A novel protein immunoassay with predetermined specificity using monoclonal antibodies against...

ELSEVIER Journal of Immunological Methods 185 (1995) 103-I 14

JOURNAL OF IMMUIGLOGICAL METHODS

A novel protein immunoassay with predetermined specificity using monoclonal antibodies against tryptic fragments:

application to HIV P24 antigen

Frideric Jean *, Bkatrice Bourcier, Solange Lebec, Michel Delaage, Jacques Barbet

Immunotech, 130 awnue de Lattre de Tassigny, BP I77F, 13276 Marseille Cedex 9, France

Received 29 July 1994; revised 3 January 1995; accepted 21 April 1995

Abstract

We have developed a new protein immunoassay method which uses antibodies raised against synthetic peptides. These synthetic peptides are selected to correspond to fragments of the protein that can be obtained by proteolytic

treatment of the protein by trypsin. Just before assay, biological samples are treated with trypsin to liberate the fragments which bind to the anti-peptide antibodies with high affinity. The exact specificity of the assay is predetermined by the amino acid sequence of the fragment which may be either conserved within a family of

antigens or, conversely, entirely specific for a particular protein. This method has been successfully employed in the development of an immunoassay for HIV P24 antigen. In that case, peptides were selected that were strongly conserved among the different HIV-l and HIV-2 strains. This methodology has permitted the development of a sensitive immunoassay with a broad specificity despite many amino acid variations between HIV strains. The methodology could be extended to other protein antigens.

Keywords: Virology; Synthetic peptide; Tryptic fragment; P24 antigen; Enzyme immunoassay

Abbreviations: ELISA, enzyme-linked immunosorbent assay; HIV, human immunodeficiency virus; AIDS, acquired immune deficiency syndrome; BSA. bovine serum albumin; BGG, bovine gamma globulin; HTG, human thyroglobulin; SMCC, 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; SMP, N-succinimidyl 3-maleimidopropionate; DMF, dimethyl formamide; PBS, phosphate-buffered saline; TFA, trifluo- roacetic acid; GT, glycyl-L-tyrosine); MES, 2 (iV-morpholino) ethane sulfonic acid: GAMIG, goat anti-mouse immunoglobulin; AchE, acetylcholine esterase.

* Corresponding author. Tel.: (33) 91 17 27 36; Fax: (33)91414358.

1. Introduction

In general, protein immunoassays require very specific antibodies with high affinity. High affinity antibodies are necessary in order to obtain a high sensitivity in the assay. Specificity, however, is

defined with respect to the measured antigen and to the medium in which it is found. One may, for instance, wish to distinguish between closely related molecules or, conversely, be able to detect any molecule of a family of related antigens. Synthetic peptides have been used as immunogens to raise antibodies with predetermined

0022-1759/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022.1759(95)00108-5

104 F. Jean et al. /Journal of Immunological Methods 185 (1995) 103-114

specificity. In this case, antibodies that exhibit a wide cross-reactivity between the synthetic peptide and the intact protein, may be used to develop specific immunoassays. However, antibodies raised against synthetic peptides often have low affinity for the corresponding epitopes of the natural protein because of important differences in their secondary, tertiary, and quaternary struc- tures (Altman et al., 1984; Burt et al., 1986; Fieser et al., 1987). Using monoclonal antibodies raised against synthetic peptides, we have developed a new protein immunoassay method that minimize this loss of affinity. In this method, synthetic peptides are selected to correspond to peptidic fragments of the protein that can be released by proteolytic treatment with trypsin (tryptic fragments). The number of such peptides is usually fairly large, and that permits the selection of peptides which precisely define the antigen to be assayed. This is most conveniently done by examining their amino acid sequences in comparison with that of related antigens, in order to precisely limit cross-reactions with other related, and non-related, antigens. In the immunoassay, the samples to be assayed are treated by trypsin to release tryptic fragments; these then bind to the antipeptide antibodies with higher affinity than the natural protein, because of structural homologies between the synthetic peptides and the tryptic fragments. In this paper we describe the application of this method to the immunoassay of the P24 HIV antigen.

P24 is the major core polypeptide of HIV-l (Barre-Sinoussi et al., 1983; Gallo et al., 1983). Its precursor P55, encoded by the GAG gene (Mues- ing et al., 1985; Ratner et al., 1985; Sanchez- Pescador et al., 19851, is cleaved by the HIV protease to form the structural core proteins: P24, P17, P7 and P6 (Peng et al., 1989). Since it is a good marker of HIV replication, P24 assays are now widely used as diagnostic tools with demonstrated clinical significance. The detection of cir- culating P24 seems to be one of the first signs of infection with HIV. P24 may be detected in some cases several weeks before seroconversion (Allain et al., 1986; Ranki et al., 19871, and in babies born to infected mothers (Furlini et al., 1989). The level of P24 in human blood correlates with

the clinical stage of the infected host and is considered as a prognostic marker (Goudsmit et al., 1986; Lange et al., 1986; Allain et al., 1987; Paul et al., 1987). The effectiveness of therapy against AIDS can be estimated by measuring the decline of the P24 concentration (Chaisson et al., 1986). This assay is used also in AIDS research for the monitoring of HIV-infected cell cultures and is more convenient and sensitive than the conventional reverse-transcriptase assay (Caruso et al., 1987; Tatsumi et al., 1990).

