detection of antibody to varicella-zoster virus by competitive and igm-antibody capture immunoassay

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Journal of Medical Virology 8:89-101 (1981) Detection of Antibody to Varicella-Zoster Virus by Competitive and IgM-Antibody Capture I mm u noassay R. S. Tedder, P. P. Mortimer, and R. Bridget Lord Department of Virology, Middlesex Hospital, London (R.S. T., R. B. LJ, and Virus Reference Laboratory, Central Public Health Laboratory, Colindale, London (P. P. M.) A simple, sensitive, and specific competitive solid phase immunoassay for antibody to varicella-zoster virus (anti-VZV) is described. The assay uses reagents that can easily be prepared and is sparing of viral antigen. Using a solid phase coated with sheep IgG from a serum raised against human p-Fc, the same reagents will accu- rately detect anti-VZV IgM. nonimmune groups that closely correlated with a history of previous VZV illness. It was not affected by the presence or absence of antibody to other herpes viruses. The IgM antibody capture assay demonstrated the presence of anti-VZV IgM in sera from patients with both varicella and zoster and gave negative results in pa- tients with infections unrelated to VZV and in healthy blood donors. The competitive assay divided sera from children and adults into immune and Key words: anti-VZV, immunity to V2V, competitive immunoassny, IgM-antibody capture immunoassay INTRODUCTION Varicella, one of the most common communicable diseases of childhood, is caused by primary infection with varicella-zoster virus (VZV). Usually it is a mild illness, but in certain situations-for instance, if the individual is immunosuppressed- primary infection with VZV may be life threatening. After varicella there is lifelong immunity to clinically apparent exogenous reinfection, though a recrudesence of the original infection may occur in the form of zoster. This is probably due to the reacti- vation of VZV, which has remained latent in the posterior root ganglia following the initial primary infection [Hope-Simpson, 19651. Primary VZV infection is usually clinically manifest, and patients with a history of varicella (or zoster) are immune. However, some of those who do not recall having had either may also be immune. Low levels of antibody persist, probably for life, after varicella [Cradock-Watson et al, 1979].-The complement fixation (CQ test is not sufficiently sensitive to detect these levels of antibody reliably [Gold and Godek, Accepted for publication July 17, 1981. Address reprint requests to R.S. Tedder, Department of Virology, Middlesex Hospital, London W 1, England. 0146a615/81/0802.~ 0 1981 Alan R. Liss, Inc.

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Journal of Medical Virology 8:89-101 (1981)

Detection of Antibody to Varicella-Zoster Virus by Competitive and IgM-Antibody Capture I mm u noassay R. S. Tedder, P. P. Mortimer, and R. Bridget Lord

Department of Virology, Middlesex Hospital, London (R.S. T., R. B. LJ, and Virus Reference Laboratory, Central Public Health Laboratory, Colindale, London (P. P. M.)

A simple, sensitive, and specific competitive solid phase immunoassay for antibody to varicella-zoster virus (anti-VZV) is described. The assay uses reagents that can easily be prepared and is sparing of viral antigen. Using a solid phase coated with sheep IgG from a serum raised against human p-Fc, the same reagents will accu- rately detect anti-VZV IgM.

nonimmune groups that closely correlated with a history of previous VZV illness. It was not affected by the presence or absence of antibody to other herpes viruses. The IgM antibody capture assay demonstrated the presence of anti-VZV IgM in sera from patients with both varicella and zoster and gave negative results in pa- tients with infections unrelated to VZV and in healthy blood donors.

The competitive assay divided sera from children and adults into immune and

Key words: anti-VZV, immunity to V2V, competitive immunoassny, IgM-antibody capture immunoassay

INTRODUCTION

Varicella, one of the most common communicable diseases of childhood, is caused by primary infection with varicella-zoster virus (VZV). Usually it is a mild illness, but in certain situations-for instance, if the individual is immunosuppressed- primary infection with VZV may be life threatening. After varicella there is lifelong immunity to clinically apparent exogenous reinfection, though a recrudesence of the original infection may occur in the form of zoster. This is probably due to the reacti- vation of VZV, which has remained latent in the posterior root ganglia following the initial primary infection [Hope-Simpson, 19651.

