FEMS Microbiology Immunology 89 (1991) 57-64 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier
57
FEMSIM 00188
A new capture test using conjugated peptides for the detection of HIV antibodies Pascale M. Richalet-S6cordel and Marc H.V. Van R e g e n m o r t e l Laboratoire d'Immunochimie, Institut de Biologie Mol~culaire et Cellulaire, Strasbourg, France Received 5 August 1991 Accepted 9 September 1991
Key words: Conjugated peptides; HIV antibodies; Enzyme immunoassay
1. SUMMARY A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gpl20 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indiCorrespondence to: M.H.V. Van Regenmortel, Laboratoire d'Immunochimie, Institut de Biologie Mol6culaire et Cellulaire, 15 rue Descartes, 67084 Strasbourg Cedex, France.
rect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies. 2. INTRODUCTION The need for a safe and easy test system for diagnosing HIV infections has led to the development of many ELISA systems using peptides as the antigenic probe [1]. Numerous studies have shown that indirect ELISA utilizing env-peptides [2-6], or carrier-bound gag-peptides adsorbed to plastic [7] is a highly sensitive and specific assay for detecting HIV antibodies. Unfortunately, however, none of the existing tests is able to recognize HIV positive sera with absolute sensi-
58 tivity and specificity. It has been reported, for instance, that ELISA may produce some false negative results with sera collected during the initial phase of antibody production [8,9] or during the final stage of AIDS [10]. In order to reduce the risk of overlooking sera containing HIV-1 antibodies, while at the same time keeping non-specific reactions and false positives to a minimum, it is customary to test blood samples first by ELISA and then to confirm each positive result by the Western blot technique (WB). The combined use of ELISA and WB methods has improved considerably the diagnosis of HIV infections, particularly in the case of seroconversions, although some false negative results [9] as well as false positive results generally involving the detection of gag-coded proteins [11] are still observed. Several groups have tried to combine different techniques to enhance the detection of HIV antibodies, for instance ELISA using recombinant antigen followed by WB [12], ELISA using several synthetic peptides followed by WB or radioimmunoprecipitation assay (RIPA) and IgM-MACRIA in case of indeterminate results [9], ELISA with whole virus followed by synthetic peptide ELISA, etc. [13]. In the present study we attempted to improve the diagnosis of HIV infections by using a new type of peptide conjugate in a capture ELISA format. Since there is a high degree of strain variation in the epitopes commonly used as synthetic HIV antigens in ELISA, as well as considerable heterogeneity in the immune response of individuals, a set of three peptides corresponding to immunodominant epitopes of gp41, gp120 and p27 ( n e d of HIV-1 was used in an attempt to decrease the number of false negative results. The proposed capture test differs from earlier tests by its use of a new type of biotinylated conjugate, of more highly diluted sera (1/1000 instead of undiluted or 1/50) and of a combination of structural epitopes from gp41 and gp120 together with a non-structural epitope from p27. This new test detected 4 0 / 4 0 HIV-1 positive sera (all sera had been tested beforehand by WB) without any false positive results (0/21), as well as 10/10 seroconverting (WB positive gp41 negative) sera.
3. M A T E R I A L S AND M E T H O D S
3.1. Selection of peptide sequences All sequences were derived from Bru subtype of HIV-1. The numbering of amino acid residues corresponds to that described by Ratner et al.-
[14]. The chosen gp41 sequence (591-611) YLKD Q Q L L G I W G C S G K L I C T T , covers the 584-618 region of the enu-glycoprotein previously described as an immunodominant region [2-4,15,16]. The gpl20 sequence (314-329) I R I Q R G P G R A F V T I G K belongs to the main neutralization determinant V3 of the envelope glycoproteins [17] and is able to elicit high levels of virus neutralizing antibodies [18]. The p27 sequence (182-198) E W R F D S R L A F H H V A R E L partly spans the sequence (185205) defined by Bahraoui et al. [19] as a B cell epitope. A Tyr residue was added at the N-terminal side of each peptide in order to allow conjugation to ovalbumin with bisdiazobenzidine.
3.2. Synthetic peptides Assembly of the protected peptide chains was carried out using the stepwise solid phase method of Merrifield (1963) on a NPS 4000 multichannel peptide synthetizer (Neosystem Laboratoires, Strasbourg, France). All amino groups were protected at the alpha amino position with the Boc group, except Trp, which was protected with the Fmoc group. The following side chain protecting groups were used: cyclohexyl ester (Asp, Glu), benzyl ether (Ser, Thr), 2-chlorobenzyloxycarbonyl (Lys), p-toluenesulfonyl (Arg, His). Boc-AAPAM-resins (0.5 mmol) were placed in each reaction vessel. The cycle used for incorporation of Boc and Fmoc amino acids has been described elsewhere [20]. Boc amino acids were coupled in the form of their benzotriazol ester, except Boc His (Tos) which was introduced as a symmetrical anhydride. The total coupling time was 45 min. Monitoring with the ninhydrin test [21] showed that most of the couplings were complete within this time. If necessary a double coupling was carried out.
59 At the end of the synthesis and after the last deprotection step, the resins were washed with ethyl ether and dried under vacuum. The high HF procedure was used for deprotection and cleavage from the resin. After lyophilisation each crude synthetic peptide was dissolved in 10% acetic acid and purified using a middle pressure chromatography apparatus (Kronwald Separation Technology, Sinsheim, F.R.G.) on a glass column filled up with RP100 material (C18, 20-45 txm). The final purity of each peptide was controlled by an analytical run on a reverse phase aquapure RP300 C8 column, 5 /xm (4.6 × 250 mm) using programmed gradient elution (2 ml/min) with the following solvents: A, 0.1 M triethylammonium phosphate (TEAP) pH 2.5; B, acetonitrile. Amino acid composition and net peptide content were determined with a Waters Picotag system (Waters Corp., Milford, MA).
3.3. Coupling procedure Bisdiazobenzidine (BDB) is a coupling agent which interacts with the N-terminal side of Tyr as well as with Lys, His and Cys residues. The BDB conjugation procedure has been described elsewhere [22]. The following procedure was used to prepare BDB: a 5 mg/ml benzidine solution was obtained by dissolving benzidine (B 3383, Sigma, St. Louis, MO) in 0.2 M HCL. NaNO 2 was added at the rate of 3.5 mg /ml of benzidine solution. The final solution was gently stirred during 2 h at 4°C. The following procedure was used to conjugate each HIV-1 peptide and N-hydroxysuccinimidobiotin to ovalbumin. During the coupling procedure, the temperature was maintained at 4°C and the mixture was continuously gently stirred. 1 mol of ovalbumin was mixed with 15 tool Of peptide and 15 mol of NHS-biotin (H 1759, Sigma), in pH 9 borate buffer 0.16 M (1 m l / m g of peptide). After 10 min, 100 #1 of BDB per 5 mg of peptide were added to the mixture. The pH was adjusted to 9 with NaOH 1 M. After 2 h, the final mixture was dialyzed for 24 h at 4°C against 3 changes of 1 1 PBS in order to eliminate unconjugated peptides and N-hydroxysuccinimido-biotin. As a first step in the BDB coupling procedure,
Lys residues present in peptides are usually protected with citraconic anhydride in order to prevent them from interacting with protein [22]. Since citraconic anhydride may irreversibly interact with Cys residues, this protecting step was not used for coupling the gp41 peptide which contained both Cys and Lys residues, but was applied in the case of the gpl20 peptide.
