Journal of Virological Methods, 38 (1992) 229-242 0 1992 Elsevier Science Publishers B.V. / All rights

VIRMET

229 reserved

/ 0166-0934/92/$05.00

01346

Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7 M.D. Laviada,

M. Babin, J. Dominguez

Instituto National de Investigaciones

and J.M. Sanchez-Vizcaino

Agrarias, Departamento

(Accepted

20 January

de Sanidad Animal, Madrid

(Spain)

1992)

Summary A sandwich enzyme-linked immunosorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the nine different AHSV serotypes. African horsesickness virus; Monoclonal antibody; ELISA; Diagnosis

Introduction African horsesickness (AHS) is a highly fatal infectious disease of equidae caused by an orbivirus which is transmitted by arthropods of various Culicoides species. AHS produces important economic damage in affected countries because of direct costs of horse deaths and vaccination as well as indirect costs such loss of trade and international competition. In August 1987, an outbreak of AHS took place in Spain, where this disease had not been detected since the first epizootic in 1966. Three provinces of the Correspondence to; M.D. Laviada, Instituto National de Investigaciones Sanidad Animal, Embajadores no. 68, 28012 Madrid, Spain.

Agrarias,

Departamento

de

230

central area (Madrid, Toledo and Avila) were affected. Several outbreaks were confirmed in a number of southern provinces of Spain and in Portugal from 1988 causing a large number of horse deaths. In order to detect a new outbreak in a short period of time to apply effective control measures before the spread of the disease, a rapid and specific diagnostic test is necessary. The current methods of diagnosis are virus isolation in tissue culture or mice, with the subsequent virus identi~cation by seroneutralization. It is laborious and time-consuming, and takes about 10 days or more to get the result. The complement fixation test is the usual method for detecting antibodies to AHSV, but antibody detection has only a limited value for diagnosis since the rapid course of the disease causes death before the development of a significant titer of antibodies. An ELISA test for the detection of AHSV using F(ab’), fragments of a specific polyclonal hyperimmune antiserum has been described previously (Du Plessis et al., 1990). Likewise, a group-specific sandwich ELBA which employs poiyclonal antisera for the rapid detection of AHSV antigens in spleen tissues and supernatant fluids has been adapted recently (Hamblin et al., 1991). However, the use of more uniform and easily obtained reagents would be desirable to make a more standardized test. We describe a new ELBA for detection of AHSV in horse spleens or ceil culture supernatants by the use of 2 monoclonal antibodies (MAb) directed against a major viral protein of the inner layer .(VP7) by a biotin-avidin amplified assay.

Materials and Methods Viruses, cells and spleen samples The strain of AHSV used was isolated from an infected Spanish horse spleen in 1989, and belongs to serotype 4. The strains of equine herpesvirus and equine arteritis virus used as negative controls, were kindly provided by C. Gomez Tejedor (INIA, Madrid). The virus was propagated in Vero cells in Minimal essential medium (MEM) supplemented with Earle’s salt, 5% fetal calf serum (FCS), 2 mM L-glutamine, 50 pg ml- ’ streptomycin and 50 IU ml-’ penicillin. A group of 167 splenic samples from suspected infected horses (82 from horses which died during the 1990 outbreak and 85 from horses dead or killed 2 mth after the last AHS death) were kindly provided by the National AHSV Reference Laboratory of Algete (Madrid), as well as seven negative spleens from the non-infected area. Virus purification AHSV

