Detection of feline immunodeficiency virus p24 antigen and p24-specific antibodies by monoclonal antibody-based assays

Journal of lirolo@~al ITethotl5

ELSEVIER

Journal of Virological Methods 46 (1994) 2X7-301

Detection of feline immunodeficiency virus p24 antigen and p24-specific antibodies by monoclonal antibody-based assays Stefania Abramob,

Lombard?*, Alessandro Polib, Claudia Massi”, Francesca Lucia Zaccaro”, Agostino Bazzichi,’ Gino Malvaldi” Mauro Bendinelli”, Carlo Garzelli”

“Department

of Biomedicine.

“Departmenr

of Animal

Section

qf Virology,

P&ho&y

Viu San Zeno, 37, 56127 Piss, Italy.

Univrrsify

of Pisrr, 56127 Pisrr, Ital)

(Accepted 21 June 1993)

Abstract

A panel of monoclonal antibodies (mAbs) detecting distinct B-cell epitopes on p24 core viral protein of feline immunodeficiency virus (FIV) were employed to develop immunoassays to measure p24 concentration in culture and serum samples, to localize p24 in FIV-infected cells and tissues, and to detect anti-p24 antibodies in cat sera. In its optimized configuration the p24 capture assay detected as little as 0.25 ng/ml of protein. The assay was found at least as sensitive as the reverse transcriptase activity assay in FIV-infected lymphocyte cultures and proved capable of detecting p24 antigen in acid pretreated sera from a high proportion of FIVinfected cats. The mAbs were also successfully used to detect the p24 antigen in permeated FIV-infected cells by flow cytometry and in tissue sections from FIV-infected cats by immunohistochemical staining. Anti-p24 antibodies in FIV-infected cat sera were assayed by a competitive capture ELISA which readily identified occasional false positive results provided by a standard ELISA using purified whole FIV-coated wells. Key

~rvrds:

FIV; Feline AIDS; Monoclonal

* Corresponding author. 0166.0934/94/$07.00 SSDI

Fax: + 39 50 555477

47 1994

0166-0934(93)E0084-F

antibody

Elsevier Science B.V. All rights reserved

1. Introduction Feline immunodeficiency virus (FIV) was first isolated by Pedersen and colleagues (1987) and has since been shown to share numerous biological and pathoiogical features with the human immutlode~ciency virus (HIV). It is therefore considered a valuable animal model for studies on anti-lentiviral vaccination strategy and drug evaluation, as well as for understanding immunodeficiency virus pathogenesis (Pedersen, in press; Schellekens & Horzinek, 1990). Genetic analysis of FIV has shown that the structural genes gng, pal and cnv are similar to those of other lentiviruses (Morikawa et al., 1991; Oimsted et al., 1989; Phillips et al., 1990; Talbot et al., 1989) and several proteins encoded by these genes have been identified. The gug gene encodes a polypeptide precursor with an apparent M, of 50 Kd. when this protein is cleaved by the protease coded by the pol gene, the major core protein p24 and at least two additional proteins, pl5 and ~10. are produced (Egberink et al., 1990: Steinman et al., 1990). For diagnostic purposes, in lentivirus infections p24 detection seems particuIarly important: for example, in HIV-infected patients the presence of persistent antigenemia is considered a sensitive prognostic marker of disease progression towards AIDS (De Wolf et al.. 1987; Goudsmith et al., 1986; Paul et al., 1987; Pedersen et al., 1987b). The development of sensitive and specific immunodiagnostic tests for detection of antibodies to FIV (O’Connor et al., 1989) or to pl7 and p24 FIV proteins (Reid et al.? 1991) has been reported. Recently, monoclonal antibodies (InAbs) to FIV p24 gqg gene product have been produced that detect four distinct B cell epitopes, all of which immunogenic also in the natural host species (Lombardi et al., 1993). In this report, we describe the use of such anti-p24 mAbs for developing sensitive immunoassays to measure the p24 viral antigen in culture and serum samples, to localize the viral core antigen in cells and tissue sections and to detect anti-p24 antibodies.

