Journal of Viro~ogicul~et~o~, 11 (1985) 177-187

Elsevier

JVM 00411

177

EVALUATION OF COMMERCIAL METHODS OF ENZYME

IMMUNOASSAY (EIA) FOR THE MEASUREMENT OF RUBELLA-SPECIFIC

IgM

JO& M. ECHEVARR~A, CARMEN SAINZ, FERNANDO DE ORY and RAFAEL NAJERA

Servicio de Virologin, Centro National de Microbiologia, Virologia e Inmunologia Sanitarias, Mojadohonda,

Madrid, Spain

(Accepted 15 February 1985)

Four commercial EIA methods for measuring rubella-specific IgM (three indirect tests and one anti-p

capture test) were evaluated, using sucrose gradient centrifugation and hemagglutination inhibition as the

reference method. Evaluation was conducted with the aid of four serum panels, including 53 primary rubella

cases, 30 healthy pregnant women, 21 sera positive for rheumatoid factor(s) (RF) and 35 sera from 29 cases

of heterophil-positive infectious mononucleosis with EBV-specific IgM detected by immunofluorescence.

All EIA methods were more sensitive than the reference method when applied to very early samples (1-5

days post-exanthema) and no differences in sensitivity were found between them. On the other hand, we

observed a significant incidence of false-positive results ifan indirect EIA method is applied to RF-positive

samples. False positivity is significantly reduced, but not totally eliminated, when samples are preabsorbed

with anti-human IgG serum and, in all cases, the absorbance values obtained were low. In contrast, there

were no false-positive results using an anti-p capture method, even in sera from cases of infectious

mononucleosis. The basis for choosing between an indirect method and an anti-p capture method for

the diagnosis of congenital and post-natal rubella virus infection is discussed.

rubella enzyme immunoassay specific IgM congenital infections

INTRODUCTION

Identification of rubella-specific IgM antibodies in serum is accepted as the most

suitable method for rapid diagnosis of the disease (World Health Organization,

1981a), as it is, in general terms, the only criterion valid for distinguishing between

primary and secondary infections by this virus (Hortsmann et al., 1969; World Health

Organization, 1981b; Echevarriaet al., 1983). As a result of the need forrapiddetection

of primary rubella in pregnant women, several different methods of enzyme immu-

noassay (EIA) for detection of these antibodies have been commercialized in recent

years. The majority are based on the indirect EIA technique (Voller and Bidwell,

1976), and a few on the capture of p-chains in solid phase (EIA anti-p) (Duermeyer and

van der Veen, 1978). The unquestionable simplicity which these assays introduce in

diagnostic methodology make possible their use in routine laboratories, which lack

0166-0934/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)

178

experience and comparative techniques (World Health Organization, 1981b). The

sensitivity of indirect EIA methods for detection of rubella-specific IgM in serum may

be affected by the presence of sufficiently high levels of rubella-specific IgG (Cradock-

Watson et al., 1976), and their specificity by the existence of rheumatoid factors (RF)

of the IgM class or other autoantibodies (Vejtrop, 1980). To obtain reliable results, the

elimination of these components from serum before testing is recommended (World

Health Organization, l981b).

Non-specific interference is less readily detected in the u-chain capture methods,

which makes these assays particularly suitable for this type of analysis (Meurman,

1983). The possible interference of infectious mononucleosis-associated heterophil

antibodies (HA) has been reported (Morgan-Capner et al., 1983), but has not yet been

confirmed.

We evaluate here some of the EIA methods for detection of rubella-specific IgM

which are commercially available in Spain. Two separate studies have been carried

out: in the first, sera from a group of patients with laboratory-confirmed primary

rubella are analysed by three indirect EIA assays and one u-chain capture method. In

addition, sera are tested from a group of healthy pregnant women with different

rubella antibody levels as determined by hemagglutination inhibition (HI). Sucrose

gradient ultracentrifugation/HI (SG-C-HI) has been used as a reference method.

In the second study, one indirect EIA and the anti-u-chain capture methods are

assayed with serum samples containing rheumatoid factors, heterophil antibodies or

both, with the object of evaluating the level of interference of these antibodies in the

two techniques.