was purified

by a combination

of the Triton

X-100 method

of

231

Huismans et al. (1987) and the Freon extraction method (Huismans, 1979). Briefly, confluent Vero cell cultures in 375 cm2 roller bottles were inoculated with AHSV using a multiplicity of infection (moi) of 0.05. The cultures were incubated at 37°C until 80-100% CPE was observed, usually between 224 days post-inoculation. Bottle contents (cells and medium) were centrifuged 1500 x g 30 min and cells were resuspended in 2 mM Tris HCl pH 8.4 with 1% Triton X100 using 2.5% of the original volume. After 1 hour at 4°C nuclei were removed by centrifugation at 720 x g for 5 min. The virus particles in the supernatant were collected by centrifugation through a 3 ml cushion of 40% (w/w) sucrose in 2 mM Tris-HCl (pH 8.4) for 90 min at 27000 rpm in a Beckman SW27 rotor. The pellet was resuspended in 2 ml of 2 mM Tris-HCl (pH 8.4) and extracted with Freon (1,1,2-trichlorotrifluoroethane) one to two times followed by buffer extraction of the Freon phase. The water phases were pooled and the virus dissociated from the cellular material was pelleted by centrifugation for 90 min through a 3 ml cushion of 40% sucrose. The pellet was re’suspended in 2 mM Tris-HCl (pH 8.4) and centrifuged on 440% sucrose gradient for 45 min at 27 000 rpm in a Beckman SW27 rotor. The gradient was fractionated and virus was detected by absorbance at 260 nm, pelleted as before and stored at -70°C in 2 mM Tris-HCl (pH 8.4). The purity of the virus was verified by gel electrophoresis stained with Coomassie blue. Protein concentration was determined by the method of Lowry (1951). When indicated, the virus was inactivated by ultraviolet (UV) radiation for 10 min at 10 cm from the focus. Virus inactivation was checked by cell culture and antigenicity by indirect ELISA. Monoclonal

antibody production

Female BALB/c mice were immunized by subcutaneous (s.c.) injection of 50 pg of purified AHSV emulsified in complete Freund’s adjuvant. Two more doses of 50 pg in incomplete Freund’s adjuvant were given S.C. 21 and 42 days after the first injection. On days 102 and 103, mice were injected with 80 pg of UV inactivated purified AHSV, intravenously and intraperitoneally, respectively, according to the method of Stahli et al. (1983). On day 104, the fusion was carried out. The spleen cells were fused with the X63 Ag8.653 myeloma cell line following a procedure similar to that described by Kohler and Milstein (1975). Culture supernatants from the wells with growing hybridoma colonies were assayed by an indirect ELISA as described below. All hybridomas were cloned by limited dilution at least twice. Cloned hybridomas were injected into pristane primed mice for ascites production (Coll, 1987). MAbs from ascites were purified by aflinity chromatography on Protein A-Sepharose CL-4B (Pharmacia, Uppsala, Sweden) as described by Ey et al. (1978).

232

Characterization

of A4Abs specificity

The binding of MAbs to viral proteins separated by Immunoblotting analysis. electrophoresis and transferred to nitrocellulose paper was performed by the method of Towbin et al. (1979) with minor modifications. Proteins of AHSV semipurified as described by House et al. (1990) were separated by SDS-PAGE in 15% acrylamide-NJV’dialyltartardiamide (DATD) gels (Escribano y Tabares, 1987). Proteins obtained from non-infected cell cultures by the same method were used as control. Separated proteins were transferred to a nitrocellulose membrane filter at a constant current of 280 mA for 6 h at 4°C. Immunoblotting of nitrocellulose membrane cut into strips was carried out as described (Escribano et al., 1990) using ascites at l/20 dilution, peroxidaseconjugated rabbit-anti-mouse immunoglobulin at l/500 dilution and incubation times of 1 h at 37°C. Immunoadsorption assay. The specificity of the MAbs was determined by immunoadsorption of the corresponding viral antigen on MAb-coated plates, by a procedure described by Melero and Gonzalez-Rodriguez (1984). Labeled antigens were prepared from Vero cells infected with AHSV at a multiplicity of infection of 0.08 and grown for the last 4 hours in the presence of 500 &i of 35S-methionine (Amersham) per ml in‘ Eagle minimal essential medium lacking methionine. At 30 h post infection, labeled infected cells were harvested and dissociated by incubation at 37°C for 30 min in virus dissociation buffer (VDB: 50 mM Tris HCl (pH 7.5) 5 mM disodium EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate). Nondissociated material was removed by centrifugation at 45 000 rpm for 90 min in a Beckman SW55 rotor through a 0.5 ml cushion of 4% sucrose. Labeled non-infected cells were processed in the same way and used as negative control. Labeled antigen samples (5 x lo5 cpm) were added to each well for the immunoadsorption assay (Melero and Gonzalez-Rodriguez, 1984). Biotinylation

of monoclonal antibodies

MAbs were biotinylated following a procedure similar to that described by Dominguez et al. (1990). Biotin conjugates were titrated and diluted to the optimal working dilution just prior to use. Indirect ELISA Indirect ELISA was carried out as described previously (Sanchez-Vizcaino et al., 1982), using a semipurified AHSV (obtained as described before without the step of centrifugation on sucrose gradient) to coat Dynatech M-129B ELISA plates. Plates coated with uninfected cell antigen were used as controls. A 25% FCS in PBS buffer was used to saturate plate remaining binding sites before incubation with hybridoma supernatants or ascites fluids, which were

