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Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1
Journal of Immunological Methods, 128 (1990) 165-175 Elsevier
165
JIM 05512
Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1 Jacqueline Cogniaux
1, N a d i n e
De Schepper 1, Marie-Louise Blondiau 1, Bernard C o m e t 2, Peter Horal 3 and Anders Vahlne 3
1 Institut Pasteur du Brabant, Brussels, Belgium, and 2 Laboratoire des Macromol~cules aux Interfaces, Universit$ Libre de Bruxelles, Brussels, Belgium, and 3 Department of Clinical Virology, University of GiJtebor~ GiJtebor~ Sweden
(Received 21 September 1988, revised received 10 August 1989, accepted I December 1989)
Five mouse hybridomas which produce monoclonal antibodies against the p17 core protein of HIV-1 have been isolated. Cross-competition assays and mapping with synthetic peptides demonstrate that two closely related epitopes are identified by these antibodies. Directed against two neighbouring peptides at the carboxy-terminal end of the molecule, they can be used for the selective detection of p17 polypeptide in a viral extract or in an infected cell lysate by a solid-phase sandwich enzyme immunoassay. Key words: HIV-1; Monoclonal antibody; p17 core protein; ELISA, sandwich
Introduction
The human immunodeficiency virus (HIV) is the established etiological agent of the acquired immunodeficiency syndrome (AIDS) (Barr6Sinoussi et al., 1983; Gallo et al., 1984; Levy et al., 1984). The structural proteins of this retrovirus have been identified (Kitchen et al., 1984) and their genomic origins clearly established (Montagnier et al., 1985; Devare et al., 1986). The core components, coded by the gag gene, are synthesized as a precursor polyprotein of 55 kDa (p55) which is subsequently cleaved into p17, p24 and p13 proteins. Two of them have been localized inside the virus particle by immuno-electron microscopy (Gelderblom et al., 1987): a monoclonal antibody against p24 proved to label con-
Correspondence to: J. Cogniaux, Institut Pasteur du Brabant, 642, rue Engeland, B-1180 Brussels, Belgium.
sistently the core region of the virion, while a p17-specific antibody led to a spherical label confined to the submembrane portion of the virion. This p17 protein, derived from the amino terminus of the p55 precursor, is myristylated at its N terminal end (Rein et al., 1986). The five monoclonals described in this report identify two different epitopes on the p17 core protein of HIV. Cross-competition assays indicate that the two epitopes are closely linked and mapping with synthetic peptides confirm that they are situated in two neighbouring regions at the carboxy terminal end of the molecule. The use of monoclonal antibodies in capture enzyme immunoassays have been shown to be valuable for the detection and quantification of antigens, the greatest sensitivity being achieved by combining antibodies to different non-overlapping epitopes (Heinz et al., 1986). We demonstrate here that the specific detection of the p17 protein is possible with a solid-phase sandwich enzyme ira-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 munoassay (ELISA) using two of our monoclonals selected for their reactivity against two different epitopes.
Materials and methods
Cells and virus The Hut-78 established adult human T cell line chronically infected with the AIDS-associated retrovirus (ARV) isolated in San Francisco was kindly provided by Dr. Jay Levy (Levy et al., 1984, 1985). The ARV-4 isolate produced by these cells grown in roller bottles was concentrated from the culture medium in a hollow fiber concentrator (Amicon, H1 x 50 cartridge) and centrifuged at 45,000 x g for 1.5 h. This virus pellet was then purified by equilibrium centrifugation in a linear metrizamide gradient (4-36% in Tris-HC1 10 mM pH 8, NaC1 100 mM, EDTA 1 raM). The virus band was found at a density of 1.12 g / m l as established by the position of the reverse transcriptase in the gradient. The HTLV-IIIB strain of HIV (Popovic et al., 1984) produced in Molt 3 cells and purified as previously described, was kindly provided by Dr. C. Briick (Smith-Kline Biologicals, Rixensart, Belgium). Enzyme-linked immunosorbent assay (ELISA) The purified virus preparation was extracted once with a three-fold volume of diethyl ether or, preferentially, disrupted with 2% octyl-fl-glucopyranoside (OGP, Sigma) and dialysed against phosphate-buffered saline (PBS) before use as antigen. The protein content was estimated by the method of Bradford (Bio-Rad protein assay reagent). 50 #1 containing 250-500 ng of viral antigen in 0.02 M carbonate buffer pH 8.8 were incubated overnight at 4°C in a 96 well Immunoplate I (Nunc, Denmark). After saturation of the plastic with 3% serum albumin (BSA) in phosphate-buffered saline (PBS), the culture supernatants to be screened were incubated in the coated wells for 2 h at 37°C. After washing with 0.05% Tween 20 in PBS, 50/L1 of peroxidase-conjugated goat anti-mouse globulin (Amersham) diluted 1/1000 in 1% BSA in PBS were incubated in each well for 2 h at 37 ° C. Colour development of the
bound peroxidase conjugate was achieved with citrate-phosphate buffer pH 5.2 and 0.015% H202.
Production of monoclonal antibodies For fusions 1 and 2, BALB/c mice were inoculated with 100 /~g of purified virus (ARV-4), partly subcutaneously and partly intraperitoneally, in complete Freund's adjuvant. They were boosted intraperitoneally after 3 weeks and 6 weeks, before a last injection was given intravenously 4 days before fusion. A similar immunization process was followed with detergenttreated (OGP) virus HTLV-III/B for fusion no. 3. The conventional technique (Fazekas de St.Groth and Scheidegger, 1980) was used to obtain hybrids between Sp2-0 myeloma cells and splenocytes. Each colony supernatant was screened for antiHIV-1 antibodies in an ELISA using the antigen corresponding to the initial immunogen. An additional screening was performed using sonicated cell suspensions as antigens (Hut-78 cells or Hut78/ARV-4 producing cells at 5 x 106/ml in PBS, 2 X 10 -4 M PMSF) to eliminate hybrids reacting with cellular components. Each well was coated with 10-15 pg of the clarified sonicate diluted in carbonate buffer pH 8.8. Western blot immunoassay Purified HIV-1 (10-15/~g/lane) or cell lysates (5 X 107 cells/ml, of PBS containing 1% Triton X-100, 0.5% deoxycholate, 0.2 mM PMSF) were submitted to electrophoresis in 12% polyacrylamide gels. The resolved polypeptides were transferred to nitrocellulose at 50 V for 16 h. After saturation with a mixture of 2% bovine serum albumin (BSA) and 3% gelatin as described (Cogniaux and Jacquemin, 1985) or with 5% non-fat dry-milk (Sarugadharan et al., 1985), individual strips were reacted overnight with 1/2 dilutions of the culture supernatants to be tested. Bound antibody was detected with 1/200 biotinylated sheep anti-mouse i m m u n o g l o b u l i n ( A m e r s h a m , RPN1021), 1/1000 peroxidase-labelled streptavidin (Amersham, RPN1231) and 4-chloro-l-naphthol as substrate. Purification and labelling of the monoclonal antibodies Immunoglobulins were purified from the culture fluids of different hybrids by chromatography
167 on Affi-gel-Protein A (MAPS kit from Bio-Rad). About 200 ml of culture supernatant were slowly run through 1 ml of gel and the immunoglobulins were eluted using 3 ml of pH 3.5 buffer. They were immediately neutralized with 1 M Tris-HC1, pH 8.5 and dialysed. Radioiodination was performed on 10-13/xg of IgG using the chloramine-T method. The labelled protein was filtered through a 10 ml Sephadex G-25 column saturated with 1% BSA in PBS. Biotin was conjugated to two of the monoclonal IgG antibodies as described by Goding (1980). The protein was adjusted to 1 mg/ml and dialysed against 0.1 M NaHCO 3 before mixing with biotin succinimide ester (1 mg/ml dimethylsulphoxide). The mixture (200 /~g of biotin for 1 mg IgG) was left at room temperature for 4 h and then dialysed against PBS containing 0.001% merthiolate.
Radioimmunoprecipitation 100/~1 of culture supernatant or 20 #1 of ascitic fluid were incubated for 2 h at room temperature with 500,000 cpm of 125I-viral lysate in 100 #l of RIPA buffer. (50 mM Tris-HC1 pH 7.9, 0.2 M NaC1, 2% Triton X-100, 0.1% Tween 80, 0.2 mM PMSF, 0.02% NAN3). Immunocomplexes were precipitated with 100 #1 of a 10% (v/v) suspension of protein A-Sepharose (Pharmacia) to which was coupled 5 /~g of goat anti-mouse Ig (Kpl, affinity purified antibody). After incubation for 16 h at 4 ° C, the Sepharose beads were washed twice with 1 ml of 'high salt washing buffer' and twice with 'low salt washing buffer' (Schneider et al., 1984). The immunoprecipitated polypeptides were separated by SDS-PAGE and visualized by autoradiography using X-ray film (Kodak X-O-Mat).