Primary VZV infection is usually clinically manifest, and patients with a history of varicella (or zoster) are immune. However, some of those who do not recall having had either may also be immune. Low levels of antibody persist, probably for life, after varicella [Cradock-Watson et al, 1979].-The complement fixation (CQ test is not sufficiently sensitive to detect these levels of antibody reliably [Gold and Godek,

Accepted for publication July 17, 1981.

Address reprint requests to R.S. Tedder, Department of Virology, Middlesex Hospital, London W 1, England.

0 1 4 6 a 6 1 5 / 8 1 / 0 8 0 2 . ~ 0 1981 Alan R. Liss, Inc.

90 Tedder, Mortimer, and Lord

19651, although other tests may be more satisfactory. Both neutralisation [Asano and Takahashi, 19781 and immune adherence haemagglutination [Gershon et al, 19761 assays appear to be sufficiently sensitive and specific, but are laborious.

A number of solid-phase anti-VZV assays have been described using as substrates either VZV infected tissue culture [Williams et al, 1974; Cradock-Watson et al, 19791 or VZV antigen absorbed to plastic [Arvin and Koropchak, 1980; Campbell-Benzie et al, 19811. It has been shown that it is possible to modify these solid-phase assays to detect anti-VZV IgM, but the presence of rheumatoid factor (RF) in specimens may lead to false-positive results. Furthermore, cross-reactivity with antibody to herpes simplex virus (HSV) has been found in one study [Cradock-Watson et al, 19791, and the possibility exists that this may occur for all such solid-phase indirect immunoassays.

We describe here the development of a solid-phase competitive immunoassay for anti-VZV that is simple and sensitive, but free from apparent cross-reactivity with other human herpes viruses. Initially, a '251-labeled antibody (RIA) was used, but later the assay was modified to incorporate a horseradish peroxidase (HRP0)-conjugated antibody (EIA). The 1z51-labeled reagent was also used successfully in a solid-phase IgM antibody capture radioimmunoassay (MACRIA), and the results of testing acute sera from patients with varicella and zoster by this assay are described.

METHODS

Sera

care, and their parents were interviewed to elicit evidence of previous varicella in their children. The sera and VZV histories were kindly made available by Dr. Charles Holme, Department of Paediatrics, Charing Cross Hospital, and Dr. Joan Edwards, PHLS, Virus Reference Laboratory. Sera were also obtained from staff at the Middle- sex Hospital attending for a routine preemployment medical examination. At this inter- view a consecutive series of 100 staff members were asked about previous varicella and zoster.

Hambling, PHLS Leeds, were assayed for anti-VZV IgM. Nine sera strongly reactive for RF (GMT 1 in 277, range 64-512, Wellcotest, Wellcome Reagents Ltd) and sera from 105 first time blood donors (North London Blood Transfusion Centre, NLBTC) were also tested. In addition, sera of patients suffering from acute infections other than VZV were assayed for anti-VZV IgM (Table In).

Hyperimmune animal anti-HSV antisera were obtained from NIAID, Bethesda, Maryland (guinea pig), from the PHLS Division of Microbiological Reagents and Quality Control (rabbit), and from the Department of Virology, Middlesex Hospital Medical School (rabbit).

Virus Antigens

VZV and HSV antigens were prepared as freeze-dried extracts of infected tissue culture by the PHLS Division of Microbiological Reagents and Quality Control. The cytomegalovirus (CMV) antigen was a glycine extract of infected human embryonic lung cells kindly provided by Dr. S. Sutherland, Department of Virology, Middlesex Hospital Medical School.