2. Materials

and methods

2. I. Vii-us and cell cultures The FIV Petaluma strain was propagated in persistently infected Crandell feline kidney (CJFK) cells or FL4 cells (kind gift of DJ. J. Yamamoto), as previously described (Yamamoto et al., 1988; Yamamoto et al., 1991). Virus was concentrated from tissue culture supernatants by ultrafiltration (Minitan, Millipore, USA) and purified by density gradient centrifugation (Montetaro et al., 1982). Purified FIV was disrupted in phosphate buffered saline (PBS) containing 0.25% sodium dodecyl sulfate (SDS) at 4’C for 2 h with gentle stirring. 2.2. Anti-FIV

monoclonal

untihodies

The preparation and characterization of the panel of anti-FIV mAbs used has been reported in detail elsewhere (Lombardi et al., 1993). The mAbs, namely

S. Lombard

ct al.lJournal

of Virological Methods 46 ilY94)

287-~301

289

AEll, CD8, AAIO, DFlO, DA2, DF3, all of IgGl isotype, specifically bind antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence (IFA) and react with FIV p24 gag gene product in immunoblots. The mAbs were secreted into ascites in BALB/c mice and purified by binding to protein-G columns (Pharmacia, Sweden). 2.3. Biotinylution

of’mAbs

Purified mAbs were dialyzed overnight against biotinylation was performed by incubating 60 ~1 ester at 1 mg/ml in dimethyl-sulphoxide with 1 ml ml in NaHCOs at room temperature (r.t.) for 2 extensively dialyzed against PBS. They maintained

0.1 M NaHCOs, pH 8.4, at 4°C; of biotin-N-hydroxyl-succinimide of purified mAb diluted at I mg/ h. Biotinylated mAbs were then binding activity to FIV antigens.

2.4. Cut sera A panel of 57 cat sera was used. The panel included 14 sera from uninfected specific pathogen free (SPF) cats, vaccinated against feline panleukopenia virus, herpesvirus, calicivirus and rabies virus (Leucoferin and Quadricat; Rhone-Merieux; IFFA CREDO, Como, Italy), 19 sera from FIV-seropositive field cats, and 24 sera from SPF cats experimentally infected with the FIV Pisa-M2 isolate. This isolate was originally obtained from a clinically healthy, feline leukemia virus-negative, cat and has since been freed of the initially present feline syncytium forming virus by two consecutive passages of cell-free plasma in cats. The preparation used was propagated in vivo by monthly passages in SPF cats (Matteucci et al., 1993). 2.5. Cupture

ELISA

fbr p24 antigen

Plates (Probind, Falcon, Italy) were coated overnight with 0.5 ,Llgpurified mAb in 100 ~1 carbonate buffer pH 9.6. After 4 washes with PBS containing 0.05% Tween 20 (PBS-Tw), plates were post-coated with 150 ~1 of PBS containing 1% bovine serum albumin (BSA) (PBS-BSA) for 1 h. Test samples (100 ~1) containing 0.5% Triton X-100 were added to the wells and incubated for 2 h. After 4 washes, 0.1 pg biotin-conjugated mAb in 100 ~11of PBS containing 1% skim milk, 5% fetal calf serum and 0.05% Tween 20 (dilution buffer) were added and incubated 1 h. The plates were then washed and further incubated 1 h with 100 ~1 of a horseradish peroxidase (HPRO)-conjugated anti-biotin (Sigma, St. Louis, MO, USA) diluted 1:lOOO in PBS-Tw-BSA. The enzyme reaction was carried out with 100 ~1 tetramethylbenzidine (KPL, Gaithersburg, MD) and stopped with 100 ~1 0.1 N H$O,; the absorbance was measured at 450 nm. All steps were performed at r.t.. Duplicate wells containing two-fold dilutions of recombinant p24 (kindly provided by Dr. 0. Jarrett‘) ranging from 100 ng/ml to 0.03 ng/ml served for standard curve.

2.6. Detection

qf’p24 antigen in acid pretreated

cat sera

Unless otherwise speciIied, immediately prior to ~24 antigen ELISA determination sera were submitted to acid treatment, as previously described by Ascher et al. (1992), to disrupt immune complexes. Sera were thawed at the time of the assay, and 100 ~1 of the specimen w’ds mixed with 50 ~1 of 1.5 M glycine (PI-I 1.85) vortexed, incubated 1 h at 37”C, and neutralized with 50 ~1 of 1.5 M Tris (pH 9.0). Detection of p24 antigen was performed according to the capture ELISA described above with minor modifications. In brief, 200 161of samples were added in duplicate to the wells coated with 200 ~1 of AEl I mAb and incubated overnight at r.t.. Biotinylated DFIO mAb, HPRG-conjugated anti-biotin, and substrate were added in volumes of 200 pl/ well. Samples giving optical readings 2 or more times greater than the mean of 14 sera from FIV-negative SPF cats were considered as positives.