MATERIALS AND METHODS

Seru. Sera were selected from samples received by our laboratory for diagnosis and

were stored at -20°C until use. The sera were grouped into four panels:

Pane/ I consisted of 82 sera from 53 cases of primary rubella, taken at different times

during the course of the infection (Table I). All patients had clinical case histories

consistent with rubella, and were diagnosed by detection of seroconversion by HI

and/or the presence of rubella-specific IgM by SGC/HI. Out of the 53 cases, 41 were

pregnant women.

Panel 2 consisted of 30 sera from 30 healthy pregnant women, all with rubella

antibodies as determined by HI (Table 1). There were no cases of recent exanthematic

illness, nor of known contact with rubella patients.

Panel 3 consisted of 21 sera from patients with various infectious diseases in which

the presence of rheumatoid factor (RF) above a 1: 10 dilution was detected by latex

agglutination. By HI, 19 of these were positive for rubella antibodies and two were

negative (Table 3). None of these patients had clinical symptoms suggestive of rubella.

179

Panel 4 consisted of 35 sera from 29 cases of infectious mononucleosis (IM) all of

which contained HA as well as specific anti-Epstein-Barr virus (EBV) IgM detectable

at a 1 : 10 dilution. ltn addition, RF was detected at dilutions of > 1 : 5 in 19 of these sera

(Table 4).

Serological methods

Hemagglutination inhibition (HI). A micromethod described previously was used

{Echevarria et al., 1983) after removing non-specific inhibitors from the sera with

kaolin. Dextrose-gelatin-Verona1 (DGV) buffer was used as dituent in all steps of the

reaction and 0.25% pigeon erythrocytes were used for detection of free antigen.

Immunoglobuiin separation. Immunoglobulins were separated byultracentrifugation

in sucrose gradients, according to the method described by Palmer et al. (1977).

Briefly, 300 ul of serum diluted 1 : 2 in PBS (pH =I: 7.2) were layered on top of a

continuous 10-500/o sucrose gradient (4.8 ml in Beckman Ultra-Clear 13 X 51 mm

tubes). Gradients were centrifuged 18 h at 38,000 rpm (Beckman SW50 rotor) and 0.5

ml fractions were recovered from the bottom of the gradient. Fractions 2 and 3 were

tested by single radial diffusion for presence of low levels of IgG (LC-Partigen-IgG,

Behring Institute) and titrated for rubella-speci~c antibodies by HI, starting from the

undiluted material. Regarding the usual distribution of IgM through the gradient,

we assume that the IgM concentration in theseundilutedfractionscorrespondsroughly

to a 1 : 8 dilution of the original serum, and HI results are therefore expressed as IgM

antibody titers accordingly (Echevarria et al., 1983).

Commercial kits tested. The following methods were evaluated: the indirect methods

Enzygnost Rubella (Behring Institute), Rubazyme M (Abbott Scientific Laboratories)

and Rubelisa M (Microbiological Associates) and, as a u-chain capture method,

Rubenz M (Northumbria Biologicals). The first indirect method utilises U-shaped

microtiter plates as the solid phase, the second, plastic beads, and the third, plastic

tubes. The anti-u-chain capture kit uses flat-bottomed microplates and a monoclonal

antibody to rubella hemagglutinin conjugated to peroxidase. The manufacturer’s in-

structions were followed rigorously in the performance of all tests.

Serum panels 1 and 2 were tested by all four methods, while panels 3 and 4 were

tested only by the Enzygnost and Rubenz M techniques. In the Enzygnost method, sera

from panels 1 and 2 were examined without previous treatment to eliminate IgG,

which the manufacturer proposes as an optional measure or to confirm test results.

Panels 3 and 4 were analysed with and without pretreatment with the anti-human IgG

serum provided by the manufacturer and following accompanying instructions (RF

Absorbens, Behring Institute).

Other methods. Rheumatoid factors were detected by latex agglutination (Latex RF,

180

Behring Institute), heterophita~glutinins by the Paul-BunelI-D~vidsohn test (Mono-

Slide Test, BioMerieux), IgG anti-EB viral capsid antigen (EBV-VCA) by indirect

immunofluorescence, according to the method of Henle and Henle (1966) using the

EB-3 cell line. Anti-EBV-VCA specific IgM was determined by indirect immunofluo-

rescence, using commercially available reagents (EBV-M, Litton Bionetics).

RESULTS

Table 1 shows the results obtained with the sera from the rubella patients of panel 1

and those of the healthy pregnant women of panel 2. The former are grouped

according to time elapsed since the appearance of exanthema. Results are expressed as

the percentage of positive cases per group by each technique. As can be seen, correla-

tion of the results in these 82 sera between SGC/HI and the EIA methods is 88-95%,

and correlation in the negative control group is loo%, since no false-positive results

appeared in any of the techniques tested.