Competitioe binding of the monocional antibodies The virus proteins were bound to the wells of a polyvinylchloride microtitre plate (125 ng/well, in 0.02 M carbonate buffer pH 8.8). After saturation of the free sites with 3% BSA for 2 h, serial dilutions of the unlabelled immunoglobulin, each in a volume of 50 #1, were added for 2 h at 37 ° C. 25 #1 (50,000 cpm) of iodinated antibody were then added and the mixtures further incubated for 2 h at 37°C.
After thorough washing, the individual wells were separated with scissors and counted in an autogamma counter (Berthold).
Peptide synthesis Based on the original sequencing (Muesing et al., 1985), eight 20-25 mer HIV-1 pl7-encoded peptides, overlapping by 5-6 amino acids, were synthesized utilizing automated solid-phase peptide synthesis on a Applied Biosystems 430A peptide Synthesizer. The peptides were synthesized using the t-Boc synthesis strategy protocol suggested by the manufacturer, all liquid reagents were from Applied Biosystems (Foster City, CA, U.S.A.). Amino acids used were from Nova Biochem (Switzerland). p-methylbenzhydryl amine (Peptides Int., Louisville, U.S.A.) was used as polymer resin. Following each amino acid coupling a sample was taken and a quantitative ninhydrin assay was performed. Only if the coupling efficiency exceeded 99% of each amino acid, was the peptide accepted for further processing. After each completed synthesis, the peptide was cleaved from the resin and the protective side chains removed by hydrogen fluoride (HF) (AGA, Sweden). During HF cleavage, anisole and dimethylsulphide (DMS) (both chemicals from Merck, F.R.G.) were added as scavengers. Immediately after HF cleavage the peptide-resin mixture was resuspended in trifluoroacetic acid (TFA) (Janssen Chemica, Belgium) and the peptide precipitated in diethyl ether (Merck). Finally, the precipitated peptide was dried under vacuum in a desiccator.
Peptide antigen coating The individual peptides were coated onto Nunc high-binding microtiter plates (Nunc, Denmark) as follows: 100 /~l poly-L-lysine (Sigma, U.S.A.), diluted 1/100 in phosphate-buffered saline (PBS), were added to each well and allowed to react for 30 min at room temperature. After discarding the poly-L-lysine, 50 /~l of the peptide antigen (dissolved in 10% acetic acid and made up to a concentration of 10/xg/ml in PBS coating buffer) were then added, promptly followed by the addition of 50 /tl 0.5% glutaraldehyde (Sigma). The mixture was allowed to react for 15 rain at room temperature after which the wells were washed
168 twice with PBS and subsequently with 200 #l of 100 m M glycine (Merck) in 0.1% bovine serum albumin (BSA) made up in water. After 30 min at room temperature the plates were again washed twice with PBS. Free protein binding sites were saturated by adding 200 #l/well of 3% BSA (made up in PBS) and incubated in a humidifier at 37 o C overnight.
manufacturer, were added and allowed to react for 1 h at 37 ° C in a humidifier. After four washes, 200 #1 of phosphatase substrate (Sigma), diluted in diethanolamine (DEA) (Fluka Chemic) (1 mg of substrate/ml of DEA) were added per well. After 20 min incubation at room temperature absorbance values were read at 405 nm.
Peptide ELISA
Results
Each of the five hybridoma culture supernatants, assayed for peptide reactivity, was tested against each p17 synthetic peptide at a dilution of 1 / 1 0 (in PBS containing 1% BSA and 0.05% Tween) using 100 #1 sample/well. After incubation for 1 h at 3 7 ° C in a humidifier the plates were washed four times in PBS containing 0.05% Tween 20. Subsequently, 100 #1 of alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (Jackson), at the dilution suggested by the
Of the several hundred hybridoma fluids tested from three independent fusions, five were found to secrete antibody specific for a protein with a molecular weight of 17 kDa, as shown by Western blot on purified HIV-1 preparations (data not shown). An additional thin band at 55 kDa was also recognised. Fusions 1 and 2, carried out after immunization of mice with intact virions (ARV-4) exclusively gave this type of specificity. The third
1
2
3
4
5
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a b
a b
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a b
6
7 ab
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a
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b
m
G P 16 0
i
I
U
m
!i
P r 55 P 40
Ill
P 24
I
I
i
m
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i
P 17
Fig. 1. Immunoblot of the proteins from cell lysates recognizedby the different Mabs: (1) 3Hll-C1; (2) 3D3-B6; (3) 8A6-A7; (4) 6D6-F6; (5) 6B6-H9; (6) Control human serum (HIV-1 negative) (7) Control human serum (HIV-1 positive). Lane a: ARV4-producing Hut78 cells; Lane b: Hut78 control cells.
169 fusion, performed after immunization with disrupted virions, gave rise to the 3D3 monoclonal, together with a series of anti-p24 antibodies which are not described here. The identity of the protein recognized by these hybridomas as a virus-specific product was confirmed by comparing their reactivities on lysates of Hut-78 control cells and Hut-78 (ARV-4) infected cells: only the virus-producing cells contained reactive antigens in immunoblotting (Fig. 1). In addition to the 17 kDa and the 55 k D a proteins detectable in a purified virus preparation, a third band at 40 k D a was visible in the cell lysate. A similar pattern was described previously by Chassagne et al. (1986), who suggested that p40 corresponds to an intermediate cleavage product of the gag precursor p55. The monoclonal antibodies were also able to immunoprecipitate the p17 protein from 125I-labelled virions, disrupted by 2% octylglucopyranoside (Fig. 2). These results indicated that the five hybrids, cloned twice in soft agar, produced monoclonal antibodies against the p17 internal core protein of the virion and against its precursor p55. Thus they were able to recognize their target after denaturation of the protein (Western blot) as well as in its native form (immunoprecipitation). The maximal amount of antibody bound to a constant amount of antigen can be considered as a direct expression of its affinity for the antigen (Frankel and Gerhard, 1979). The plateau of absorbance values (A492) reached using increasing dilutions of ascitic fluid in the ELISA permitted the different monoclonals to be ranked in the following order of affinity: 8A6-A7 > 3 D 3 - F l l > 3 H l l - C 1 > 6B6-H9 > 6D6F6. The characteristics of the five hybrid clones are summarized in Table I.
Cross-competition The epitope specificity of the monoclonals was examined in competition assays using the viral antigen adsorbed on microtest plates. The binding of each iodinated IgG in the presence of an excess of 'cold' antibody is shown in Table II. The radioactivity bound in the presence of an irrelevant monoclonal (anti-HTLV-I p24) determined the 100% binding value. Two groups of anti-pl7 antibodies were distinguished, each competing slightly
M.W.
1
2
3
4
5
6
7
8
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I
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P~ P!7 Fig. 2. Radioimmunoprecipitation of 125I-HIV antigens followed by SDS-PAGE analysis and autoradiography. The precipitates obtained with each monoclonai from hybrid culture fluid were analysed in the following lanes: 1 = Mab 8A6-A7; 2 = Mab 3D3-B6; 3 = Mab 6D6-F6; 4 = Mab 3Hll-C1; 5 = Mab 6B6-H9; 6 = negative control; 7 = Mab anti-p24; 8 = Mab HIV-positive human serum. M.W. indicates the molecular weight markers used. From top to bottom, phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme(14,400). with one another (Table II). This conclusion was confirmed by the competition curves established between variable amounts of each monoclonal IgG and a constant amount of 125I-3Hll-C1 (Fig. 3A) or 125I-6D6-F6 (Fig. 3B). TABLE I CHARACTERISTICSOF THE MONOCLONALS Antigenic specificity was determined by immunoblotting. Biotinylated anti-mouse subclass reagents were used in an ELISA for subclass determination. The plateau of absorbance values (A492) reached using increasing dilutions of ascitic fluid in the ELISA permitted evaluations of the relative affinities of the monoclonals. Fusion Hybridoma Specificity Subclass A492 no. (plateau value) 1 2 3
8A6-A7 3Hll-C1 6D6-F6 6B6-H9 3D3-Fll
p17,p55 p17,p55 p17,p55 p17,p55 p17,p55
IgG2b IgG1 IgG1 IgG3 IgG1
1.82 1.36 0.554 0.725 1.73
170 T A B L E II EPITOPE SPECIFICITY S T U D I E D BY C O M P E T I T I V E B I N D I N G Each unlabelled monoclonal was incubated with the antigen for 2 h before further incubation with each individual iodinated IgG. A n irrelevant monoclonal (V G10-A3, anti-p24 HTLV-I) was used as unlabelled competitor to determine the 100% value in each case. Competitor antibody
Percentage of iodinated IgG b o u n d
6B6-H9 6D6-F6 8A6-A7 3Hll-C1 3D3-B6 VG10-A3
6B6-H9 *
6B6-F6 *
9.5% 113% . 100%
0.6% 1.5% 51% 76% . 100%
.