Several HIV isolates have been described in the literature, reflecting the genetic variability of retroviruses isolated from both different individu- als and also from the same patient (Devare et al., 1986; Fisher et al., 1988; Saag et al., 1988; Zagury et al., 1988). The great number of mutations observed in the different HIV strains reflects the errors made by the reverse transcriptase during DNA synthesis (Preston et al., 1988; Roberts et al., 1988). Obviously, the high mutation rate, which allows the virus to escape the immune system, also makes difficult the production of anti-viral antibodies effective against all HIV strains. This is, of course, particularly true in the case of antibody production against P24 for immunoassays. Moreover, in the case of P24 immunoassays the early effectiveness of diagnosis depends on the sensitivity. Thus, antibodies with high affinity are required.

To apply our method to the immunoassay of P24 antigen, monoclonal antibodies against P24 synthetic peptides were first produced. These synthetic peptides correspond to highly conserved regions of the viral protein located within two trypsin cleavage sites. One of these tryptic fragments is composed of two peptides linked by a natural disulfide bridge. Using two monoclonal antibodies directed against the two peptides of this tryptic fragment, we were able to develop an ELISA with one antibody immobilized on a microtiter plate and the other conjugated to acetylcholine esterase (AchE) as a tracer.

In a P24 immunoassay, natural antibodies against viral proteins in the serum of an HIV infected person can interfere by masking the epitopes of the antibodies used in the assay (Allain et al., 1986). Usually, this interference requires

F. Jean et al. /Journal of Immunological Methods 185 (1995) 103-I 14 105

the dissociation of the P2bantibody complexes by acid treatment just before assaying the serum samples (Mathiesen et al., 1988). The effect of tryptic digestion on this interference has also been evaluated.

adding the lyophilized peptide to the activated protein solution to give a molecular ratio of 30: 1. After 16 h of incubation at 4°C immunogens were purified by size exclusion chromatography on G25S (Pharmacia, Uppsala, Sweden) using pH 7.2 phosphate buffered saline (PBS) for elution.

2. Materials and methods 2.3. Peptide tracer synthesis

2.1. Tryptic peptide selection

P24 amino acid sequence analysis of 18 HIV-l strains and eight HIV-2 strains, extracted from the GENBANK database, was performed using the BISANCE software (Dessen et al., 1990).

Peptides were selected according to the follow- ing criteria: they should correspond to a tryptic fragment, or part of a tryptic fragment, they should be at least ten amino acids long, they should be well conserved (at least 80% homology) among HIV-l and, if possible, HIV-2 strains.

Peptide synthesis was performed by Neosystem (Strasbourg, France).

2.2. Imrnunogen synthesis

Synthetic peptides with a cysteine residue were cross-linked to three different carrier proteins: bovine serum albumin (BSA; Merck, Darmstadt, Germany), bovine gamma globulin (BGG; Merck, Germany), and human thyroglobulin (hTG; Sigma, St. Louis, MO).

SMCC in solution at 20 mg/ml in DMF was added to a solution of glycyl-L-tyrosine (Sigma, USA) at 20 mM in 0.1 M, pH 7.0 sodium phosphate to give a molar ratio of 1:l. The reaction mixture was incubated for 2 h at 20°C the pH was then lowered to 2 by addition of 1 N HCI. The mixture was purified by HPLC on reverse phase Cl8 (Microbondapak; Waters, Marlbor- ough, USA) using a solution of 0.05% TFA in water, with a linear gradient of acetonitrile for elution. The fractions containing glycyl-t_-tyrosine, derivatized with SMCC (GT-SMCC), were pooled and evaporated under nitrogen to give a precipitate. After centrifugation the supernatant was discarded and the pellet dissolved in DMF. GT-SMCC in DMF at 25 mM was then added to the peptide at 1 mM in 0.1 M 2(N-morpholino) ethane sulfonic acid (MES, Sigma, USA) at a molar ratio of 2:1, and, after 18 h of incubation at 4°C the tracers were purified by HPLC as above. Peptide tracers were radiolabeled with ‘2’1 by the chloramine-T method (Greenwood and Hunter, 19631, and purified again by HPLC under the same conditions as above.

Carrier proteins were first derivatized with succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC; Pierce, Rockford, USA) or N-succinimidyl 3-maleimidopropionate (SMP; Fluka, Buchs, Switzerland). The cross-linking reagent at 25 mg/ml in dimethyl formamide (DMF) was added to the carrier protein at 5 mg/ml in 0.1 M sodium phosphate pH 7.0 buffer, to a molecular ratio of 5O:l. The reaction mixture was incubated for 4 h at 37°C and then separated by size exclusion chromatography (Bio-Gel P2; Bio-Rad, Richmond, CA) using 0.1 M, pH 6.0 sodium phosphate buffer for elution. Peptides were cross-linked to the derivatized protein by

2.4. Immuniza lions

BALB/c mice were immunized sequentially with monthly intraperitoneal injections of 50 pg of the peptide-carrier protein conjugate in 250 ~1 of PBS emulsified in one volume of complete Freund’s adjuvant, for the priming, and in incom- plete Freund’s adjuvant for the booster immunizations. Mice were bled ten days after each injection to measure the antipeptide antibody titer and affinity. Four days before fusion a booster injection of 50 pg of peptide-carrier conjugate was administered intravenously.


