Short communication

Adaptation of a sandwich enzyme-linked immunosorbent assay todetermine the concentration of bovine leukemia virus p24 andoptimal conditions for p24 expression in short-term cultures of

peripheral blood mononuclear cells

M. van den Heuvel a,*, D. Portetelle b, B. Jefferson a, R.M. Jacobs a

a Department of Pathobiology, University of Guelph, Guelph, Ont., Canada N1G 2W1b Department of Microbiology, Gembloux, Belgium

Received 3 December 2002; received in revised form 14 April 2003; accepted 15 April 2003

Abstract

Bovine leukemia virus (BLV) is a common retroviral infection of cattle. Infection is accompanied by integration of BLV into the

host cell genome and is persistent for the life of the individual as is the presence of anti-BLV antibodies. Lymphosarcoma occurs in a

small fraction of infected adult individuals but otherwise there is little or no associated disease. Viremia is undetectable, however,

BLV is expressed readily once infected cells are cultured in vitro. A sandwich enzyme-linked immunosorbent assay (sELISA) was

optimized, using murine monoclonal antibodies, to quantify the major internal structural protein (p24) produced in short-term

cultures of peripheral blood mononuclear cells (PBMCs). Optimal production of BLV p24 was achieved utilizing RPMI

supplemented with 10% fetal bovine serum (FBS), pH 7, and 5�/106 cells per ml. Cultures were terminated at 24 h. The sELISA was

linear between 30 and 900 ng/ml and the limit of detection was 1.2 ng/ml. At three concentrations of p24, intra- and inter-assay

coefficients of variation (CV) varied between 9.2 and 13.3 and 5.1 and 12.9%, respectively.

# 2003 Elsevier Science B.V. All rights reserved.

Keywords: Bovine leukemia virus; p24; Sandwich enzyme-linked immunosorbent assay; Short-term culture; Optimization

Bovine leukemia virus (BLV) is the etiological agent

of lymphosarcoma in adult cattle, a relatively rare

neoplasm of B-cells (Burny et al., 1985). BLV infects

cattle worldwide. Infections are life-long, a consequence

of the retroviral life cycle where the BLV provirus

integrates into the host cell genome (Burny et al., 1978,

1988). The majority of infected cattle are asymptomatic

but, approximately one-third of infected cattle develop

persistent lymphocytosis (PL) (Burny et al., 1988), a

polyclonal expansion of B-cells. Less than 5% of BLV-

infected cattle develop lymphosarcoma, although ap-

proximately two-thirds of cattle with lymphosarcoma

have a history of PL (Burny et al., 1985).

In asymptomatic BLV-infected cows with a normal

number of peripheral blood B-cells, from 0.1%9/1.8 to

9.2%9/19 (mean9/S.D.) of peripheral blood lympho-

cytes (PBLs) are BLV-infected, whereas in cows with PL

BLV-infected cells comprise from 13.9%9/6.6 to 66%9/

4.8 of PBLs (Mirsky et al., 1996). BLV is demonstrable

by the polymerase chain reaction from nuclear extracts

of PBLs, but viral gene products are not readily

detectable by immunological methods such as Western

blotting or radioimmunoassay (Ferrer, 1980; Kettmann

et al., 1980, 1982; Mirsky et al., 1996), suggesting that in

vivo, BLV is either not present in blood or is present in

minute quantities below the sensitivity of current detec-

tion methodologies.

However, since infection with BLV results in a

persistent anti-BLV antibody titer, it is thought that

some constant but low level of expression must occur

perhaps compartmentalized to such organs as lymph

nodes, spleen, or bone marrow. Several studies have

* Corresponding author. Tel.: �/1-519-824-4120x54907; fax: �/1-

519-763-1450.

E-mail address: mvandenh@uoguelph.ca (M. van den Heuvel).

Journal of Virological Methods 111 (2003) 61�/67

www.elsevier.com/locate/jviromet

0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0166-0934(03)00148-4

determined that BLV expression is up-regulated rapidly

if PBLs are cultured (Mirsky et al., 1996; Gupta and

Ferrer, 1982; Miller et al., 1969; Taylor and Jacobs,

1993; Zandomeni et al., 1992).The difficulty in detecting viral proteins in freshly

isolated peripheral blood mononuclear cells (PBMCs) is

thought to be due to the presence of a host factor which

blocks transcription of the integrated provirus (Gupta

and Ferrer, 1982; Taylor and Jacobs, 1993; Zandomeni

et al., 1992). This phenomenon has also been observed

in other retroviral infections, notably HTLV I and II

and HIV I (Castro et al., 1991; Garcia-Blanco andCullen, 1991), making BLV an interesting model to

study mechanisms of host response to retroviruses.

