Radioimmunoassay of adjuvant-associated porcine parvovirus using a monoclonal antibody in a nitrocellulose membrane system

Journal of Virological Methods, 12 (1985) 193-198

193

Elsevier JVM 00449

RADIOIMMUNOASSAY PARVOVIRUS

OF ADJUVANT-ASSOCIATED

USING A MONOCLONAL

NITROCELLULOSE

JONATHAN

MEMBRANE

B. KATZ

and RICHARD

PORCINE

ANTIBODY

IN A

SYSTEM

A. VAN DEUSEN

Biologics Virology Laboratory, National Veterinary Services Laboratories, Veterinary Services, Animal and Planr Health Inspection Service, U.S. Department of Agriculture, P.O. Box 844, Ames, IA 50010, U.S.A. (Accepted

6 August

A quantitative

1985)

and simple

indirect

radioimmunoassay

(PPV), employing

a monoclonal

IRIA was equally

sensitive to live or inactivated

bound

radioactivity

commercially radioactivity

and PPV quantity

used adjuvants,

directed

were prepared

between

in adjuvants

of 16 commercial

vitro estimation

according

radioimmunoassay

hydroxide

bound

At fixed adjuvant

radioactivity

to commercial

parvovirus

for porcine

to nitrocellulose

(AH) and carboxyvinyl

manner.

mass in adjuvanted

porcine

was developed

PPV adsorbed

PPV. There was a linear relationship

parvovirus

membrane.

The

between membrane-

(HA) units of virus. Two

polymer

(CP), reduced

concentrations,

bound

there were, never-

and HA units of PPV. Known

vaccine formulations.

PPV vaccines was estimated

of antigenic

(IRIA)

against

within a range of IO-83 hemagglutinating

aluminum

in a concentration-dependent

theless, linear relationships content

antibody

amounts

Using these standards,

by IRIA. The IRIA may be one practical

of PPV the PPV

method of in

vaccines.

adjuvanted

vaccines

INTRODUCTION

Potency expensive, procedures

evaluation procedures

of adjuvanted vaccines customarily requiring host animals or laboratory

may include

challenge

protection,

or assays

involves lengthy, animal surrogates. for antibodies

often These

by plaque

reduction, indirect fluorescent antibody, hemagglutination inhibition, orenzyme-linked immunosorbent assay methods. Although there is currently no in vitro method for the complete assessment of adjuvanted vaccine efficacy, protein immunoassay techniques have enabled direct measurement of selected antigens (Hawkes et al., 1982; Furuya et al., 1984; Porter and Porter, 1984). The ability of nitrocellulose to bind biological macromolecules, as noted by Tsuchida et al. (1973), has been utilized by Southern (1975) and others (Hawkes et al., 1982; Flores et al., 1983) for rapid immobilization of the antigen to be measured. Kostenbader and Cliver (1983) demonstrated that viral adsorption to nitrocellulose could be quantitative. With the availability of an anti-porcine parvovirus (PPV) monoclonal antibody, it was of interest to 0166-0934/85/$03.30

0 1985 Elsevier Science Publishers

B.V. (Biomedical

Division)

194

determine

if complex,

radioimmunoassay. nal antibody

adjuvanted

PPV vaccines could be evaluated

The assay described

directed

against

lose. Bound monoclonal

PPV present

antibody

for viral content

below employs an anti-PPV in vaccine specimens

by

mouse monoclo-

dotted on nitrocellu-

is then detected using 1251-labelled sheep anti-mouse

immunoglobulin. MATERIALS

AND

METHODS

Reference virus PPV virus (NADL-8 Services Laboratories, embryonic

swine kidney

strain, Diagnostic Virology Ames, IA) was expanded cells. Virus was concentrated

Laboratory, National in a rapidly growing in semipurified

Veterinary culture of

form through

brief sonication of infected cells, pH elevation to 8.6, and differential centrifugation by the method of Dr.W. Mengeling (1985, National Animal Disease Center, Ames, IA; pers. comm.). The resulting material had 4,000 hemagglutination (HA) units per 50 pl and was frozen at -80°C until use. One HA unit was defined as the minimum quantity of virus per 50 ul which produced complete agglutination when added to an equal volume of 0.5% (v/v) guinea pig red blood cells suspended in 0.01 M phosphate buffered saline (PBS), pH 7.4. Formalin-inactivated and binary ethyleneimine-(BEI) inactivated testing.

PPV were also stored similarly

to await indirect

radioimmunoassay

(IRIA)

Anti-PPV monoclonal antibody Several hybridoma cell lines producing monoclonal anti-PPV antibodies were obtained (Mengeling et al., 1984) recloned, and expanded to produce ascites in BALB/c mice using standard procedures (Van Deusen and Whetstone, 1981). Ascites from clone 3C9Dll (MAB) had a PPV hemagglutination-inhibition titer of 256, and was functional in IRIA. Optimal signal-to-background ratios were achieved at a 1 : 50 dilution of MAB in blocking solution. Blocking solution in this and all subsequent steps consisted of 5% (w/v) generic non-fat dry milk and 0.02% (w/v) sodium azide in PBS. 1251-labelled second antibody *251-labelled sheep anti-mouse Corporation, Arlington Heights, dilution minimal

immunoglobulin was purchased from Amersham IL, and used within 4 wk of production. A 1 : 300

of isotopic second antibody background.

in blocking

solution

yielded optimal

signal with

Adjuvants and formulations of adjuvant-associated virus preparations Aluminum hydroxide (AH), carboxyvinyl polymer (CP), and a pyran copolymer (AA) were obtained either from firms producing USDA-licensed inactivated PPV vaccines or from Dr. R. Levings, Biologics Virology Laboratory, NVSL, Ames, IA.

