fuid) were assayed in a solid-phase RIA for binding to a purified HIV lysate adsorbed to assay wells at the following concentrations: 0.1 kg (o), 0.2 kg (m) and 0.4 pg (0).

(~24). The immunoglobulin subclass secreted by MB12 was determined by RIA and Ouchterlony analysis and found to be an IgG 1. The specificity of the MB12 reactivity to the HIV p24 can be seen in Western blot (Fig. 1). Monoclonal antibody MB12 strongly reacts with the affinity purified HIV ~24, the HIV purified preparation and with p24 and the core poly-protein precursor, pp55, in the extract

100

0

LOG

1 COMPETITOR

2

3

PROTEIN(ng)

Fig. 3. Titration of MAb MB12 in LICRIA for HIV ~24. Decreasing amounts of HIV lysate were allowed to bind in a liquid phase to MAb MB12 at a 1:60 (a), 1:80 (0) and 1:lOO (0) final dilution of cell culture supernatant fluid. MAb not bound to HIV p24 in the reaction well, was detected by using 0.2 kg of the same viral lysate coated to assay wells as described in Materials and Methods. Inset: Effects of different incubation times and temperature on the sensitivity of the LICRIA. MAb MB12 at a 1: 100 final dilution was used to precipitate decreasing amounts of HIV lysate in 96-well PVC microtiter plates in the following conditions: 1 h at 37°C (o), 2 h at r.t. (0) and lb16 h at 4°C (0). MAb MB12 not bound to p24 in the competitor antigen well was detected using 0.2 ug of HIV lysate coated to assay wells as described in Materials and Methods.

205

from the immortalized and infected T-cell clones H9/HTLV-III and CEMiLAV 1. The epitope recognized by MB12 was confirmed to be type specific since no crossreactivity was shown with HTLV-I, HTLV-II, bovine leukemia virus (BLV) and visna by ELISA and Western blot analysis (data not shown).

Optimization

of LICRIA for HIV p24

MAb MB12 was used in a type specific LICRIA for the HIV p24 detection. In order to optimize MAb binding at limiting antigen levels so as to obtain maximum sensitivity in the competition RIA, several dilutions of tissue culture supernatant containing MAb MB12 IgG were tested in a solid phase RIA for their ability to bind HIV. As shown in Fig. 2 increasing levels of MAb binding were observed with increasing amounts of purified HIV lysate adsorbed to assay wells. With 0.2 pg of the purified HIV preparation per well on the solid phase, 8- to 9-fold less MAb was required to achieve the same level of binding to 0.1 pg of the HIV lysate. Since the binding of MAb MB12 was similar using either 0.4 or 0.2 pg of the viral lysate, we decided to use the purified HIV preparation at the concentration of 0.2 kg per well in the detection plates as the source of p24 for a type-specific LICRIA utilizing MAb MB12. The purified HIV lysate was then used as competitor antigen to precipitate MAb MB12 in a liquid phase in reaction wells so inhibiting the binding of free MAb MB12 to the antigen in the detection wells. MAb MB12 was used at a final dilution ranging from 1:60 to 1:lOO because of the linear response in the titration curve described in Fig. 2. Dilutions from the upper portion of the curve were avoided because of the expected lower sensitivity of the assay at these antibody concentrations. At the same time, since at least 6000 total counts bound to the detection antigen were required for a good reproducibility of the RIA, dilutions corresponding to the lower portion of the titration curve were not used. As shown in Fig. 3, the sensitivity obtained with a 1:lOO MAb MB12 dilution was greater than that obtained with a dilution of 1:80 or 1:60. In fact, with the 1:lOO MAb final dilution, the 50% binding of MB12 to the detection antigen could be inhibited by only 18 ng of the purified HIV lysate diluted in assay buffer and could be completely inhibited by 300 ng of the same viral preparation. Normal mouse IgG, at a range of concentrations from 0.05-100 ng, did not demonstrate any binding to the competitor or to the detection antigen. Consequently, we decided to use in the LICRIA for HIV p24 a 1:lOO final MAb MB12 dilution, with a range from 6000-10000 total counts bound to the detection antigen in absence of competitor protein.