Characterization and purification of factors that

decrease BLV production require an assay to monitor

BLV production. A sandwich enzyme-linked immuno-

sorbent assay (sELISA), based on the method used to

detect the 51 kDa envelope glycoprotein (gp51), de-

scribed by Portetelle et al. (1989), was evaluated for itsability to detect the more strongly expressed major core

protein, p24 (24 kDa), as a measure of viral production

in short-term cultures of PBMCs.

Fetal lamb kidney (FLK) cells (Jensen et al., 1990)

infected with BLV were cultured in Dulbecco’s modified

Minimum Essential Medium (DMEM) supplemented

with 10% heat inactivated fetal bovine serum (FBS)

(Life Technologies, Burlington, Ontario) and 0.05 mg/ml final concentration of gentamycin (Fisher Scientific,

Nepean, Ontario). Cell cultures were trypsinized (Life

Technologies) for 10 min at room temperature (RT) and

washed in phosphate buffered saline (PBS) pH 7. Cell

pellets, held at 4 8C, were lysed with a 10% aqueous

solution of n -octyl-bD-glucopyranoside (Sigma�/Al-

drich, Oakville, Ontario), then clarified by centrifuga-

tion at 12 000�/g for 10 min to be used as a source ofBLV p24.

Murine monoclonal anti-p24 antibodies (D. Porte-

telle, Gembloux, Belgium) were isotyped according to

the manufacturer’s instructions (Serotec, Raleigh, NC).

The 4?G9 and 4?F5 antibodies were found to be IgG1

isotype, while 2?C1 was an IgG2a isotype.

An immunosorbent column was prepared using the

2?C1 antibody according to manufacturer’s instructions(AvidChrome-Hydrazide, Sigma�/Aldrich). Briefly, the

monoclonal antibody was desalted and oxidized for 30

min at RT with 0.1 ml of 0.1 M NaIO4 prior to coupling

to the cartridge matrix. The column was incubated at

RT for 15 min, then rinsed with PBS to elute unbound

antibody.

The FLK lysate was applied to the column at a flow

rate of 0.2 ml/min, followed by two washes with PBS toremove unbound proteins. The p24 was eluted with 0.1

M NaAc, pH 3.5 and collected in 1.5 ml microfuge tubes

containing 500 ml 2 M Tris�/buffered saline, pH 8.8.

Protein in the eluate was monitored at 280 nm. Aliquots

of the protein-containing peaks were pooled and stored

frozen at �/20 8C for use as a standard in the p24

sELISA. The protein concentration in the pooled

fractions was determined by a dye-binding microproteinprocedure using albumin standards.

Two BLV-seropositive Holstein cows infected natu-

rally and one BLV-seronegative cow were housed

separately according to Canadian Council on Animal

Care Guidelines. BLV-status was confirmed by repeated

agar gel immunodiffusion tests. One of the BLV-infected

cows also had PL as determined by repeated hematolo-

gical examinations.Venous blood was collected into acid citrate antic-

oagulant and separated by centrifugation for 25 min at

800�/g at 20 8C. The buffy coat was diluted 1:5 with

sterile PBS. Fifteen milliliters of buffy coat cell suspen-

sion were layered onto 15 ml of Histopaque (Sigma�/

Aldrich) and centrifuged at 400�/g for 30 min at 20 8C.

PBMCs at the interface were removed, diluted 1:5 with

PBS, centrifuged at 100�/g for 10 min then washedtwice with PBS. The cells were diluted to 2�/106 viable

cells per ml of RPMI medium supplemented with 10%

heat inactivated FBS, L-glutamine (0.3 g/l) and 0.02%

amikacin. Each well of a 24-well plate (Falcon, VWR

Canlab, Mississauga, Ontario) contained a total volume

of 2 ml. Plates were incubated at 37 8C for 24 h then

centrifuged at 100�/g to pellet the cells which were then

washed twice with PBS. The cells were re-suspended in 1ml PBS, counted and the viability determined by trypan

blue exclusion. The remaining cells were pelleted then

lysed with 100 ml 10% n-octyl-bD-glucopyranoside in

water. They were kept on ice for 10 min, then 900 ml of

PBS were added to the lysates. The plates were

centrifuged at 500�/g for 3 min to pellet cellular debris.