195

Formulations

resembling

proprietary

except that known amounts ul final mixture)

adjuvanted

PPV vaccines

of PPV reference virus (80,60,40,20,

were added to form standards

ence AH and CP, supplied

for IRIA testing.

known

contained

concentrations

were prepared

gel in EMEM-FBS.

referin

(v/v). Each of these

40 HA units of PPV per 10 ul final product.

Finally,

a series of

of PPV (80, 60, 40, 20, 10, 0 HA units per 10 ul final mixture)

in each of 4 media: 0.01 M PBS, Eagle’s Minimal

5% (v/v) fetal bovine

Additionally,

as 3% (w/v) and 10% (w/v) gels in water, were prepared

0.01 M PBS at 20%, 15%, lo%, 5%, 2.5% and 0% concentrations references

were constructed, 10,O HA units per 10

serum (EMEM-FBS), The FBS was tested

antibody

at a 1 : 2 dilution

different

media provided

against

IRIA technique Nitrocellulose membranes

and found

100 TCID,,

additional

references

(Biorad

Essential

Medium with

10% AH gel in EMEM-FBS, negative

and 5% CP

for neutralizing

of PPV. These dilutions

PPV

of PPV in

for IRIA evaluation.

Laboratories,

Richmond,

CA) were rinsed in

deionized water, dried, and sliced into 1 cm* squares. Then ul aliquots of the test material were dotted onto the squares, dried, and the squares placed individually in wells of a 24-well disposable

tissue culture

plate. One ml of blocking

solution

was

added per well for 1 h. This and all other reactions were at room temperature and were conducted with gentle rotary mixing on an orbital shaker. Following aspiration of blocker, 250 ul of MAB diluted in blocker was added per well and incubated 90 min followed by four lo-min washes with 1 ml blocker per well. Then 250 ul of L251-sheep anti-mouse immunoglobulin in blocker was added per well. After 90 min this reagent was aspirated, followed by four IO-min washes with blocker as before. Each square was then removed and counted in a Packard Multi-Prias gamma counter (Downers Grove, IL) for 10 min. RESULTS

The IRIA was used to measure from 0.8 HA units up to 800 HA units of PPV applied to nitrocellulose measured,

squares.

However,

and the IRIA exhibited

for routine

use, a range of lo-80

a linear relationship

between

bound

HA units was radioactivity

and HA units of virus within these limits (Fig. 1). Live, formalin-inactivated, and BEI-inactivated virus preparations were equally well recognized. Reference suspensions of high-titered pseudorabies virus grown in swine kidney cells went unrecognized by this MAB-specific system, as did high-titered reference suspensions of transmissible gastroenteritis virus grown in swine testicle cells (both viruses supplied by Biologics Virology Laboratory, NVSL, Ames, IA). This information indicated that the assay was insensitive to typical swine host cell antigens and nutrient serum proteins which might be commonly ture.

employed

in producing

virus-laden

As shown in Fig. 2, AH gel and CP gel both significantly

fluids for vaccine

manufac-

(P< 0.05) depressed

IRIA

196

0;

0

,

,

,

,

,

,

,

IO

20

30

40

50

60

70

, 80 ADJUVANTPERCENTAGE &GEL INOOIMPBSI

HA UNITS OF PPV

Fig. 1. Titration

of PPV in 0.01 M PBS by IRIA.

Fig. 2. Effect of adjuvant duplicate

measurements

percentages

level on IRIA (background

of AH gel (o---o)

sensitivity

Each point is a mean of triplicate

measurement

subtracted)

of 40 HA units of PPV. All points

are means

of 40 HA units of virus in the presence

of

of variable

or CP gel (o-o).

to PPV in a concentration

dependent

manner,

but did not significantly

elevate background or eliminate the linearity of the recognition given adjuvant concentration tested (Fig. 3). Figure 4 presents

measurements

titrations

of standard

amounts

tive formulations resembling several commercial the slope of the IRIA titration and particularly

phenomenon

of PPV prepared

at the

in adjuvant/addi-

vaccines. All formulations decreased affected detection of higher levels of

PPV, indicating reduction in sensitivity. The formulation associated with the greatest depression in bound radioactivity at any given PPV level was one with a high level of

0

IO

20

30

40

50

60

70

80

HA UNITSOF PPV

Fig. 3. Titration in EMEM-5%

of PPV by IRIA in the presence of PBS (A---A), EMEM with 5% FBS (a--a), FBS (o---o),

or 5% CP gel in EMEM-5%

FBS (o-o).