3.4. Capture test The following ELISA procedure was used to measure the binding of conjugated peptides to coated antibodies from human sera. Microtiter plates (Falcon, U.S.A.) were coated at room temperature for 15 h with 20 p,g/ml protein G (Pharmacia LKB, Uppsala, Sweden) in 0.05 M carbonate buffer, pH 9.6. This high concentration of protein G obviated the use of a blocking step for remaining sites on the plastic. After three washings of microtiter plates with phosphatebuffered saline (PBS) containing 0.05% Tween 20 (PBS-T), human sera diluted 1:1000 in PBS-T were added for 30 min at 37°C. After repeated washings, conjugated peptides diluted in PBS-T were added for 30 min at 37°C. One of the three conjugated peptide s was added in each well. Each conjugate was used at a dilution that gave the best ratio OD (HIV positive sample)/OD (normal human sera). After repeated washings, positive reactions were detected by incubation with peroxidase labelled streptavidin (horseradish peroxidase-streptavidin, Amersham, U.K.) diluted 1:6000 in PBS-T, for 30 min at 37°C. The final reaction was visualized by addition of tetra methyl benzidine (TMB) (3,3' 5.5' benzidine, Boehringer, F.R.G) diluted 1 : 10 in citrate buffer, for 10 min at 37°C. OD values were measured at 405 nm after the reaction had been stopped with 50 txl of 1 M HC1. No thimerosal or sodium azide was used. The volume of mixture added at each step of the capture test was 100/xl per well. In order to determine the cut-off line of the assay, 21 sera from normal individuals were tested with the conjugated (591-611) gp41, Y(314-329) gp120 and Y(182-198) p27 peptides. Using sera diluted 1:1000, the capture test procedure gave average absorbances of 0.10 (SD: 0.033), 0.28 (SD: 0.041) and 0.23 (SD: 0.036) respectively.
60 C o n t r o l s e r a w e r e c o n s i d e r e d positive w h e n t h e O D values w e r e h i g h e r t h a n the a v e r a g e O D v a l u e plus 3 SD, i.e. r e s p e c t i v e l y 0.20, 0.40 a n d 0.35 O D units.
A m i n o acid analysis of two s a m p l e s o f e a c h p e p tide s h o w e d that t h e p u r i f i e d p r o d u c t s h a d t h e e x p e c t e d c o m p o s i t i o n ( d a t a not shown).
3.5. Sera
3.6. Correlation with a commercially available assay T w e n t y o f t h e 40 s e r o p o s i t i v e s e r a as well as t h e 10 s e r o c o n v e r t i n g a n d 19 of t h e 21 n o r m a l h u m a n s e r a w e r e t e s t e d with a c o m m e r c i a l E L I S A kit ( L a b s y s t e m s O Y , H e l s i n k i , F i n l a n d ) , which utilizes a single gp41 p e p t i d e c o n j u g a t e d to bovine s e r u m a l b u m i n as a n t i g e n i c p r o b e [4]. In this assay, t h e m a n u f a c t u r e r s r e c o m m e n d that s e r a b e d i l u t e d 1 / 5 0 . T h e m i c r o p l a t e r e a d e r was b l a n k e d a g a i n s t t h e r e a g e n t blank. T h e following f o r m u l a was u s e d to c o n v e r t t h e a b s o r b a n c e s to e n z y m e i m m u n o a s s a y units ( E I U ) : E I U = ( O D o f t h e test s a m p l e / O D o f t h e positive c o n t r o l ) x 100. A s the O D o b t a i n e d with t h e positive c o n t r o l was 1.00, we s i m p l i f i e d t h e f o r m u l a to E I U = ( O D of the test s a m p l e ) x 100. T h e cut-off line o f t h e assay was 35 E I U , c o r r e s p o n d i n g to an O D o f 0.35. T o facilitate t h e c o m p a r i s o n with o u r test, the results o f t h e i n d i r e c t E L I S A w e r e c o n v e r t e d into O D values. T h e c u t - o f f line o f t h e assay was thus 0.35 OD.
4. R E S U L T S
4.1. Synthetic peptides T h e d e g r e e o f p u r i t y of t h e p e p t i d e s a s s e s s e d by H P L C was at least 95% for t h e ( 5 9 1 - 6 1 1 ) gp41 p e p t i d e a n d 85% for t h e Y ( 3 1 4 - 3 2 9 ) g p l 2 0 a n d Y ( 1 8 2 - 1 9 8 ) p27 p e p t i d e s . T h e n e t p e p t i d e cont e n t was u s e d as a b a s e for m o l a r i t y calculation.
":':':':':':':"
A
S e r a f r o m 40 s e r o p o s i t i v e a n d 10 s e r o c o n v e r t i n g (gp41 n e g a t i v e ) p a t i e n t s , a n d o f 21 seronegative healthy individuals were examined. The s e r a o r i g i n a t e d f r o m t h e B l o o d T r a n s f u s i o n Center, S t r a s b o u r g , I n s t i t u t d e V i r o l o g i e , S t r a s b o u r g , and from the New York Blood Center. All sera h a d b e e n t e s t e d b e f o r e h a n d by W B . T h e s e r a h a d b e e n h e a t e d at 56°C for 30 min a n d w e r e cent r i f u g e d for 10 min at 4000 r p m just b e f o r e the assay.
O
[ (591-611)
gp41
6 O
° ° • ••° ° ° °
B
[ Y(314-329)
gp121~
E °~, ° °° ° •°°°° ° • °°° , • ~°
E3 0
•° •
Y(182-198)
C
p27
•°°°° ° °°°°° ° •°° ° °
. ;-;. ",',"
NHS
HIV positive
.:.
seroconverting
Fig. 1. Capture assay showing binding with peptide conjugates. Anti-peptide IgG activity was measured in 21 normal human sera (NHS), 40 HIV positive sera and 10 seroconverting (WB positive, gp41 negative) sera. All sera were diluted 1:1000. Antibody levels were expressed in OD units at 405 nm after 10 min of incubation of streptavidin-peroxidase with substrate. (A) Binding of (591-611) gp41 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.10) plus 3 SD (3 x 0.033), i.e. ~ 0.20 OD unit. (B) Binding of the Y(314-329) gpl20 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.28) plus 3 SD (3x0.041), i.e. ~ 0.40 OD unit. (C) Binding of the Y(182-198) p27 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.23) plus 3 SD (3 x0.036), i.e. ~ 0.35 OD unit.