An sELISA used for measuring BLV gp51 (Portetelle

et al., 1989) was adapted for evaluation. Each well of a96-well ELISA plate (Nunc Immunosorb, Life Technol-

ogies) was coated with 300 ng 4?G9 in 100 ml PBS and

incubated at 4 8C for 4�/6 h on a shaking table. The

plate was then washed twice with washing buffer. Excess

binding sites were occupied by adding 100 ml of

saturation buffer (10% bovine serum albumin in PBS,

pH 7) to each well and incubating for 10 min prior to

adding 100 ml of blocking buffer. Triplicate 100 mlaliquots of protein from the clarified cell lysate of each

PBMC culture were added to individual wells. Duplicate

samples of the p24 standard were also applied and these

were diluted five times in triple dilutions, yielding

standards from 11 to 900 ng. The plate was incubated

overnight at 4 8C on a shaking table. The following day,

the plate was washed three times with washing buffer,

then 200 mg each of peroxidase conjugated 2?C1 and4?F5 in 100 ml washing buffer were added to each well

and incubated at 4 8C for 1 h on a shaking table. The

plate was washed four times with washing buffer prior

to adding 100 ml of tetra-methyl benzidine (Kirkegaard

M. van den Heuvel et al. / Journal of Virological Methods 111 (2003) 61�/6762

and Perry Laboratories, Gaithersburg, MD) to each

well. After 5�/10 min, the reaction was stopped by

adding 100 ml of 2 N H2SO4 to each well. The

absorbence at 450 nm for each sample was read on a

microplate reader (Ceres, Biotech Instruments, Wi-

nooski, VT). A calibration program (Kineti-Calc II,

KCJR, EIA Software, Ceres) was used to determine p24

(ng) in each sample based on the p24 standards. It was

noted in pilot experiments that cell viability decreased

under some manipulations, so as to account for the loss

of p24 expression associated with dying or dead cells,

the total amount of p24 detected in 100 ml of cell lysate

was divided by the number of viable cells harvested,

yielding ng of p24 per 105 viable cells.

A series of experiments were carried out in triplicate

to determine the optimal conditions for p24 production

by BLV-infected PBMCs. Variables examined were

culture medium, cell concentration, pH, % FBS, and

duration of cell culture.

The data were summarized using descriptive statistics.

Least squares regression analysis was used to assess

linearity. Differences between treatments were assessed

using analysis of variance (ANOVA) and the Bonferroni

post-test. Differences were considered significant if P 5/

0.05.

The BLV p24 sELISA was linear over the range of

30�/900 ng/ml (r�/0.95, Fig. 1). Also, over this range a

close linear relationship (r�/0.993) was demonstrated

when known and predicted concentrations of BLV p24

were plotted indicating that interferences were not

influencing recovery (data not shown). The binding

capacity of the antibody-coated wells approached sa-

turation at 1 mg/ml of affinity-purified p24. To deter-

mine the lower limit of detection, defined as the lowest

value which was significantly different from zero (P 5/

0.05), of the p24 sELISA, eight aliquots of the p24

standard were applied in triple dilutions across a 96-well

plate (12 dilutions). The limit of detection was 1.2 ng/ml.

There were no significant differences between the

RPMI, Dulbecco’s and Joklik’s modified Minimum

Essential Medium supplemented with 10% heat inacti-

vated FBS (data not shown). The cell seeding rate was

arbitrarily set at 5�/106 cells per ml since there was no

significant effect of seeding rate on p24 production at

cell densities between 105 and 107 per ml (data not

shown). Maximal p24 production was observed at pH of

7 (Fig. 2), which was significantly higher than all other

pH values (P 5/0.05) with the exception of pH 5.5.

There was a significant decrease in p24 production when

the pH was increased above 7.5 (P 5/0.01). The higher

pH values resulted in cell viability of less than 10%,

while the optimal pH values yielded cell viability

consistently over 75%. Cell viability tended to be

decreased at pH 5.5 relative to pH 7 (data not shown).