In Figs. 1 and 2, the mean values of rubella antibodies are expressed with respect to

the time course of the evolution of the disease. As the system for the expression of

results is different for each method, the lower limits and the higher positive values

obtained with each technique have been aligned to render the curves comparable: i.e.,

cut-off values of Rubazyme and Enzygnost are 1 .O and 0.2 respectively; these values

are in the same point on the vertical left axis in Fig. 1 and aligned to the cut-off value of

HI-SGC (titer 8) in the right vertical axis. As can be seen, the onset as well as the peak

of the specific IgM curve are detected later by the SGCfHI rather than by the EIA

methods.

The sera from panel 1 showing discrepancies between the results obtained by

SGC/HI and EIA are listed in Table 2, together with results from other samples from

the same patients. These discrepancies were seen in eight sera which were taken either

very early (1-3 days post-exanthema) or very late (54 days post-exanthema) in rubella

cases confirmed by seroconversion in HI and/or by the presence of specific IgM in

samples taken at other times during the course of the infection. They were negative in

SGC/HI but positive in one or more EIA methods.

Tables 3 and 4 show data from the analysis of panels 3 and 4 with the Enzygnost

Rubella (indirect) and Rubenz M (anti-~-capture) methods. The latter test gave no

positive result with the samples in these panels, while with the former, 10 positives were

seen when whole serum was used (4 in the panel with RF, as seen in Table 3, and bout

of the group of IM cases, as seen in Table 4). This number was reduced to two positives

when IgG was previously removed by absorption with anti-human IgG serum in the

IM group and to zero in the RF group. Table 5 lists individually the sera from which

these positive results were obtained. The two sera which have remained persistently

positive after IgG removal are from cases of IM which contain rubella antibodies at

medium titer by HI and, in one, also RF was detected at a titer of 1 : 20. These results

were reproduced in two analyses performed on separate days.

TA

BL

E

I

Rub

ella

-spe

cifi

c Ig

M

(per

cent

ages

of

po

sitiv

es)

mea

sure

d by

su

cros

e gr

adie

nt

ultr

acen

trif

ugat

ion

and

hem

aggl

utin

atio

n in

hibi

tion

(SG

C-H

I)

and

four

E

IA

met

hods

in

82

sera

fr

om

53 p

rim

ary

rube

lla

infe

ctio

ns

(pan

el

1) a

nd

30 s

era

from

he

alth

y pr

egna

nt

wom

en

(pan

el

2).

DPE

n N

o.

of

sera

SGC

-HI

Enz

ygno

st

Rub

azym

e R

ubel

isa

Rub

enz

M

Pane

l I

Bef

ore

rash

5

0 0

0 0

0

o-

2 10

0

50

40

60

50

3-

5 9

77.8

10

0 10

0 10

0 10

0

6-25

44

10

0 10

0 10

0 10

0 10

0

226

5 60

80

10

0 80

75

b

Unk

now

n 9

88.9

88

.9

88.9

83

.3’

83.3

’

Tot

al

82

75.6

85

.4

85.2

85

.1

72.3

Cor

rela

tion

with

10

0 88

.5

88.7

88

.8

95.6

XX

-HI

Pane

l 2

Con

trol

s 30

0

0 0

0 0

Cor

rela

tion

with

10

0 10

0 10

0 10

0 10

0

SGC

-HI

d D

PE

=

days

po

st-e

xant

hem

a.

h Fo

ur

sera

te

sted

.

‘ Si

x se

ra

test

ed.

__ HI-SOC

---- Enrygnort ( maon “OIWJJ,

- - - R”barym*

- - --._ -.-._

Fig. 1. Rubella-specific IgM in 53 cases of primary rubella infection correlated with the time of infection

(days post-exanthema). Comparison between three methods: Enzygnost, Rubazyme and SGC/HI (mean

values).

Fig. 2. Rub~iia-specific IgM in 53 cases of primary rubella infection correlated with the time of infection

(days post-exanthema). Comparison between three methods: Rubelisa, Rubenz M and SGCYHI (mean

values).

TA

BL

E

2

Rub

ella

-spe

cifi

c Ig

M

in s

era

from

pa

nel

I: d

iscr

epan

t re

sults

be

twee

n %

X/H

I an

d fo

ur

EIA

m

etho

ds.

