single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis a virus

ELSEVIER

Journal of Virological Methods

Journal of Virological Methods 68 (1997) 81- 87

Single-antibody in situ enzyme immunoassay for infectivity titration of Hepatitis A virus

Steven Borovec *, Eric Uren

Virology Group, Reseurch and Development, CSL Limited, Bioplasma Division, 189-209 Camp Road. Broadmeadows 3047, Australia

Received 10 April 1997; received in revised form 21 June 1997; accepted 23 June 1997

Abstract

Hepatitis A virus (HAV) establishes a persistent infection in cultured cells, with minimal effect on host cell metabolism. As a result, the virus produces very little, if any, cytopathic effect (CPE), even with cell culture-adapted strains. This feature precludes the use of a plaque or standard endpoint assay (using CPE as an indicator of infection) for the titration of infectious virus. The radioimmunofocus assay (RIFA) is the standard method for HAV titration, though this method is labour intensive and requires the use of radioisotopes. To this end, a single-antibody in situ enzyme immunoassay (EIA) has been developed, using binding of a perioxidase-labelled monoclonal antibody to fixed cell monolayers as an indicator of infection. This novel assay is highly reproducible, can be read by eye, and is

suitable for high throughput situations. Furthermore, the assay has been validated against the RIFA making it suitable for use in studies validating the safety of therapeutic biologicals for human use. 0 1997 Elsevier Science B.V.

Keywords: Hepatitis A virus; Titration; Enzyme immunoassay; Plasma

1. Introduction

HAV is the prototype of the hepatovirus genus within the family Picornaviridae. The 27-nm particle is nonenveloped and contains a single strand of positive-sense RNA as the genome. The replication cycle of HAV is unusually protracted in cultured cells, possibly due to a combination of

* Corresponding author. Tel.: + 61 3 9246545; fax: + 61 3 92465469.

limiting amounts of viral RNA (Anderson et al., 1988) and an overproduction of viral protease (Borovec and Anderson, 1993). Stability studies reveal the mature virion to be resistant to solvents (Siegl et al., 1981), high temperatures (Parry and Mortimer, 1984), and extremes of pH (Scholz et al., 1989).

HAV is the causative agent of type A hepatitis

in humans. Laboratory findings include elevated liver enzymes in the serum, bilirubin in the urine, and increasing titres of specific antibody. The

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82 S. Borouec, E. Uren i Journal of Virological Methods 68 (1997) 81-87

disease is usually self-limiting though older pa- tients may progress to a fulminant hepatitis result- ing in death (Gust and Feinstone, 1988). A 224 week viraemic phase is noted (generally prior to the onset of symptoms) and so the possibility exists for transmission of HAV by the administra- tion of blood products, examples having occurred recently in Italy, Belgium, Germany, Ireland (Vermylen and Peerlinck, 1994) and elsewhere.

To validate the efficacy of viral inactivation procedures incorporated into plasma product manufacturing processes, it was necessary to de- sign an infectivity assay for HAV which was simple, robust, highly reproducible, sensitive and amenable to rapid processing of samples. Further- more, it was essential that the method be tolerant to the high concentrations of plasma proteins and salts often found in intermediate and final products under test.

2. Materials and methods

2.1. Cell cultures

A continuous African green monkey kidney cell line, BS-C-1, was purchased from ATCC at passage number 44. Cells were cultured in Eagle’s minimal essential medium (MEM; CSL Bio- sciences, Australia) supplemented with non-essential amino acids, glutamine, HEPES, and 10% foetal calf serum. Cells were used between passage number 45 and 60.

2.2. Virus

Supplies of HAV (strain HM175A.2) were obtained from the Hepatitis research unit, Macfar- lane Burnet Centre for Medical Research, Melbourne (coded HAV022), or produced by CSL Bioplasma (coded HAV032). Both batches of virus were grown and purified according to the method of Bishop et al. (1994). Briefly, BS-C-1 cells established in multi-layered cell factories are infected with HAV, maintained for 5 days, and removed by trypsinisation. The infected cells were washed, lysed with a nonionic detergent and the cytoplasmic fraction adjusted to contain 1% (w/v)

sodium dodecyl sulphate before purification by discontinuous sucrose-glycerol density gradient ultracentrifugation. The virus-containing fractions were pooled, aliquotted and frozen at - 70°C until use.

