Journal of Virological Methods, 8 (1984) 99-109

Elsevier

JVM 00293

99

A COMPARISON OF RUBAZYME-M AND MACRIA FOR THE DETECTION OF RUBELLA-SPECIFIC IgM

JENNIFER M. BEST, SARA J. PALMER, PETER MORGAN-CAPNER and JULIAN HODGSON

Department of Virology, Sr. Thomas’ Hospital and Medical School, London SE1 7EH, and Department of

Microbiology. King’s College Hospital and Medical School, London SE5 8RX. U.K.

(Accepted 14 November 1983)

One-hundred and eighty-six carefully selected sera were tested for rubella-specific IgM by Rubazyme-M

(Abbott Diagnostics)and an M-antibody capture radioimmunoassay (MACRIA). Eleven of these sera were

from cases of infectious mononucleosis, six of which gave positive results in MACRIA, while one gave a

positive result in Rubazyme-M. Of the remaining 175 sera, 158 gave concordant results whilst 17 sera gave

discordant results; these 17 were also tested by serum fractionation. Problems were encountered with all

assay systems used. It is therefore recommended that the results of all tests for rubella-specific IgM should

be interpreted with caution.

rubella rubella-specific IgM Rubazyme-M MACRIA

INTRODUCTION

The detection of rubella-specific IgM has an established place in the diagnosis of

both postnatal and congenitally-acquired rubella. Serum fractionation with haemag-

glutination inhibition (HI) tests performed on the fractions obtained has been the

standard method for detecting rubella-specific IgM since the late 1960s. The methods

of fractionation commonly used are sucrose density gradient fractionation (SDGF;

Best et al., 1969; Al-Nakib et al., 1975) and gel filtration (GF; Morgan-Capner et al.,

1980). The disadvantages of these methods are the time taken and the labour required

in titrating the several fractions obtained from each serum, the limitation on the

number of sera that can be tested and, in the case of SDGF, the cost of the ultracentri-

fuge. During the last few years several radioimmunoassay (RIA) and enzyme-immu-

noassay (EIA) tests have been described for the detection of rubella-specific IgM

(Pattison, 1982) and EIA kits are now being marketed by several commercial compa-

nies. One of these tests, Rubazyme-M (Abbott Diagnostics) is being distributed

widely, including distribution to laboratories with no previous experience of the

serological diagnosis of rubella. We have therefore compared this test with M-antibo-

dy capture radioimmunoassay (MACRIA) (Mortimer et al., 1981) and with serum

fractionation. Sera tested had been collected over many years and were selected in

0166-0934/84/$03.00 C 1984 Elsevier Science Publishers B.V

100

order to present as many problems as possible; they included only 18 sera collected

between days 9 and 21 after onset of rash, the time at which the highest levels of

rubella-specific IgM are present. Tests were carried out in two laboratories, the staff of

which have had extensive experience of rubella serodiagnosis.

MATERIALS AND METHODS

Sera

One-hundred and eighty-six sera were evaluated. These included sera from confirm-

ed cases of rubella collected at different times after onset of rash, sera found to

contain no rubella-specific IgM in other tests, including 10 sera with high (>SOOIU) HI

antibody levels, and sera that had previously presented problems for determining the

presence of rubella-specific IgM, such as sera that had previously given conflicting or

equivocal results in different assays (Table 1). Twenty sera from babies with congeni-

tal rubella were also evaluated; sera had been obtained at birth or at intervals of up to 9

mth after birth. The diagnosis of congenital rubella had been confirmed in each case

serologically (presence of rubella-specific IgM or persistence of antibodies beyond 6

mth of age) or by isolation of rubella virus. The six other neonates tested had

symptoms compatible with congenital rubella (e.g. cataract, failure to thrive) but no

serological evidence of intrauterine infection. Twenty-seven sera were tested from

seven rubella vaccinees who had received either Cendehill or RA27/3 vaccines;

twenty-one of these sera were serial samples from four vaccinees.

Eighteen sera contained IgM antibodies specific for other viruses; 11 sera were from

cases of infectious mononucleosis (heterophil antibody and EB IgM positive), eight of

which had previously been shown to be reactive in MACRIA (Morgan-Capner et al.,

1983), two contained CMV-specific IgM, one varicella-zoster specific IgM, three

hepatitis A IgM, while one serum had given a false positive result in a hepatitis A IgM

(HAVAB-M; Abbott Diagnostics) test.