Semm specimens were taken from 38 mentally handicapped children in home

Sera from 30 cases of varicella and 29 cases of zoster, provided by Dr. M H

Anti-Varicella-Zoster Immunoassay 91

Preparation of Labeled Anti-VZV IgG

anti-VZV titre (CF 1 in 2,000) by chromatography on a DE52 anion-exchange column (Whatman Ltd) as previously described [Tedder and Wilson-Croome, 19801. Thirty microgrammes of IgG were iodionated with Bolton and Hunter reagent (Amersham International) to a specific activity of about 15 pCi per pg in accordance with the manufacturer's instructions. Alternatively, 2 mg of the same IgG was coupled with HRPO by a modification of the method of Nakane and Kawaoi [1974] described else- where [Smith and Tedder, 19811.

Solid-Phase Anti-VZV lmmunoassay

VZV antigen was coated onto polystyrene tubes (GW3, Seward Ltd) for the ra- dioimmunoassay and onto flat-bottomed eight-well strips (Removawells, Dynatech Ltd) for the enzyme immunoassay. One hundred microlitres of antigen, diluted 1500 in 20 mM Tris buffer, pH 7.6, containing 0.1% sodium azide (TAB), was incubated for 72 hours at room temperature in the tubes or wells. These were then aspirated, washed twice with TAB, and then completely filled with TAB containing 0.1 % bovine serum albumen (TABSA). After at least 1 hour at room temperature the solid phase was emptied to near dryness and stored, sealed, at 4"C, with 0.1-0.2 ml TABSA remaining in situ. This solid phase was stable for at least 3 months at 4°C.

in the following manner: Twenty-five microliters of standard or test sera were placed in the solid phase which had been aspirated to dryness just prior to use. Seventy-five microlitres of labeled anti-VZV IgG was added, mixed, and incubated overnight at room temperature. The solid phase was then washed and the binding of the labeled anti-VZV IgG measured. Sera giving 50% or greater inhibition of the maximum ex- pected binding were considered to contain significant levels of anti-VZV.

For the RIA, '"I-anti-VZV (25 nCi per 75 pl, 30,000 cpm) was added in TAB containing 2% bovine serum albumen (BSA) and 20% pooled normal human sera negative for anti-VZV (NHS). After washing, the tubes were counted in a 16-channel gamma counter (NE 1600, Nuclear Enterprises Ltd).

For the EIA, HRPO-anti-VZV (about arbitrary units per 75 pl) [Smith and Ted- der, 19811 was added in 20 mM phosphate-buffered salin pH 7.2 (PBS) containing 0.01% Tween 20 (PBST). After washing with PBST, 100 pl substrate (8 mg 2.2'- azinodi-(3ethylbenzthiazoline sulfonic acid), ABTS, in 10 ml citrate phosphate buf- fer, 0.15 M, pH 4.05, containing 100 4 2 0 volumes Hz02) were added and incubated in the dark at mom temperature for 30 minutes. The reaction was stopped by adding 100 p1 sodium fluoride solution (10 mg in 100 ml distilled water) to the wells. The assay was read visually by comparing colour release in test wells with the colour given by a serum known to cause 50% inhibition of HRPO-conjugate binding.

Immunoglobulin G was prepared from a convalescent zoster serum with a high

The assay for anti-VZV was a simultaneous competitive immunoassay carried out

IgM Antibody Capture Radioimmunoassay (MACRIA) for Anti-VZV IgM

Specular-finish polystyrene beads 6.4 mm in diameter (Northumbria Ltd) were coated with anti-human p-Fc-specific sheep IgG (Seward Ltd). The beads were shaken in anti-p IgG diluted 1500 in 50 mM carbonate buffer pH 9.6 for 1 hour at room temperature. They were then incubated overnight and stored in the same solution at 4°C.