8A6-A7 *
3Hll-C1 *
82% 74% 9.2% 20%
52% 64% 2% 1.36%
63.6% 59.5% 3.3% 2.5% 4.2% 100%
. 100%
The binding of 3Hll-C1 was completely inhibited by approximately 100 ng of 8A6-A7 or 3Hll-C1 IgG (Fig. 3.4) but only 30% inhibition was reached using as much as 450 ng of 6D6-F6 or 6B6-H9. Conversely, the binding of labelled 6D6-F6 was completely inhibited by its 'cold' counterpart or by 6B6-H9, but only to a limited extent by 8A6-A7 or 3Hll-C1 (Fig. 3B). 6B6-H9 antibody was even more active in competition than the autologous 6D6-F6, especially at low doses, a finding which can be explained by the higher avidity of 6B6-H9 for the same epitope (see Table I).
3D3-B6 *
100%
The cross-competition experiments suggest that two antigenic regions, which are probably closely situated on the p17 protein, can be defined by the two groups of monoclonal antibodies. This conclusion has been confirmed by using eight synthetic peptides covering the whole sequence of the p17 protein (Fig. 4) in a peptide ELISA. As seen in Table III, three hybridomas (8A6-A7, 3Hll-C1, 3D3-Fll) showed strong reactivity against the p17-G peptide. The other two hybridomas (6D6-F6 and 6B6-H9) were strongly reactive with the p17-H peptide. No hybridoma exhibited reactivity to more than one peptide.
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Fig. 3. Binding assay of a given a m o u n t of radioiodinated Mab l g G in the presence of variable a m o u n t s of each monoclonal IgG. 125 125 8A6-A7 (*); 3 H l l - C 1 (o); 6D6-F6 (e); 6B6-H9 (¢0- A: binding of I - 3 H l l - C 1 . B: binding of I-6D6-F6.
171
IP-1-AjIP17 P17-c
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AAADTGHSSQVSQNYPIVQN I ALDK]EEEQNKSK KKAQQAAADT 749
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632 Fig. 4. Schematic presentation of the eight, overlapping p17 peptides and their designation. Also shown are the amino acid sequences for the two reactive peptides (pl7-G and pl7-H). Below the first and last amino acid of the sequence are shown the corresponding base pair number according to the original sequencing data (see materials and methods section).
4"
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0.5
Detection of p 1 7 / p 5 5 in cell lysates by a standard solid-phase ELISA
d
HIV-1 c a n m u l t i p l y in h u m a n l y m p h o c y t e cultures f r o m i n f e c t e d i n d i v i d u a l s a n d p r o d u c t i v e infection c a n b e m e a s u r e d with a s a n d w i c h imm u n o a s s a y that detects viral antigens in small v o l u m e s of cell lysates ( M c D o u g a l et al., 1985). W e have used o n e a n t i - p l 7 m o n o c l o n a l as c a p t u r e antibody and a second anti-pl7 antibody directed against a different e p i t o p e as d e t e c t o r a n t i b o d y . Cell suspensions f r o m v i r u s - p r o d u c i n g cultures o r u n i n f e c t e d c o n t r o l s were a d j u s t e d to 2 × 1 0 6 / i l l l in the original culture m e d i u m a n d a d d e d with 1% Triton-X100. 50 /~1 v o l u m e s of cell lysates were d i s t r i b u t e d in the wells of m i c r o t e s t p l a t e s previo u s l y c o a t e d with 200 ng I g G f r o m M a b 8 A 6 - A 7 in c a r b o n a t e b u f f e r p H 9.6 a n d s a t u r a t e d w i t h 1% s e r u m a l b u m i n a n d 4% n e w b o r n calf s e r u m in PBS. T h e p r o t e i n b o u n d was t h e n d e t e c t e d b y a b i o t i n c o n j u g a t e o f M a b 6 D 6 - F 6 I g G followed b y s t r e p t a v i d i n c o n j u g a t e d to p e r o x i d a s e , a n d staining.
I
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NUMBER
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Fig. 5. Detection of p17/p55 in serial dilutions of cell extracts by a 'sandwich' ELISA with biotinylated Mab 6D6-F6. Curves a and b: virus-producing Molt 3 extract captured by the anti-pl7 Mab 8A6-A7 (a) or by the unrelated VG10-A3 (b). Curves c and d: control Molt 3 extract captured by the anti-pl7 (c) or by the unrelated Mab (d).
T h e d o s e - r e s p o n s e curve o b t a i n e d with serial d i l u t i o n s of a v i r u s - i n f e c t e d cell extract i n d i c a t e d a linear r e l a t i o n s h i p b e t w e e n a b s o r b a n c e values at 492 n m a n d the d o s e o f i n f e c t e d cells b e t w e e n 105 a n d 0.6 × 1 0 4 / w e l l (Fig. 5, curve a). T h e s a m e e x t r a c t a d s o r b e d to wells c o a t e d w i t h a n u n r e l a t e d m o n o c l o n a l ( a n t i - p 2 4 H T L V - 1 ) gave a low signal (Fig. 5, curve b), s i m i l a r to the b a c k g r o u n d ob-
TABLE III OPTICAL DENSITY READINGS (405 NM) OBTAINED USING EACH OF THE FIVE HYBRIDOMA COMBINATIONS An unrelated HTLV-1 clone (VG10-A3) was also included as a negative control. Hybridoma
Peptide designation pl7-A
pl7-B
pl7-C
p-17D
pl7-E
p-17F
pl7-G
pl7-H
8A6-A7 3Hll-C1 6D6-F6 6B6-H9 3D3-Fll HTLV-1 VG10-A3
0.09 0.14 0.13 0.00 0.11 0.01
0.14 0.15 0.19 0.00 0.17 0.00
0.11 0.20 0.23 0.02 0.10 0.00
0.11 0.16 0.15 0.00 0.12 0.00
0.08 0.12 0.16 0.01 0.07 0.00
0.12 0.12 0.16 0.01 0.12 0.01
1.90 > 2.00 0.23 0.02 2.00 0.01
0.08 0.08 > 2.00 1.15 0.08 0.01
172 TABLE IV APPLICATION O F T H E HIV-CAPTURE I M M U N O A S S A Y TO LYSATES F R O M C U L T U R E S O F H U M A N LYMPHOCYTES Groups I and II were cultures from seropositive individuals; group III were cultures from seronegative individuals.
(A) Control assay (B) p17/p55 assay (C) p24/p55 assay Culture
Capture Ab
Biotinylated detector Ab
Anti-HTLV-1 Mab VG10-A1 Anti-HIV p17 Mab 8A6-A7 Anti-HIV-p24 Mab 9A6-H12
Anti-HIV p17 Mab 6D6-F6 Anti-HIV p17 Mab 6D6-F6 Polyclonal human anti-HIV IgG
Absorbance (A 492) A Control assay
B p17/p55 assay
C p24/p55 assay
0.254 + 0.233 + 0.214 + 0.256 +
0.003 0.002 0.006 0.006
0.578 + 0.007 0.588 + 0.009 1.084 + 0.051 0.422 + 0.005
0.443 0.678 0.465 0.896
0.241 + 0.002 0.257 + 0.008 0.299 + 0.003
0.313 + 0.066 0.211 + 0.023 0.312 + 0.001
0.869 -t- 0.076 0.360 + 0.016 0.399 + 0.008
0.228 + 0.003 0.291 + 0.001 0.226 + 0.008
0.299 + 0.009 0.299 -t- 0.009 0.221 + 0.012
0.237 + 0.008 0.237 + 0.008 0.175 + 0.010
0.294
0.347
0.243
Group 1 TJ (2nd) a VG VK TS (2nd) ~
-1-0.018 + 0.069 + 0.045 + 0.021
Group II TS (lst) a PS TJ (lst)
Group 11I MC AJ SJ ' positive' threshold value
a 1st and 2nd correspond to successive samples taken from the same lymphocyte culture at 4 days intervals.
served with control uninfected cells in wells coated with the HIV-1 specific 8A6-A7 antibody (curve c) or the anti-HTLV-1 antibody (curve d). This immunoassay has been applied to a small number of human lymphocyte cultures from seropositive individuals, set u p in order to study materno-foetal transmission of HIVs (SprecherGoldberger et al., 1988). Lymphocyte extracts, made up with 2 × 106 cells/ml culture medium and 1% Triton X-100, were tested in parallel in the 'sandwich ELISA' described earlier (Table IV, B) and with an anti-p24 Mab as capture antibody and biotinylated human 'HIV-positive' IgG as detector antibody (Table IV, C). Coating with the unrelated VG10-A3 Mab controlled the specificity of the capture stage (Table IV, A). Absorbance readings greater than five standard deviations above the mean of the results in three uninfected lymphocyte cultures were considered positive. A good correlation between the two assays was observed for the samples of group I (positive) and
group III (negative). Results obtained with the three specimens in group II indicated that using a polyclonal anti-HIV detector antibody offered greater sensitivity in the detection of viral antigens.