61
4.2. Conjugated peptides Due to interference of the spectrum of N-hydroxysuccinimido-biotin with that of amino acid residues, amino acid analysis of conjugated peptide preparations could not be interpreted quantitatively. A dilution of each conjugate was chosen that gave the best ratio OD (HIV positive sample)/OD (normal human sera). The gp41, gpl20 and p27 peptide conjugates were used at a dilution of 1/1000, 1/18000 and 1/4000 respectively. 4.3. Capture test All sera were tested in the capture test against the three conjugated peptides. As protein G is an IgG Fc receptor [23], the investigation was restricted to the anti-HIV-1 IgG response. Tests were repeated at least twice in independent ELISA experiments. The (591-611) gp41 peptide conjugate recognized 40/40 of the HIV positive sera. None of the 21 normal sera were recognized nor any of the 10 seroconverting sera (Fig. 1A). The Y(314-329) gpl20 peptide conjugate recognized 35/40 (87.5%) of the HIV positive sera and 9/10 (90%) of the seroconverting (gp41 negative) sera. There were also no false positive results with the 21 normal human sera (Fig. 1B). The Y(177-198) p27 peptide conjugate recognized 31/40 (77.5%) of the HIV positive sera and
• .....:.:.:......
..,..
E tO O
NHS
HIV positive
seroconverting
Fig. 2. Binding in an indirect commercial E L I S A (Labsystems) using a single gp41 peptide as probe. Anti-peptide IgG activity was measured in 19 normal h u m a n sera (NHS), 20 HIV positive sera and 10 seroconverting (gp41 negative) sera. As r e c o m m e n d e d by the manufacters, all sera were diluted 1 : 50. Antibody levels are expressed in O D at 405 n m after 30 min of incubation of anti-human IgG enzyme conjugate with substrate. T h e four seroconversion sera giving O D values in the range of false positives correspond to samples 3, 7, 8 and 9 (Table 2).
10/10 of the seroconverting (gp41 negative) sera. No false positive results were observed with the 21 normal human sera (Fig. 1C). 4.4. Indirect ELISA The commercial assay using a gp41 peptide recognized 20/20 of the HIV positive sera and 10/10 of the seroconverting (WB positive, gp41 negative) sera. However, 4/19 (21%) of the
Table 1 Reactivity of H I V positive and seroconverting (WB positive, gp41 negative) sera in the capture assay and in a commercialized indirect E L I S A Type of serum
HIV positive Seroconve rting Normal h u m a n
Total number of sera (n 1) 40 10 21
% Positive n I sera in capture assay with gp41 gpl20 100 0 0
87.5 90 0
p27 77.5 100 0
N u m b e r of sera tested with commercial gp41 peptide (n 2)
% Positive n 2 sera in commercial assay
20 10 19
100 100 21
Sera from patients were diluted 1 : 1000 in the capture test and 1:50 in the commercial indirect E L I S A (Labsystems, Helsinki). Values with peptides in the capture assay are expressed as % positive sera among total tested sera (n l) and values with the commercial test as % positive sera of n 2. T h e sera were considered positive in the capture test, when the O D values were > (OD for 21 NHS + 3 SD) after 10 min of incubation of streptavidin-peroxidase with substrate, or in the Labsystem test, when the E I U were > 35 after 30 min incubation of anti-human IgG enzyme conjugate with substrate. Tests were repeated at least twice in independent E L I S A experiments.
62
normal human sera were also positive. Each of the false positive results remained positive in repeated tests (Fig. 2). The results of the capture tests and of the commercial indirect E L I S A are compared in Table I.
5. D I S C U S S I O N The new capture test is a highly sensitive assay since it was able to detect specific antibodies in 100% of the H I V positive sera (by WB technique) when the sera were diluted 1/1000. This highworking dilution was no doubt responsible for the low non-specific background noise and the absence of any false positives. In addition, when the conjugated p27 peptide was used as probe, sera that were WB + and gp41- were also detected as H I V positive (Table 2). The commercial assay that was tested in parallel was also fairly sensitive since it detected 100% of the 30 tested H I V positive sera which included 10 seroconversion sera. However, the specificity of the commercial assay was not as good as that of the capture test since the level of false posi-
tives was 21%, in spite of a centrifugation step (4000 rpm, 10 min) prior to testing. Presumably the way the sera had been stored (many had been kept for several years and had been repeatedly thawed and refrozen) as well as the low serum dilution (1/50) used in the assay are responsible for the high percentage of false positives. Similar problems are likely to be encountered with other available tests based on synthetic peptides which also entail the use of sera used of low dilution. Our data indicate that it is necessary to combine the results obtained with at least two peptide conjugates (gp41 and p27 peptides) in order to detect 100% of H I V positive and of seroconversion (WB + / g p 4 1 - ) sera. Although the commercial test based on a single conjugated gp41 peptide detected 100% of the seroconversion sera, it should be noted that the O D levels reached by four of these samples (sera 3, 7, 8 and 9 in Table 2) were similar to O D values of false positives (Fig. 2); the specificity of these four results is thus in doubt. A m o n g the six seroconversion sera that gave an O D > 2.0 in the commercial test, three (samples 1, 2 and 10) were strongly recognized in the capture test while three (4, 5 and 6) gave values very close to the cut-off level (not shown).
Table 2 Comparison of results obtained with the seroconverting sera in the capture test and the commercial indirect ELISA, with those obtained in Western blot tests Seroconverting
ELISA
serum
Capture assay
1 2 3 4 5 6 7 8 9 10
Bands revealed in Commercial
gp41
gpl20
p27
gp41 peptide
-
+ + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + +
WB
p18, p25, p55, p68, gp160 p18, p25, p55, p68, gp120, gp160 p18, p25, gp120, gp160 p25, p55, gp120, gp160 p25, gp120, gp160 p18, p25, gp120, gp160 p25 p18, p25, p55 gpl20, g p l 6 0 gpl20, g p l 6 0
The E L I S A were considered positive ( + ) when the OD was above the cut-off threshold. The WB were considered as positive when one or more bands corresponding to HIV~I separated proteins were revealed with enzyme labelled human anti-IgG in the DuPont assay. Sera 7 -1 0 which are not simultaneously positive for both p25 and gp120/ 160 correspond possibly to the beginning of seroconversion. In the commercial test three of these sera (7, 8 and 9) gave O D values close to that of false positives. Sera 7, 8 and 10 gave a strong response in the capture test.