233

diluted in the same buffer. Peroxidase labeled rabbit anti-mouse IgGs (Biodiagnostic, UK) was added as conjugate antibody. 200 ~1 of substrate solution (80.6 mM 3-dimethylaminobenzoic acid (DMAB), 1.56 mM 3-methyl2 benzothiazolinona hydrazone hydrochloride in 0.1 M phosphate buffer with 0.025% H202) were added to each well to develop the reaction. Color development was stopped by adding 50 ,ul of 3 N H2S04 after 10 to 15 min. Plates were read at 620 nm using a Titertek Multiskan reader (Flow, Ayrshire, UK). Titer was defined as the dilution of the MAb that gave an absorbance value (AeZO with AHSV antigen minus A 620 with uninfected cell antigen) of 0.11 (average value of negative ascites = 0.02). Double antibody sandwich ELISA

(DAS-ELISA)

A sandwich enzyme immunoassay was developed for capture and detection of AHSV following essentially the methods described before (Dominguez et al., 1990; Coll, 1988). Dynatech M-129B ELISA plates were coated with 1 pg/well of purified 5G5 and 3D2 MAbs diluted in PBS by overnight incubation at 4°C. Plates were washed 5 times with washing solution and incubated for 1 h at 37°C (otherwise as indicated) with 100 yl/well of two-fold dilutions from l/l of spleen homogenate samples or AHSV infected cell culture supernatant or purified virus in dilution buffer (PBS, 1% BSA). After 5 washes with washing solution, the plates were incubated for 1 h at 37°C with 100 &well of the working optimal dilution of biotin labeled 5G5 MAb. After 5 more washes, the plates were incubated with 100 pi/well of avidin-peroxidase (Vector Laboratories, Inc. Burlingame, CA) diluted l/2000 in dilution buffer for 45 min at room temperature. Plates were washed 5 times and the enzymatic reaction developed as described for indirect ELISA. Competition

assays for epitope determination

Using the DAS-ELISA technique, competitive binding assays were carried out with the biotin-labeled MAbs against unlabeled MAbs. Plates were coated with MAbs 5G5 and.3D2 and incubated with 100 pi/well of AHSV cell culture supernatant to saturate the MAbs binding sites. After virus incubation, 100 ~1 of optimal working dilution of biotinylated antibodies containing different concentrations of purified MAbs (60, 30, 15, 7.5, 3.75, 1.8, 0.9 and 0.4 pg ml- ‘) were added. The results were expressed as inhibition percentage using the following formula: Eabsorbance (A& in the absence of unlabeled antibody AezO in the presence of unlabeled antibody] x 100/A620 in the absence of unlabeled antibody. Spleen homogenate

sample preparation

Splenic tissues from horses were homogenized

in MEM in a Dounce

glass

234

1 ABCDEF

A

MW

B

C

D

VP292

-

46

-

30

-

VP5 -

VP7-

Fig. 1. Immunoadsorption assay (1) and Western immunoblotting (2) of MAbs 5G5 and 3D2 with AHSV proteins. Panel (1). Lanes A and B: polyclonal anti-AH% mouse serum with labeled antigens from AHSV infected and uninfected Vero cells, respectively. Lanes C and D: MAb 5G5 with labeled antigens from AHSV infected and uninfected Vero cells, respectively. Lanes E and F: MAb 3D2 with labeled antigens from AHSV infected and uninfected Vero cells, respectively. Panel (2). Lane A: polyclonal anti-AHSV mouse serum with proteins of semipurified AHSV. Lanes B and C: MAb 5G5 with pfoteins of semipurified AHSV and proteins from non-infected cell culture, respectively. Lanes D and E: MAb 3D2 with proteins of semipurified AHSV and proteins from non-infected cell culture, respectively.