Assay conditions In order to enhance the sensitivity of the LICRIA, different concentrations of for the binding of a 1:lOO MAb a purified HIV 1ysate were used as competitors MB12 final dilution to the detection antigen under three different conditions of time and temperature. In the experiment shown in the inset of Fig. 3, the MAb-com-

206

petitor mixture was incubated in 96-well PVC microtiter plates (Reaction Plates) for 1 h at 37”C, for 2 h at 25°C and for 16-18 h at 4°C. The sensitivity of the RIA was the same after incubating the MAb-competitor mixture for 2 h at 25°C or after incubation for 16-18 h at 4°C. Incubation for 1 h at 37°C resulted in the least sensitive RIA. For practical reasons, we decided to incubate the MAb MB12 with competitor antigen for 16-18 h at 4°C. Triton X-100, an ionic detergent, was used to disrupt HIV virions and to release ~24, the major structural protein of the virus. To determine a concentration of Triton X-100 which would not cause any non specific binding of MAb to detection antigen, final concentrations ranging from 1% to 0.01% of Triton X-100 were used instead of competitor antigen in the competition RIA. No interference was shown with any of the Triton X-100 concentrations used. Besides, by using HIV lysate as competitor diluted in Triton X-100 at a 0.5% final concentration, no difference in sensitivity was shown with respect to the dilutions of the same competitor in assay buffer (data not shown). All competitor antigen preparations used in the LICRIA were therefore treated with 1% Triton as well as 0.05% Tween 20. Specificity and sensitivity of LICRIA An affinity purified HIV p24 was used as standard in the LICRIA. Complete inhibition of the MAb MB12 binding to the detection antigen was achieved in the LICRIA by 160 ng of the purified p24 while as little as 2 ng was required for 20% competition, with some competition observed at 800 pg. As shown in Fig. 4, the extract of the immortalized and infected T-cell clone Hc)/HTLV-III at a concentration of 100 )*g was able to inhibit completely the binding of the MAb MB12 to the detection antigen, while 7.5 kg achieved a 50% com-

LOG

COMPETITOR

PROTEIN

(rig)

Fig. 4. Specific reactivity of affinity purified HIV ~24. purified HIV lysate and H9/HTLV-III cell extract in a LICRIA. MAb MB12 at a I:100 final dilution was used to precipitate ~24 in the following competitors: affinity purified HIV ~24 (+), HIV lysate (0). and H9IHTLV-III extract (0). H9 extract (0) was used as negative control. Unbound MAb MB12 was detected using 0.2 kg of purified HIV lysate as described in Materials and Methods.

207

4

2

6

DAYS AFTER

INFECTiON

LOG

COMPETITOR

PRCWiN

(ng)

Fig. 5. Correlation of the LICRIA for the HIV p24 with RT. The RT assay (A) and the LICRIA (B) were performed on HIV infected and uninfected H9 cells as described in Materials and Methods. (A) A = HIV infected H9 cells; n = H9 uninfected cells; (B) l = HIV lysate; 0 = HIV infected H9 cell extract and Osupernatant on day 13: 0 = HIV infected H9 cell extract and qsupernatant on day 9; P’ = HIV infected H9 cell extract and V supernatant on day 6; V = HIV infected H9 cell extract and v supernatant on day 4; 3 = H9 uninfected cell extract and n supernatant.