Thus, RPMI containing 5�/106 cells per ml at pH 7 was

utilized in subsequent experiments.

The effects of varying percent heat inactivated FBS

and time in culture on cell viability were assessed by

two-way ANOVA in three replicate experiments (Table

1). Protein supplementation and time in culture were

significantly related to viability (P 5/0.002); there was

no significant interaction for these two treatments. The

highest viability (96%) occurred at 24 h with no FBS

(P 5/0.002), but p24 production tended to be less

Fig. 1. The linearity of the BLV p24 sELISA. Affinitiy-purified p24 was serially diluted. Data points represent the mean9/S.D. (n�/4). The assay

appeared linear over the range of 30�/900 ng (r�/0.95). The lowest value that was significantly (P 5/0.01) different from zero, or the limit of

detection, was 1.2 ng/ml.

M. van den Heuvel et al. / Journal of Virological Methods 111 (2003) 61�/67 63

without the addition of 10% FBS (Fig. 3). There were no

significant differences in viability between cultures with

5, 10, 15, or 20% FBS (Table 1). Increasing FBS above

10% was associated with a significant decrease (Fig. 3,

P 5/0.05) in p24 production and a trend toward

decreasing viability (Table 1). Presumably, the balance

between stimulatory and inhibitory factors that may be

present in FBS (Zandomeni et al., 1992) was altered

such that, above 10%, inhibition of p24 production

resulted. The p24 production tended to increase with the

duration of culture, however, the differences between 24,

36, 48, and 72 h in culture were not significant (Fig. 4).

Viability was highest at 12 and 24 h and significantly

decreased at subsequent times (Table 1). It appeared

that 24 h cultures utilizing RPMI supplemented with

10% FBS resulted in a reasonable balance of cell

viability and p24 production.

To test the reproducibility of the optimized assay, 108

PBMCs were collected, as described above, from two

BLV-infected cows one of which had PL and produced

high amounts of p24 and another with a normal white

blood cell count that produced relatively low amounts

of p24 in culture. PBMCs were cultured in 100 ml of

RPMI supplemented with 10% heat inactivated FBS for

24 h, then lysed, processed as described above and

stored in aliquots at �/20 8C. The affinitity-purified p24

standard was included as a third test sample. Replicates

of each of the three samples were used to test the intra-

assay and inter-assay variability. Coefficients of varia-

tion (CV) were 9.2�/13.3% for intra-assay (Table 2) and

5.1�/12.9% for inter-assay (Table 3) testing.

A commercially available test for HIV p24 (NEN Life

Sciences, MA) has an intra-assay CV of 2�/4% and inter-

assay CV of 13�/23%. Therefore, this BLV p24 sELISA

Fig. 2. The effect of pH of culture medium on expression of p24 by PBMCs in a 24 h culture from a BLV-infected cow with PL. Mean values (9/S.D.,

n�/3) of p24 produced per 105 viable cells. Cells were cultured in RPMI with 10% heat inactivated FBS. * Indicates significant decline relative to pH

7.0 (P 5/0.01).

Table 1

Effects of time (h) and heat inactivated FBS (%) on viability (mean%9/S.D., n�/3) of cultured PBMCsa

FBS (%) in medium Time (h) in culture

12 24 36 48 72

0 77%9/12.5 969/3.6 729/15.5 719/21.6 759/5.3

5 499/7.7 709/14.8 479/9.5 459/12.3 369/4.2

10 689/5.6 759/8.7 429/10.5 319/12.9 369/3.1

15 479/15.6 529/17.1 409/13.2 439/8.1 339/3.5

20 509/9.7 689/21.5 519/12.6 239/5.7 529/9.5

a Viability was assessed by trypan blue exclusion. Data were analyzed using two-way ANOVA and the Bonferroni post-test. Significant effects of

FBS and time (P 5/0.002). No significant FBS�/time interaction. Viability in 0% FBS was significantly greater than 5, 10, 15 or 20% FBS (P 5/

0.002). There were no significant differences between 5, 10, 15 or 20% FBS. Viability was not different between 12 and 24 h but at 24 h viability was

significantly greater than 36, 48, or 72 h (P 5/0.002).