Flow cytometry analysis of mAb binding to FIV persistently infected FL4 cells was performed by using an Epics Elite cell analyzer (Coulter Electronics, Hialeah, Fla.). To permeate cell membranes, cells (5 x 105) were incubated for 10 min at 4°C with 2 ml of PBS containing 1% p-formaldehyde. After 2 washes with PBS containing 0.5% BSA, cells were incubated with 1.5 ml of absolute methanol at -20°C for 10 min. After washing, cells were incubated at 4°C with an appropriate dilution of mAbs in PBS containing 5% fetal bovine serum and 0.1% sodium azide. After 1 h, cells were washed and a fluorescein-conjugated goat anti-cat IgG antibody (Sigma) was added for 30 min; finally, after further washings, cells were fixed in PBS containing 1% p-formaldehyde, 2% glucose, and 0.1% sodium azide. Standard alignment using fluorescent microspheres was done prior to data acquisition. A 525 nm bandpass filter was used for detection of green fluorescence. The data were first collected in a two parameter histogram of size versus granularity. After electronically removing from analysis any debris by selective gating, the data were transferred to a single parameter log fluorescence histogram. Data from approximately 10 000 cells were collected for each experimental condition.

For immunoperoxidase staining, formalin-fixed, paraffin-embedded FIV-infected FL4 cells or 4 pm thick tissue sections, were air dried overnight at 37°C dewaxed in xylene, rehydrated and stained by hematoxylin for 20 min. After 3 washes with 0.05 M Tris-HCl pH 7.6 containing 0.15 M NaCl (Tris buffer), sections were incubated 30 min with Tris buffer containing 5% normal goat serum, blotted dry, and then incubated overnight at 37°C with different dilutions of anti-p24 mAbs in Tris buffer. Endogenous peroxidase activity was eliminated by a IO-min wash in methanol containing 3% hydrogen peroxide. The sections were then sequentially incubated at 37°C for 30 min with biotinylated affinity purified goat anti-mouse IgG (Vector Lab, Burlingame. CA, USA) diluted 1:200 in Tris buffer and for 1 h with peroxi-

S. Lombardi rt al.!Journal of Virological Methods 46 (lY94)

287 ~301

291

dase-labelled streptavidin (Biogenex Lab, San Ramon, CA, USA) diluted 1:lOO in Tris buffer. Between each incubation step, sections were washed 3 times for 5 min with Tris buffer. A 0.06% 3,3_diaminobenzidine tetrachloride and 0.2% hydrogen peroxide solution in Tris buffer or 3-amino, 9-ethyl carbazole (Biomeda, Fostercity, CA) were used as chromogens. Formalin-fixed, paraffin-embedded FIV-infected FL4 cells and tissues from SPF FIV-negative cats served as positive and negative controls, respectively. A non-related mAb was included in each assay as further negative control. For immunofluorescence (IFA), acetone-fixed FL4 cells were tested by the antip24 mAbs as previously described (Lombardi et al., 1993). Peripheral blood lymphocytes (PBL) from uninfected SPF cats were used as negative controls. 2.9. Competitiw

capture ELISA

,fbr unti-p24

cut antibodies

A competitive capture immunoassay was developed to measure anti-p24 antibodies in cat sera. For this purpose, ELISA plates were coated with AEI 1 mAb and postcoated as described above. After 4 washes with PBS-Tween, 100 ~1 of disrupted FIV (50 ng/ml) were added to the wells; after 2 h, plates were washed and 50 ~1 of biotinylated-DFlO mAb (2 pg/ml) together with 50 ~1 of serially diluted cat sera were added. After 2 h, plates were washed 4 times with PBS-Tween and 100 /II of goat HPRO-labeled anti-biotin serum (Sigma) diluted 1: 1000 in PBS-BSA-Tween were added to each well for I h. The plates were then developed as described above. Ml steps were performed at r.t.. Test samples were considered positive when a reduction of at least 50% of the absorbance binding value of labelled DFlO mAb was observed as compared to the binding of labelled DFlO in presence of sera from FIV-negative SPF cats. 2.10. Stun&vu