63 F u r t h e r m o r e , sera 7 and 8 whose W B profiles revealed no antibodies reacting with the glycoproteins gp41, g p l 2 0 nor with the precursor protein gpl60, gave a strong response in the capture test while being in the range of some false positives in the commercial assay. It seems that the sensitivity of the W B technique is insufficient for detecting some antibodies reacting with g p l 2 0 and g p l 6 0 whereas both the capture and commercial tests react with peptides f r o m the g p l 2 0 and gp41 sequences. O n the o t h e r hand, antibodies directed against g p l 6 0 which contains the gp41 sequence do not recognize the 591-611 peptide conjugate although they bind to peptides from the gp41 region. Since there is published evidence [24] that the c o n f o r m a t i o n of the region 5 9 3 - 6 0 3 in gp41 is important for recognition by antibodies directed to the native protein, it seems reasonable to assume that the structure of the gp41 conjugated peptide used in the capture test resembles that of the same region in the native protein. This particular peptide conjugate may thus be a very sensitive p r o b e for detecting the a p p e a r a n c e of early antibodies to gp41. A n o t h e r potential advantage of the capture test is the ability to detect I g M or I g A antibodies without interference f r o m the major I g G response. It is k n o w n [25] that the presence of I g G interferes with the detection of o t h e r antibody subclasses and capturing h u m a n antibodies by means of /x or a - c h a i n specific antiglobulins should be able to o v e r c o m e this problem. T h e detection of I g M and I g A antibodies is likely to be important for the detection of antibodies prod u c e d early after H I V infection as well as for diagnosis of m o t h e r to child c o n t a m i n a t i o n with H I V . Since I g M and I g A do not cross the placenta, their presence in the blood of the n e w b o r n indicates vertical transmission [26].
ACKNOWLEDGEMENTS We are grateful to Dr. S. PlauE (Neosystem, Strasbourg) for helpful discussions. W e thank Dr. M.L. N o r t h (Centre de Transfusion Sanguine, Strasbourg), Dr. A.R. N e u r a t h (New Y o r k Blood
Center) and Dr. M.P. Schmith (Institut de Virologie, Strasbourg) for g e n e r o u s gifts of patient sera. W e thank Ms. Susanne W e n c k e r for help in typing the manuscript. This work was supported by the Institut National de la SantE et de la R e c h e r c h e MEdicale, the Conseil REgional d' Alsace and by Neosystem Laboratoires S.A., Strasbourg.
REFERENCES [1] Modrow, S. and Wolf, H. (1990) Use of synthetic peptides as diagnostic reagents in virology. In: Immunochemistry of Viruses II. (Van Regenmortel, M.H.V. and Neurath, A.R., Eds.), pp. 83, Elsevier Science Publishers, Amsterdam. [2] Gnann, J.W. Jr., Schwimmbeck, F.L., Nelson, J.A., Truax, A.B. and Oldstone, B.A. (1987) Diagnosis of AIDS by using a 12-amino acid peptide representing an immunodominant epitope of the human immunodeficiency virus. J. Infect. Dis. 156, 261-267. [3] Smith, R.S., Naso, R.B., Rosen, J., Whalley, A., Hom, Y-L., Hoey, K., Kennedy, C.J., McCutchan, J.A., Spector, S.A. and Richman, D.D. (1987) Antibody to a synthetic oligopeptide in subjects at risk for human immunodeficiency virus infection. J. Clin. Microbiol. 25, 1498-1504. [4] Niirv~inen, A., Korkolainen, M., Suni, J., Korpela, J., Kontio, S., Partanen, P., Vaheri, A. and Huhtala, M-L. (1988) Synthetic e n r gp41 peptide as a sensitive and specific diagnostic reagent in different stages of human immunodeficiency virus type 1 infection. J. Med. Virol. 26, 111-118. [5] Modrow, S., HEflacher, B., Mellert, W., Erfle, V., Wahren, B. and Wolf, H. (1989) Use of synthetic oligopeptides in identification and characterization of immunological functions in the amino acid sequence of the envelope protein of HIV-1. J. Acquired Immune Deficiency Syndromes 2, 27-31. [6] Cumming, S.A., McPhee, D.A., Maskill, W.J., Kemp, B.E., Doherty, R.R. and Gust, I.D. (1990) Use of a conserved immunodominant epitope of HIV surface glycoprotein gp41 in the detection of early antibodies. AIDS 4, 83-86. [7] Modrow, S., HEflacher, B., Mertz, R. and Wolf, H. (1989) Carrier-bound synthetic peptides. Use as antigen in HIV1 ELISA tests and in antiserum production. J. lmmunol. Meth. 118, 1-7. [8] Saah, A.J., Farzadegan, H, Fox, R., Nishanian, P., Rinaldo, C.R., Jr., Phai, J.P., Fayey, J.L., Lee, T-H., Polk, B.F. and The Multicenter AIDS Cohort Study (1987) Detection of early antibodies in human immunodeficiency virus infection by enzyme-linked immunosorbent assay, Western blot, and radioimmunoprecipitation. J. Clin. Microbiol. 25, 1605-1610.
64 [9] Ayres, L., Avillez, F., Garcia-Benito A., Deinhardt, F., Giirtler, L., Denis, F., L6onard, G., et al. (1990) Multicenter evaluation of a new recombinant enzyme immunoassay for the combined detection of antibody to HIV-1 and HIV-2. AIDS 4, 131-138. [10] Maskill, W.J., Crofts, N., Waldman, E., Healey, D.S., Howard, T.S., Silvester, C. and Gust, I.D. (1988) An evaluation of competitive and second generation ELISA screening tests for antibody to HIV. J. Virol. Meth. 22, 61-73. [11] Tribe, D.E., Reed, D.L., Lindell, P., Kenealy, W.R., Ferguson, B.Q., Gybulski, R., Winslow, D., Waselefsky, D.M. and Petteway, S.R. (1988) Antibodies reactive with human immunodeficiency virus gag-coded antigens (gagreactive only) are a major cause of enzyme-linked immunosorbent assay reactivity in a blood donor population. J. Clin. Microbiol. 26, 641-647. [12] Downie, J.C., Howard, R., Bowcock, B. and Cunningham, A.L. (1989) HIV-1 antibody testing strategy, evaluation of ELISA screening and Western blot profiles in a mixed low risk/high risk patient population. J. Virol. Meth. 26, 291-304. [13] De Cock, K.M., Porter, A., Kouadio, J., Maran, M., Gnaore, E., Adjorlolo, G., Lafontaine, M-F., Bretton, G., Gershy Darner, G-M., Odehouri, K., George, J.R. and Heyward, W.L. (1990) Rapid and specific diagnosis of HIV-1 and HIV-2 infections, an evaluation of testing strategies. AIDS 4, 875-878. [14] Ratner, L., Haseltine, W., Patarca, R., Livak, K.J., Starcieh, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn, E.A., Baumeister, K., Ivanoff, L., Petteway, S.R. Jr., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R. and Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277-284. [15] Wang, J.J., Steel, S., Wisniewolski, R. and Wang C.Y. (1986) Detection of antibodies to human T-lymphotropic virus type II by using a synthetic peptide of 21 amino acid residues corresponding to a highly antigenic segment of gp41 envelope protein. Proc. Natl. Acad. Sci. USA 83, 6159-6163. [16] Chiodi, F., von Gegerfeldt, A., Albert, J., Feny6, E.M., Gaines, H., von Sydow, M., Biberfeld, G., Parks, E. and Norrby, E. (1987) Site-directed ELISA with synthetic peptides representing the HIV transmembrane glycoprotein. J. Med. Virol. 23, 1-9.