M. van den Heuvel et al. / Journal of Virological Methods 111 (2003) 61�/6764

yielded inter-assay results comparable to the commercial

HIV p24 test, but had greater within-assay variation.

The latter deficiency could be compensated for by

taking the mean of a larger number of replicates.

The BLV p24 sELISA was unable to detect p24 in

freshly isolated PBMCs, and was just capable of

differentiating expression of p24 in cultured PBMCs

from an asymptomatic BLV-infected cow from back-

ground. Lysates of cells from seronegative sources were

always negative for p24, indicating that no cross-

reaction was occurring between the antibodies and

cellular proteins. Thus, this sELISA is not useful as a

diagnostic test for BLV but, it appeared satisfactory to

determine differences in expression of BLV p24 in

PBMCs from a cow with PL.

The BLV p24 sELISA lends itself well for larger

experiments where the effects of several variables can be

simultaneously assessed. PBMCs were cultured in 24-

Fig. 3. The effect of heat inactivated FBS (%) supplementation of RPMI on expression of p24 by PBMCs in a 24 h culture from a BLV-infected cow

with PL. Mean values (9/S.D., n�/3) of p24 produced per 105 viable cells. Symbols over bars indicate significant reduction relative to 10% FBS (*,

P 5/0.01; �/, P 5/0.05).

Fig. 4. The effect of time (h) in culture on expression of p24 by PBMCs from a BLV-infected cow with PL. PBMCs were cultured in RPMI with 10%

heat inactivated FBS. Mean values (9/S.D., n�/3) of p24 produced per 105 viable cells. There were no significant differences in p24 between 24, 36,

38, or 72 h, however, p24 at 12 h was significantly decreased (*, P 5/0.01).

M. van den Heuvel et al. / Journal of Virological Methods 111 (2003) 61�/67 65

well plastic plates, which were easily harvested, washed

and processed in the plate, using a centrifuge culture

plate adaptor. The test is relatively fast; requiring about

6 h of labor over 2.5 days. In addition, the use of a 96-

well plate for analysis allows up to 28 samples to be

assayed in triplicate on one plate, therefore, limiting the

error between plates.

The BLV p24 sELISA has specificity and ease of

measurement offered by monoclonal antibodies and a

colourimetric end-point. In contrast, earlier assays have

utilized polyclonal antibodies and/or radioisotopes (Ban

et al., 1991; Cowley et al., 1992; Schmerr et al., 1980).

The assay is less cumbersome than earlier ones based

upon biological viral characteristics such as syncytium

induction (Esteban et al., 1985; Schmerr et al., 1980) or

reverse transcriptase activity (Graves et al., 1977).

In conclusion, the BLV p24 sELISA has potential

usefulness for determining viral expression in cultured

PBMCs from BLV-infected symptomatic (i.e. PL) cattle

but was challenged by the low levels of p24 produced in

cultured PBMCs from hematologically normal BLV-

infected cattle. As anticipated, the assay did not detect

BLV p24 in PBMCs isolated freshly.

Acknowledgements

This research was funded by grants from the Natural

Sciences and Engineering Research Council of Canada

and the Ontario Ministry of Agriculture, Food, and

Rural Affairs. M. van den Heuvel was the recipient of a

Dairy Farmers of Ontario doctoral scholarship.

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Intra-assay variability in optical density (OD450 nm) of the BLV p24 sELISA

Sample Number of replicatesb Mean OD450 nm Standard deviation (S.D.) CV (%)

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Lysate from a cow with PL 20 0.642 0.062 9.8

Affinity-purified p24 17 0.608 0.056 9.2

a Lysate refers to the contents of cultured PBMCs lysed by the addition of a 10% aqueous solution of n -octyl-bD-glucopyranoside.b Indicates that a sample was repeated 17 or 20 times on one occasion.

Table 3

Inter-assay variability in optical density (OD450 nm) of the BLV p24 sELISA

Sample Number of replicatesb Mean OD450 nm S.D. CV (%)

Lysate from an asymptomatic cowa 6 0.092 0.022 5.1

Lysate from a cow with PL 6 0.555 0.052 9.4

Affinity-purified p24 20 0.548 0.062 12.9

a Defined in Table 2.b Indicates that a sample was repeated in triplicate on 6 or 20 occasions over the duration of the study.

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