Cas

e no

. Se

rum

no

. D

PE

H

I

(tite

r)

SG’J

HI

(tite

r)

Enz

ygno

st

(AA

)

Rub

azym

e

(ind

ex)

Rub

elis

a

(AA

)

Rub

enz

M

(AU

)

1 I 2”

7

13

149

II

22

23”

12

24

25”

14

29

30”

18

38”

39

23

49

50”

25

53

54”

6 1 15 1 13

19

54 3 13 3 12

<4

<8

0.33

8,00

0 64

1.

65

<4

<8

0.54

128

16

1.64

<4

<8

(-)

32

32

1.08

<4

<8

0.38

1,02

4 16

0.

66

<4

<8

0.28

256

32

0.57

1,02

4 32

0.

52

256

<8

(-)

16

<8

0.31

128

8 0.

34

8 <8

0.

21

128

64

0.94

(-)

0.30

13

2.76

6 1.

68

115

1.09

2 0.

7 1

(-)

2.76

6 2.

55

48

(-)

1.85

28

2.65

7 2.

20

72

1.21

6 0.

47

32

1.88

5 1.

27

88

1.11

6 0.

32

15

2.42

1 0.

83

85

2.46

0 0.

41

24

1.01

1 (-

) (-

)

1.02

0 0.

36

5

I.41

5 0.

39

17

1.84

4 0.

39

5

2.55

6 1.

99

37

a A

noth

er

sam

ple

from

th

e sa

me

patie

nt.

b D

PE

=

days

po

st-e

xant

hem

a;

AU

=

arbi

trar

y un

its.

184

TABLE 3

Rubella-specific IgM by indirect and anti-u-capture El As in 21 sera with RF (Panel 3). Samples are grouped

according to rubella HI antibody titer.

HI no. Rubenz M Enzygnost (WS) Enzygnost (AS)

+ - + - +

<4 2 0 2 0 2 0 2

4- 8 6 0 6 0 6 0 6

16- 32 6 0 6 2 4 0 6

64-512 I 0 7 2 5 0 7

Total 21 0 21 4 17 0 21

WS = whole serum; AS = serum absorbed with rabbit anti-human IgG serum

TABLE 4

Rubella-specific IgM by indirect and anti-u-capture EIAs in 35sera from IM cases, all positive for heterophil

antibodies and EB virus-specific IgM (Panel 4). Samples are grouped according to the presence of RF and

rubella HI antibodies.

RF HI no. Rubenz M Enzygnost (WS) Enzygnost (AS)

+ + - +

+ <4 6 0 6 I 5 0 6

16-512 13 0 13 4 9 I 12

_ <4 9 0 9 0 9 0 9

32-256 7 0 7 I 6 1 6

WS = whole serum; AS = serum absorbed with rabbit anti-human ISC serum.

DISCUSSION

The results obtained with panels 1 and 2 indicate a correlation of 88-95% between

the EIA techniques tested and the reference method in the detection of rubella-specific

IgM in sera from primary rubella patients. The 100% negative results observed in the

panel of sera from healthy pregnant women, randomly selected, independent of their

antibody titer in HI, show comparability between the techniques.

The discrepant results are limited to the eight sera stated in Table 2. The results

obtained clearly indicate a higher sensitivity for the EIA methods with respect to

SGC/HI, as can be also seen in the early portions of the curves in Figs. 1 and 2. Under

these conditions, all four EIA methods gave essentially identical results.

185

TABLE 5

Rubella-specific IgM by indirect and anti-p-capture EIA: individuai results in 10 sera from groups 3 and 4

yiving positive values in Enzygnost. Cut-off values are: 5 AU for Rubenz M and AA = 0.20 for Enzygnost.

Sera that remain positive in Enzygnost after IgG removal are underlined.