ELISA,fbr

anti-FIV

cut untihodies

ELISA plates were coated overnight with 0.5 /lg/well SDS-disrupted whole FIV in carbonate buffer (pH 9.6). The plates were subsequently post-coated with PBSBSA for 1 h. Cat sera, serially diluted in dilution buffer, were added to the plates and incubated 1 h. Bound IgG antibodies were revealed by a HRPO-conjugated mouse mAb anti-cat IgG (Sigma) diluted in PBS-BSA-Tween. All steps were performed at r.t. The enzyme reaction was carried as described above. Post-coating was done with 150 pi/well; virus, samples, conjugate, substrate, and H2S04 were added in volumes of 100 pi/well.

3. Results 3.1. Development

of capture ELISA

.for p24 untigen

To optimize the configuration of the capture assay, each purified anti-p24 mAb was used to prepare both antibody-coated plates and biotin conjugates. The results

Table 1 Optimization

of capture

mAb used to coat wells

Riotin-conjugated AEI

AEll CD8 DFIO AA10 DA2 DF3 “Data

ELISA conliguration

1

0.150” 0.190 1.679 1.452 O.&O 0.210 are expressed

as optical

for FIV p24

MAb CD8

DFIO

AA10

DA2

DF3

0.080 0.075 1.079 1.141 0.060 0.176

1.975 1.197 0.200 0.395 0.150 0.351

1.615 0.x93 0.567 0.603 0.453 0.x97

0.140 0.195 0.30s 0.513 0.137 0.349

0.150 0.142 0.443 0.397 0.322 0.079

density

at 450 nm

5 2.6

4

z-s

t f 3

5

.-c

24 2i Q 1

c c 0.5

0

Uninfected

FIV-infected

Fig. I. Capture ELISA for FIV ~24. Detection of FIV $4 in sera from SPF FIV-negative (A). field FIVpositive (0) and cxperimcntally FIV-Infected (m) cats. The shadowed arca includes values lower than the cutoff limit set at 0.5 n&ml ~24, which corresponds to 2 times the mean of optical density (O.D.) of 14 SPF cat sera. The insert shows the standard curve as determined using recombinant ~24. The horizontal line represents the binding value of hiotinylated DFIO mAb in the absence of ~24.

S. Lombardi et al./Journal of Virological Methods 46 (1994) 287-301

293

of all the possible combinations among the 6 anti-p24 mAbs, obtained with a concentration of 50 ng/ml of FIV, are given in Table 1. The highest sensitivity was achieved by using the AEll mAb to coat the wells and the DFlO mAb for conjugate preparation. In other experiments, serial dilutions of recombinant p24 were prepared and tested in the optimized FIV-capture ELBA to determine the lower sensitivity limit of the assay. As shown in Figure 1 (insert), the assay can detect 0.25 ng/ml of ~24. When used to test the supernatants of FIV-infected lymphocyte cultures, the p24 capture ELISA was found to be at least as sensitive as well as less cumbersome than the reverse transcriptase (RT) activity assay (data not shown). 3.2. Detection of p24 antigen in sera from FIV-in&ted

cuts

To examine whether the p24 assay could be used for detection of the antigen in the circulation of FIV-infected cats, sera from 16 FIV-seropositive and 14 uninfected SPF cats were tested as described in Materials and Methods by capture ELISA with or without acid pretreatment. As shown by Table 2, p24 was detected in 2/16 (12.5%) untreated sera whereas the acidification procedure yielded 8/16 (50%) positive results. Fig. 1 summarizes the results obtained by testing a large number of acidified sera. p24 antigen was shown in 8 out of 19 (42%) FIV-positive field cats and in 12 out of 24 (50%) experimentally infected cats at concentrations ranging between 0.6 and 5.0 ng/ml. All the 14 uninfected cats examined were negative. 3.3. Flow cytometry The six anti-p24 Table 2 Detection

mAbs

were investigated

of FIV p24 in cat sera with and without

Cat no.