Discussion
A monoclonal antibody specific for p17/18 has been described previously by Chassagne et al. (1986). The p55-gag precursor and a p40 intermediate product was also recognized by this monoclonal, a finding similar to that reported here. Four monoclonals, specific for p24 but non reactive with p55, have been described (Di Marzo Veronese et al. 1985). They have proved to be useful for immunohistology of lymph nodes in patients with HIV infection (Tenner-Racz et al., 1987). Among other monoclonals, raised against
173 the gag proteins of a British isolate of HIV, three recognized p55:p18, and three p18 alone (Ferns et al., 1987). In this report, immunization of mice with intact virions gave rise to hybrids secreting anti-p17 antibodies, whereas the injection of disrupted particles led essentially to the production of anti-p24 hybridomas, suggesting that some antigenic determinants of p17 are localized on the surface of the virion and readily accessible to the immune system of the host. This suggestion has been confirmed by a recent observation of Gelderblom et al. (1988), showing by immunoelectron microscopy that the p17 epitopes can be localized on the surface of the virion or budding HIV-1. The cross-competition experiments described here led us to conclude that two closely located epitopes could be distinguished on the p17 core protein by five monoclonal antibodies. Synthetic peptides offer an excellent tool for the mapping of Mab reactivity, and determining whether or not different clones are directed against the same epitope region. However, it should be noted that only antibodies directed against continuous epitopes lend themselves to mapping in this fashion. We were able to demonstrate that the monoclonals 6D6-F6 and 6B6-H9 were reactive with the carboxy-terminal peptide H (118-138), whereas 8A6, 3 H l l and 3D3 showed reactivity with the neighbouring peptide G (100-122) in complete agreement with the results of the competition experiments (P. Horal et al., 1988). A region of homology between the primary sequence of thymosin a-1 and the gag protein p17 has been detected by Satin et al. (1986). A 30amino acid synthetic peptide (HGP 30), analogous to that region and spanning residues 86-115 of the gag protein, has been shown to elicit antibodies which recognize the p17 protein by immunoblotting and to neutralize several strains of HIV in vitro (Naylor et al., 1987). Interestingly, peptide G (position 100-122) partially overlaps this HGP 30 peptide. The possibility that our anti-p17 monoclonals might neutralize the infectivity of HIV-1 in vitro now needs to be considered. McDougal et al. (1985) have described a solid-phase sandwich ELISA for the detection of the HIV-1 antigens in supernatants of LAV-infected human lymphocyte
cultures. Using HIV-positive human sera, this technique has been applied successfully for the detection of various types of antigen with monoclonal antibodies, acting as either capture antibody a n d / o r detector antibody (Mizuchi et al., 1984; Soos et al., 1984; Heinz et al., 1986). Their effectiveness was shown to depend primarily on their avidity and the highest sensitivity was achieved by combining antibodies to different non-overlapping epitopes (Heinz et al., 1986). In our hands, the monoclonal 8A6-A7 can be used as capture antibody and 6D6-F6 as detector antibody, although they recognize two neighbouring peptides, H and G, at the carboxy terminal end of the molecule. The specificity of this assay was confirmed by the absence of antigen binding when the unrelated anti-HTLV-1 Mab was used to coat the plates. The analysis of ten samples from human lymphocyte cultures suggested that the presence of viral proteins could be detected by two anti-pl7 monoclonals in a 'sandwich ELISA' although greater sensitivity was obtained using an anti-p24 monoclonal as capture antibody, followed by a biotinylated polyclonal IgG which could bind to many epitopes on p24 and p55 proteins. However, our system permits the selective detection of p17 and has been used by one of us (B.C.) to study the insertion o f different HIV proteins into lipid vesicles. Using this kind of assay for the quantification of p17/p55 in a cell lysate or in a virus preparation would require purified preparation of the proteins as standards and these are not yet available. However, different preparations of viral antigens can be compared and relative amounts of p17 can be evaluated since the absorbance values in ELISA are directly proportional to the antigen dilution over a given concentration range.
Acknowledgements We gratefuUy acknowledge the secretarial ~help of J. Herinckx. This work was supported by a grant from the Fonds de la Recherche Scientifique M&licale (Belgium) and a predoctoral fellowship to B.C. from the Institut pour 1' Encouragement de la Re-
174 cherche Scientifique dans l'Industrie et l'Agriculture. N.D. was supported by Smith-Kline RIT.
References Barr6-Sinoussi, F., Chermann, J.C., Rey, F., Nugeyre, M.T., Charmaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier, L. (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 220, 86. Chassagne, J., Verrelle, P., Dionet, CI., Clavel, F., Barr6Sinoussi, F., Chermann, J.C., Montagnier, L., Gluckman, J.C. and Klatzmann, D. (1986) A monoclonal antibody against LAV gag precursor: use for viral protein analysis and antigenic expression in infected cells. J. Immunol. 136, 1442. Cogniaux, J. and Jacquemin, P.C. (1985) Production of monoclonal antibodies against HTLV-I proteins recognizing surface epitopes of live infected cells. Leukemia Res. 9, 1117. Devare, S.G., Srinivasan, A., Bohan, C.A., Spira, T.J., Curran, J.W. and Kalyanaraman, V.S. (1986) Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products. Proc. Natl. Acad. Sci. U.S.A. 83, 5718. Di Marzo Veronese, F., Sarngadharan, M.G., Rahman, R., Markham, P.D., Popovic, M., Bodner, A.J. and Gallo, R.C. (1985) Monoclonal antibodies specific for p24, the major core protein of human T-cell leukemia virus type III. Proc. Natl. Acad. SCi. U.S.A., 82, 5199. Fazekas de St.Groth, S. and Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1. Ferns, R.B., Tedder, R.S. and Weiss, R.A. (1987) A characterization of monoclonal antibodies against the human immunodefieiency virus (HIV) gag products and their use in monitoring HIV isolate variation. J. Gen. Virol. 68, 1543. Frankel, M. and Gerhard, W. (1979) The rapid determination of binding constants for antiviral antibodies by a radioimmunoassay. An analysis of the interaction between hybridoma proteins and influenza virus. Mol. Immunol. 16, 101. Gallo, R.C., Salahuddin, S.Z., Popovic, M., Shearer, G.M., Kaplan, M., Haynes, B.F., Palker, T.J., Redfield, R., Oleske, J., Safai, B., White, G., Foster, P. and Markham, P.D. (1984) Frequent detection and isolation of cytopathic retrovirus (HTLV-III) from patients with AIDS and at risk for AIDS. Science 224, 500. Gelderblom, H.R., Hausmann, E.H.S., Ozel, M., Pauli, G. and Koch, M.A. (1987) Fine structure of human immunodeficiency virus (HIV) and immunolocalization of structural proteins. Virology 156, 171. Gelderblom, H., Winkel, T., Reupke, H., Rabanus, J.P., Biedrig, M., (3oldstein, A., Sarin, P. and Pauli, G. (1988) Localization of p17 epitopes by immunoelectron mi-
croscopy in and on HIV-1. Abstracts IV International Conference on AIDS, Stockholm, no. 1006, p. 114. Goding, J.W. (1980) Antibody production by hybridomas. J. Immunol. Methods 39, 285. Heinz, F.X., Tuma, W., Gnirakhoo, F. and Kunz, C. (1986) A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of Tick-borne encephalitis virus. J. Biol. Stand. 14, 133. Horal, P., Cogniaux, J., Deschepper, N. and Vahlne, A. (1988) Fine mapping of monoclonal antibodies directed against the HIV-1 gag proteins using synthetic peptides. Abstracts IV International Conference on AIDSn Stockholm, no. 1656, p. 89. Kitchen, L.W., Barin, F., Sullivan, J.L., McLane, M.F., Brettier, B., Levine, P.H. and Essex, M. (1984) Aetiology of AIDS-antibodies to human T-cell leukaemia virus (type III) in haemophiliacs. Nature 312, 367. Levy, J.A. and Shimabukuro, J. (1985) Recovery of AIDS-associated retroviruses from patients with AIDS or AIDS-related conditions and from clinically healthy individuals. J. Infectious Dis. 152, 734. Levy, J.A., Hoffman, A.D., Kramer, S.M., Landis, J.A. and Shimabukuro, J.M. (1984) Isolation of lymphocytopathic retroviruses from San Francisco patients with AIDS. Science 225, 840. McDougal, J.S., Cort, S.P., Kennedy, M.S., Cabridilla, C.D., Feorino, P.M., Francis, D.P., Hicks, D., Kalyanaraman, V.S. and Martin L.S. (1985) Immunoassay for the detection and quantitation of infectious human retrovirus, lymphadenopathy-associated virus (LAV). J. Immunol. Methods 76, 171. Mizuchi, A., Kitagawa, N., Miyachi, Y. and Lio, M. (1984) Monoclonal antibodies to human chorionic gonadotropin and their application to two-site sandwich radioirnmunoassay. J. Immunol. Methods 74, 369-374. Montagnier, L., Clavel, F., Krust, B., Chamaret, S., Rey, F., Barr~-Sinoussi, F. and Chermann, J.C. (1985) Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus. Virology 144, 283. Muesing, M.A., Smith, D.H., Cabradilla, C.D., Benton, C.V., Lasky, L.A. and Capon, D.J. (1985) Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature 313, 450-458. Naylor, P.H., Nayior, C.W., Badamchian, M., Wada, S., Goldstein, A.L., Wang, S-S., Sun, D.K., Thronton, A.H. and Satin, P.S. (1987) Human immuno-deficiency virus contains an epitope immunoreactive with thymosin a-1 and the 30-amino acid synthetic p17 group-specific antigen peptide HGP-30. Proc. Natl. Acad. Sci. U.S.A. 84, 2951. Popovic, M., Sarngadharan, M.G., Read, E. and Gallo, R.C. (1984) Detection, isolation and continuous production of cytopathic retrovirnses (HTLV-III) from patients with AIDS and pre-AIDS. Science 224, 497. Rein, A., McChire, M.R., Rice, N.R., Luftig, R.B. and Schultz, A.M. (1986) Myristylation site in Pr65 g~g is essential for virus particle formation by Moloney murine leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 83, 7246.