petition in the RIA. Thus lOOO-fold more protein was required to achieve the same level of competition in cell extracts as that seen in the affinity purified HIV ~24. The density gradient purified HIV detergent lysate at a concentration of 18 ng was able to achieve a 50% competition so indicating that approximately 40% of the total viral proteins of the HIV preparation is ~24. The similar slopes and complete competition of the curves generated by the affinity purified HIV ~24, the purified HIV lysate and H9/HTLV-III cell extract indicate that a common antigenic determinant is detected in each of the three preparations by MAb MB12 in this RIA. Specificity of the LICRIA is further demonstrated by the lack of competition observed with as much as 100 pg of the uninfected T-cell clone H9. Correlation

of the LICRtA

for p24 and RT activity in infected cells

Fig. 5 shows the time course of HIV infected H9 versus uninfected H9 cells. The H9 cells were infected with HIV, as described in Materials and Methods, and supernatants from both infected and uninfected cell cultures were assayed for the presence of RT activity and for the presence of HIV p24 by LICRIA. In addition the cell pellets obtained from the centrifugation of infected and uninfected cultures were assayed for the presence of HIV p24 by LICRIA. The results plotted in Fig. 5, panel A, demonstrate a positive RT activity on day 13 while viral p24 is already detected six days after H9 cell infection (panel B). Also, on day six the LICRIA shows an equal expression of p24 in both cell extract and supernatant from infected cell cultures, but on day nine and 13 an enhanced expression of p24 is detectable in the cell extract with respect to the supernatant. Uninfected H9 cells were completely negative in both LICRIA and RT assay. In other experiments RT activity was always regularly detectable later than p24 (data not shown), thus in-

dicating that measuring viral activity as represented by the amount of ~24, the major structural protein, is more sensitive than RT assay. In addition, the possibility of using in the LICRIA the extract of infected cells as a source of p24 instead of the supernatant of the infected cell culture increases its sensitivity further.

Discussion The development of the LICRIA for the detection of HIV p24 offers advantages with respect to other tests available to detect replicating HIV in permissive cells. The use of antisera has limited the sensitivity and reproducibility of several tests and only recently Higgins et al. (1986) have demonstrated that using in a sandwich ELISA two monoclonal antibodies recognizing a different epitope of the HIV p24 the replicating virus could be detected in permissive ccl1 cultures three days earlier than the RT activity. The new MAb MB12 to HIV p24 used alone in the LICRIA, makes the immunoassay highly sensitive and reproducible. Our results, in fact, show that an affinity purified HIV p24 is able to give a 20% competition in the LICRIA at a concentration of 2 ng per well and still to compete at only 800 pg per well. Besides, the LICRIA widely anticipates the RT activity thus indicating that the amount of ~24, as expression of virus replication is, in comparison to the enzymatic activity, an earlier marker of infection. The easy methodology, the familiarity of many laboratories with RIA methods, the use of 96well microtiter plates and the relatively low cost of [‘251]protein A make this assay valuable for testing conveniently large numbers of samples. The LICRIA for HIV p24 is in addition a truly quantitative immunoassay. In fact, using the affinity purified HIV p24 as a standard in the LICRIA we were able to quantitate p24 in the purified HIV lysate and in the HIV infected H9 cell extract finding that 2.5-fold and lOOO-fold respectively more proteins than the pure HIV p24 are required to achieve a 50% competition in the RIA. The possibility by the LICRIA to recognize very small differences in the quantity of p24 has obviously important implications. The new immunoassay, in fact, could be useful to study viral replication in permissive cell cultures and to define the in vitro activity of antiviral drugs. Since minor differences can be detected in the gag gene of different HIV isolates (Higgins et al., 1986) there is the possibility that the LICRIA using a monoclonal antibody, can recognize even small differences in the structure of a protein with important diagnostic implications. In fact, variable epitopes might be used as markers for certain field isolates and so if subtle but detectable differences in the p24 appear, such markers would be valuable to trace the spread of particular subtypes of AIDS viruses from one individual to others within and across the limit of geographic areas.

Acknowledgements We thank Dr. M.G. Sarnga~haran,

Dr. R.C. Gallo, Dr. F. Barre-Sinoussi

and

209

Dr. J.C. Chermann for helpful discussions; Dr. E. Brocchi for testing the MAb MB12 reactivity to visna virus and bovine leukemia virus. The advice and encouragement of Dr. J. Schlom are gratefully acknowledged. This work was partially supported by Consiglio Nazionale delle Ricerche, Contratto Finalizzato 850089152 and Minister0 della Pubblica Istruzione.

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