2.5. Tryptic cleauage

20 ~1 of trypsin (Sigma, USA) at 10 mg/ml in 1 M Tris-HCl; 0.1 M CaCl,; pH 7.5 were added to 200 ~1 of normal human serum with different concentrations of rP24 (Transgene, Strasbourg, France). After 2 h of incubation at 37°C the enzymatic reaction was stopped with 20 ~1 of soybean inhibitor (Sigma, USA) at 8.5 mg/ml in distilled water.

2.6. Antiserum characterization

100 ~1 of mouse antiserum at different dilu- tions in PBS with 0.1% BSA were incubated for one night with 100 ~1 of radiolabeled peptide tracer (25000 cpm) at 4°C. Antibody-antigen complexes were then precipitated by the addition of 50 ~1 of normal human plasma and 250 ~1 of polyethyleneglycol 6000 at 20% in PBS. After a 15 min centrifugation at 3000 X g and 4°C the supernatant was discarded and the activity in the pellet was measured in a gamma counter.

Affinities and specificities were evaluated by incubating 100 ~1 of mouse antiserum diluted in PBS, 0.1% BSA, to the concentration giving 50% of the maximum tracer binding, with 50 ~1 of radiolabeled peptide tracer (25 000 cpm) and different concentrations of the antigen to be tested, diluted in PBS, 0.1% BSA. Incubation, PEG pre- cipitation, and determination of activity were performed as above. The affinity was calculated by the slope of the line obtained when bound radioactivity/free radioactivity (B/F) was plotted against bound radioactivity (B) (Scatchard, 1949).

2.7. Hybridoma supernatant screening

Goat anti-mouse immunoglobulin (GAMIG; Immunotech, Marseille, France) and peptides were coated directly onto microtiter plate wells (Falcon 3911; Becton Dickinson, Franklin Lakes, USA) by incubating 50 ~1 of GAMIG solution at 10 pg/ml in PBS or the peptide at 5 pg/ml in carbonate buffer during one night at 4°C. The wells were then saturated with 250 ~1 of PBS, 1% BSA for 3 h at 37°C.

50 ~1 of hybridoma supernatant were incu-

bated in GAMIG-coated wells for 3 h at room temperature. After one wash with 300 ~1 of 9 g/l NaCl-0.005% Tween 20 wash solution, 50 ~1 of radiolabeled peptide tracer (25000 cpm) in PBS, 0.1% BSA were deposited in each well and incubated overnight at 4°C. The wells were then washed three times and the bound activity determined.

Alternatively, 50 ~1 aliquots of hybridoma supernatant were incubated overnight in peptide- coated wells, and then washed once with the wash solution, before 50 ~1 of radiolabeled GAMIG (25000 cpm) were added to each well. After 3 h of incubation at room temperature, wells were washed three times with wash solution and bound activity was determined.

2.8. Monoclonal antibody radiolabeling

Monoclonal antibodies were radiolabeled with “‘1 by the chloramine-T method and purified by size exclusion chromatography on PDlO columns (Pharmacia, Sweden) using PBS, 0.1% BSA, for elution.

2.9. Monoclonal antibody biotinylation and coating

Monoclonal antibodies at about 1 mg/ml in 20 mM borate, 150 mM NaCl pH 8.2, were derivatized with 8% (w/w) of NHS-biotin (Sigma, USA) at 10 mg/ml in DMF. After 15 min of incubation at room temperature, the reaction was stopped by the addition of 1 M NH&l solution to a final concentration of 0.1 M, and biotinylated antibodies were separated from excess reagents by dialy- sis against PBS.

Antibody-coated tubes were then prepared by incubating overnight at 4°C 1 ml of biotinylated antibody at 1 Fg/ml in PBS, 0.1% BSA in avidin-coated tubes (Immunotech, France). The contents of the tubes were aspirated just before use. Antibody-coated wells were prepared in the same way, but with only 300 ~1 per well.

2.10. Monoclonal antibody pair selection

All possible monoclonal antibodies pairs were tested by incubating 100 ~1 aliquots of different

F. Jean et al. /Journal of Immunological Methods 185 (1995) 103-114 107

concentrations of rP24, trypsinized as above in serum, with 100 ~1 of each monoclonal antibody “‘1 tracer, in each monoclonal antibody-coated tube, overnight at 4°C. Tubes were then washed once with 2 ml of wash solution and the bound activity was measured.

2.11. Monoclonal antibody-acetylcholinesterase conjugate synthesis

The monoclonal antibody-enzyme tracer was prepared by conjugation of the Fab’ fragment with acetylcholine esterase (AchEI derivatized with SMCC. The preparation of F(ab’>? fragments, Fab’ fragments, incorporation of maleimido groups into AchE and coupling with Fab’ fragments were performed according to Grassi et al. (19891, except that F(ab’), fragments were purified by cation exchange chromatography using FPLC with a Mono S HR5X5 column (Pharmacia, Sweden) eluted with 50 mM, pH 4.2 acetate at 1 ml/min with a linear gradient of NaCl. Enzymatic activity was measured at each step of the preparation by the calorimetric method of Ellman et al. (1961). One Ellman unit corresponds to the amount of enzyme producing an absorbance increase of one absorbance unit at 25°C in 1 min in 1 ml and for an optical path of 1 cm.