[17] Javaherian, K., Langlois, J., McDanal, C., Ross, K.L., Eckler, L.I., Jellis, C.L., Profy, A.T., Rusche, J.R., Bolognesi, D.P., Putney, S.D. and Matthews, T.J. (1989) Principal neutralizing domain of the human immunodeficiency virus type 1 envelope protein. Proc. Natl. Acad. Sci. USA 86, 6768-6772. [18] Neurath, A.R., Strick, N. and Lee, S.Y. (1990) B-cell epitope mapping of human immunodeficiency virus envelope glycoproteins with long (19- to 36-residue) synthetic peptides. J. Gen. Virol. 71, 85-95. [19] Bahraoui, E., Yagello, M., Billaud, J-N., Sabatier, J-M., Guy, B., Muchmore, E., Girard, M. and Gluckman, J-C. (1990) Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees. AIDS Res. Hum. Retrov. 6, 1087-1098. [20] Plau6, S. and Briand, J.P. (1988) Solid-phase peptide synthetis. In: Synthetic Polypeptides as Antigens. (Burdon, R.H. and Van Knippenberg, P.H., Eds.), pp. 41-94, Elsevier, Amsterdam. [21] Kaiser, E., Colescott, R.L., Bossinger, C.D. and Cook, P.I. (1970) Color test for detection of free terminal amino group in the solid phase synthesis of peptides. Anal. Biochem. 34, 595-598. [22] Muller, S. (1988) Peptide-carrier conjugation. In: Synthetic Polypeptides as Antigens. (Burdon, R.H. and Van Knippenberg, P.H., Eds.), pp. 95-130, Elsevier, Amsterdam. [23] Bj6rck, L. and Kronvall, G. (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J. Immunol. 133, 969-974. [24] Oldstone, M.B.A., Tishon, A., Lewicki, H., Dyson, N.J., Fehrer, V.A., Assa-Munt, N. and Wright, P.E. (1991) Mapping the anatomy of the immunodominant domain of the human immunodeficiency virus gp41 transmembrane protein: peptide conformation analysis usi0g monoclonal antibodies and proton nuclear magnetic resonance spectroscopy. J. Virol. 65, 1727-1734. [25] Weiblen, B.J., Schumacher, R.T. and Hoff, R. (1990) Detection of IgM and IgA HIV antibodies after removal of IgG with recombinant protein G. J. Immunol. Meth. 126, 199-204. [26] Rakusan, T.A., Parrott, R.N. and Sever, J.L. (1991) Limitations in the laboratory diagnosis of vertically acquired HIV infection. J. Acquired Immune Deficiency Syndromes 4, 116-121.
57
FEMSIM 00188
A new capture test using conjugated peptides for the detection of HIV antibodies Pascale M. Richalet-S6cordel and Marc H.V. Van R e g e n m o r t e l Laboratoire d'Immunochimie, Institut de Biologie Mol~culaire et Cellulaire, Strasbourg, France Received 5 August 1991 Accepted 9 September 1991
Key words: Conjugated peptides; HIV antibodies; Enzyme immunoassay
1. SUMMARY A new capture test utilizing conjugated peptides has been developed for the detection of antibodies elicited against HIV-1. Human sera diluted 1:1000 were incubated in ELISA plates precoated with protein G. The captured IgG were allowed to react with three synthetic peptides corresponding to the gp41 sequence (591-611) YLKDQQLLGIWGCSGKLICTT, the gpl20 sequence (314-329) IRIQRGPGRAFVTIGK and the p27 sequence (182-198) EWRFDSRLAFHHVAREL. The peptides were used in the form of N-hydroxysuccinimido-biotin ovalbumin conjugates. Peroxidase-labelled streptavidin was used to detect antigen-antibody complexes. The sensitivity and specificity of detection of antibodies were analyzed with 40 HIV positive sera, 10 seroconverting sera and 21 normal human sera (NHS). The results were compared with a commercial indirect ELISA in which a single conjugated gp41 peptide was used as antigenic probe. This indiCorrespondence to: M.H.V. Van Regenmortel, Laboratoire d'Immunochimie, Institut de Biologie Mol6culaire et Cellulaire, 15 rue Descartes, 67084 Strasbourg Cedex, France.
rect ELISA recognized 100% of the HIV positive and the seroconverting sera. The new capture test using the gp41 conjugated peptide also recognized 100% of the HIV positive sera but was more specific since it gave no false positive results whereas the indirect test did. The gp120 and p27 conjugated peptides detected 35/40 (87.5%) and 31/40 (77.5%) of HIV positive sera respectively and also detected 9/10 (90%) and 10/10 (100%) of the seroconverting sera respectively, without any false positive results (0/21). The proposed new capture test is a very sensitive and specific assay for detecting HIV antibodies. 2. INTRODUCTION The need for a safe and easy test system for diagnosing HIV infections has led to the development of many ELISA systems using peptides as the antigenic probe [1]. Numerous studies have shown that indirect ELISA utilizing env-peptides [2-6], or carrier-bound gag-peptides adsorbed to plastic [7] is a highly sensitive and specific assay for detecting HIV antibodies. Unfortunately, however, none of the existing tests is able to recognize HIV positive sera with absolute sensi-
58 tivity and specificity. It has been reported, for instance, that ELISA may produce some false negative results with sera collected during the initial phase of antibody production [8,9] or during the final stage of AIDS [10]. In order to reduce the risk of overlooking sera containing HIV-1 antibodies, while at the same time keeping non-specific reactions and false positives to a minimum, it is customary to test blood samples first by ELISA and then to confirm each positive result by the Western blot technique (WB). The combined use of ELISA and WB methods has improved considerably the diagnosis of HIV infections, particularly in the case of seroconversions, although some false negative results [9] as well as false positive results generally involving the detection of gag-coded proteins [11] are still observed. Several groups have tried to combine different techniques to enhance the detection of HIV antibodies, for instance ELISA using recombinant antigen followed by WB [12], ELISA using several synthetic peptides followed by WB or radioimmunoprecipitation assay (RIPA) and IgM-MACRIA in case of indeterminate results [9], ELISA with whole virus followed by synthetic peptide ELISA, etc. [13]. In the present study we attempted to improve the diagnosis of HIV infections by using a new type of peptide conjugate in a capture ELISA format. Since there is a high degree of strain variation in the epitopes commonly used as synthetic HIV antigens in ELISA, as well as considerable heterogeneity in the immune response of individuals, a set of three peptides corresponding to immunodominant epitopes of gp41, gp120 and p27 ( n e d of HIV-1 was used in an attempt to decrease the number of false negative results. The proposed capture test differs from earlier tests by its use of a new type of biotinylated conjugate, of more highly diluted sera (1/1000 instead of undiluted or 1/50) and of a combination of structural epitopes from gp41 and gp120 together with a non-structural epitope from p27. This new test detected 4 0 / 4 0 HIV-1 positive sera (all sera had been tested beforehand by WB) without any false positive results (0/21), as well as 10/10 seroconverting (WB positive gp41 negative) sera.
