A highly sensitive enzyme-linked immunosorbent assay to quantitate antibodies to Epstein-Barr virus membrane antigen gp340

Journat of Virologicat Methods, 9 (1984) 201-208

201

Elsevier JVM 00337

A HIGHLY

SENSITIVE

QUANTITATE ANTIGEN

ENZYME-LINKED

ANTIBODIES

IMMUNOSORBENT

TO EPSTEIN-BARR

ASSAY TO

VIRUS MEMBRANE

gp340

BEVERLEY

J. RANDLE

and M.A. EPSTEIN

Department of Pathology, University of Bristol, The Medical School, University Walk, Bristol, 858 ITD, U.K. (Accepted

19 June

1984)

An enzyme-linked Epstein-Barr

be a thousand-fold was possible during

Epstein-Barr

assay (ELISA) antigen

has been developed

(MA) glycoprotein,

more sensitive than conventional

to follow accurately

a course

ELISA

immunosorbent

(EB) virus membrane

the sequential

for the detection

indirect immunofluorescence production

of antibodies

to

The assay was found to

8~340, in tamarins.

tests and consequently

of specific antibodies

it

to gp340 by tamarins

of immunization.

Epstein-Barr

virus membrane

antigen

antibodies

to EBV, MA gp340

Epstein-Barr

virus

virus vaccine

fNTRODUCTION

Recent substantial against Epstein-Barr

progress in the development of a prototype subunit vaccine (EB) virus infection (Epstein and Morgan, 1983; Epstein, 1984)

was greatly facilitated by the availability of a highly specific, quantitative radioimmunoassay (North et al,, 1982a) which permitted the elaboration of an efficient preparation method for the antigen required (Morgan et al., 1983). The vaccine is based on a high molecular

weight glycoprotein

component

(gp340) of EB virus membrane

anti-

gen (MA) and although immunogenicity studies have been undertaken with gp340 (North et al., 1982b; Morgan et al., 1984), these studies could not be fully exploited in the absence of a sensitive test to quantitate antibody responses. A rapid enzyme-linked immunosorbent assay (ELISA) has therefore been developed for antibodies to gp340 in cottontop tamarins, the animal of choice for work with EB virus in vivo (Epstein, 1984). The present paper gives details of t’his assay and illustrates the use of the ELISA to monitor production of specific antibodies to gp340 by a tamarin on immunization with the vaccine preparation.

0166-0934/X4/$03.00

S 1984 Elsevier Science Publishers

B.V.

202 MATERIALS

AND METHODS

Preparation

of gp340 for coating ELISA

gp340 was prepared (Hoffman

by affinity

plates

chromatography

using monoclonal

et al., 1980; the cell line was kindly provided

MD, U.S.A.).

An antibody

immunosorbent

column

antibody

by Dr. G. Hoffman, was prepared

72A 1

Baltimore,

by cyanogen

bro-

mide coupling of Sepharose CL-4B (Pharmacia Fine Chemicals, London, England) with 72Al antibody isolated by ammonium sulphate precipitation of 40 ml ascitic fluid from BALB/c mice bearing 72Al tumours. An extract of B95-8 cell (Miller et al., 1972) membranes in 0.5% (w/v) n-octylglucopyranoside (og) (Sigma Chemical Co., Poole, Dorset, England) in phosphate buffered saline (PBS) was loaded on to an affinity column of 72Al Sepharose. After extensive washing with ogPBS buffer the column was eluted with 50 mM diethylamine, pH 11.5, in ogPBS and immediately neutralized with 1 M Tris HCI, pH 8. The eluted material was concentrated and applied to a Sephacryl S-300 column (Pharmacia Fine Chemicals) in ogPBS and fractions were monitored by measurement of optical density at 280 nm. The peak of material eluting immediately after the void volume was collected. The material was electrophoresed to determine the purity of the preparation and stored until use in 50 ug aliquots in ogPBS at -70°C. Full details of this procedure are given by Randle et al. (1984). Collection

of test samples for titration

in ELISA

Supernatants containing 72Al monoclonal antibody were collected from rapidly growing cultures of the 72A1 cell line. The supernatant pool was found to have a titre of 1: 32 when reacted with B95-8 cells in an indirect immunofluorescence assay. Supernatants

were also collected

from cultures

of the KLHl

Randle, unpubl. data) which secretes a mouse IgGl antibody limpet haemocyanin. KLH 1 was used to control for non-specific in the ELISA. Tamarin

Supernatants

were stored

sera were collected

cell line (prepared

by

recognizing keyhole binding of antibody

at -2O’C until use.