175 Sarin, P.S., Sun, D.K., Thornton, A.H., Naylor, P.H. and Goldstein, A.L. (1986) Neutralization of HTLV-III/LAV replication by antiserum to thymosin a-1. Science 232, 1135. Sarngadharan, M.G., Di Marzo Veronese, F., Lee, S. and Gallo, R.C. (1985) Immunological properties of HTLV-III antigens recognized by sera of patients with AIDS and AIDS-related complex and of asymptomatic carriers of HTLV-III infection. Cancer Res. (suppl.), 45, 4574s. Schneider, J., Yamamoto, N., Hinuma, Y. and Hunsmann, G. (1984) Sera from adult T-cell leukemia patients react with envelope and core polypeptides of adult T-cell leukemia virus. Virology, 132, 1.
Soos, M., Taylor, S.J., Gard, T. and Siddle, K. (1984). A rapid, sensitive two-site immunometric assay for TSH using monoclonal antibodies: investigation of factors affecting optimisation. J. Imrnunol. Methods 73, 237-249. Termer-Racz, K., Racz, P., Dietrich, M., Kern, P., Janossy, G., Veronese-Dimarzo, F., Klatzmann, D., Gluckman, J.C. and Popovic, M. (1987) Monoclonal antibodies to human immunodeficiency virus: their relation to the patterns of lymph node changes in persistent generalized lymphoadenopathy and AIDS. AIDS 1, 95.
165
JIM 05512
Characterization of monoclonal antibodies against the p17 core protein of the human immunodeficiency virus 1 Jacqueline Cogniaux
1, N a d i n e
De Schepper 1, Marie-Louise Blondiau 1, Bernard C o m e t 2, Peter Horal 3 and Anders Vahlne 3
1 Institut Pasteur du Brabant, Brussels, Belgium, and 2 Laboratoire des Macromol~cules aux Interfaces, Universit$ Libre de Bruxelles, Brussels, Belgium, and 3 Department of Clinical Virology, University of GiJtebor~ GiJtebor~ Sweden
(Received 21 September 1988, revised received 10 August 1989, accepted I December 1989)
Five mouse hybridomas which produce monoclonal antibodies against the p17 core protein of HIV-1 have been isolated. Cross-competition assays and mapping with synthetic peptides demonstrate that two closely related epitopes are identified by these antibodies. Directed against two neighbouring peptides at the carboxy-terminal end of the molecule, they can be used for the selective detection of p17 polypeptide in a viral extract or in an infected cell lysate by a solid-phase sandwich enzyme immunoassay. Key words: HIV-1; Monoclonal antibody; p17 core protein; ELISA, sandwich
Introduction
The human immunodeficiency virus (HIV) is the established etiological agent of the acquired immunodeficiency syndrome (AIDS) (Barr6Sinoussi et al., 1983; Gallo et al., 1984; Levy et al., 1984). The structural proteins of this retrovirus have been identified (Kitchen et al., 1984) and their genomic origins clearly established (Montagnier et al., 1985; Devare et al., 1986). The core components, coded by the gag gene, are synthesized as a precursor polyprotein of 55 kDa (p55) which is subsequently cleaved into p17, p24 and p13 proteins. Two of them have been localized inside the virus particle by immuno-electron microscopy (Gelderblom et al., 1987): a monoclonal antibody against p24 proved to label con-
Correspondence to: J. Cogniaux, Institut Pasteur du Brabant, 642, rue Engeland, B-1180 Brussels, Belgium.
sistently the core region of the virion, while a p17-specific antibody led to a spherical label confined to the submembrane portion of the virion. This p17 protein, derived from the amino terminus of the p55 precursor, is myristylated at its N terminal end (Rein et al., 1986). The five monoclonals described in this report identify two different epitopes on the p17 core protein of HIV. Cross-competition assays indicate that the two epitopes are closely linked and mapping with synthetic peptides confirm that they are situated in two neighbouring regions at the carboxy terminal end of the molecule. The use of monoclonal antibodies in capture enzyme immunoassays have been shown to be valuable for the detection and quantification of antigens, the greatest sensitivity being achieved by combining antibodies to different non-overlapping epitopes (Heinz et al., 1986). We demonstrate here that the specific detection of the p17 protein is possible with a solid-phase sandwich enzyme ira-
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
166 munoassay (ELISA) using two of our monoclonals selected for their reactivity against two different epitopes.
Materials and methods
Cells and virus The Hut-78 established adult human T cell line chronically infected with the AIDS-associated retrovirus (ARV) isolated in San Francisco was kindly provided by Dr. Jay Levy (Levy et al., 1984, 1985). The ARV-4 isolate produced by these cells grown in roller bottles was concentrated from the culture medium in a hollow fiber concentrator (Amicon, H1 x 50 cartridge) and centrifuged at 45,000 x g for 1.5 h. This virus pellet was then purified by equilibrium centrifugation in a linear metrizamide gradient (4-36% in Tris-HC1 10 mM pH 8, NaC1 100 mM, EDTA 1 raM). The virus band was found at a density of 1.12 g / m l as established by the position of the reverse transcriptase in the gradient. The HTLV-IIIB strain of HIV (Popovic et al., 1984) produced in Molt 3 cells and purified as previously described, was kindly provided by Dr. C. Briick (Smith-Kline Biologicals, Rixensart, Belgium). Enzyme-linked immunosorbent assay (ELISA) The purified virus preparation was extracted once with a three-fold volume of diethyl ether or, preferentially, disrupted with 2% octyl-fl-glucopyranoside (OGP, Sigma) and dialysed against phosphate-buffered saline (PBS) before use as antigen. The protein content was estimated by the method of Bradford (Bio-Rad protein assay reagent). 50 #1 containing 250-500 ng of viral antigen in 0.02 M carbonate buffer pH 8.8 were incubated overnight at 4°C in a 96 well Immunoplate I (Nunc, Denmark). After saturation of the plastic with 3% serum albumin (BSA) in phosphate-buffered saline (PBS), the culture supernatants to be screened were incubated in the coated wells for 2 h at 37°C. After washing with 0.05% Tween 20 in PBS, 50/L1 of peroxidase-conjugated goat anti-mouse globulin (Amersham) diluted 1/1000 in 1% BSA in PBS were incubated in each well for 2 h at 37 ° C. Colour development of the
bound peroxidase conjugate was achieved with citrate-phosphate buffer pH 5.2 and 0.015% H202.