2.12. Immunoassay

250 ~1 of standard solution or serum sample were deposited in tubes containing 250 pg of lyophilized trypsin, 125 ~1 of 1 M, pH 2 glycine- HCl, 1.5% Triton X-100 buffer were then added to each tube, followed by 125 ~1 of 1 M Tris-HCl, pH 8.8, 20 mM CaCl,, 0.1 g/l of thimerosal to

18 30 82 100

bring the pH back to neutrality. Antibody-antigen complexes were dissociated by incubating the sample for 30 min at 37°C and pH 2 just before addition of the 1 M, pH 8.8 Tris-HCl buffer. 200 ~1 of each treated standard or sample were then pipetted into antibody-coated wells and the plates were incubated for 2 h at 4-6°C. After five washes with 300 ~1 of wash solution, 200 ,ul of antibody- AchE conjugate at 4 U Ellman/min per ml in 0.16 M, pH 6.2 H,PO,, 0.2 M NaCl, 5 g/l of lactose, 0.15 g/l of thimerosal, 5 g/l of BSA, and 0.1 g/l of normal mouse immunoglobulin were pipetted into each well. The plates were incubated for 3 h at 4-6°C. The wells were then washed again five times with 300 ~1 of wash solution and 200 ~1 of Ellman enzymatic colori- metric substrate were distributed to each well. After 30 min in the dark, at room temperature, with constant shaking, the enzymatic reaction was stopped with 50 ~1 of 0.1 mM 9 amino-1,2,3,4-te- tra-hydroacridine hydrochloride hydrate (Aldrich, Strasbourg, France1 in distilled water and optical densities were measured at 405 nm.

3. Results

3.1. Selection of tryptic peptides

The tryptic digest map of P24 of the reference strain HIV-l BRU (Fig. 1) revealed 22 trypsin cleavage sites. According to the criteria that we had previously established, three peptides were selected and synthesized. GllC peptide: GSDIAGTTSTC (one letter

amino acid code) corresponds to amino acids 101-110 which are the amino terminus of the tryp-

132 143 153 167 173 193 22i

+ JI & 1L 4 c & *JI + & GllC Nl7K T18K

* + + + + + + + + * *

25 70 97 131 140 154 162 170 182 203 229

-s-s-

Fig. 1. Tryptic digest map of the reference strain HIV-l BRLJ. Arrows indicate the trypsin cleavage sites and numbers correspond

to the positions of lysines and arginines.


tic fragment 101-131. It was synthesized with an extra cysteine at the carboxy terminus to allow derivatization of the sulfhydryl group.

N17K peptide: NWMTETLLVQNANPDCK is the complete tryptic fragment corresponding to amino acids 183-199.

T18K peptide: TLEEMMTACQGVGGPGH - K corresponds to amino acids 210-227 of the carboxyterminal end of the tryptic fragment 204- 227.

The last two peptides N17K and T18K possess a natural cysteine and they are linked by a disulfide bridge in the native P24 protein. Neither GllC nor T18K peptides correspond to whole tryptic fragments, but only to their most conserved regions.

Amino acid sequence analysis (Fig. 2) showed that the GllC sequence was strictIy conserved among the different strains of HIV-l and HIV-2 so far studied; peptides N17K and T18K showed, at most, one mutation in HIV-l strains and three mutations in HIV-2 strains by comparison with

the HIV-l BRU prototype strain. For none of these peptides have any mutations involving arginine or lysine substitutions been reported. Such mutations might affect trypsin cleavage and peptide release.

3.2. Immunization and monoclonal antibody production

Sera of immunized mice were characterized by their antipeptide titer. This titer correspond to the dilution of the serum that gave 50% of maximum peptide tracer binding.

The use of GllC conjugated with a carrier protein as immunogen in different protocols did not yield a high antibody titer, even after six months of repeated immunizations. This probably reflects the poor immunogenicity of this peptide. We were thus unable to obtain monoclonal antibodies against this peptide.

By contrast, both N17K and T18K peptides, conjugated with carrier proteins, induced a high antibody response with titers of above 1:lOOO. Higher titers were obtained by immunization with peptides conjugated to different carrier proteins (BSA, BGG, and hTG) with two different cross-

Strains GllC N17K TIBK

1.BRU G S D I A G T T S T NWMTETLLVQNANPDCK TLEEMMTACQGVGGPGHK 1_HXS2 .. ..__ .... ___...._......_ .............. _._... ,_JH3 _ .__ ... __. . .._ ......... .._. ..................

,_CDC45,______-___ _.........._ ........ . ..............

,_oy, . .._..~._. _..........__ . .._ ._ .......... .._...

,_SF2 .......... _......._..___ .._ ..................

,_HAN ....... . . . _.........._.___ _ .......... .._ ._...

1_“45S ...... .._. .... .._ .......... ..................

,_NDK _._.__~_._ . .._._._....~_._. _......_...~ ......

,-BH,o .... . . .._ ._..._._._..~.._. _._..._..__.._._ ..

1_BH5 ..... . .._ ... . ......... _._. _......_....._ .... ,_pv22 _.....~ ... _....._._...~ . .._ ......... ..~_ ._... 1_WM& __..__~ ...... .._......~ ......... .._...~ ......

,_EL, _._ _..~ ... _ ................ _______________~__ ,_MAL . .._ .. .._. .._._ ............ _______________~__ ,_M,, .__. ...... _R_______________ ..... .._...~ ...... 1_RF .._...~ _.. _____~___________ _______________~__ 1.22 .. _ .... _ .. G--_________-____ -______-~____-_S__

Fig. 2. Amino acid sequence comparison of 17 HIV-l and 8 HIV-2 strains with HIV-l BRU for the GllC, N17K, and T18K

peptides. -: identical amino acid; * : deletion.