1 2 3 4 5 6 I 8 9-16 Positive/total

for reactivity

to FIV-infected

cells by

prior acid treatment

p24 in serum (ng/ml) pre-acidification

post-acidification

0.6 4 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5 <0.5

I .4 5 0.8 1.8 2.5 0.9 1.2 0.8 <:0.5

2116

8/16

Sera from FIV-positive cats were tested by capture ELISA for p24 detection with and without prior acid treatment. Sera giving optical readings 2 or more times greater than the mean of sera from uninfected cats, which corresponds to a cut-off value of 0.5 ng/ml ~24, were considered positive.

R

AA10

DA2 n-4

Fluorescence

Intensity

Binding activity of anti-p24 mAbs to FIV-infected cells as assessed by flow cytometry. Permeated FL4 cells persistently infected with Petaluma strain were incubated with the indicated anti-p24 mAbs (----- ) or unrelated mAb (. - - -).

flow cytometry using persistently infected FL4 cells. As shown in Fig. 2, all the mAbs stained a high proportion of permeated FL4 cells, but &ailed to stain unpermeated FIV-infected cells (data not shown). Stained FL4 cells ranged between 57% (mAb AEl 1) and 86% (mAb DF3). An unrelated mAb failed to stain the cells. Thus, these results clearly indicate that the mAbs react with gag products in infected cells and that the four epitopes recognized by such mAbs are expressed in the cytoplasm but not on the surface of cells.

Fig. 3. Detection of p24 antigen by AEI I mAb based-immunoperoxidase in formalin fixed and paraffin embedded, persistently FIV-infected FL4 cells and in tissue sections from FIV-infected cats. A Intracytoplasmic staining of FIV-p24 in FL4 cells (500X); B lack of immunoreactivity in FL4 cells, when a nonrelated mAb was used (500X); C large macrophage (arrow), immunoreactive for FIV-p24 protein, in the follicle mantel of a mesenteric lymph node (500X); D p24 positive staining in the cytoplasm of epithelial cells of renal tubules in the kidney (500X).

S. Lombardi et al./Journal of Virological Methods 46 (1994) 287-301

295

296

S. Lombardi

et al./Journal

qf Virological Method.7 46 (1994) 287-301

3.4. Immunohistochemistry The panel of anti-p24 mAbs was first analyzed for reactivity with acetone-fixed or formalin-fixed paraffin-embedded FIV-infected FL4 by IFA and immunoperoxidase. As already reported, by IFA no differences were observed in the staining pattern exhibited by the 6 mAbs; in particular, a diffuse granular cytoplasmatic fluorescence was observed in approximately 60% of FIV-infected FL4 cells (Lombardi et al., 1993). By immunoperoxidase on paraffin-embedded FL4 cells, the best results were obtained with the AEll mAb, which stained approximately 25% of cells (Fig. 3A). A non-related mAb, used as negative control, gave no staining (Fig. 3B). None of the anti-p24 mAbs reacted with PBL from uninfected cats by either IFA or immunoperoxidase (data not shown). On tissue sections from FIV-infected cats, only the AEl 1 mAb was studied. This

1.2

1

0.8

0.6

0.4

0.2

0 10

100

Reciprocal Fig. 4. Competition capture ELBA for anti-p24 (A) and 5 FIV-infected (W) cats were allowed DFlO mAb for binding to FIV p24 bound to positive where they reduced by at least 50% the sera. O.D., optical density.

1,000

10,000

of serum dilution

antibody in to compete AEll mAb binding of

cat sera. Dilutions of sera from 3 uninfected with a constant amount of biotin-labelled coated wells. Samples were considered as DFIO mAb in the presence of FIV negative

S. Lombardi

et ul.lJournal

of Vimlogical

Mrthvds

46 11994) 287--301

291

mAb detected the p24 antigen in the cytoplasm of rare small lymphocytes and macrophages of lymph nodes (Fig. 3C). The antibody also stained clusters of tubular epithelial cells (Fig. 3D) and rare interstitial inflammatory cells and glomerular cells in the kidney, as well as scattered mononuclear cells in the bone marrow (not shown). The AEll mAb showed no reactivity when tested on tissue preparations from uninfected SPF cats. 3.5. Anti-p24 antibodies in cat sera The competition binding ELISA described in Materials and Methods was used to detect anti-p24 antibodies in cat sera. The representative results presented in Fig. 4 show that the binding of biotinylated DFlO mAb was inhibited by sera from FIV-