Production of monoclonal antibodies For fusions 1 and 2, BALB/c mice were inoculated with 100 /~g of purified virus (ARV-4), partly subcutaneously and partly intraperitoneally, in complete Freund's adjuvant. They were boosted intraperitoneally after 3 weeks and 6 weeks, before a last injection was given intravenously 4 days before fusion. A similar immunization process was followed with detergenttreated (OGP) virus HTLV-III/B for fusion no. 3. The conventional technique (Fazekas de St.Groth and Scheidegger, 1980) was used to obtain hybrids between Sp2-0 myeloma cells and splenocytes. Each colony supernatant was screened for antiHIV-1 antibodies in an ELISA using the antigen corresponding to the initial immunogen. An additional screening was performed using sonicated cell suspensions as antigens (Hut-78 cells or Hut78/ARV-4 producing cells at 5 x 106/ml in PBS, 2 X 10 -4 M PMSF) to eliminate hybrids reacting with cellular components. Each well was coated with 10-15 pg of the clarified sonicate diluted in carbonate buffer pH 8.8. Western blot immunoassay Purified HIV-1 (10-15/~g/lane) or cell lysates (5 X 107 cells/ml, of PBS containing 1% Triton X-100, 0.5% deoxycholate, 0.2 mM PMSF) were submitted to electrophoresis in 12% polyacrylamide gels. The resolved polypeptides were transferred to nitrocellulose at 50 V for 16 h. After saturation with a mixture of 2% bovine serum albumin (BSA) and 3% gelatin as described (Cogniaux and Jacquemin, 1985) or with 5% non-fat dry-milk (Sarugadharan et al., 1985), individual strips were reacted overnight with 1/2 dilutions of the culture supernatants to be tested. Bound antibody was detected with 1/200 biotinylated sheep anti-mouse i m m u n o g l o b u l i n ( A m e r s h a m , RPN1021), 1/1000 peroxidase-labelled streptavidin (Amersham, RPN1231) and 4-chloro-l-naphthol as substrate. Purification and labelling of the monoclonal antibodies Immunoglobulins were purified from the culture fluids of different hybrids by chromatography
167 on Affi-gel-Protein A (MAPS kit from Bio-Rad). About 200 ml of culture supernatant were slowly run through 1 ml of gel and the immunoglobulins were eluted using 3 ml of pH 3.5 buffer. They were immediately neutralized with 1 M Tris-HC1, pH 8.5 and dialysed. Radioiodination was performed on 10-13/xg of IgG using the chloramine-T method. The labelled protein was filtered through a 10 ml Sephadex G-25 column saturated with 1% BSA in PBS. Biotin was conjugated to two of the monoclonal IgG antibodies as described by Goding (1980). The protein was adjusted to 1 mg/ml and dialysed against 0.1 M NaHCO 3 before mixing with biotin succinimide ester (1 mg/ml dimethylsulphoxide). The mixture (200 /~g of biotin for 1 mg IgG) was left at room temperature for 4 h and then dialysed against PBS containing 0.001% merthiolate.
Radioimmunoprecipitation 100/~1 of culture supernatant or 20 #1 of ascitic fluid were incubated for 2 h at room temperature with 500,000 cpm of 125I-viral lysate in 100 #l of RIPA buffer. (50 mM Tris-HC1 pH 7.9, 0.2 M NaC1, 2% Triton X-100, 0.1% Tween 80, 0.2 mM PMSF, 0.02% NAN3). Immunocomplexes were precipitated with 100 #1 of a 10% (v/v) suspension of protein A-Sepharose (Pharmacia) to which was coupled 5 /~g of goat anti-mouse Ig (Kpl, affinity purified antibody). After incubation for 16 h at 4 ° C, the Sepharose beads were washed twice with 1 ml of 'high salt washing buffer' and twice with 'low salt washing buffer' (Schneider et al., 1984). The immunoprecipitated polypeptides were separated by SDS-PAGE and visualized by autoradiography using X-ray film (Kodak X-O-Mat).
Competitioe binding of the monocional antibodies The virus proteins were bound to the wells of a polyvinylchloride microtitre plate (125 ng/well, in 0.02 M carbonate buffer pH 8.8). After saturation of the free sites with 3% BSA for 2 h, serial dilutions of the unlabelled immunoglobulin, each in a volume of 50 #1, were added for 2 h at 37 ° C. 25 #1 (50,000 cpm) of iodinated antibody were then added and the mixtures further incubated for 2 h at 37°C.
After thorough washing, the individual wells were separated with scissors and counted in an autogamma counter (Berthold).
Peptide synthesis Based on the original sequencing (Muesing et al., 1985), eight 20-25 mer HIV-1 pl7-encoded peptides, overlapping by 5-6 amino acids, were synthesized utilizing automated solid-phase peptide synthesis on a Applied Biosystems 430A peptide Synthesizer. The peptides were synthesized using the t-Boc synthesis strategy protocol suggested by the manufacturer, all liquid reagents were from Applied Biosystems (Foster City, CA, U.S.A.). Amino acids used were from Nova Biochem (Switzerland). p-methylbenzhydryl amine (Peptides Int., Louisville, U.S.A.) was used as polymer resin. Following each amino acid coupling a sample was taken and a quantitative ninhydrin assay was performed. Only if the coupling efficiency exceeded 99% of each amino acid, was the peptide accepted for further processing. After each completed synthesis, the peptide was cleaved from the resin and the protective side chains removed by hydrogen fluoride (HF) (AGA, Sweden). During HF cleavage, anisole and dimethylsulphide (DMS) (both chemicals from Merck, F.R.G.) were added as scavengers. Immediately after HF cleavage the peptide-resin mixture was resuspended in trifluoroacetic acid (TFA) (Janssen Chemica, Belgium) and the peptide precipitated in diethyl ether (Merck). Finally, the precipitated peptide was dried under vacuum in a desiccator.
Peptide antigen coating The individual peptides were coated onto Nunc high-binding microtiter plates (Nunc, Denmark) as follows: 100 /~l poly-L-lysine (Sigma, U.S.A.), diluted 1/100 in phosphate-buffered saline (PBS), were added to each well and allowed to react for 30 min at room temperature. After discarding the poly-L-lysine, 50 /~l of the peptide antigen (dissolved in 10% acetic acid and made up to a concentration of 10/xg/ml in PBS coating buffer) were then added, promptly followed by the addition of 50 /tl 0.5% glutaraldehyde (Sigma). The mixture was allowed to react for 15 rain at room temperature after which the wells were washed
168 twice with PBS and subsequently with 200 #l of 100 m M glycine (Merck) in 0.1% bovine serum albumin (BSA) made up in water. After 30 min at room temperature the plates were again washed twice with PBS. Free protein binding sites were saturated by adding 200 #l/well of 3% BSA (made up in PBS) and incubated in a humidifier at 37 o C overnight.
manufacturer, were added and allowed to react for 1 h at 37 ° C in a humidifier. After four washes, 200 #1 of phosphatase substrate (Sigma), diluted in diethanolamine (DEA) (Fluka Chemic) (1 mg of substrate/ml of DEA) were added per well. After 20 min incubation at room temperature absorbance values were read at 405 nm.
Peptide ELISA
Results
Each of the five hybridoma culture supernatants, assayed for peptide reactivity, was tested against each p17 synthetic peptide at a dilution of 1 / 1 0 (in PBS containing 1% BSA and 0.05% Tween) using 100 #1 sample/well. After incubation for 1 h at 3 7 ° C in a humidifier the plates were washed four times in PBS containing 0.05% Tween 20. Subsequently, 100 #1 of alkaline phosphatase-conjugated goat anti-mouse immunoglobulin (Jackson), at the dilution suggested by the
Of the several hundred hybridoma fluids tested from three independent fusions, five were found to secrete antibody specific for a protein with a molecular weight of 17 kDa, as shown by Western blot on purified HIV-1 preparations (data not shown). An additional thin band at 55 kDa was also recognised. Fusions 1 and 2, carried out after immunization of mice with intact virions (ARV-4) exclusively gave this type of specificity. The third
1
2
3
4
5
a D
a b
a b
ab
a b
6
7 ab
m
a
m
b
m
G P 16 0
i
I
U
m
!i
P r 55 P 40
Ill
P 24
I
I
i
m
i
i
P 17
Fig. 1. Immunoblot of the proteins from cell lysates recognizedby the different Mabs: (1) 3Hll-C1; (2) 3D3-B6; (3) 8A6-A7; (4) 6D6-F6; (5) 6B6-H9; (6) Control human serum (HIV-1 negative) (7) Control human serum (HIV-1 positive). Lane a: ARV4-producing Hut78 cells; Lane b: Hut78 control cells.