homogenizer to form a 50% w/v tissue suspension. After centrifugation at 2000 x g for 5 min, the supernatant was used as. sample for the DAS-ELISA test. Spleen homogenate supernatants were filtered through.a 0.45 pm and 0.22 pm sterile disposable filters (Minisart, Sartorius) for virus isolation. 1 ml of each splsnic homogenate was inoculated onto confluent monolayers of Vero cells and incubated at 37°C. After 1.5 h of virus adsorption, the inoculum was removed, and replaced by fresh medium with 5% FCS. Cell cultures were observed daily for development of CPE for 5 days. In case CPE was not observed, 3 blind passages were carried out before the sample was considered as negative.

235

Results Selection of anti-VP7

A4Abs

Two anti-VP7 MAbs, 3D2 and 5G5, were selected from a panel of 46 antiAHSV MAbs containing 17 anti-VP7 MAb, as determined by Western immunoblotting and immunoadsorption assays. 5G5 reacted only with nondenatured VP7 used in the immunoadsorption assay, suggesting that it binds to a conformational epitope, whereas 3D2 bound to denatured VP7 in the immunoblotting analysis (Fig. 1). The combination of these two MAbs for the solid-phase and one (5G5) for the conjugate was chosen after testing in DAS-ELBA all possible combinations among the six purified MAbs anti-VP7 that had shown higher activity in the indirect ELBA. The epitopes recognized by MAbs 5G5 and 3D2 were analyzed by a competition ELISA using plates coated with MAb captured AHSV. As shown in Fig. 2, these two MAbs did not compete for the same viral epitope. Rather, they reacted with 2 different and independent sites on the VP7. The 2 MAbs were obtained as mouse ascites, purified by affinity chromatography on protein A-Sepharose, and titrated by indirect ELISA. Titers were l/2000 for 3D2 and l/lo6 for 5G5.

A 100

6 % inhibition

% inhibition

1

loo1

\

60.

60-

40-

\

2Oi

O\ 6

1.6

3

0.76

0.37

0.16

0.09

unlabeled MAb (ug/well) -

302

-6Q6

0.04

20

10

6

2.6

1.26

0.82

0.31

0.16

unlabeled MAb (ug/well) -

3D2

-6@S

Fig. 2. Binding of biotin-labeled 5G5 (A) or 3D2 (B) to solid-phase captured AHSV in the presence of 5G5 (+) and 3D2 (.). The results are expressed as % inhibition using the formula: (A620 in the absence of unlabeled antibody - &0 in the presence of unlabeled antibody) x 100/A 6~~ in the absence of unlabeled antibody.

236

absorbance 251

at 620 nm

absorbance

at 620 nm

05-1 0:

--h ~/~_ 8 16

l/cell

32

84

culture -

128

256

512

m?1204840988192

supernatant 37’

+

49

dilution

860 430

215 107

virus protein

53

26

13

concentration -

37.

6.7

3.3

1.7

0.8

hg/ well)

*4a

Fig. 3. Estimation of ELISA sensitivity by titration of AHSV infected cell culture supernatant containing 8 x lo6 TCIDSO ml-’ (A) and titration of purified AHSV (B). Samples were incubated at 37°C for 1 h (.) or at 4°C overnight (*). Non-infected cell culture supernatant was used as negative control (+). The bracketed lines represent SD.

Assay characterization The sensitivity of the ELISA was determined by testing dilutions of a stock of AHSV infected cell culture supernatant containing 8 x lo6 TCIDso ml-‘. The sensitivity, defined as the lowest virus concentration that can be measured over the background with 99.5% confidence, was estimated by the tdistribution of triplicate determinations of negative samples. An absorbance of 0.15 was considered as the detection limit. That means about 2.5 x lo3 TCIDSO per well (Fig. 3A). This assay was not a strict indicator of virus concentration because it also reacted with non-infectious AHSV antigen present in the sample. For this reason, the protein concentration of purified virus was determined using the ELISA. Concentrations of 10 ng/well of purified virus were detected (Fig. 3B). Sensitivity can be slightly increased incubating the samples at 4°C overnight. The specificity of the ELISA was confirmed by the lack of reactivity with the uninfected cell culture fluid or equine herpes virus (EHV- 1) and equine arteritis virus (EAV) infected cell cultures (lo5 TCIDSO/well). A mean background value of 0.13 (SD = 0.02) was obtained in absence of antigen. These results were obtained using a dilution buffer without Tween 20. When a 0.05% of Tween 20 was added, the sensitivity of the assay was decreased at least 8-fold without lowering the background (Fig. 4). The inclusion of tensioactive substances in the dilution buffer such as 2% SDS and 2% NP 40, or 3 M NaCl to increase the ionic force, and the incubation of samples in 0.5 M