F. Jean e! al. /Journal of Immunological Methods 185 (19951 103-114 109

linking reagents (SMCC and SMP). The carrier their capacity to bind to the peptide-coated to protein was changed every time the antibody titer microwells or to the ‘=I peptide tracer. Six of the was found to be stable over at least 1 month. This anti-N17K monoclonal antibodies and fifteen of change resulted in a ten-fold increase in the the anti-T18K monoclonal antibodies bound to anti-peptide antibody titer. The different cross- the peptide coated wells but did not bind to the linking reagents were used to avoid immunization peptide conjugated with glycyl+-tyrosine via against these reagents. SMCC or via SMP.

Characterization of an immune serum (Fig. 3) revealed that antibodies raised against N17K and against T18K exhibited high affinity for trypsinized P24, equivalent to the affinity for the immunizing peptide (Kd = 10e9 M), and poor affinity for the native whole protein. Moreover, in the case of both peptides, the antibody affinity was the same for the peptide and for the peptide conjugated with glycyl-L-tyrosine via the same cross-linking reagents as those used for immunogen synthesis.

3.3. Selection of monoclonal antibodies

Four mice immunized with N17K and three mice immunized with T18K by four monthly intraperitoneal injections of peptide-carrier conjugate, as described in the materials and methods section, were selected for hybridoma production, on the basis of their anti-peptide antibody titer ( > 1:lOOO) and affinity (K, > lo9 M). Seven monoclonal antibodies against N17K and 20 monoclonal antibodies against T18K were selected for

All monoclonal antibodies were tested in pairs for simultaneous binding to P24 tryptic fragments with one biotinylated antibody immobilized on avidin-coated tubes and the other one radiolabeled with I*?. Maximum binding was obtained with the anti-N17K monoclonal antibody 117 immobilized on coated tubes and the anti-T18K monoclonal antibody 44 used as tracer. Interest- ingly, 117 is one of the anti-N17K antibodies that does not bind to the tracers N17K-SMCC-GT and N17K-SMP-GT.

In no case was there simultaneous binding of two monoclonal antibodies raised against the same peptide.

The principle of the assay was to use trypsin cleavage to release the dipeptide containing N17K linked to Tl8K via a disulfide bridge, to bind the

30

1x10" 1 o-‘Q I@’ IO' IO-’ 1x10~

Concentration (M)

. P24 . N17K

. P24, trypsvvzed . N17K-GlyTyr

5 j ~_~~ _ ._. Y _~ ~..

1x10-” IV’Q 10-s 104 lo-’ 1x104

Concentration (M)

. P24 * Tlt3K

. P24, trypswed . TlWGlyTyr

Fig. 3. Characterization of sera from mice immunized with N17K (A) and T18K (B) by competitive binding inhibition experiments,

with “‘1 peptide tracers.


dipeptide to anti-N17K antibody-coated wells and to reveal the presence of the bound dipeptide with an anti-T18K tracer (Fig. 4).

3.4. Sample treatment

In order to simplify the assay and improve the stability of the enzyme, trypsin was distributed and lyophilized in tubes to provide the amount required for the treatment of 2.50 ~1 of serum.

By trypsinizing serum samples containing 12’1- labeled P24 in the presence or absence of anti-P24 antibodies and then analyzing the efficiency of proteolysis by SDS-PAGE and autoradiography, we were able to demonstrate that anti-P24 antibodies were protective against the proteolysis of P24 (Fig. 5). Using the same method, we also demonstrated that this protective effect could be abolished by prior acid treatment of one volume of sample with one-half volume of 1 M glycine- HCI, pH 2, buffer, followed by 30 min of incubation at 37°C and the addition of one-half volume of 1 M Tris-HCl, pH 8.8, to bring the pH back to neutrality (Fig. 5). The addition of glycine-HCI buffer without heating was not sufficient for the proteolysis of P24 in the presence of anti-P24 antibodies (not shown).

Furthermore, we observed that the activity of

T 18K

Anti N17K

I

Fig. 4. ELISA reaction scheme showing the anti-N17K monoclonal antibody immobilized on the solid phase and the anti-

T18K conjugated with acetylcholinesterase (Enz.).

ABCDEFCHI

kDa

46.0, ^

Fig. 5. Autoradiography of ‘2SI-labeled P24 diluted in normal

human serum (lanes A, B, and C), ‘251-labeled P24 diluted in

human serum with anti-P24 natural antibodies (lanes D, E,

and F), and 1’51-labeled human immunoglobulin G diluted in

normal human serum (lanes G, H, and I) after SDS polyacryl-

amide gel electrophoresis analysis under non-reducing condi-

tions (15% gel). Lanes A, D, and G: after treatment with

trypsin. Lanes B, E, and H: after acid treatment followed by

trypsinization. Lanes C, F. and I: without any treatment.

trypsin, diluted in serum and measured by the spectrophotometric method of Walsh and Wilcox (1970), was five-fold higher, if one volume of 0.5 M, pH 2, glycine-HCI buffer was first added to one volume of serum sample, as compared to the activity after the addition of one volume of pH 7.2 PBS (Fig. 6).