169 fusion, performed after immunization with disrupted virions, gave rise to the 3D3 monoclonal, together with a series of anti-p24 antibodies which are not described here. The identity of the protein recognized by these hybridomas as a virus-specific product was confirmed by comparing their reactivities on lysates of Hut-78 control cells and Hut-78 (ARV-4) infected cells: only the virus-producing cells contained reactive antigens in immunoblotting (Fig. 1). In addition to the 17 kDa and the 55 k D a proteins detectable in a purified virus preparation, a third band at 40 k D a was visible in the cell lysate. A similar pattern was described previously by Chassagne et al. (1986), who suggested that p40 corresponds to an intermediate cleavage product of the gag precursor p55. The monoclonal antibodies were also able to immunoprecipitate the p17 protein from 125I-labelled virions, disrupted by 2% octylglucopyranoside (Fig. 2). These results indicated that the five hybrids, cloned twice in soft agar, produced monoclonal antibodies against the p17 internal core protein of the virion and against its precursor p55. Thus they were able to recognize their target after denaturation of the protein (Western blot) as well as in its native form (immunoprecipitation). The maximal amount of antibody bound to a constant amount of antigen can be considered as a direct expression of its affinity for the antigen (Frankel and Gerhard, 1979). The plateau of absorbance values (A492) reached using increasing dilutions of ascitic fluid in the ELISA permitted the different monoclonals to be ranked in the following order of affinity: 8A6-A7 > 3 D 3 - F l l > 3 H l l - C 1 > 6B6-H9 > 6D6F6. The characteristics of the five hybrid clones are summarized in Table I.
Cross-competition The epitope specificity of the monoclonals was examined in competition assays using the viral antigen adsorbed on microtest plates. The binding of each iodinated IgG in the presence of an excess of 'cold' antibody is shown in Table II. The radioactivity bound in the presence of an irrelevant monoclonal (anti-HTLV-I p24) determined the 100% binding value. Two groups of anti-pl7 antibodies were distinguished, each competing slightly
M.W.
1
2
3
4
5
6
7
8
I
I
I
I
I
I
I
I
|
P~ P!7 Fig. 2. Radioimmunoprecipitation of 125I-HIV antigens followed by SDS-PAGE analysis and autoradiography. The precipitates obtained with each monoclonai from hybrid culture fluid were analysed in the following lanes: 1 = Mab 8A6-A7; 2 = Mab 3D3-B6; 3 = Mab 6D6-F6; 4 = Mab 3Hll-C1; 5 = Mab 6B6-H9; 6 = negative control; 7 = Mab anti-p24; 8 = Mab HIV-positive human serum. M.W. indicates the molecular weight markers used. From top to bottom, phosphorylase B (92,500), bovine serum albumin (66,200), ovalbumin (45,000), carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500) and lysozyme(14,400). with one another (Table II). This conclusion was confirmed by the competition curves established between variable amounts of each monoclonal IgG and a constant amount of 125I-3Hll-C1 (Fig. 3A) or 125I-6D6-F6 (Fig. 3B). TABLE I CHARACTERISTICSOF THE MONOCLONALS Antigenic specificity was determined by immunoblotting. Biotinylated anti-mouse subclass reagents were used in an ELISA for subclass determination. The plateau of absorbance values (A492) reached using increasing dilutions of ascitic fluid in the ELISA permitted evaluations of the relative affinities of the monoclonals. Fusion Hybridoma Specificity Subclass A492 no. (plateau value) 1 2 3
8A6-A7 3Hll-C1 6D6-F6 6B6-H9 3D3-Fll
p17,p55 p17,p55 p17,p55 p17,p55 p17,p55
IgG2b IgG1 IgG1 IgG3 IgG1
1.82 1.36 0.554 0.725 1.73
170 T A B L E II EPITOPE SPECIFICITY S T U D I E D BY C O M P E T I T I V E B I N D I N G Each unlabelled monoclonal was incubated with the antigen for 2 h before further incubation with each individual iodinated IgG. A n irrelevant monoclonal (V G10-A3, anti-p24 HTLV-I) was used as unlabelled competitor to determine the 100% value in each case. Competitor antibody
Percentage of iodinated IgG b o u n d
6B6-H9 6D6-F6 8A6-A7 3Hll-C1 3D3-B6 VG10-A3
6B6-H9 *
6B6-F6 *
9.5% 113% . 100%
0.6% 1.5% 51% 76% . 100%
.
8A6-A7 *
3Hll-C1 *
82% 74% 9.2% 20%
52% 64% 2% 1.36%
63.6% 59.5% 3.3% 2.5% 4.2% 100%
. 100%
The binding of 3Hll-C1 was completely inhibited by approximately 100 ng of 8A6-A7 or 3Hll-C1 IgG (Fig. 3.4) but only 30% inhibition was reached using as much as 450 ng of 6D6-F6 or 6B6-H9. Conversely, the binding of labelled 6D6-F6 was completely inhibited by its 'cold' counterpart or by 6B6-H9, but only to a limited extent by 8A6-A7 or 3Hll-C1 (Fig. 3B). 6B6-H9 antibody was even more active in competition than the autologous 6D6-F6, especially at low doses, a finding which can be explained by the higher avidity of 6B6-H9 for the same epitope (see Table I).
3D3-B6 *
100%
The cross-competition experiments suggest that two antigenic regions, which are probably closely situated on the p17 protein, can be defined by the two groups of monoclonal antibodies. This conclusion has been confirmed by using eight synthetic peptides covering the whole sequence of the p17 protein (Fig. 4) in a peptide ELISA. As seen in Table III, three hybridomas (8A6-A7, 3Hll-C1, 3D3-Fll) showed strong reactivity against the p17-G peptide. The other two hybridomas (6D6-F6 and 6B6-H9) were strongly reactive with the p17-H peptide. No hybridoma exhibited reactivity to more than one peptide.
x
p
E Do ~
E Q. o v
30
g
O
O--
O .ID
i
O
20
O ~
10
5
i
P,
450
112
28
Competing
7 Mab IgG
4SO (ng/well)
IIZ
Competing
ZO
7 Mab
IgG
(ng/well)
Fig. 3. Binding assay of a given a m o u n t of radioiodinated Mab l g G in the presence of variable a m o u n t s of each monoclonal IgG. 125 125 8A6-A7 (*); 3 H l l - C 1 (o); 6D6-F6 (e); 6B6-H9 (¢0- A: binding of I - 3 H l l - C 1 . B: binding of I-6D6-F6.
171
IP-1-AjIP17 P17-c
!.5 a
AAADTGHSSQVSQNYPIVQN I ALDK]EEEQNKSK KKAQQAAADT 749
I
1.0
632 Fig. 4. Schematic presentation of the eight, overlapping p17 peptides and their designation. Also shown are the amino acid sequences for the two reactive peptides (pl7-G and pl7-H). Below the first and last amino acid of the sequence are shown the corresponding base pair number according to the original sequencing data (see materials and methods section).
4"
O
0.5
Detection of p 1 7 / p 5 5 in cell lysates by a standard solid-phase ELISA
d
HIV-1 c a n m u l t i p l y in h u m a n l y m p h o c y t e cultures f r o m i n f e c t e d i n d i v i d u a l s a n d p r o d u c t i v e infection c a n b e m e a s u r e d with a s a n d w i c h imm u n o a s s a y that detects viral antigens in small v o l u m e s of cell lysates ( M c D o u g a l et al., 1985). W e have used o n e a n t i - p l 7 m o n o c l o n a l as c a p t u r e antibody and a second anti-pl7 antibody directed against a different e p i t o p e as d e t e c t o r a n t i b o d y . Cell suspensions f r o m v i r u s - p r o d u c i n g cultures o r u n i n f e c t e d c o n t r o l s were a d j u s t e d to 2 × 1 0 6 / i l l l in the original culture m e d i u m a n d a d d e d with 1% Triton-X100. 50 /~1 v o l u m e s of cell lysates were d i s t r i b u t e d in the wells of m i c r o t e s t p l a t e s previo u s l y c o a t e d with 200 ng I g G f r o m M a b 8 A 6 - A 7 in c a r b o n a t e b u f f e r p H 9.6 a n d s a t u r a t e d w i t h 1% s e r u m a l b u m i n a n d 4% n e w b o r n calf s e r u m in PBS. T h e p r o t e i n b o u n d was t h e n d e t e c t e d b y a b i o t i n c o n j u g a t e o f M a b 6 D 6 - F 6 I g G followed b y s t r e p t a v i d i n c o n j u g a t e d to p e r o x i d a s e , a n d staining.
I
I
10
5 CELL
I
2.5
•
T
1,25
NUMBER
0.6
0.3
( xlO'4 / well
)
Fig. 5. Detection of p17/p55 in serial dilutions of cell extracts by a 'sandwich' ELISA with biotinylated Mab 6D6-F6. Curves a and b: virus-producing Molt 3 extract captured by the anti-pl7 Mab 8A6-A7 (a) or by the unrelated VG10-A3 (b). Curves c and d: control Molt 3 extract captured by the anti-pl7 (c) or by the unrelated Mab (d).