2.3. Monoclonal antibody

The HAV-specific antibody K3-4C8 (MacGre- gor et al., 1983) was used as a perioxidase-linked, protein A purified monoclonal antibody. The antibody was stored at - 20°C in buffered glycerol. Studies by Stapleton et al. (1993) have shown this antibody is neutralising and binds an epitope formed upon the assembly of pentameric capsid subunits.

2.4. Titration qf injkctious virus by in situ EIA

Microtitre plates (96 well, flat-bottomed, tissue culture grade, Nunc Inc, Naperville, IL) were seeded with 100 ,ul of a suspension of BS-C-l cells prepared in growth medium ( * 104.’ cells/ml). After incubation at 37°C (in 5% CO, until conflu- ency was just achieved) 100 ,~l dilutions of virus sample (prepared in MEM supplemented with 2% FCS) were added to test wells (eight wells per dilution), without removal of growth medium. To ensure detection of an endpoint, 3-fold serial dilutions of each sample were prepared to inoculate two microtitre plates (i.e. 20 dilutions in total). Two columns of mock-infected wells were included on each plate, as well as one extra column on the first plate for blanking the plate reader (where used). Monolayers were then incubated for a further 14 days, after which the medium was removed. Cells were fixed using 100 ~1 per well of a 1: 1 mixture of acetone and methanol (precooled to 4°C) for 5 min. The fixative was removed, and monolayers allowed to dry (uncovered) at room temperature for at least 2 h.

The presence of virus-specific antigen was detected using a l/800 dilution of monoclonal antibody (prepared in PBS, 1% [w/v] Carnation* skim milk powder; 100 ,~l per well). Antibody was added to test and mock-infected wells, but not to blanking wells. After incubation at 37°C for 1 h, the antibody solution was removed and monolay-

S. Borovec, E. Uren /Journal oj Virologic,al h4rtltod.v 68 (1997) 81-87 83

ers washed three times (at 300 pi/well) with PBS

plus 0.1% [w/v] Tween 20, using an automated plate washer. Immediately after washing, bound

antibody was detected using hydrogen peroxide (BDH, 30% [w/v]; l/2400) and O-Phenylenedi-

amine (OPD; Sigma) prepared in a citrate/phos-

phate buffer (final OPD concentration = 0.6

mg/ml; 100 pljwell). The colour change was

monitored visually, and stopped by the addition of 100 ~1 1 M sulphuric acid per well, after

approximately 15 min. Where indicated, plates

were read spectrophotometrically at 492 mm. A positive/negative (P/N) ratio was determined us-

ing mock-infected wells as the negative value.

Wells are easily scored by eye as either positive or negative. with no intermediate reactions

having been noted. The titre of infectious virus was determined by the method of Spearman and

Karber (Schmidt and Emmons, 1989) which is based on the 50% tissue culture infective dose (TCID,,,) endpoint. Using the assay format described, infectious virus titre could be expressed to a 95% confidence level, with the unit being TCID,,,/ml of undiluted sample.

This method for virus quantification is a modified plaque assay developed by Lemon et al. (1983) later modified by Anderson et al. (1987). Briefly, dilutions of sample (prepared in MEM plus 2% FCS) were allowed to infect BS- C- 1 monolayers established on solvent-resistant coverslips. Cells were then covered with an agarose overlay and incubated for 7 days. After removal of the overlay, cells were fixed with acetone and allowed to dry overnight. Foci of infection were then detected using a “‘I-linked monoclonal antibody, and visualised by autora- diography.

followed by incubation for 10 days were used to titrate the monoclonal antibody and establish the optimal concentration. After staining with

antibody, OD readings were taken and P/N val-

ues calculated. Fig. 1 demonstrates that the most specific reaction was gained using a dilution of

1:800.

3.2. Relationship between PIN nalue and

multiplicity of infection

Serial, 3-fold dilutions of virus were assayed

as described in Materials and methods. After an incubation of 10 days, monolayers were stained with antibody (1:800) and OD readings taken.