92 Tedder, Mortimer, and Lord

Two microliters of standard or test sera was placed in polystyrene 5 X 4 well trays (Abbott Ltd). One hundred ninety-eight microliters of PBS containing 0.05% Tween 20 and 5% foetal calf serum (PBSTF) was added to each well. The coated beads were washed three times in PBS and then added, one to each well, for 3 hours at 37°C. They were washed and, after the addition to each well of 200 pl of VZV antigen, diluted 1 5 0 in PBST incubated for 20 hours at 4°C. The beads were then washed three times with PBST and 200 p1 12'I-anti-VZV IgG (80 nCi per 200 pl, 100,OOO cpm) in PBST containing 5% each of NHS, sheep and foetal calf serum were added. After incubation for 3 hours at 37°C the beads were washed, and the binding of l'I-anti-VZV IgG was measured. Reactivity of sera in MACRIA was expressed in arbitrary units per ml by reference to a curve derived from the reactivity of standard sera containing known amounts of anti-VZV IgM.

Additional Tests for Herpes Group Antibody

Antibodies to HSV, VZV, and CMV were measured by complement fixation. Three minimum 100% haemolytic doses of guinea pig complement were incubated overnight in lucite plates at 4OC with doubling dilutions of heat-inactivated sera and the appropriate viral antigen. The antibody level was indicated by the serum dilution that gave 50% inhibition of haemolysis.

immunofluorescence. The tests were done by Dr. Joan Edwards, PHLS V i m Refer- ence Laboratory.

RESULTS

Competitive RIA

tially serum taken from a patient 2 weeks before the onset of varicella), between 6% and 8% of the labeled IgG bound to the solid phase. Sera containing high levels of anti-VZV reduced the binding of labeled IgG to 0.15%. Reactive sera or pools of sera were progressively diluted in antibody-negative serum, and the inhibition of binding was determined. Their titres were expressed as the reciprocal of that dilution estimated to give 50% inhibition (Fig. 1). In experiments to determine the optimum conditions for coating the solid phase (not detailed here), it was apparent that there was an in- verse relationship between the quantity of antigen used for coating and the resulting 50% inhibitory titres of given sera.

anti-VZV were measured by CF and RIA. There was reasonable correlation between the two assays, although sera exhibited higher titres in RIA than in CF (Fig. 2).

Three hyperimmune animal anti-HSV sera tested undiluted and at dilutions up to 1 in 256 were not reactive in the assay. There was no evidence of subthreshold (up to 49%) inhibition.

IgM and IgG antibodies to the EBV capsid antigen were detected by indirect

When '"I-anti-VZV was incubated with serum containing no VZV antibody (ini-

Under the conditions described, the titres of 18 individual human sera containing

Reactivity of Human Sera in Competitive RIA

Sera from the 38 children showed a wide range of reactivity in the competitive RIA (Fig. 3). Reactivity in this assay (>50% inhibition) closely correlated with a history of previous varicella. By this test 15 children appeared to have antibody and 23 did not. Of the 15 antibody-positive children, 12 had a history of varicella. In contrast, only two of 23 antibody-negative children had a history of varicella. One of

Anti-Varicella-&ter Immunoassay 93

Dilvlbn

Fig. 1. The effect on reactivity in competitive RIA of making serial half log,, dilutions of positive sera in anti-VZV-negative serum. Sera from an adult with a past history of varicella (left curve) and an adult convalescing from recent zoster (right curve), and a pool of sera from a random group of adults with a history of varicella (centre curve) were serially diluted. The RIA titres were expressed as the re- ciprocal dilution giving 50% inhibition of l*sl-anti-VZV binding (arrowed).

t

.

CF Titrc c 2 2 8 32 128 512 2048 I , . . I . . . . . . 1

Fig. 2. Relationship between the titre of anti-VZV determined by two methods (CF and RIA) in 18 human sera.

these was reported as possibly having had varicella in infancy; the other was said to have had varicella recently. The serum from the latter child repeatedly gave 20-30% inhibition in RIA and did not react in the VZV MACRIA.