by venepuncture

before and at intervals

during

the

administration of gp340 in artificial liposomes (North et al., 1982b). The sera were stored at -7O“C until use. Two human sera were collected, one from an individual with high levels of antibody to EB virus MA, and the second from an individual with negligible reactivity to EB virus MA, as assayed by indirect immunofluorescence. Determination

of optimum

conditions for ELISA

(I) Conditions for antigen coating 96-well microtitre plates (Dynatech,

Guernsey,

C.I.) were coated with 50 ul of gp340

203

solution per well at concentrations of 1 pg or 5 ug per ml in a 15 mM carbonate135 mM bicarbonate buffer, pH 9.6. The plates were incubated overnight at 4°C. The coating solution was removed by a suction pipette and the plates were then incubated with either 100 ul of PBS or 100 ul of blocking solution (10% (v/v) foetal calf serum in PBS) for 2 h at room temperature. Plates coated with two concentrations of gp340 under two different conditions were then reacted with dilutions of 72A 1 or KLH 1 culture supernatants. PBS or blocking solution was then removed and duplicate 50 ul test samples were applied to the coated wells and incubated for 1 h at 4°C. After this, the test samples were removed and the wells were washed twice with 200 ul of PBS Tween (0.05% (v/v) Tween 20 in PBS) (Sigma Chemical Co.) per well. 50 ul of rabbit anti-mouse IgG peroxidase conjugate (Miles Research Laboratories, Stoke Poges, England), diluted l/500 in BSA buffer (0.5% (w/v) bovine serum albumin in PBS), were then placed in the wells and incubated for 30 min at 4’C. The peroxidase conjugate was removed and the wells were washed three times with 200 ul PBS Tween. After washing, the presence of bound second layer antibody peroxidase conjugate was assessed by addition of 100 ul of substrate solution per well. This consisted of 4 mM orthophenylene diamine (Sigma Chemical Co.) in 25 mM citrate/50 mM phosphate buffer, the substrate being dissolved immediately before use and with 20 ul of 30% hydrogen peroxide (Sigma Chemical Co.) added to 50 ml of the solution. The enzyme reaction was stopped after 6 minutes by the addition of 50 pl of 2.5 M sulphuric acid per well. The optical density of the enzyme product was then measured at 492 nm in a Titertek Multiskan (Flow Laboratories, Irvine, Scotland). (‘2) renditions for detection ofbound tamarin antibody by pe~~xidase conjugate reagents

96-well microtitre plates were incubated with 50 ul per well of a gp340 solution at a concentration of 5 &ml in carbonate/bicarbonate buffer pH 9.6. After incubation overnight at 4”C, the coating solution was replaced by 100 ul blocking solution per well and incubated at room temperature for 2 h. A tamarin serum, known to recognise gp340 by immunoprecipitation of lactoperoxidase labelled gp340 (Epstein and Morgan, 1984) was used as a test sample to determine the optimal detection method for tamarin antibody bound to gp340 in the ELISA. After removal of the blocking serum, 45 ul aliquots of tamarin serum were incubated for 1 h at 4’C in wells coated with gp340. The wells were washed with PBS Tween as above and then treated with one of the following peroxidase reagents which provided detection systems: (i) 50 ul of l/200 dilution of rabbit anti-human IgG peroxidase conjugate (Miles Research Laboratories) in BSA buffer per well for 30 min at 4°C; (ii) 50 pi of l/IO dilution of rabbit antibody to tamarin Ig (prepared by Dr. J.R. North, Department of Pathology, University of Bristol) per well for 30 min at 4”C, followed after washing with PBS Tween with 50 1.11 of l/200 dilution of goat anti-

204

rabbit

IgG peroxidase

conjugate

(Miles Research

Laboratories)

in a BSA buffer for

30 min at 4% After incubation reagent

detected

the wells were washed with PBS Tween and the bound peroxidase by enzyme

reaction

as above.

Titration of tamarin antibody to gp340 by ELISA Volumes was diluted prepared,

of sera for assay were limited and consequently 15 pi of each test serum 1 : 10 in BSA buffer and, from this start sample, a series of dilutions was

Plates were coated with gp340 at a concentration blocking solution as above. Duplicate

45 ul samples

of 5 &ml

of test sera were incubated

and then reacted with

for 1 h at 4°C in wells coated

with gp340. The wells were washed twice with PBS Tween and then incubated with 50 ul aliquots of l/200 dilution of rabbit anti-human IgG peroxidase conjugate in BSA buffer for 30 min at 4°C. After incubation, the wells were washed three times with PBS Tween and the bound above.

peroxidase

conjugate

45 ul duplicate samples of two human also assayed by ELISA on each occasion done at different times.

was then detected

by enzyme reaction

as

sera, prepared as a series of dilutions, were to provide a standard caIibration for assays

RESULTS

Determination of optimum conditions for ELISA Experiments

were performed

to establish

the optimum

conditions

for antigen

coating, for the detection of bound antibody by a second layer, peroxidase-labelled reagent, and for standardization between assays. The purity of gp340 sampies used in the experiments was determined by electrophoresis in a 5% (w/v) SDS polya~rylamide gel. Figure 1 shows that such preparations contained approximately 95% gp340 with a 5% contamination with low molecular weight polypeptides. Initially, a series of dilutions of 72A 1 culture supernatant was incubated in microtitre wells coated with gp340 at various concentrations. The plates were examined to define conditions where high levels of enzyme reaction were obtained in wells containing the 72Al antibody and low levels in those to which the non-specific control KLHl antibody had been added. The titre of a test sample was defined by the last dilution which gave a value above 0.5 optical density at 492 nm, the value obtained when reacting the non-specific antibody in the ELISA. 72A1 culture supernatant was found to have an end-point titre of 1: 10,000. The greatest sensitivity for the detection of bound antibody was found when wells

205 C

CIP340

1

2

Fig. 1. Coomassie the ELISA.