3. M A T E R I A L S AND M E T H O D S
3.1. Selection of peptide sequences All sequences were derived from Bru subtype of HIV-1. The numbering of amino acid residues corresponds to that described by Ratner et al.-
[14]. The chosen gp41 sequence (591-611) YLKD Q Q L L G I W G C S G K L I C T T , covers the 584-618 region of the enu-glycoprotein previously described as an immunodominant region [2-4,15,16]. The gpl20 sequence (314-329) I R I Q R G P G R A F V T I G K belongs to the main neutralization determinant V3 of the envelope glycoproteins [17] and is able to elicit high levels of virus neutralizing antibodies [18]. The p27 sequence (182-198) E W R F D S R L A F H H V A R E L partly spans the sequence (185205) defined by Bahraoui et al. [19] as a B cell epitope. A Tyr residue was added at the N-terminal side of each peptide in order to allow conjugation to ovalbumin with bisdiazobenzidine.
3.2. Synthetic peptides Assembly of the protected peptide chains was carried out using the stepwise solid phase method of Merrifield (1963) on a NPS 4000 multichannel peptide synthetizer (Neosystem Laboratoires, Strasbourg, France). All amino groups were protected at the alpha amino position with the Boc group, except Trp, which was protected with the Fmoc group. The following side chain protecting groups were used: cyclohexyl ester (Asp, Glu), benzyl ether (Ser, Thr), 2-chlorobenzyloxycarbonyl (Lys), p-toluenesulfonyl (Arg, His). Boc-AAPAM-resins (0.5 mmol) were placed in each reaction vessel. The cycle used for incorporation of Boc and Fmoc amino acids has been described elsewhere [20]. Boc amino acids were coupled in the form of their benzotriazol ester, except Boc His (Tos) which was introduced as a symmetrical anhydride. The total coupling time was 45 min. Monitoring with the ninhydrin test [21] showed that most of the couplings were complete within this time. If necessary a double coupling was carried out.
59 At the end of the synthesis and after the last deprotection step, the resins were washed with ethyl ether and dried under vacuum. The high HF procedure was used for deprotection and cleavage from the resin. After lyophilisation each crude synthetic peptide was dissolved in 10% acetic acid and purified using a middle pressure chromatography apparatus (Kronwald Separation Technology, Sinsheim, F.R.G.) on a glass column filled up with RP100 material (C18, 20-45 txm). The final purity of each peptide was controlled by an analytical run on a reverse phase aquapure RP300 C8 column, 5 /xm (4.6 × 250 mm) using programmed gradient elution (2 ml/min) with the following solvents: A, 0.1 M triethylammonium phosphate (TEAP) pH 2.5; B, acetonitrile. Amino acid composition and net peptide content were determined with a Waters Picotag system (Waters Corp., Milford, MA).
3.3. Coupling procedure Bisdiazobenzidine (BDB) is a coupling agent which interacts with the N-terminal side of Tyr as well as with Lys, His and Cys residues. The BDB conjugation procedure has been described elsewhere [22]. The following procedure was used to prepare BDB: a 5 mg/ml benzidine solution was obtained by dissolving benzidine (B 3383, Sigma, St. Louis, MO) in 0.2 M HCL. NaNO 2 was added at the rate of 3.5 mg /ml of benzidine solution. The final solution was gently stirred during 2 h at 4°C. The following procedure was used to conjugate each HIV-1 peptide and N-hydroxysuccinimidobiotin to ovalbumin. During the coupling procedure, the temperature was maintained at 4°C and the mixture was continuously gently stirred. 1 mol of ovalbumin was mixed with 15 tool Of peptide and 15 mol of NHS-biotin (H 1759, Sigma), in pH 9 borate buffer 0.16 M (1 m l / m g of peptide). After 10 min, 100 #1 of BDB per 5 mg of peptide were added to the mixture. The pH was adjusted to 9 with NaOH 1 M. After 2 h, the final mixture was dialyzed for 24 h at 4°C against 3 changes of 1 1 PBS in order to eliminate unconjugated peptides and N-hydroxysuccinimido-biotin. As a first step in the BDB coupling procedure,
Lys residues present in peptides are usually protected with citraconic anhydride in order to prevent them from interacting with protein [22]. Since citraconic anhydride may irreversibly interact with Cys residues, this protecting step was not used for coupling the gp41 peptide which contained both Cys and Lys residues, but was applied in the case of the gpl20 peptide.
3.4. Capture test The following ELISA procedure was used to measure the binding of conjugated peptides to coated antibodies from human sera. Microtiter plates (Falcon, U.S.A.) were coated at room temperature for 15 h with 20 p,g/ml protein G (Pharmacia LKB, Uppsala, Sweden) in 0.05 M carbonate buffer, pH 9.6. This high concentration of protein G obviated the use of a blocking step for remaining sites on the plastic. After three washings of microtiter plates with phosphatebuffered saline (PBS) containing 0.05% Tween 20 (PBS-T), human sera diluted 1:1000 in PBS-T were added for 30 min at 37°C. After repeated washings, conjugated peptides diluted in PBS-T were added for 30 min at 37°C. One of the three conjugated peptide s was added in each well. Each conjugate was used at a dilution that gave the best ratio OD (HIV positive sample)/OD (normal human sera). After repeated washings, positive reactions were detected by incubation with peroxidase labelled streptavidin (horseradish peroxidase-streptavidin, Amersham, U.K.) diluted 1:6000 in PBS-T, for 30 min at 37°C. The final reaction was visualized by addition of tetra methyl benzidine (TMB) (3,3' 5.5' benzidine, Boehringer, F.R.G) diluted 1 : 10 in citrate buffer, for 10 min at 37°C. OD values were measured at 405 nm after the reaction had been stopped with 50 txl of 1 M HC1. No thimerosal or sodium azide was used. The volume of mixture added at each step of the capture test was 100/xl per well. In order to determine the cut-off line of the assay, 21 sera from normal individuals were tested with the conjugated (591-611) gp41, Y(314-329) gp120 and Y(182-198) p27 peptides. Using sera diluted 1:1000, the capture test procedure gave average absorbances of 0.10 (SD: 0.033), 0.28 (SD: 0.041) and 0.23 (SD: 0.036) respectively.