10% AH gel

All points are means of duplicate

measurements. Fig. 4. Titrations is denoted triplicate

of PPV in the presence of 8 simulated

next to each titration. measurements.

All preparations

commercial

adjuvants.

made in EMEM-%%

Primary

adjuvant

FBS. All points

component are means

of

197

AH gel (up to 20% v/v). Experiments units

of PPV/lO

supernatants were essentially

measured.

negative

for PPV by IRIA, indicating

AH-based

preparation,

much PPV as the intact

measured

and

of both preparations

virus removal

substantially

by ultracentrifu-

lower in PPV than did

while the 2,000 X g CP supematant

CP preparation.

of 60 HA intact;

of the same preparations

The 85,000 X g supernatants

The 2,000 X g AH supernatant

the intact

in which preparations

of 2,000 X g and 85,000 X g centrifugations

were also concurrently gation.

were conducted

ul in 10% AH gel and in 5% CP gel were measured

These results indicated

contained

that a portion

as

of the

PPV in the AH formulation behaved as if it was associated with the AH particulate, and was removed with it by the low speed centrifugation, which easily pelleted AH but not CP. Commercial vaccines utilizing high levels of AH were also tested intact, and supernatants

of these vaccines centrifuged

well. It was found

2,000 X g supernatants parent products.

at 2,000 X g and 85,000 X g were analyzed

that 85,000 X g supernatants contained

substantially

The 2,000 X g supernatants

as much PPV as did the respective tants were again low in detectable

contained

minimal

less PPV than the intact

of AA and CP-based

intact products, PPV.

although

as

PPV, and that the AH-based

vaccines

contained

the 85,000 X g superna-

DISCUSSION

The IRIA demonstrated linearity over a practical range of PPV concentrations also detected very low and very high levels of PPV. The system recognized formalin-inactivated,

and BEI-inactivated

PPV equally

well, indicating

and live,

that the epi-

tope recognized by the MAB was not destroyed by the inactivants, at least for analytical purposes. When selecting a MAB for use in IRIA, one produced against an inactivated antigen might potentially measure a denatured epitope which is not antigenically protective. It would be desirable to use monoclonal antibodies which recognized only antigens which induced protective in vivo responses. The absolute specificity of monoclonal antibodies renders them highly useful when analyzing complex

fluids containing

cellular,

the basis of many conventionally

nutrient prepared

It was hypothesized that adjuvants many vaccines might noncompetitively tants and/or

enzymes

in enzyme-linked

serum, and viral proteins. cell-culture

origin

Such fluids form

vaccines.

and/or additives (e.g. thimerosal) present in inhibit, adsorb, or sterically hinder the reaccolorometric

assay systems which have been

used for measuring purified antigens (Furuya et al., 1984; Porter and Porter, 1984). These potential enzymatic complications might be expected to be lessened with a radioimmunological approach. IRIA is also exquisitely quantitative and unaffected by optical characteristics of nitrocellulose or material adsorbed to it. However, the radiological approach does involve a relatively unstable, expensive, and potentially hazardous immunoreagent. The cause of the significant decrease in bound radioactivity as a function of AH and CP concentration is enigmatic. There are at least three possible explanations of the

198

observed bound

decrease.

Firstly,

there may be some detachment

virus from the nitrocellulose

tendency

of nitrocellulose

particuiar,

during

to bind macromolecules,

the fine particulate

of adjuvant

the IRIA procedure.

AH adjuvant

its affinity

is unknown.

and adjuvant-

Despite

the general

for CP, AA, and, in

However,

results

of the

centrifugation experiments indicated that some or all of the AH was retained on the membrane, inasmuch as intact vaccines contained more measurable PPV than did the 2,000 X g supernatants

of the same vaccines,

which were essentially

ly, some virus may be loosely adjuvant-associated detach

into the liquid medium

during

Lastly, high levels of adjuvant noclonal

antibody

tive formulations

binding

affect IRIA sensitivity,

Second-

but not nitrocellulose-bound,

the IRIA even if adjuvant

may sterically

in the appropriate

AH-free.

itself remains

and bound.

hinder the MAB or the labelled anti-mosteps. Because specific adjuvant/addi-

as shown in Fig. 4, measurement

mass in a specific vaccine would require knowledge tion of that product.

of the adjuvant/additive

of antigenic composi-

Nevertheless, using homologous adjuvant/additive preparations containing known quantities of a reference antigen, it may be possible to assess some intact, adjuvanted, inactivated vaccines using IRIA to estimate antigenic mass. This would be one approach vaccines,

to rapid, practical, and economical in vitro potency testing of adjuvanted inactivated vaccines, or subunit vaccine products. The effects of specific

adjuvants

and additives

must be carefully

considered

for each vaccine in view of their

effects on IRIA sensitivity. ACKNOWLEDGEMENTS

We thank Mr. S.K. Hanson and Ms. I. Peterson for encouragement and excellent technical assistance, and Dr. R. Levings and Dr. W. Mengeling for encouragement and advice. REFERENCES

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