Reactivity in the anti-VZV CF test correlated with reactivity in the competitive anti-VZV RIA. All 12 sera reactive by CF at titres > 1 in 2 (GMT 1 in 11.3, range 2-128) were also reactive in RIA (Table I). Three sera were reactive by RIA but not

94 Tedder, Mortimer, and Lord

E 2 x t e u) 0

i

5 10 15 2 lZ5I binding,lO cpm

Fig. 3. binding (cprn). Reactivity of zoster serum and antibody negative serum (NHS) arrowed. Hatched area indi- cates history of varicella.

Reactivity of sera from 38 children in the competitive anti-VZV RIA expressed as "'I-anti-VZV

by CF: These sera gave 61 %, 71 %, and 81 % inhibition, respectively. Two of the children concerned had a history of previous varicella. The reactivity in tests for anti- body to HSV, CMV, and EBV did not seem to affect the 1251 binding associated with anti-VZV negative sera (Fig. 4).

One of the difficulties associated with assessing immunity to VZV is the low levels to which specific antibody declines in adult sera. For a serological test to be of value, it should be able to separate adult s eq into immune and nonimmune groups. To determine whether this was possible with the competitive RIA, 208 consecutive sera, taken from hospital staff seen at preemployment medical intemiews, were tested for anti-VZV. The reactivities of these sera, expressed as percentage inhibition of maxi- mum binding, clearly fell into two groups with few exhibiting intermediate values (Fig. 5). The presence of anti-VZV detected by RIA (>50% inhibition) correlated well with a history of previous varicella and zoster (Table 11).

Competitive EIA

released by each serum was compared visually with that released by a standard serum known to give 50% of the maximum colour release. There was close agreement be- tween the two assays. Of the 15 sera positive by RIA, 12 were positive by EIA. The 23 sera negative by RIA were all negative by EIA.

MACRIA for Anti-VZV IgM

Sera strongly reactive for anti-VZV IgM bound some 20-fold more label that did an anti-VZV-negative serum. When, in the first stage, these sera were serially diluted in PBSTF, label binding was maximum at a dilution of 1:lOO (Fig. 6).

The children's sera previously tested by RIA were also tested by EIA. The colour

Anti-Varicella-Zoster Immunoassay 95

t i . .

. .

. . !

- I ' I

i .

Y $ i

"

Y

I I I I

'VZV- *HSV- ' C M ~ 'EBV-

anti-Herpes antibody

I !

. 9

i t

Fig. 4. sence of antibody to viruses of the herpes group. The data from these sera are shown four times, di- vided on each occasion into two groups depending upon the presence or absence of antibody to VZV (detected by CF), HSV (detected by CF), CMV (detected by CF), and EBV (detected by indirect immu- nofluorescence). Serum reactivity expressed in binding of T-anti-VZV IgG.

Reactivity in the competitive anti-VZV RIA of 38 children's sera related to the presence or ab-

Inhibition

Fig. 5 . Reactivity in RIA of 208 consecutive adult sera expressed as percent inhibition of binding of "'1-anti-VZV I&.

To quantify the anti-VZV IgM reactivity of sera, all specimens were tested at a single constant dilution of 1: 100 in PBSTF. A pool of strongly MACRIA-reactive acute phase varicella and zoster sera was prepared and considered to contain 100 arbi- trary units of anti-VZV IgM per ml. Standards containing known amounts of anti- VZV IgM per ml of serum were prepared by making appropriate dilutions of this pool

% Tedder, Mortimer, and Lord

20

io3 ' io4 ' Dilution of l is t stage

Fig. 6. serum (0 ) to their dilution in the first stage.

The relationship of reactivity in VZV-MACRIA of anti-VZV IgM positive pool (0) and negative

in NHS. A curve was derived from the reactivities of these standards (Fig. 7), and MACRIA-positive sera were assigned a level of anti-VZV IgM in arbitrary units by reference to this curve.