Fig. 2 shows that dilution of virus was propor-

tional to the OD value gained, presumably due to the lower number of particles produced in wells infected at a low multiplicity (high dilution). Eight replicates of each dilution were tested, with a similar profile being seen for each, though the end point differed due to the proba- bility of finding a single particle in each respec-

tive well.

P/N a

I:50 1:Hlo I200 1:400 I:800 moo l3200 M400

3. Results

3.1. Establishing optimal antibody concentration

Cell monolayers infected with HAV at a multiplicity of infection (m.o.i) of 0.1 TCID,,/cell,

Fig. 1. Determination of optimum antibody concentration.

Wells of a microtitre plate established with BS-C-1 cells were

infected with HAV at a m.o.i. of 0.1 TCID,,,/cell. Blank and

control wells were included. Monolayers were incubated for IO

days and stained with varying dilutions of antibody K3-4C8

(12 wells per dilution), and optical densities read at 492 nm.

The resultant P/N values for each dilution were then averaged.

84 S. Borovec, E. Wren /Journal of’ Virological Methods 68 (1997) 81-87

OD 492nm

0.5

0.45

0.4

0.35

0.3

0.25

0.2

0.15

0.1

0.05

0

1 5 7 9 11 13 15 17 19 M San@0 EMion

Fig. 2. Effect of multiplicity of infection on OD reading. Serial 3-fold dilutions of virus were plated onto BS-C-I cells (8 wells per dilution) and incubated for 10 days. Monolayers were fixed and stained with antibody at 1:800. M; average of all mock-infected wells.

3.3. Relationship between P/N value and assay incubation period

Monolayers were inoculated with virus dilutions and incubated for varying periods (3, 5, 10, 14 or 20 days) before fixation and staining with antibody. OD values for each dilution were averaged from 8 wells. Fig. 3 shows that a consistent OD value is obtained with an incubation of 14 days, with no benefit from a longer

OD 492nm

“‘“~

0.5

0.4

0.3

0.2

0.1

0

1 2 3 4 5 6 7 9 9 San-@ Dilution

Fig. 3. Effect of incubation period on assay response. The five bars for each sample dilution represent increasing periods of assay incubation (from left: 3, 5, 10, 14, or 20 days).

incubation being noted. A consistent intensity of colour in all positive wells is important for accurate reading of the assay.

3.4. Inter- and intra-assay variability, and comparison with RIFA

To test the robustness of the assay, a number of titrations were carried out using different aliquots of the same virus stock, different stock, cells at varying passage level, and multiple operators. In addition, parallel titration by RIFA was performed in three instances as a direct comparison between the two methods. Analysis of all EIA titres shown in Table 1 demonstrates an average inter-assay variation of 1.8-fold for the duration of the study. Average intra-assay variability (i.e. multiple assay of virus from the same aliquot, performed by the same operator using cells at identical passage level) is 1.7-fold. Direct comparison with RIFA titres shows slightly higher sensitivity for the EIA, with the RIFA showing a similar intra-assay variation (1.6-fold).

4. Discussion

While the detection of viral antigen and nucleic acid is generally amenable to efficiently processing large sample numbers, the existence of a simple and sensitive assay for infectious HAV in a mi- croplate format has been lacking. The need for such an assay is particularly useful in the validation of virus inactivation and removal processes utilised in the manufacture of blood products. For these studies, the detection of antigen and/or nucleic acid is not appropriate since these con- stituents in isolation are either noninfectious (capsid protein), or of a greatly reduced infectivity (naked, full-length, genomic RNA) and therefore not indicative of the efficacy of a virus inactivation procedure. While it is possible to titrate infectivity on the basis of viral antigen produced de novo, such methods are labour-intensive, requiring the liberation and transfer of antigen to a separate plate for detection by EIA (Hart et al., 1994), immunofluroescence or immuoperoxidase (Nadala and Loh, 1990).