237

2.5

absorbance

at 620 nm

2

1.5

1

0.5

04

I

I

I

I

I

I

l/l

l/2

l/4

l/6

l/l6

l/32

sample

1

l/64

I

l/l28

dilutions

Fig. 4. ELBA titration curves of a positive spleen sample diluted in sample dilution buffer with (0) or without ( .) 0.05% Tween 20. Negative spleen. sample with (A) or without (+). Tween 20 was used as control of backgrounds.

MgC& (pH 4) or VDB buffer prior to be tested also decreased the sensitivity, as did the dry of MAbs onto the solid-phase and the performance of the assay in one step (simultaneous incubation of labeled MAb and samples) (data not shown). Interassay coefficient of variation (CV), defined as (mean/SD) x 100, was obtained analyzing 44 samples in duplicate in three separate assays and dividing the samples into three concentration groups. It varied between 4.212.0%. Intraassay variation was calculated testing six times in the same assay a total of 15 samples of high, medium and low absorbance. The CV varied between 7.3~8.6%. These results are summarized in Table 1. Detection of AHSV

in infected spleens

The ELISA was evaluated for its ability to detect AHSV in infected spleens. A total of 167 samples of spleen homogenates from suspected infected horses

238 TABLE 1 Interassay and intraassay

variation of the AHSV ELISA test for equine spleen homogenates

Sample

A620

n

Group mean

CV (%)

Interassay variation? High Medium Low

I .3-2.00 0.8-l .29 0.330.79

5 11 21

1.75 1.05 0.50

4.2c 9.1’ 12.0c

Intraassay High Medium Low

1.3-2.00 0.8-1.29 0.34.79

3 4 8

1.75 1.13 0.55

7.4d 8.6* 7.3d

variationb:

“A total of 44 spleen homogenates were analyzed in duplicate in three separate assays and the results grouped by the absorbance at 620 nm. bEach sample as analyzed six times in the same assay and the results grouped by the absorbance at 620 nm. ‘Mean of the CV of independent duplicates for each group of spleen homogenates. dMean of the CV of each group.

belonging to the Spanish infected area were analyzed by this technique at several dilutions starting at l/l, and compared with virus isolation in cell culture. Results are shown in Fig. 5, where the value of each individual sample is represented. 34.8% (15) of positive cell culture samples gave an absorbance value higher than 2.00, whereas a mean of negative values of 0.13 was obtained with 7 negative spleens from the Spanish non-infected area.

positive

doubtful negotive

vrus

isolation

+ .

, virus

isolation

-

Fig. 5. ELISA detection of AHSV in horse spleen homogenates compared with virus isolation in cell cultures. The value of each individual sample at dilution l/l is represented by a spot. Horizontal dotted lines mark the separation between absorbance values interpreted as negative, doubtful or positive result.

239

To define the cut-off between negative and positive samples a normal distribution of negative samples was assumed. Therefore, an absorbance value below 0.20 (mean + 3 SD) would be negative with 99% confidence. As shown in Fig. 5, between absorbances of 0.20 and 0.40 the sample could be positive or negative, and must be confirmed by other techniques. The sample was considered positive if it gives an absorbance value greater than 0.40. According to these results, a sensitivity of 97.4%, defined as (number of true positives - number of false negatives)/number of true positives, and a specificity of lOO%, defined as (number of true negatives - number of false positives)/ number of true negatives, were obtained.