Sera were examined from 27 cases in which there was a firm diagnosis of vari- cella and from 3 in which it was suspected. The age range of the patients was 13/4-78 years, and the day of collection in relation to the onset of the rash, in the 15 cases where this was precisely known, ranged from the 6th to the 30th day. Positive MA- CRIA results were obtained in 25 of the 27 definite and all 3 of the suspected cases (mean = 28.1 units per ml, range 1.6-> 100, n = 28). Of the two remaining cases, the serum from one gave an equivocal (0.9 units) and the other a negative ((0.3 units) result.

Sera from 18 definite and 10 suspected cases of zoster were examined. The ages of the patients ranged from 13 to 85 years, and the number of days from appearance of lesions to serum collection, where known, ranged from 1 to 19 days. Sera from 17 of the 18 definite and 6 of the 10 suspected cases of zoster were MACRIA-positive (mean 34.6 units per ml, range 1.3- > 100, n = 23). The sera from the two other definite cases gave equivocal results (0.8 and 0.4 units).

The reactivities of sera from patients with infections other than varicella and zoster were slight and did not reach significant levels (( 1.0 unit per ml, T/N ratio 2.5:1, Table III).

DISCUSSION

The purpose of this study was to investigate the use of a CF-grade VZV antigen and labeled anti-VZV IgG as reagents in solid-phase immunoassays for total serum

Anti-Varicella-Zoster Irnrnunoassay 97

-,

20-

n 0 -

P - D - .€

10-

n

I 100 10 3 1 03

30

20 2

% 6

10

2

Units per ml anli-Vz IgM

Fig. 7. anti-VZV IgG binding and as the ratio between binding of test and negative Semm (TIN ratio).

Reactivity in MACRIA of sera containing known quantities of anti-VZV IgM, expressed as '"I-

TABLE I. Relationship Between RIA Antibody and Reactivity in VZV CF in 38 Children's Sera

VZV CF t i tn

< 2 (Neg) 2 4 8 16 32 64 128 vzv RIA Ab -I- ve 3 2 2 1 5 1 1 Ab - ve 23 .

TABLE II. Reactivity in Anti-VZV RIA of 100 Adult Sera Related to History of Previous Varicella and a s t e r

Anti-VZV RIA

VZV history + ve - ve Total

Yes 59 0 59 No 18 6 24 Don't know 15 2 17

Total 92 8 100

TABLE Ul. Reactivity of 147 Non-VZV Sera in VZV-MACRIA

Reactivity in VZV MACRIA ~~

2 1 unit 0.4-0.9 (0.3 unit Total no. Sera tested per ml unit per ml per ml of sera

6 3 9 Blood donors - 4 101 105

1 4 5 HSV acute sera - 5 2 7 CMV acute sera - 1 2 3 EBV acute sera -

6 6 Rubella acute sera - -

6 6 6 6

R.F. positive -

Hepatitis A acute sera - - Hepatitis B acute sera - -

98 Tedder, Mortimer, and Lord

and for IgM-specific antibody to VZV. The results suggest that these two reagents may be the basis for simple, sensitive, and more accurate serological tests for anti-VZV than have previously been described.

fixation tests will detect anti-VZV in the sera of most children who have a history of varicella. Levels of antibody detected in this way decline, and CF tests are insuffi- ciently sensitive to be of use in defining serological evidence of previous VZV infec- tion in adult sera [Gold and Godek, 1965; Yamada et al, 19791. Although most of the sera from children with a history of previous infection that we studied reacted by CF, four did so only at a level at which nonspecific reactions might be expected to occur. In fact, out of the 15 children’s sera that were anti-VZV-positive by RIA, 7 would be considered as nonimmune by CF because their titres were below 1 : 8.