To take advantage of the effect of this acid treatment on both trypsin activity and antigen-antibody complex dissociation, we designed the fol- lowing protocol: 250 ~1 of serum sample were added to the tubes containing the lyophilized trypsin, then 125 ~1 of 1 M glycine-HCl pH 2 buffer were added to lower the pH, and 1.5% Triton Xl00 to inactivate the virus. The dissociation of antigen-antibody complexes was possibly achieved at this stage by the incubation of the samples for 30 min at 37°C. Finally, 125 ~1 of 1 M Tris-HCl pH 8.8 were added to return the pH to neutrality.

The incubation of the sample in anti-T18K coated wells was performed immediately after neutralization, without the addition of any inhibitor, since earlier experiments performed to assess the need for prolonged trypsinization and

F. Jean et al. /Journal of Immunological Methods 185 (1995) 103-114 111

09

08

E 07

2 NI

d 06

2 e

2 05 Q

04

03 L I I I r

3 20 40 60

Seconds

80 100

_ Dtluted wth PBS - - Diluted with 0 5 M gly-HCI pH 2

Fig. 6. Enzyme kinetics of trypsin in solution in human serum,

diluted with PBS or with 0.5 M glycine-HCl, pH 2. The

kinetics were measured with N-benzoyl-L-arginine ethyl ester

as substrate according to Walsh et al. (1970). The enzymatic

activity, corresponding to the reaction rate was: 7.35E-4 AU/s

with PBS and 3.89E-3 AU/s with glycine-HCI.

termination by the trypsin inhibitor prior to the addition of the reaction mixture to the antibody- coated wells, had shown that such treatment gave identical results (data not shown).

3.5. Quality of the assay

In order to optimize the assay, several parame- ters were evaluated including temperature, number of steps, volume and length of incubations, composition of the tracer buffer, and the number of washings. The best results were obtained using a two-step protocol, as described above, since simultaneous incubation of the serum samples and the tracer led to an increase of the non- specific and a decrease of the specific binding. A typical standard curve is shown in Fig. 7.

The sensitivity of the assay was evaluated by assaying the zero standard 12 times and the 8.8 pg/ml standard 12 times. Sensitivity was defined as the concentration corresponding to the average optical density obtained for the zero standard plus three standard deviations (Fig. 8); it was estimated to be 2.5 pg/ml.

In order to assess the specificity of the test, the

0 100 200 300 400

P24 concentration (pg/ml)

Fig. 7. ELISA standard curve.

culture supernatants of three different HIV strains, HIV-l BRU, HIV-l NDK, and HIV-2 ROD were assayed. The level of virus replication in these cultures was previously estimated by the reverse transcriptase assay. All three culture supernatants were diluted before testing to a concentration corresponding to 100 cpm/ml in the reverse transcriptase assay. The P24 (P26 for HIV-21 concentrations measured for these dilu-

0 035

E 0 030

0” P

2 0025 m

2

5 0 020

0015

1

0010 -i 2 4 6 8 10

P24 concentration (pg/ml)

Fig. 8. Sensitivity of the assay. 12 measurements were per-

formed for the zero and the 8.8 pg/ml standards. The value

for the average absorbance for the zero standard plus three

standard deviations is shown on the curve (dashed line) and

corresponds to 2.5 pg/ml.

112 F. Jean et al./Journal of Immunological Methods 185 (1995) 103-114

tions were 76 pg/ml for HIV-l BRU, 71 pg/ml for HIV-l NDK and 36 pg/ml for HIV-2 ROD respectively. These results indicate cross-reactivity between HIV-l BRU and NDK of near 100% and of approximately 50% with HIV-2 ROD, assuming a constant relationship between P24 concentration and reverse transcriptase activity.

4. Discussion

The aim of this study was to develop a sensitive immunoassay with a broad specificity for the P24 HIV antigen. Our approach was based on the detection of a peptide fragment, generated by tryptic cleavage of the P24 antigen, using antibodies raised against carefully selected synthetic peptides. With this method, it was possible to take advantage of synthetic peptides as immunogens, without the loss of affinity often observed when anti-peptide antibodies are employed to bind to the whole native protein. In the present case, this loss of affinity was illustrated again in that most antibodies raised against N17K and T18K peptides did not bind to the native P24 antigen with high affinity, as was observed when the sera of immunized mice were characterized. By contrast, anti-T18K and anti-N17K antibody affinity for the tryptic P24 peptides was close to the affinity of the T18K and N17K synthetic peptides, respectively. The fact that some monoclonal antibodies did not recognize the peptide tracer, but bound to the peptide-coated wells, probably reflects the fact that the peptides derivatized with GT-SMCC or GT-SMP have a structure different from that of the immobilized peptide and, possibly, different from that of the immunogen. Fur- thermore, since the antibody 117, coated on the solid phase in the assay, binds only to the immobilized N17K, we may hypothesize that the structure of the tryptic fragment is closer to the immobilized synthetic peptide than to the peptide tracer.

The native P24 binds to anti-peptide antibodies with very poor affinity, but to the trypsinized P24 with an affinity close to that of the synthetic peptides. We may conclude, therefore, that P24 cleavage by trypsin is sufficiently complete to

liberate most of the peptide fragments from the native protein. The efficiency of the trypsinization in the serum was also evaluated in immunoassays by comparing the results obtained with P24 first trypsinized in buffer and then diluted in serum, with results obtained with P24 diluted in serum and then trypsinized. In both cases we obtained the same standard curves and we conclude that the cleavage yield was very good and did not depend on the P24 concentration in the serum.