1.4

1.2

1

5: z d 6

0.8

0.6 I \

0.4

0.2

0

I

50

100

200

400

800

Reciprocal of serum dilution Fig. 5. Anti-FiV antibody assay run on 8 uninfected cat sera by either standard ELISA for anti-FIV antibody (A) or competitive capture ELISA for anti-p24 antibody (D). Dotted and dashed lines rcpresent two sera giving false positive results when tested by standard anti-whole FIV assay. O.D., optical density.

infected cats in a dose-dependent manner, but not by sera from SPF cats. Anti-p24 serum titers in FIV-positive cats ranged between 120 and 1:640. The competition capture ELISA was also used to examine occasional sera from uninfected cats that gave false positive results when tested for anti-FIV antibodies by standard ELISA with whole virus. As shown in Fig. 5, the competitive capture ELISA clearly discriminated false-positive from truly positive sera.

4. Discussion We have recently described a panel of mAbs that detect four distinct B epitopes of FIV p24 core antigen (Lombardi et al., 1993). In the present study these mAbs were used to develop a quantitative capture ELISA for detecting p24 antigen in cell cultures and serum samples. The optimized configuration of this assay consisted in the use of the AEl 1 mAb to coat the wells and the DFlO mAb for conjugate preparation. These mAbs have been shown to detect two distinct epitopes of p24 (Lombardi et al., 1993). The assay has a lower sensitivity limit of approximately 0.25 ng/ml of p24 and the assay is now routinely employed in our laboratory to monitor FIV growth in cell cultures. Assays for FIV-antigen with similar sensitivity have been developed by Siebelink et al. (1990) and by Tilton et al. (1990). and employed to detect FIV growth in cultured feline lymphocytes. Our assay, however, has been also validated as a sensitive ‘tool’ to measure antigenemia in infected animals: in fact, 20 out of 43 sera from FIV-infected cats proved to be p24-positive. Similar to what observed for HIV (Ascher et al, 1992; Nishanian et al., 1990) an acidification step, aimed to dissociate antigen-antibody complexes, highly enhanced the recovery of FIV p24 antigen. When 16 FIV antibody-positive sera were tested in capture ELISA with or without prior acid pretreatment 8 (50%) and 2 (12.5%) were found to be positive, respectively. The possibility of quantitating p24 in serum seems particularly important, as in HIV infection the appearance of persistent HIV antigenemia has been associated with progression to AIDS and has acquired the status of routine prognostic marker (De Wolf et al., 1987; Goudsmith et al., 1986; Paul et al., 1987). A reliable measurement of FIV antigen in serum antigen might prove useful for monitoring infection and disease development, and for evaluating prognosis and antiviral drug therapy also in FIV-infected cats. Studies are now in progress to study the prevalence of FIV antigen in serum and saliva at different stages of infection; in cats saliva is much simpler to collect than blood and therefore saliva may be exploited for FIV diagnostic and epidemiological purposes (Poli et al., 1992). The available data wouid indicate that FIV antigen test is at least as sensitive as FIV isolation (l~atteucci et al., 1993). The anti-p24 mAbs were also employed to detect the viral antigen in FIV-infected cells by flow cytometry. It was found that only permeated FIV-infected FL4 cells are stained by the mAbs, thus indicating that the corresponding epitopes are present in the cytoplasm but not on the surface of infected cells. These results are apparently in contrast with data by Nishino et al. (1992) who by flow cytometry detected a faint reaction with an anti-p24 mAb on the surface of FIV-infected cells. In our analysis,