Discussion ELISAs are being widely used for the diagnosis of viral diseases because of their high sensitivity and because they permit rapid processing of large numbers of samples. The results are also obtained within a few hours, a time very much shorter than that required for virus isolation. This is especially important for diseases like AHS, in which it is necessary to make the diagnosis as soon as possible in order to apply control measures to avoid the spread of the disease. We described the use of 2 MAbs in a biotin-avidin amplified ELISA to detect AHSV. These MAbs recognize 2 non-overlapping epitopes on the VP7, an inner structural viral protein. The epitope bound by MAb 5G5 seems to be conformational, since it is only recognized by the immunoadsorption assay, and not when the protein’is denatured in SDS-PAGE (Fig. 1). Because this MAb had a high titer in binding to AHSV and labeled well with biotin, it was chosen for conjugation. On the other hand, MAb 3D2 binds to a nonconformational epitope and does not recognize well the native virus. However, when both MAbs were used together in the solid-phase, a clear cooperative effect in capturing AHSV was observed. Preliminary results of competitive binding assays between 6 MAbs anti-VP7 suggest that there is one antigenically dominant site in this protein since most of the MAbs obtained are directed against the same epitope (data not shown). Anti-VP7 MAbs have been selected to optimize the ELISA because VP7 is a major protein and a putative group specific antigen, possibly with little variability between serotypes as is the case in bluetongue virus (BTV), the prototype orbivirus (Huismans and Erasmus, 1981). Comparison of the AHSV-4 VP7 sequence with that of BTV-10 have revealed an overall similarity of 44% (Roy et al., 1991). In fact, isolates of serotypes 1,2,3,4,7,8 and 9 have been recognized by our MAbs 5G5 and 3D2 by an indirect immunofluorescence assay (Dr. Mebus, personal communication). Nevertheless, isolates of different serotypes and from different geographical origin should be examined in order to demonstrate the degree of conservation of these epitopes among AHSV. Although VP7 is considered to be an inner viral protein, the ELISA detects intact virus particle. The addition of dissociating agents as tensioactive such as

240

0.05% Tween 20 (Fig. 4), 2% NP 40, 2% SDS, or the treatment with MgCl* at low pH that has been shown to disrupt the outer protein layer of BTV (Huismans et al., 1983) diminished the assay sensitivity. However, a denaturing effect of these substances on the epitope recognized by the MAbs cannot be ruled out as a cause of the binding reduction. It is probably that regions of VP7 are also exposed on the virus surface, as shown for BTV using immunogold labeling (Hyatt and Eaton, 1988), surface labeling and reactivity with monoclonal antibodies (Lewis and Grubman, 1990). This assay allows the detection of about 10 ng/well of purified virus or 2.5 x lo3 TCIDSO/well infected cell cultures in only 3 h. Other similar assay reported by Du Plessis et al. (1990) takes more time and uses polyclonal antibodies. Other differences between the two assays could be due to the use of ophenylenediamine (OPD) as substrate for the ELISA since about 2-fold higher sensitivity was found when compared to DMAB-MBTH in simultaneous experiments (not shown), when detection limit was defined as reported (an absorbance value twice that of background). On the other hand, the sensitivity defined as the amount of protein detected in purified virus could be influenced by the quality of virus purification, by the presence of foreign proteins from infected cells. This assay can detect AHSV in infected spleens with a sensitivity of 97.4% (n = 43), although doubtful results must be confirmed by other techniques like virus isolation in cell culture. The equine spleen has been shown to be the suitable sample for routine diagnosis since it contains the highest amount of virus (Tessler, 1972). Although virus could not be isolated from most of the samples that fall into the doubtful range, the ELISA values could be explained by the presence of inactivated virus in the spleen, since the samples were obtained from an infected area and time and storage conditions between necropsia and tissue culture inoculation were not known. Attempts to detect the AHSV genome by means of the polymerase chain reaction are in progress to verify that hypothesis. The use of MAbs that can be produced in large amounts with uniform quality will facilitate the standardization of this assay, compared to those using polyclonal antisera. The ELISA described here, therefore, provides a valuable test for use for the routine diagnosis and rapid detection of AHSV in biologicalsamples.

Acknowledgements We thank H. Hooghuis of National AHSV Reference Laboratory of AIgete, Madrid for the AHS spleen samples, Dr. J.M. Escribano and Dr J. Co11 for their useful discussions -and suggestions, and J. Bustos for her excellent technical assistance. This work has been supported by the Instituto National de Investigaciones Agrarias (INIA) project number 9152 and by Cedeti project number 96 11.

241

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