Because N17K and T18K were known to be linked by a disulfide bond, the possibility of de- signing a ‘sandwich’ assay, by using two antibodies against these two peptides was taken into account from the beginning of the project. This possibility is obviously another advantage of the method, permitting us to raise independently the antibodies to be used in the immunometric assay. The existence of this disulfide bond, even after trypsinization, was demonstrated by the preparation of such a ‘sandwich’ assay with anti-T18K and anti-N17K antibodies after proteolytic cleavage.

We expected that interference by natural anti P24 antibodies would be avoided by trypsinization of the samples. However, the presence of such antibodies protected the bound P24 from proteolysis. Moreover, we demonstrated that trypsin activity in the serum samples increased five fold after acid treatment, probably because of some inhibitor inactivation. We then decided to ameliorate proteolysis by systematically lower- ing the pH of the sample. The dissociation of the antigen-antibody complexes can be brought about as in classical P24 immunoassays, merely by incubating the sample at acid pH for 30 min at 37°C. We have, however, taken advantage of the resis- tance of antibodies to trypsinization by trypsinizing the sample immediately after its neutralization in the anti-N17K-coated tubes without af- fecting the immunoreactivity of the 117 antibody.

The high level of sensitivity achieved with this assay, demonstrates again the strong affinity of the antibodies for the dipeptides generated by trypsin cleavage of P24: the structure of the dipeptides is closer to the synthetic peptides used as immunogens than it is to the native protein.

F. Jean et al./Journal of Immunological Methods 185 (1995) 103-114 113

Furthermore, by careful selection of the peptides to be synthesized, we were able also to develop an immunoassay that detects P24 from HIV-l NDK and P26 from HIV-2 ROD despite differences of 4.3% and 31.6% respectively from the prototype HIV-l BRU P24 amino acid sequence. Since N17K and T18K amino acid sequences are identical in 13 HIV-l strains and since P26 from HIV-2 ROD is also recognized by our antibodies (even with one amino acid substitution in N17K and two substitutions in T18K) most HIV-l and HIV-2 strains would probably be detected by this immunoassay.

Further investigations to evaluate the specificity of this assay for other HIV strains, and to assess its clinical usefulness are in progress.

Using trypsin or other endoproteases, the same methodology may be applied to other protein immunoassays where antibodies with high affinity against particular needed.

Acknowledgements

amino acid sequences are

C. Daniel is gratefully acknowledged for excel- lent technical assistance, H. Rickenberg for careful reading of the manuscript and comments, and H. Cailla for very helpful discussions.

References

Allain, J.-P., Laurian, Y., Paul, D.A., Senn, D. and Members of the AIDS-Haemophilia French Study Group (1986) Serological markers in early stages of human immunodeficiency virus infection in haemophiliacs. Lancet ii, 1233.

Allain, J.-P., Laurian, Y., Paul, D.A., Verroust, F., Leuther, M., Gazengel, C., Senn, D., Larrieu, M.J. and Bosser C. (1987) Long term evaluation of HIV antigen and antibodies to P24 and GP41 in patients with hemophilia. Potential clinical importance. New Engl. J. Med. 317, 1114.

Altman, A., Cardenas, J.M., Houghten, R.A., Dixon, F.J. and Theofilopoulos, A.N. (1984) Antibodies of predetermined specificity against chemically synthesized peptides of human interleukin 2. Proc. Natl. Acad. Sci. USA 81, 2176.

Barre-Sinoussi, F., Chermann, J.-C., Rey, F., Nugeyre, M.T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Brun-Vezinet, 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, 868.

Burt, D.S., Hastings, G.Z. and Stanworth, D.R. (1986) Use of synthetic peptides in the production and characterization of antibodies directed against predermined specificities in rat immunoglobulin E. Mol. Immunol. 23, 181.

Caruso, A., Terlenghi, L., Ceccarelli, R., Verardi, R., Foresti, I., Scura, G., Manta, N., Bonfanti, C. and Turano, A. (1987) Liquid competition radioimmunoassay for the detection and quantitation of the HIV ~24. J. Virol. Methods 17, 199.

Chaisson, R.E., Allain, J.-P., Leuther, M. and Volberding, P.A. (1986) Significant changes in HIV antigen level in the serum of patients treated with azidothymidine. New Engl. J. Med. 315, 1610.

Dessen, P., Fondrat, C., Valencien, C. and Mugnier, C. (19901 BISANCE: a french service for access to biomolecular sequence databases. CABIOS 6, 355.

Devare, S.G.. Srinivasan, A., Bohan, C.A., Spira, T.J.. Curran, J.W. and Kaiyanaraman, V.S. (1986) Genomic diversity of the acquired immunodeficiency syndrome retroviruses is reflected in alteration of its translational products. Proc. Natl. Acad. Sci. USA 83, 5718.

Ellman, G.L., Courtney, K.D., Andres, V. and Featherstone, R.M. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88.

Fieser, T.M., Tainer, J.A., Geysen, H.M., Houghten, R.A. and Lerner. R.A. (19871 Influence of protein flexibility and peptide conformation on reactivity of monoclonal anti- peptide antibodies with a protein alpha-helix. Proc. Natl. Acad. Sci. USA 84, 8568.

Fisher, A.G., Ensoli, B., Looney, D., Rose, A., Gallo, R.C., Saag, M.S., Shaw, G.M., Hahn, B.H. and Wong-Staal, F. (1988) Biologically d. iverse molecular variants within a single HIV-l isolate. Nature 334, 444.