S. Lombardi et al.lJournal

qf Virological Methods 46 (1994) 2X7-301

299

dead cells and debris were not included, and these showed indeed a slight fluorescence that might account for the faint reaction reported by Nishino et al.. The availability of an anti-p24 mAb that stains a high proportion (86%) of FIV-infected cells as detected by flow cytometry, could be useful to investigate the cell types preferentially infected by FIV in vivo and to develop a sensitive flow cytometrybased neutralization assay (in preparation). The anti-p24 mAbs also allowed the localization of the core viral antigen in the cytoplasm of acetone-fixed or formalin-fixed paraffin-embedded FIV-infected cells by immunohistochemistry. In particular, by IFA on acetone-fixed cells all the antip24 mAbs similarly stained a high proportion of infected cells (approximately 60%) while by immunoperoxidase on formalin-fixed paraffin-embedded cells the AE11 mAb gave the best results by detecting the p24 protein in 25% of infected cells. Such reduction in sensitivity is not surprising since it is well known that tissue antigens may be denatured or altered by formalin fixation and paraffin inclusion. In tissue sections, AEll mAb proved useful to localize the p24 protein in infected cells: for example, we demonstrated FIV p24 in cells from lymph nodes, kidney and bone marrow. A further advantage of the availability of an anti-p24 mAb based immunohistochemical test is that post-mortem diagnosis of FIV infection and retrospective studies that may improve our understanding of feline AIDS epidemiology are now possible. A competition capture ELISA based on AEl l/DFlO mAbs was also developed for titrating cat antibodies to FIV p24 antigen. In this assay various dilutions of cat serum were allowed to compete with a constant amount of biotin-labelled mAb. FIV-infected cats inhibited the binding of biotinylated DFlO mAb with titers ranging between 1:20 and 1:640; similar titers have been previously reported by using recombinant p24 (Reid et al., 1991). Our competition capture ELISA proved useful to eliminate false positive results that may arise when FIV-negative cat sera are tested on purified whole FIV-coated wells (Siebelink et al., 1990). In addition, as in the case of HIV infection of humans (Allain et al., 1987; Lange et al., 1986; Weber et al., 1987), the possibility of detecting antibodies against individual viral proteins may be important for studies concerning the pathogenesis of the disease.

5. Acknowledgements This work was supported by grants from Minister0 della Sanita - Istituto Superiore di Sanita, ‘Progetto Allestimento Modelli Animali per I’AIDS’, Rome, Italy. We are grateful to Dr. 0. Jarrett, University of Glasgow, and Dr. J. Yamamoto, University of Southern California, Los Angeles, for providing us with reagents. 6. References Allain. J.P., Laurian, Y., Paul, D.A., et al. (1987) Long-term evaluation of HIV antigen and antibodies to p24 and gp41 in patients with hemophilia. New Engl. J. Med. 317, 11141121. Ascher, D.P.. Roberts, C. and Fowler. A. (1992) Acidification modified p24 antigen capture assay in HIV