T h e d o s e - r e s p o n s e curve o b t a i n e d with serial d i l u t i o n s of a v i r u s - i n f e c t e d cell extract i n d i c a t e d a linear r e l a t i o n s h i p b e t w e e n a b s o r b a n c e values at 492 n m a n d the d o s e o f i n f e c t e d cells b e t w e e n 105 a n d 0.6 × 1 0 4 / w e l l (Fig. 5, curve a). T h e s a m e e x t r a c t a d s o r b e d to wells c o a t e d w i t h a n u n r e l a t e d m o n o c l o n a l ( a n t i - p 2 4 H T L V - 1 ) gave a low signal (Fig. 5, curve b), s i m i l a r to the b a c k g r o u n d ob-
TABLE III OPTICAL DENSITY READINGS (405 NM) OBTAINED USING EACH OF THE FIVE HYBRIDOMA COMBINATIONS An unrelated HTLV-1 clone (VG10-A3) was also included as a negative control. Hybridoma
Peptide designation pl7-A
pl7-B
pl7-C
p-17D
pl7-E
p-17F
pl7-G
pl7-H
8A6-A7 3Hll-C1 6D6-F6 6B6-H9 3D3-Fll HTLV-1 VG10-A3
0.09 0.14 0.13 0.00 0.11 0.01
0.14 0.15 0.19 0.00 0.17 0.00
0.11 0.20 0.23 0.02 0.10 0.00
0.11 0.16 0.15 0.00 0.12 0.00
0.08 0.12 0.16 0.01 0.07 0.00
0.12 0.12 0.16 0.01 0.12 0.01
1.90 > 2.00 0.23 0.02 2.00 0.01
0.08 0.08 > 2.00 1.15 0.08 0.01
172 TABLE IV APPLICATION O F T H E HIV-CAPTURE I M M U N O A S S A Y TO LYSATES F R O M C U L T U R E S O F H U M A N LYMPHOCYTES Groups I and II were cultures from seropositive individuals; group III were cultures from seronegative individuals.
(A) Control assay (B) p17/p55 assay (C) p24/p55 assay Culture
Capture Ab
Biotinylated detector Ab
Anti-HTLV-1 Mab VG10-A1 Anti-HIV p17 Mab 8A6-A7 Anti-HIV-p24 Mab 9A6-H12
Anti-HIV p17 Mab 6D6-F6 Anti-HIV p17 Mab 6D6-F6 Polyclonal human anti-HIV IgG
Absorbance (A 492) A Control assay
B p17/p55 assay
C p24/p55 assay
0.254 + 0.233 + 0.214 + 0.256 +
0.003 0.002 0.006 0.006
0.578 + 0.007 0.588 + 0.009 1.084 + 0.051 0.422 + 0.005
0.443 0.678 0.465 0.896
0.241 + 0.002 0.257 + 0.008 0.299 + 0.003
0.313 + 0.066 0.211 + 0.023 0.312 + 0.001
0.869 -t- 0.076 0.360 + 0.016 0.399 + 0.008
0.228 + 0.003 0.291 + 0.001 0.226 + 0.008
0.299 + 0.009 0.299 -t- 0.009 0.221 + 0.012
0.237 + 0.008 0.237 + 0.008 0.175 + 0.010
0.294
0.347
0.243
Group 1 TJ (2nd) a VG VK TS (2nd) ~
-1-0.018 + 0.069 + 0.045 + 0.021
Group II TS (lst) a PS TJ (lst)
Group 11I MC AJ SJ ' positive' threshold value
a 1st and 2nd correspond to successive samples taken from the same lymphocyte culture at 4 days intervals.
served with control uninfected cells in wells coated with the HIV-1 specific 8A6-A7 antibody (curve c) or the anti-HTLV-1 antibody (curve d). This immunoassay has been applied to a small number of human lymphocyte cultures from seropositive individuals, set u p in order to study materno-foetal transmission of HIVs (SprecherGoldberger et al., 1988). Lymphocyte extracts, made up with 2 × 106 cells/ml culture medium and 1% Triton X-100, were tested in parallel in the 'sandwich ELISA' described earlier (Table IV, B) and with an anti-p24 Mab as capture antibody and biotinylated human 'HIV-positive' IgG as detector antibody (Table IV, C). Coating with the unrelated VG10-A3 Mab controlled the specificity of the capture stage (Table IV, A). Absorbance readings greater than five standard deviations above the mean of the results in three uninfected lymphocyte cultures were considered positive. A good correlation between the two assays was observed for the samples of group I (positive) and
group III (negative). Results obtained with the three specimens in group II indicated that using a polyclonal anti-HIV detector antibody offered greater sensitivity in the detection of viral antigens.
Discussion
A monoclonal antibody specific for p17/18 has been described previously by Chassagne et al. (1986). The p55-gag precursor and a p40 intermediate product was also recognized by this monoclonal, a finding similar to that reported here. Four monoclonals, specific for p24 but non reactive with p55, have been described (Di Marzo Veronese et al. 1985). They have proved to be useful for immunohistology of lymph nodes in patients with HIV infection (Tenner-Racz et al., 1987). Among other monoclonals, raised against
173 the gag proteins of a British isolate of HIV, three recognized p55:p18, and three p18 alone (Ferns et al., 1987). In this report, immunization of mice with intact virions gave rise to hybrids secreting anti-p17 antibodies, whereas the injection of disrupted particles led essentially to the production of anti-p24 hybridomas, suggesting that some antigenic determinants of p17 are localized on the surface of the virion and readily accessible to the immune system of the host. This suggestion has been confirmed by a recent observation of Gelderblom et al. (1988), showing by immunoelectron microscopy that the p17 epitopes can be localized on the surface of the virion or budding HIV-1. The cross-competition experiments described here led us to conclude that two closely located epitopes could be distinguished on the p17 core protein by five monoclonal antibodies. Synthetic peptides offer an excellent tool for the mapping of Mab reactivity, and determining whether or not different clones are directed against the same epitope region. However, it should be noted that only antibodies directed against continuous epitopes lend themselves to mapping in this fashion. We were able to demonstrate that the monoclonals 6D6-F6 and 6B6-H9 were reactive with the carboxy-terminal peptide H (118-138), whereas 8A6, 3 H l l and 3D3 showed reactivity with the neighbouring peptide G (100-122) in complete agreement with the results of the competition experiments (P. Horal et al., 1988). A region of homology between the primary sequence of thymosin a-1 and the gag protein p17 has been detected by Satin et al. (1986). A 30amino acid synthetic peptide (HGP 30), analogous to that region and spanning residues 86-115 of the gag protein, has been shown to elicit antibodies which recognize the p17 protein by immunoblotting and to neutralize several strains of HIV in vitro (Naylor et al., 1987). Interestingly, peptide G (position 100-122) partially overlaps this HGP 30 peptide. The possibility that our anti-p17 monoclonals might neutralize the infectivity of HIV-1 in vitro now needs to be considered. McDougal et al. (1985) have described a solid-phase sandwich ELISA for the detection of the HIV-1 antigens in supernatants of LAV-infected human lymphocyte
cultures. Using HIV-positive human sera, this technique has been applied successfully for the detection of various types of antigen with monoclonal antibodies, acting as either capture antibody a n d / o r detector antibody (Mizuchi et al., 1984; Soos et al., 1984; Heinz et al., 1986). Their effectiveness was shown to depend primarily on their avidity and the highest sensitivity was achieved by combining antibodies to different non-overlapping epitopes (Heinz et al., 1986). In our hands, the monoclonal 8A6-A7 can be used as capture antibody and 6D6-F6 as detector antibody, although they recognize two neighbouring peptides, H and G, at the carboxy terminal end of the molecule. The specificity of this assay was confirmed by the absence of antigen binding when the unrelated anti-HTLV-1 Mab was used to coat the plates. The analysis of ten samples from human lymphocyte cultures suggested that the presence of viral proteins could be detected by two anti-pl7 monoclonals in a 'sandwich ELISA' although greater sensitivity was obtained using an anti-p24 monoclonal as capture antibody, followed by a biotinylated polyclonal IgG which could bind to many epitopes on p24 and p55 proteins. However, our system permits the selective detection of p17 and has been used by one of us (B.C.) to study the insertion o f different HIV proteins into lipid vesicles. Using this kind of assay for the quantification of p17/p55 in a cell lysate or in a virus preparation would require purified preparation of the proteins as standards and these are not yet available. However, different preparations of viral antigens can be compared and relative amounts of p17 can be evaluated since the absorbance values in ELISA are directly proportional to the antigen dilution over a given concentration range.
Acknowledgements We gratefuUy acknowledge the secretarial ~help of J. Herinckx. This work was supported by a grant from the Fonds de la Recherche Scientifique M&licale (Belgium) and a predoctoral fellowship to B.C. from the Institut pour 1' Encouragement de la Re-
174 cherche Scientifique dans l'Industrie et l'Agriculture. N.D. was supported by Smith-Kline RIT.
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