60 C o n t r o l s e r a w e r e c o n s i d e r e d positive w h e n t h e O D values w e r e h i g h e r t h a n the a v e r a g e O D v a l u e plus 3 SD, i.e. r e s p e c t i v e l y 0.20, 0.40 a n d 0.35 O D units.
A m i n o acid analysis of two s a m p l e s o f e a c h p e p tide s h o w e d that t h e p u r i f i e d p r o d u c t s h a d t h e e x p e c t e d c o m p o s i t i o n ( d a t a not shown).
3.5. Sera
3.6. Correlation with a commercially available assay T w e n t y o f t h e 40 s e r o p o s i t i v e s e r a as well as t h e 10 s e r o c o n v e r t i n g a n d 19 of t h e 21 n o r m a l h u m a n s e r a w e r e t e s t e d with a c o m m e r c i a l E L I S A kit ( L a b s y s t e m s O Y , H e l s i n k i , F i n l a n d ) , which utilizes a single gp41 p e p t i d e c o n j u g a t e d to bovine s e r u m a l b u m i n as a n t i g e n i c p r o b e [4]. In this assay, t h e m a n u f a c t u r e r s r e c o m m e n d that s e r a b e d i l u t e d 1 / 5 0 . T h e m i c r o p l a t e r e a d e r was b l a n k e d a g a i n s t t h e r e a g e n t blank. T h e following f o r m u l a was u s e d to c o n v e r t t h e a b s o r b a n c e s to e n z y m e i m m u n o a s s a y units ( E I U ) : E I U = ( O D o f t h e test s a m p l e / O D o f t h e positive c o n t r o l ) x 100. A s the O D o b t a i n e d with t h e positive c o n t r o l was 1.00, we s i m p l i f i e d t h e f o r m u l a to E I U = ( O D of the test s a m p l e ) x 100. T h e cut-off line o f t h e assay was 35 E I U , c o r r e s p o n d i n g to an O D o f 0.35. T o facilitate t h e c o m p a r i s o n with o u r test, the results o f t h e i n d i r e c t E L I S A w e r e c o n v e r t e d into O D values. T h e c u t - o f f line o f t h e assay was thus 0.35 OD.
4. R E S U L T S
4.1. Synthetic peptides T h e d e g r e e o f p u r i t y of t h e p e p t i d e s a s s e s s e d by H P L C was at least 95% for t h e ( 5 9 1 - 6 1 1 ) gp41 p e p t i d e a n d 85% for t h e Y ( 3 1 4 - 3 2 9 ) g p l 2 0 a n d Y ( 1 8 2 - 1 9 8 ) p27 p e p t i d e s . T h e n e t p e p t i d e cont e n t was u s e d as a b a s e for m o l a r i t y calculation.
":':':':':':':"
A
S e r a f r o m 40 s e r o p o s i t i v e a n d 10 s e r o c o n v e r t i n g (gp41 n e g a t i v e ) p a t i e n t s , a n d o f 21 seronegative healthy individuals were examined. The s e r a o r i g i n a t e d f r o m t h e B l o o d T r a n s f u s i o n Center, S t r a s b o u r g , I n s t i t u t d e V i r o l o g i e , S t r a s b o u r g , and from the New York Blood Center. All sera h a d b e e n t e s t e d b e f o r e h a n d by W B . T h e s e r a h a d b e e n h e a t e d at 56°C for 30 min a n d w e r e cent r i f u g e d for 10 min at 4000 r p m just b e f o r e the assay.
O
[ (591-611)
gp41
6 O
° ° • ••° ° ° °
B
[ Y(314-329)
gp121~
E °~, ° °° ° •°°°° ° • °°° , • ~°
E3 0
•° •
Y(182-198)
C
p27
•°°°° ° °°°°° ° •°° ° °
. ;-;. ",',"
NHS
HIV positive
.:.
seroconverting
Fig. 1. Capture assay showing binding with peptide conjugates. Anti-peptide IgG activity was measured in 21 normal human sera (NHS), 40 HIV positive sera and 10 seroconverting (WB positive, gp41 negative) sera. All sera were diluted 1:1000. Antibody levels were expressed in OD units at 405 nm after 10 min of incubation of streptavidin-peroxidase with substrate. (A) Binding of (591-611) gp41 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.10) plus 3 SD (3 x 0.033), i.e. ~ 0.20 OD unit. (B) Binding of the Y(314-329) gpl20 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.28) plus 3 SD (3x0.041), i.e. ~ 0.40 OD unit. (C) Binding of the Y(182-198) p27 conjugated peptide: the cut-off line corresponds to the upper limit of a normal population giving an average OD of 21 NHS (0.23) plus 3 SD (3 x0.036), i.e. ~ 0.35 OD unit.
61
4.2. Conjugated peptides Due to interference of the spectrum of N-hydroxysuccinimido-biotin with that of amino acid residues, amino acid analysis of conjugated peptide preparations could not be interpreted quantitatively. A dilution of each conjugate was chosen that gave the best ratio OD (HIV positive sample)/OD (normal human sera). The gp41, gpl20 and p27 peptide conjugates were used at a dilution of 1/1000, 1/18000 and 1/4000 respectively. 4.3. Capture test All sera were tested in the capture test against the three conjugated peptides. As protein G is an IgG Fc receptor [23], the investigation was restricted to the anti-HIV-1 IgG response. Tests were repeated at least twice in independent ELISA experiments. The (591-611) gp41 peptide conjugate recognized 40/40 of the HIV positive sera. None of the 21 normal sera were recognized nor any of the 10 seroconverting sera (Fig. 1A). The Y(314-329) gpl20 peptide conjugate recognized 35/40 (87.5%) of the HIV positive sera and 9/10 (90%) of the seroconverting (gp41 negative) sera. There were also no false positive results with the 21 normal human sera (Fig. 1B). The Y(177-198) p27 peptide conjugate recognized 31/40 (77.5%) of the HIV positive sera and
• .....:.:.:......
..,..
E tO O
NHS
HIV positive
seroconverting
Fig. 2. Binding in an indirect commercial E L I S A (Labsystems) using a single gp41 peptide as probe. Anti-peptide IgG activity was measured in 19 normal h u m a n sera (NHS), 20 HIV positive sera and 10 seroconverting (gp41 negative) sera. As r e c o m m e n d e d by the manufacters, all sera were diluted 1 : 50. Antibody levels are expressed in O D at 405 n m after 30 min of incubation of anti-human IgG enzyme conjugate with substrate. T h e four seroconversion sera giving O D values in the range of false positives correspond to samples 3, 7, 8 and 9 (Table 2).