Blue stain of 5% (w/v) SDS polyacrylamide

gp340 was isolated

with diethylamine, the void volume I%galactosidase

by affinity

was separated was collected

chromatography

by gel filtration and concentrated

(116 kd), phosphorylase-b

on Sephacryl

gel of gp340 sample used for coating plates in using 72AI Sepharose.

The material,

S-300. The peak running

(lane 2). Marker

proteins

immediately

eluted after

(lane I) were myosin (200 kd),

(97 kd) and bovine serum albumin

(68 kd). Arrow

indicates

origin.

were coated with gp340 at 5 &ml serum in PBS blocking

and subsequently

reacted with 10% (v/v) foetal calf

solution.

Two methods were available for detection of tamarin antibody binding to gp340. First, bound antibody was detected by reaction with an anti-human IgG peroxidase conjugate. Secondly, to increase possible specificity and sensitivity, the bound tamarin antibody was reacted with an intermediate, unlabelled rabbit anti-tamarin antibody and bound rabbit antibody was then detected with an anti-rabbit IgG peroxidase conjugate. It was found that both peroxidase reagents detected tamarin antibody bound to gp340. However, the double sandwich method using the intermediate rabbit antibody, gave high levels of background reaction in gp340 coated wells incubated with a non-specific antibody. Consequently an anti-human IgG peroxidase conjugate was used to detect tamarin antibody binding to gp340 in the ELISA. Finally, when the two human sera of low and high gp340 reactivity were incorporat-

206

ed into

the ELISA

between

assays performed

was defined

to provide

calibration

at different

curves

and standards

times, the end-point

for comparison

titre for the low standard

serum in excess of 1 : 10,000.

as 1 : 400 and that for the high standard

Measurement of titres of antibodies to gp340 in tamarin serum Samples of blood were collected from a tamarin before, and at intervals during, immunization with artificial liposomes containing gp340. Sera were prepared and a series of dilutions made to establish the titre of these samples. The diluted sera were incubated in wells of microtitre plates coated with gp340. Bound tamarin antibody was detected by incubation of the gp340 coated wells with an anti-human immunoglobulin G peroxidase conjugate and subsequent development for bound peroxidase reagent by enzyme reaction. The results are given in Fig. 2 which shows the optical density of the enzyme product at 492 nm plotted against the dilution of the serum under test. The end-point titres of

. prebleed bleed I . bleed2 u bleed3

. bleed 4

I___,

1

1

1.100

1:I0

1:lOOO

Serum dilution Fig. 2. Titration liposomes dilution collected

of sera from

containing

a tamarin

~~340. The optical

before density

and at intervals of the enzyme

of the serum sample under test. Five serum samples at intervals

to determine

during

the end-point

immunization.

during

product

were tested: a prebleed

An optical density of0.5. indicated

titre of the samples.

immunization

with artificial

at 492 nm is plotted

against

the

sample and four sera

by the broken line, was used

207

the sera before and after immunization of antibody

were detected

liposomes

containing

strikingly

were then calculated.

in the sera. On immunization

gp340, the end-point

with a maximum

negligible

levels

with artificial

titre of the sera was found

titre of 1 : 1,000 noted

serum

Initially

of the tamarin at the fourth

to increase bleed.

DISCUSSION

The ELISA developed for the measurement of specific antibody to the gp340 glycoprotein subunit of EB virus MA, was found to be rapid and very sensitive. The assay was able to detect antibodies to gp340 at dilutions one thousand-fold greater than could be detected by standard typed seronegative culture supernatant,

immunofluorescence

techniques.

A human serum,

by immunofluorescence assay, gave a titre of 1: 400 and 72Al of titre 1 : 32 by indirect fluorescence, was found to have an

end-point titre 1 : 10,000. This is in marked contrast to the recent finding of Luka et al. (1984) that ELISA and immunofluorescence assays had very similar sensitivities when applied to human sera recognising MA. When the ELISA was used to monitor the production of specific antibodies to gp340 in a tamarin immunized with gp340 incorporated into artificial liposomes, the assay was able to detect specific antibody production after two doses. The titre of the tamarin serum was found to increase with immunization and reached a maximum end-point titre of 1 : 1,000 after the final dose of gp340 incorporated into artificial liposomes. Thus, an ELISA has been developed which is suitable to monitor with great sensitivity the sequential production of specific antibodies to gp340 by tamarins, and which will therefore provide an important of vaccination procedures in vivo.

and rapid technique

for the assessment

ACKNOWLEDGEMENTS

The authors thank Dr. Andrew Morgan for helpful discussions and Dr. Kay Howe for the use of a Titertek Multiskan ELISA reader. The work was supported by the Medical Research Council, London (SPG8007184), and the Cancer Research Campaign, London Kong).

(out of funds donated

by the Bradbury

Investment

Company

of Hong

and Lymphoma

Research

REFERENCES

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M.A.,

Epstein,

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