VZV, and a number of methods have been proposed. The immune adherence haemag- glutination [Gershon et al, 19761, fluorescent [Williams et al, 19741 and neutralisation assays [Asano and Takahashi, 19781 are tests of similar sensitivity [Yamada et al, 19791, but all are laborious and therefore may not be suitable for routine use.

Solid-phase indirect immunoassays for anti-VZV using fixed infected tissue cul- ture [Cradock-Watson et al, 19791 or antigen-coated plastic [Arvin and Koropchak, 1980; Campbell-Benzie et al, 19811 have been described and are more sensitive than CF. They are capable of detecting the small amounts of IgG present in immune sera that will bind to the solid-phase antigens. In order to exclude binding of antibody to antigens other than those that are virus-specific, it is necessary to repeat each test using control tissue culture antigen. This increases the work load and requirement for reagents. In addition the interpretation of weakly positive results in indirect immunoas- says is difficult.

assays may not distinguish between them. Either it may be caused by broadly reactive antibody arising from previous exposure to VZV, or it may be due to cross-reactive oligovalent antibody that is evoked by expasure to a related but different virus such as HSV. An antigenic relationship between HSV and VZV has been indicated by rises of heterologous CF antibody in patients with primary HSV and VZV infections, although these rises were seen only in patients who had evidence of previous infection by the heterologous virus [Schmidt et al, 19691. Absorption of sera prior to testing with a homologous antigen in order to demonstrate a reaction specific for the homologous virus [Campbell-Benzie et al, 19811 is unlikely to resolve this problem.

instance, to detect antibody to hepatitis A virus [Purcell et al, 19761 and hepatitis B virus [Overby and Ling, 19761. In this study a CF-grade VZV antigen and a labeled anti-VZV IgG prepared from convalescent zoster serum were used in a competitive assay for serum anti-VZV. It was possible to achieve a 40-fold difference in label binding between VZV-immune and nonimmune sera. The competitive RIA and EIA were easy to perform and did not require a control antigen. They were considerably more sensitive than CF. A further advantage of the assay was that, because whole serum could be used in the test, the serum titres expressed were “real.” By contrast, in solid-phase indirect immunoassays, where initial high dilutions of sera have to be made in order to obtain a satisfactory result, the “real” titre, and in turn the relative sensitivity of the assay, is correctly determined by dividing the test serum titre by the initial dilution.

No readily available test exists to determine immunity to VZV. Complement

There is therefore a need for a more sensitive assay to determine immunity to

Low-level antibody reactivity against VZV can occur in two ways, and indirect

Competitive immunoassays are an alternative to indirect assays and are used, for

Anti-Varicella-Zoster Immunoassay 99

Cross-reactive antibody against minor determinants will not interfere significantly in competitive immunoassays. The maximum label binding obtained when anti-VZV negative NHS is tested in a competitive assay is governed by the “fit” achieved be- tween the labeled IgG and the solid-phase antigen. For a serum to react significantly in the assay there must be a greater than 50% inhibition of label binding. The anti- genic cross-reactivity between VZV and HSV is probably slight, and is certainly not at a level that would induce cross-reactive antibodies to more than half of the VZV spe- cific antigens present on the solid phase, the outcome necessary for an HSV serum to give a false-positive antibody reaction. This explanation of the lack of significant cross-reactivity in the competitive anti-VZV assay is supported by the failure of three hyperimmune animal anti-HSV sera to cause even minimal inhibition. Furthermore, the presence of antibody to other herpes viruses had no obvious effect on the reactivity of the children’s sera (Fig. 4). It remains to be determined whether, in patients who have been exposed previously to VZV, the competitive assay will demonstrate a heter- ologous antibody rise in response to acute HSV infection. This has been observed in CF [Schmidt et al, 1%9] and immunofluoresence [Cradock-Watson et al, 19791 tests.

The history of previous varicella or zoster correlated with the presence of anti- VZV (Fig. 3, Table 11). On its own a positive history would probably be a good index of immunity to VZV but is likely to underestimate the number of immune people, as some patients who do not recall, or who deny, having had varicella or zoster may in fact be immune.