Furlini, G., Re, M.C., Ferranti, G., Dalla Casa, P. and La Placa, M. (1989) Serum antibody pattern, antigenemia. and virus isolation in infants born to mothers seropositive for human immunodeficiency virus type 1. J. Med. Virol. 28, 129.

Gallo, R.C., Sarin. P.S., Gelman, E.P., Robert-Gurof, M., Richardson, E., Kalyanamaran, V.S., Mann, D., Sidhu, G.D., Stahl, R.E., Zolla-Pazner, S., Lcibowitch, J. and Popovic, M. (1983) Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science 220, 865.

Goudsmit, J., De Wolf, F., Paul, D.A., Epstein, L.G.. Lange, J.M.A., Krone, W.J.A., Speelman, H., Wolters, E.C., Van der Noordaa, J., Oieske, J.M., Van der Helm, H.J. and Coutinho, R.A. (1986) Expression of human immunodeficiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet ii, 177.

Grassi, J., Frobert, Y., Pradelles, P., Chercuitte, F., Gruaz, D., Dayer, J.-M. and Poubelle, P.E. (1989) Production of monoclonal antibodies against interleukin-1 alpha and 1


beta. Development of two enzyme immunometric assays (EIA) using acetylcholinesterase and their application to biological media. J. Immunol. Methods 123, 193.

Greenwood, F. and Hunter, W. (1963) The preparation of 1311-labeled human growth hormone of high specific radioactivity. Biochem. J. 89, 114.

Lange, J.M., Paul, D.A., Huisman, H.G., de Wolf, F., Van den Berg, H., Coutinho, R.A., Danner, S.A., Van der Nordaa, J. and Goudsmit, J. (1986) Persistent HIV antige- naemia and decline of HIV core antibodies associated with transition to AIDS. Br. Med. J. 293, 14.59.

Mathiesen, T., Sundqvist, V.A., Albert, J., Ohlson, E. and Wahren, B. (1988) Acid hydrolysis of serum samples to increase detection of HIV antigen. J. Virol. Methods 22, 143.

Muesing, M.A., Smith, D.H., Cabradilla, C.D., Benton, C.V., Lasky, L.A. and Capon, D. (1985) Nucleic acid structure and expression of the human AIDS/lymphadenopathy retrovirus. Nature 313, 450.

Paul, D.A., Falk, L.A., Kessler. H.A., Chase, R.M., Blaauw, B., Chudwin, D.S. and Landay, A.L. (1987) Correlation of serum HIV antigen and antibody with clinical status in HIV-infected patients. J. Med. Virol. 22, 357.

Peng, C., Ho, B.K., Chang, T.W. and Chang N.T. (1989) Role of human immunodeficiency virus type l-specific protease in core protein maturation and viral infectivity. J. Virol. 63, 2550.

Preston, B.D., Poiesz, B.J. and Loeb, L.A. (1988) Fidelity of HIV-l reverse transcriptase. Science 242, 1168.

Ranki, A., Krohn, M., Allain, J.-P., Franchini, G., Valle, S.-L., Antonen, J., Leuther, M. and Krohn, K. (1987) Long latency precedes overt seroconversion in sexually transmit- ted human-immunodeficiency-virus infection. Lancet ii, 589

Ratner, L., Haseltine, W., Patarca, R., Livak, K., Starcich, B., Josephs, S.F., Doran, E.R., Rafalski, J.A., Whitehorn,

E.A., Baumeister, K., Ivanoff, L., Petteway, S.R., Pearson, M.L., Lautenberger, J.A., Papas, T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C. and Wong-Staal, F. (1985) Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 313, 277.

Roberts, J.D., Bebenek, K. and Kunkel, T.A. (1988) The accuracy of reverse transcriptase from HIV-l. Science 242, 1171.

Saag, MS., Hahn, B., Gibbons, J., Li, Y., Parks, ES. Parks, W.P. and Shaw, G. (1988) Extensive variations of human immunodeficiency virus type-l in vivo. Nature 334, 440.

Sanchez-Pescador, R., Power, M.D., Barr, P.J., Steimer, KS., Stempien, M.M., Brown-Shimer, S.L., Gee, W.W., Re- nard, A., Randolph, A., Levy, J.A., Dina, D. and Luciw, P. (1985) Nucleotide sequence and expression of an AIDS- associated retrovirus (ARV-2). Science 227, 484.

Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51, 660.

Tatsumi, M., Jean, F., Robert, V., Chermann, J.-C. and De- vaux, C. (1990) Use of monoclonal antibodies for the detection and quantitation of HIV1 core protein ~25: comparative evaluation of in vitro HIV1 infection by im- munofluorescence, antigen capture ELISA and reverse transcriptase assays. Res. Virol. 141, 649.

Walsh, K.A. and Wilcox, P.E. (1970) Serine proteases. In: G.E. Perlmann and L. Lorand (Eds.), Methods in Enzy- mology, Vol. XIX. Academic Press, New York, P. 31.

Zagury, J.F., Franchini, G., Reitz, M., Collalti, E., Starcich, B., Hall, L., Fargnoli, K., Jacodzinski, L., Guo, H.-G., Laure, F., Arya, SK., Josephs, S., Zagury, D., Wang-Staal, F. and Gallo, R.C. (1988) Genetic variability between isolates of human immunodeficiency virus (HIV) type 2 is comparable to the variability among HIV type 1. Proc. Nat]. Acad. Sci. USA 85, 5941.

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