10/10 of the seroconverting (gp41 negative) sera. No false positive results were observed with the 21 normal human sera (Fig. 1C). 4.4. Indirect ELISA The commercial assay using a gp41 peptide recognized 20/20 of the HIV positive sera and 10/10 of the seroconverting (WB positive, gp41 negative) sera. However, 4/19 (21%) of the
Table 1 Reactivity of H I V positive and seroconverting (WB positive, gp41 negative) sera in the capture assay and in a commercialized indirect E L I S A Type of serum
HIV positive Seroconve rting Normal h u m a n
Total number of sera (n 1) 40 10 21
% Positive n I sera in capture assay with gp41 gpl20 100 0 0
87.5 90 0
p27 77.5 100 0
N u m b e r of sera tested with commercial gp41 peptide (n 2)
% Positive n 2 sera in commercial assay
20 10 19
100 100 21
Sera from patients were diluted 1 : 1000 in the capture test and 1:50 in the commercial indirect E L I S A (Labsystems, Helsinki). Values with peptides in the capture assay are expressed as % positive sera among total tested sera (n l) and values with the commercial test as % positive sera of n 2. T h e sera were considered positive in the capture test, when the O D values were > (OD for 21 NHS + 3 SD) after 10 min of incubation of streptavidin-peroxidase with substrate, or in the Labsystem test, when the E I U were > 35 after 30 min incubation of anti-human IgG enzyme conjugate with substrate. Tests were repeated at least twice in independent E L I S A experiments.
62
normal human sera were also positive. Each of the false positive results remained positive in repeated tests (Fig. 2). The results of the capture tests and of the commercial indirect E L I S A are compared in Table I.
5. D I S C U S S I O N The new capture test is a highly sensitive assay since it was able to detect specific antibodies in 100% of the H I V positive sera (by WB technique) when the sera were diluted 1/1000. This highworking dilution was no doubt responsible for the low non-specific background noise and the absence of any false positives. In addition, when the conjugated p27 peptide was used as probe, sera that were WB + and gp41- were also detected as H I V positive (Table 2). The commercial assay that was tested in parallel was also fairly sensitive since it detected 100% of the 30 tested H I V positive sera which included 10 seroconversion sera. However, the specificity of the commercial assay was not as good as that of the capture test since the level of false posi-
tives was 21%, in spite of a centrifugation step (4000 rpm, 10 min) prior to testing. Presumably the way the sera had been stored (many had been kept for several years and had been repeatedly thawed and refrozen) as well as the low serum dilution (1/50) used in the assay are responsible for the high percentage of false positives. Similar problems are likely to be encountered with other available tests based on synthetic peptides which also entail the use of sera used of low dilution. Our data indicate that it is necessary to combine the results obtained with at least two peptide conjugates (gp41 and p27 peptides) in order to detect 100% of H I V positive and of seroconversion (WB + / g p 4 1 - ) sera. Although the commercial test based on a single conjugated gp41 peptide detected 100% of the seroconversion sera, it should be noted that the O D levels reached by four of these samples (sera 3, 7, 8 and 9 in Table 2) were similar to O D values of false positives (Fig. 2); the specificity of these four results is thus in doubt. A m o n g the six seroconversion sera that gave an O D > 2.0 in the commercial test, three (samples 1, 2 and 10) were strongly recognized in the capture test while three (4, 5 and 6) gave values very close to the cut-off level (not shown).
Table 2 Comparison of results obtained with the seroconverting sera in the capture test and the commercial indirect ELISA, with those obtained in Western blot tests Seroconverting
ELISA
serum
Capture assay
1 2 3 4 5 6 7 8 9 10
Bands revealed in Commercial
gp41
gpl20
p27
gp41 peptide
-
+ + + + + + + + +
+ + + + + + + + + +
+ + + + + + + + + +
WB
p18, p25, p55, p68, gp160 p18, p25, p55, p68, gp120, gp160 p18, p25, gp120, gp160 p25, p55, gp120, gp160 p25, gp120, gp160 p18, p25, gp120, gp160 p25 p18, p25, p55 gpl20, g p l 6 0 gpl20, g p l 6 0
The E L I S A were considered positive ( + ) when the OD was above the cut-off threshold. The WB were considered as positive when one or more bands corresponding to HIV~I separated proteins were revealed with enzyme labelled human anti-IgG in the DuPont assay. Sera 7 -1 0 which are not simultaneously positive for both p25 and gp120/ 160 correspond possibly to the beginning of seroconversion. In the commercial test three of these sera (7, 8 and 9) gave O D values close to that of false positives. Sera 7, 8 and 10 gave a strong response in the capture test.
63 F u r t h e r m o r e , sera 7 and 8 whose W B profiles revealed no antibodies reacting with the glycoproteins gp41, g p l 2 0 nor with the precursor protein gpl60, gave a strong response in the capture test while being in the range of some false positives in the commercial assay. It seems that the sensitivity of the W B technique is insufficient for detecting some antibodies reacting with g p l 2 0 and g p l 6 0 whereas both the capture and commercial tests react with peptides f r o m the g p l 2 0 and gp41 sequences. O n the o t h e r hand, antibodies directed against g p l 6 0 which contains the gp41 sequence do not recognize the 591-611 peptide conjugate although they bind to peptides from the gp41 region. Since there is published evidence [24] that the c o n f o r m a t i o n of the region 5 9 3 - 6 0 3 in gp41 is important for recognition by antibodies directed to the native protein, it seems reasonable to assume that the structure of the gp41 conjugated peptide used in the capture test resembles that of the same region in the native protein. This particular peptide conjugate may thus be a very sensitive p r o b e for detecting the a p p e a r a n c e of early antibodies to gp41. A n o t h e r potential advantage of the capture test is the ability to detect I g M or I g A antibodies without interference f r o m the major I g G response. It is k n o w n [25] that the presence of I g G interferes with the detection of o t h e r antibody subclasses and capturing h u m a n antibodies by means of /x or a - c h a i n specific antiglobulins should be able to o v e r c o m e this problem. T h e detection of I g M and I g A antibodies is likely to be important for the detection of antibodies prod u c e d early after H I V infection as well as for diagnosis of m o t h e r to child c o n t a m i n a t i o n with H I V . Since I g M and I g A do not cross the placenta, their presence in the blood of the n e w b o r n indicates vertical transmission [26].
ACKNOWLEDGEMENTS We are grateful to Dr. S. PlauE (Neosystem, Strasbourg) for helpful discussions. W e thank Dr. M.L. N o r t h (Centre de Transfusion Sanguine, Strasbourg), Dr. A.R. N e u r a t h (New Y o r k Blood
Center) and Dr. M.P. Schmith (Institut de Virologie, Strasbourg) for g e n e r o u s gifts of patient sera. W e thank Ms. Susanne W e n c k e r for help in typing the manuscript. This work was supported by the Institut National de la SantE et de la R e c h e r c h e MEdicale, the Conseil REgional d' Alsace and by Neosystem Laboratoires S.A., Strasbourg.
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