In two children with a history of varicella, anti-VZV was not detected (CF-, RIA-, and EIA-negative). These may have been false-negative results.

Zoster immune globulin (ZIG) is much needed for the protection of certain sus- ceptible patients exposed to VZV, but is in short supply. With only slight changes in the quantity of antigen used to coat the solid phase and in the ratio of label to test serum, it is possible to alter considerably the sensitivity of the competitive assay. Suit- ably desensitized, these assays would prove useful to identify blood donors with high leveis of anti-VZV. At present only a few blood transfusion service centres are able to identify donors with high anti-VZV titres, and so ZIG is mainly produced from donors with a history of recent varicella or zoster. This may have led to the preparation of ZIG of low efficacy [Evans et al, 19801.

A major advantage of the IgM antibody capture assay over other IgM assays is that separation of IgM from serum prior to testing is unnecessary. MACFUAs also resist interference by high levels of specific IgG antibody and by RF, both of which may cause false reactions in indirect solid-phase immunoassays [Cohen et al, 1967; Fraser et al, 19711. In this study reactivity in MACRIA was confined to sera from patients with acute VZV illness and was not seen in the sera of a random series of blood donors, patients with miscellaneous viral infections, or patients whose serum contained RF (Table In).

varicella and is absent in most cases of zoster [Leonard et al, 1970; Schmidt and Len- nette, 1975; Gerna et al, 19791. However, others have found anti-VZV IgM in acute zoster sera, either by immunofluorescence [Ross and McDaid, 1972; Cradock-Watson et al, 19791, or RIA [Arvin and Koropchak, 19801. The 59 sera tested in this study from patients with acute varicella or zoster almost all contained anti-VZV IgM.

Cradock-Watson et al [1979] found that IgM responses were weaker in zoster than varicella patients, and that a third of their zoster patients under the age of 30 years did not make an IgM response. In our series there was no difference in the mag-

It has been suggested by various authors that anti-VZV IgM is detected only in

100 Tedder, Mortimer, and Lord

nitude of the anti-VZV IgM response made by varicella and zoster patients, and their responses did not appear to be related to age. A series of specimens will have to be examined in parallel before these conflicting findings can be resolved, but we suggest that interference by high levels of anti-VZV IgG may have suppressed IgM fluores- cence.

The anti-VZV MACRIA could, in the past, have played an important role in distinguishing between varicella and atypical or vaccine-modified variola. It could still serve to differentiate between VZV infection and the other skin lesions that mimic it. In one other respect the assays described here for anti-VZV and anti-VZV IgM may be useful.

Trials of attenuated VZV vaccines are now under way in several countries, and, for efficient use of the vaccine, routine and readily available serological tests for anti- VZV are needed. There, is then, on several counts, a growing need of accurate tests for anti-VZV, both to discriminate between susceptibility and immunity and to detect acute and recrudescent infection. A conventional VZV antigen of the type commonly used in complement fixation tests and a labeled anti-VZV human IgG derived from a high-titre zoster serum are the only reagents required for the solid-phase assays de- scribed here. Particularly if the labeled anti-VZV were in the form of an enzyme con- jugate, these assays would be a convenient and useful addition to the range of diagnos- tic tests in the clinical laboratory.

ACKNOWLEDGMENTS

We thank members of the Virology Department, Middlesex Hospital, and the Virus Reference Laboratory, Colindale, for their help and constructive criticism; also Dr. Joan Edwards, Dr. Charles Holme, Dr. Peter Hambling, and Mrs. Gillie Peake (Occupational Health, Middlesex Hospital) for serum specimens and relevant clinical details; Miss Rosemary Nyeholm for technical assistance; Miss Anna Patterson for her help with preparation of this manuscript.

Control for the provision of antigens. We are grateful to the PHLS Division of Microbiological Reagents and Quality

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