Serum no. RF Rubella anti-EBV-VCA

HI G/M

Rubenz M

(AU)

Enzygnost (AA)

WS AS

Panel 3

9 l/IO 16 ND 0 0.47 0.16

13 I/10 32 ND 0 0.36 0. I7

I8 l/20 64 ND 0 0.75 0.08

20 l/40 128 ND 0 0.43 0.00

Panel 4

9 (-) 256 32/f 0 0.27 0.22

14 l/IO <4 512/+(AS) 0 0.20 0.12

23 I/IO 64 512/+(AS) 0 0.23 0.14

25 l/20 128 l,024/+(AS) 0 0.32 0.24

27 l/IO 128 5 lZ/+(AS) 0 0.25 07

29 l/IO 512 5121ffAS) 0 0.40 0.10

WS = whole serum; AS = absorbed serum with anti-human IgG: AU = arbitrary units

In the case of RF and IM-associated antibodies (Table 5) from panels 3 and 4, it can

be seen that the simultaneous presence of RF and even low levels of rubella-specific

IgG antibodies gives rise to considerable percentage of false-positive results in the

indirect EIA method tested. To obtain reliabie results, the previous elimination of IgG

appears to be indispensable. Treatment with anti-human IgG serum appears efficient,

although in two sera (numbers 9 and 25 of panel 4) positive results were obtained even

after such treatment. In one of these (number 25) RF was detected to a titer of 1: 20 and

rubella antibodies to a titer of 1 : 128 by HI, so the positive result may be due to an

inefficient removal of IgG. In the other case (number 9) no RF was detected by the

technique employed, although rubella antibodies were seen by HI at a titer of 1: 256.

However, in these two sera the absorbance values obtained after removal of IgG were

very low, both near the cut-off value. Similar values were obtained in some early or late

sera from cases of primary rubella. Therefore, modifications of the cut-off value to a

higher level do not seem possible without loss in sensitivity, but the establishment ofa

equivocal zone inducing to repeat the test or to use another different test may be useful

to obtain reliable results.

On the other hand, no positive result was obtained when the sera from panels 3 and 4

were tested by the Rubenz M method. In this study, we did not obtain false-positive

results associated with heterophil-positive IM sera, although such results have recently

186

been reported (Morgan-Capner et al., 1983). This can probably be attributed to the

difference in techniques used in the two studies: we used an EIA method, while

Morgan-Capner et al. used RIA, whose sensitivity level is somewhat higher (5 AU for

EIA, 3.3 AU for RIA) (Mortimer et al., 1981). If, as the authors suggest, the interfer-

ence is due to the nonspecific stimulation of B lymphocytes by EBV, the levels of

specific IgM induced may be so low that they are undetectable by ETA but remain

detectable by a well-calibrated RIA. In fact, the values obtained by Morgan-Capner

and colleagues do not generally exceed 6 AU.

This hypothesis does not, however, explain the apparent false-positive results

obtained by the Enzygnost method on serum 25 of panel 4 (Table 5), a case in which the

anti-u capture method was negative. In no other case do the results indicate a higher

sensitivity for the indirect technique. Furthermore, in our experience with the Enzy-

gnost method, sera with low levels of virus-specific IgM usually give absorbance levels

1.5-3 times higher after anti-IgC treatment (unpubl. obs.) than with whole serum, and

this criterion was not met in this case, in which the reading of the absorbed serum (0.22)

is lower than that of the whole serum (0.27). Non-specific adsorption of IgM to the

solid phase may explain this result. To confirm this, the serum was fractionated by

ultracentrifugation on sucrose gradient and the heavy fractions analysed by indirect

EIA. All results were negative, indicating that the false positivity was probably due to

non-specific adsorption (Dittmar et al., 1979). Alternatively, the presence of IgM

antibodies to cellular components incorporated into the viral envelope or present free

in the antigen may explain the phenomenon (Morgan-Capner et al., 1983). Such

antibodies may affect the specificity of the indirect method, but probably do not affect

the specificity of the anti-u capture method, as it uses peroxidase-conjugated monoclo-

nal antibodies against the virus hemagglutinin as the final marker.

The methods evaluated in this study perform satisfactorily for the diagnosis of

primary rubella. The technique of u-chain capture appears to offer greatercertainty in

its results, as it does not appear to be affected by the presence of autoantibodies in the

sample. Considering that the application of this method is no more difficult than that

of the indirect EIA tests, it is clear that such a procedure is much more suitable for

diagnosis of primary rubella in pregnant women, as well as for the detection of

congenital infections by rubella virus, in which specific IgG antibodies of maternal

origin may constitute an important source of false-negative results (Cradock-Watson

et al., 1976; Schmitz et al., 1980).

ACKNOWLEDGMENTS

The authors thank T. Minguito, M.A. Bustillo and R. Diez for their excellent

technical assistance and Ms. R. Rojas for skilled secretarial help.

187

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