seropositives. J. AIDS 5, 1080-1083. De Wolf, F., Goudsmit, J., Paul, D.A.. et al. (1987) Risk of AIDS related complex and AIDS in homosexual men with persistent HIV antigenemia. Br. Med. J. (Clin. Res.) 295, 567 589. Egberink, H.F., Ederveen. J., Monteiaro, R.C., Pedersen, N.C., Horzinek, M.C. and Koolen, M.J.M. (1990) IntraceIlular proteins of feline immunode~ciency virus and their antigenic relationship with equine infectious anemia virus proteins. J, Gen. Virol. 71, 739. 743. Goudsmith. J., De Wolf, F., Paul, D.A., et al. (1986) Expression of human immunodeticiency virus antigen (HIV-Ag) in serum and cerebrospinal fluid during acute and chronic infection. Lancet 2. 177 180. Lange, J.M.A., Countinho, R.A., Krone, W.J.A., et al. (1986) Distinct IgG recognition patterns during progression of subclinical and clinical infection with iymphadenopathy associated virus;human T lymphotropic virus. Br. Med. J. (Clin. Res.) 292. 2288230. Lombardi. S., Bendinelli, M. and Garzelli, C. (1993) Detection of B epitopes on the p24 gn.g protein of feline immunodeficiency virus (FIV) by monoclonal antibodies. AIDS Res. Hum. Retroviruses 9, 141 145. Matteucci. D., Baldinotti, F., Mazzetti, P., et al. (1993) Detection of feline immunodeticiency virus in saliva and plasma by cultivation and polymerase chain reaction. J. Clin. Microbio!. 3 1, 494501. Montelaro, R.C., Lohrey, N.. Parekh, B., Blakeney, E.W. and Issel, C.J. (1982) Isolation and comparative biochemical properties of the major internal polypeptides of equine infectious anemia virus. J. Viral. 42, 102991038. Morikawa, S., Lutz. H., Aubert. A. and Bishop, D.H.L. (1991) Identification of conserved and variable regions in the envelope glycoprotein sequences of two feline immunodeliciency viruses isolated in Zurich, Switzerland. Virus Res. 21, 53 -63. Nishanian, P., Huskins. K.R., Stehn, S.. Detels, R. and Fdhey, J.L. (1990) A simple method for improved assay demonstrates that HIV ~24 antigen is present as immune complexes in most sera from HIVinfected jndividtials. J. infect. Dis. 162. 21 2X. Nishino, Y., Ohki. K., Kimura. T., Morikawa, S., Mikami. T. and Ikuta, K. (1992) Major core proteins, ~24s. of human. simian, and feline immunodeticiency viruses are partially expressed on the surface of the virus-infected cells. Vaccine 10, 677-683. O’Connor, Jr.. T.P., Tanguay, S.. Steinman, R. et al. (1989) Development and evaluation of immunoassay for detection of antibodies to the feline T-lymphotropic lentivirus (Feline Immunodeficiency Virus). J. Clin. Microbial. 27, 474479. Olmsted, R.A., Hirsch, V.M., Purcell. R.H. and Johnson, P.R. (1989) Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentivirus. Proc. Nati. Acad. Sci. (USA) 86. 4355-4360. Paul, D.A., Falk. L.A., Kessler, H.A., et al. (1987) Correlation of serum HIV antigen and antibody with clinical status in HIV-infected patients, J. Med. Virol. 22, 357-363. Pedersen. N.C. (1993) Feline immunodeliciency virus infection, In: J.A. Levy (Ed.), The Retroviridac, Vol. 2. Plenum Press. New York. m press. Pedersen, N.C.. Ho, E.W.. Brown, M.L. and Y~lmamoto. J.K. (lYX7a) Isolation ofa T-l~nlphotroptc virus from domestic cats with an immunodeficiency-like syndrome. Science 235. 790-793. Pedersen N.C.. Moller-Nielsen, C.. Vcstcrgaard, B.F.. Gerstoft. J., Krogsgaard. K. and Nielsen. J.O. (1987b) Temporal relation of antigenemia and loss of antibodies to core antigens to development of clinical disease in HIV infection. Br. Med. J. (Clin. Res.) 295, 567--5X9. Phillips T.R.. Talbott. R.L., Lament. C.. Muir. S., Lovelace. K. and Elder. J.H. (1990) Comparison of two host cell range variants of fehne immunode~ciency virus. J. Viral. 64, 46054613. Poli A.. Giannelli. C., Pistello. M.. et al. (19923. Detection of salivary antibodies in cats infected with feline immunodeticiency virus. J. Clin. Microbial. 30. 2038 ~2041. Reid. G., Rigby, M.A.. McDonald. M., Hosie. M.J.. Neil. J.C. and Jarrett. 0. (1991) lmmunodiagnosis 01 feline immunodeficiency virus infection using recombinant viral pl7 and ~24. AIDS 5 1477 1483. Schellekens. H. and HotTinek, M.C. (Eds.) (1990) Animal Models in AIDS, Elsevier, Amsterdam. Steinman R.. Dombrowski. J.. O’Connor, T., et al. (1990) Biochemical and immunological characterization of the major structural proteins of feline immunode~ciency virus. J. Gen. Viral. 71, 701 706.

S. Lomhardi

1’1 al./Journal

qf Virological

Methods

46 (1994)

287- 301

301

Talbot R.L., Sparger, E.E., Lovelace, K.M., et al. (1989) Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. (USA) 86, 5743-5747. Tilton, G.K., O’Connor, T.P. Jr., Seymour. C.L., et al. (1990) Immunoassay for detection of feline immunodeficiency virus core antigen. J. Clin. Microbial. 28, 898-904. Weber, .J.N., Weiss, R.A., Roberts, C., et al. (1987) Human immunodeficiency virus infection in two cohorts of homosexual men: neutralizing sera and association of anti-gag antibody with prognosis. Lancet I. 119-122. Yamamoto, J.K., Sparger. E.. Ho, E.W., et al. (1988) The pathogenesis of experimentally induced feline immunodeticiency virus (FIV) infection in cats. Am. J. Vet. Res. 49. 12461258. Yamamoto, J.K., Ackley. C.D., Zochlinski, H., et al. (1991) Development of IL-2-independent feline lymphoid cell lines chronically infected with feline immunodeticiency virus: importance for diagnostic reagents and vaccines. Intervirology 32, 361-375.