Table I

S. Borovec, E. Uren /Journal of Virological Methods 68 (1997) 81-87 85

Comparison of multiple titrations of HAV infectivity by EIA and RIFA

Date Operator Stock code Cell passage level Infectivity titre EIA to RIFA ratio

EIA” RIFAb

I /3:‘96 A 022 51 9.6’ 8.7’ 1.9 2,‘3:96 A 022 51 9.4’ 8.9’ 3.2 3’3196 A 022 51 9.3’ 8.7’ 4.0

21 ‘3/96 A 022 48 9.4 13’5:96 B 022 51 9.P 14!5,‘96 B 022 51 9.4d 2116196 A 022 50 9.4 2816196 C 022 41 9.4 4;7,‘96 C 022 49 9.4

22:7;96 A 032 50 8.5’

23:7i96 A 032 50 8.6”

24;7;96 A 032 50 8.7’

2;8,‘96 B 022 41 9.5’

2:8,‘96 D 022 41 9.6’

“ Log,,, TCID,,,/ml

h Log,,, Radioimmunofocus-forming units/ml

‘.d.‘.‘Titrations with same superscript are from the same aliquot.

A number of HAV infectivity assays have been developed, the most basic using plaque formation as an indicator of infection by a single virion (Anderson, 1987; Cromeans et al., 1987). In our hands, the ability of the HM175A.2 strains to cause plaques is variable and CPE is found to be generally minimal and of low reproducibility in both BS-C- 1 and FRhK-4 cell lines. Furthermore, CPE is an indirect indicator of infection and so when titrating infectious virus in potentially cytotoxic conditions, the environmental effect on cells can be mistakenly interpreted as virus-induced CPE. This is of partic- ular importance in virus inactivation validation studies where a low level of detection is desirable, so that a greater log reduction value can be demonstrated for a given inactivation process. Indeed, it was noted that cells exhibiting extensive cytotoxicity in the presence of product are still capable of supporting virus replication, and therefore exhibit specific reactivity in the EIA (data not shown).

tissue culture plates which can be infected with 0.9 ml of virus dilution. This reduces the limit of detection to 0.8 log,,TCID,,/ml.

The ability of this assay to yield high P/N values (Fig. 4) and therefore be read visually is of great advantage considering the time required to spectrophotometrically read plates and translate data into P/N values. The major factor which allows reading by eye is the extended period of assay incubation (14 days), although the use of a monoclonal antibody versus polyclonal antibody no doubt increases specificity. It is possible to shorten the incubation period, however this involves using a plate reader and setting threshold values for P/N data. In our hands it is generally possible to gain an accurate assay result after 5 days incubation using a P/N threshold of 2.0 (Fig. 3). In view of the large number of assays undertaken in our laboratory, it was decided to standardise on the longer assay incubation to minimise the man-hours required for each sample titrated.

The statistical limit of detection for the assay as described is 1.8 log,,TCID,,/ml, though this may be lowered by using monolayers of a greater surface area which are infected with larger sample volumes. A convenient method involves the use of 6-well

While it is not possible statistically to claim any greater sensitivity for the EIA as compared to the RIFA (given the low number of comparative runs carried out), it does appear that the EIA is slightly better with an average 5-fold higher titre being

86 S. Borovec, E. Uren /Journal of Virological Methods 68 (1997) 81-87

noted on direct comparison (Table 1). A 2-fold higher titre by EIA would be expected in any case due to the difference in assay type (EIA being expressed as 50% infectious dose, and RIFA being a quanta1 assay). It is possible however, that the efficiency of infection is greater for the EIA since the inoculum is in contact with the monolayer for 14 days, while for the RIFA infection is allowed to occur for 1 h before the agarose overlay is added.

Acknowledgements

We wish to thank Neil Goss, Randel Fang and Andrew MacGregor for critical reading of the manuscript. We are also grateful to Connie Broumis, Meghan Hattarki, Claire Lamble, and David Johnstone for technical assistance.

While this assay was developed for use in process validation studies, it would be equally suitable for any purpose requiring the detection of quantitation of infectious virus. For example, the method could be utilised for screening com- pounds which potentially block virus attachment to cultured cells, or for assessing the neutralising ability of immune globulin preparations. The in situ EIA method is also easily adapted for use

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single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis a virus

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