In order to assess the effect of rheumatoid factor (RF) in Rubazyme-M, a serum

containing RF was mixed in different proportions (1 : 3, 1 : 1,3 : 1) with a serum with a

high level of HI antibodies (400 IU) and a serum with a low level of HI antibodies (20

IU) and these mixtures and the RF containing serum were then tested by Rubazyme-M.

Sera were stored at -20°C at St. Thomas’ Hospital, while at King’s College Hospital

0.02% sodium azide was added before storage at 4°C.

In order to assess the sensitivity of Rubazyme-M, the MACRIA standard sera (40,

10, 3.3, and 1 arbitrary units (AU); Tedder et al., 1982) were tested by Rubazyme-M

and the high and low positive controls from the Rubazyme-M kit were tested by

MACRIA.

Rubazyme-M

This is an indirect EIA, with rubella antigen attached to polystyrene beads. Tests

were carried out according to the manufacturer’s instructions. Briefly, sera were

101

pre-incubated in duplicate at 45°C for 60 min with specimen incubation buffer to

eliminate rheumatoid factor interference. A rubella antigen coated bead was then

added to each well containing a l/200 dilution of serum and incubated at 45°C for 90

min. After washing, goat anti-human IgM conjugated with horseradish peroxidase

was added to each well and incubated at 45°C for 90 min. The orthophenyldiamine

substrate solution was added to each well after washing and the reaction was stopped

with 1 N hydrochloric acid after 30 min incubation. Absorbance was then measured at

492 nm in a Quantum analyser (Abbott Diagnostics). One negative, one high positive

and three low positive controls provided in the kit were included in each test. Results

were calculated and printed out by the Quantum Analyser. The Rubazyme-M index is

the mean of the test sample absorbance divided by the mean absorbance of the 3 low

positive controls. Providing the controls gave satisfactory results, a Rubazyme-M

index <0.910 was negative, >1.090 was positive and values in between equivocal

(+/-).

Neutralisation tests to prove whether reactivity was due to rubella-specific IgM

were performed with reagents and instructions provided by Abbott Diagnostics.

M-antibody capture RIA (MACRIA)

This assay was originally described by Mortimer et al. (1981) and has since been

modified by Tedder et al. (1982), who replaced the ‘251-labelled rabbit anti-rubella

immunoglobulin with ‘251-labelled monoclonal antibody to rubella HA. Briefly,

polystyrene beads coated with sheep or rabbit anti-u (Seward Laboratory, London,

U.K., or Dakopatts a/s, Copenhagen, Denmark) were incubated in a 1 : 40 dilution of

serum. After washing, the beads were incubated in a dilution of rubella haemaggluti-

nating antigen (HA) before washing again and adding ‘251-labelled monoclonal

anti-rubella antibody. After further incubation the beads were washed and bound

radioactivity measured in a gamma-counter. In each assay a series of control sera

containing 40, 10, 3.3 and 1 AU of rubella-specific IgM were included and a standard

calibration curve obtained. The reactivity of test sera was expressed in AU of rubella-

specific IgM as obtained from the calibration curve. In routine use levels of rubella-

specific IgM <l AU are considered negative, l-3.3 AU equivocal and >3.3 AU

positive. For the purposes of comparison, equivocal results in MACRIA or Rubazy-

me-M were considered negative.

Serum fractionation

Sera giving discordant results in Rubazyme-M and MACRIA were tested by serum

fractionation; SDGF (Al-Nakib et al., 1975) was used at St. Thomas’ Hospital, and

GF at King’s College Hospital (Morgan-Capner et al., 1980).

TA

BL

E

1

Com

pari

son

of

Rub

azym

e-M

w

ith

MA

CR

IA

Serd

N

umbe

r R

ubaz

yme-

M

test

ed

posi

tive

Rub

azym

e-M

nega

tive

Rub

azym

e-M

equi

voca

l

Con

cord

ant

Dis

cord

ant

Rec

ent

rube

lla:

18

18

0 0

18

0

MA

CR

IA

posi

tive

(9-2

1 da

ys)

Ear

ly

and

late

se

ra

from

re

cent

20

19

1

0 19

I

rube

lla:

MA

CR

IA

posi

tive

(O-X

and

21

day

s)

Rem

ote

rube

lla:

53

4 49

0

49

4

MA

CR

IA

nega

tive

Sera

pr

esen

ting

prob

lem

s fo

r 15

5

9 1

10

5

rube

lla-s

peci

fic

IgM

te

stin

g:

7 M

AC

RIA

po

sitiv

e

3 M

AC

RIA

eq

uivo

cal

5 M

AC

RIA

ne

gativ

e

Sera

gi

ving

eq

uivo

cal

9 0

7 2

9

MA

CR

IA

resu

lts

0

103

N

104

RESULTS

Excluding the sera from cases of infectious mononucleosis (Table 3) 158 of the 175

sera gave concordant results in Rubazyme-M and MACRIA (Table 1). The 17 CR

giving discordant results were tested by serum fractionation in addition to MACRIA

and Rubazyme-M (Table 2). However, it was not possible to obtain a result by SDGF

on sera 4 and 8 as IgG was present throughout the gradient; this occurred with serum 8

as it had been heat inactivated. Serum 1 taken from an adult with confirmed rubella 2

TABLE 2

Details of seventeen sera giving dlscordant results

Serum Category Serum fractionation MACRIA

(SDGF or GF) (AU)

Rubazyme-M

6

7

8

9

10

II

12

13

14

15

16

17

Early positive

Negative IgM

Negative IgM

Negative IgM

Negative IgM

High HI titre

Problem serum

(Rash 8 wk earlier)

Problem serum (5% mth

after vaccination)

Problem serum

Problem serum

Problem serum

(RF positive)

Intrauterine infection

(cord blood)

Intrauterine Infection

(2 mth)

Intrauterine infection

(26 days)

Intrauterine infection

(cord blood)

Neonate with situ

inversus and biliary

atresia (6 wk)

6/12 after vaccination

6/52 after vaccination

Impossible

+c-)’

I 2.6*’ + + +

Impossible

InhibItor

+c+,*

-c+j*

-c+j*

6.5

1.0

<I.0

1.0

<l.O

11.5

6.8**

9.0**

3.4**

14.0

21.0

14.0

1.2

S.8**

4.3

2.0**

+ + + + - - i- +/- + +/- - +

+/- +/- +/- + + +

_***

_***

_***

_***

+ +

* Results of 2nd test. ** A mean of 2-4 tests. *** Serum tested at l/200, 11500, and l/1000. 0 SDGF with immunofluorescence testing of fractions

105

TABLE 3

Results of testing sera containing heterophil antibody and EBV-specific IgM

Serum MACRIA (AU) Rubazyme-M

1

2

3

4

5

6

7

8

9

10

11

3.3

6.0

4.2

<l.O +/-

3.6

I./

2.1

4.9

25.0 +

2.2

1.5

days after onset of rash was positive by GF and by MACRIA, but not by Rubazyme-

M (Rubazyme-M index ~0.738). A similar negative result was obtained with serum 16

(Rubazyme-M index = 0.903) in which a low level of rubella-specific IgM was detected

by MACRIA and SDGF. Three sera (2, 4 and 5) gave false positive or equivocal

results in at least two Rubazyme-M runs, two of them also giving a very low equivocal

result (1 AU) in MACRIA. Serum 3 gave a false positive Rubazyme-M result on one of

three occasions. Although serum 7 was positive by Rubazyme-M and SDGF it was in

the equivocal range with MACRIA. Sera 8 and 9 were obtained from the same

pregnant woman. The first serum had been referred to us as a small amount of HI

activity had been detected in the IgM fractions obtained by GF at the referring

laboratory. The first serum (serum 8) gave equivocal results in Rubazyme-M and 6.8

AU in MACRIA. A second serum (serum 9) obtained 6 wk later contained a 2-ME

insensitive inhibitor in the IgM fractions and gave positive results in both Rubazyme-

M and MACRIA.

Sera 6 and 17 gave positive results when first tested by SDGF, but negative results

when retested respectively 18 mth and 7 yr later. It was thus impossible to determine

whether it was MACRIA or Rubazyme-M that was giving the incorrect result.

Serum 10 which contained IgM RF and had been used to prepare mixtures of sera

containing RF, was negative in Rubazyme-M, but gave 3.4 AU in MACRIA (mean of 3

results). All RF-serum mixtures were negative in Rubazyme-M.

Sera 11, 12, 13 and 14, from babies with congenital rubella, gave false negative

results in Rubazyme-M. No rubella-specific IgM had been detected in sera 13 and 14

when tested approximately 10 yr earlier by SDGF, although both sera were positive in

a recent test using prolonged incubation of serum fractions with rubella antigen

(Al-Nakib et al., 1975). Serum 15, which was negative by SDGF even when serum

fractions were tested by immunofluorescence, gave a false positive result in MACRIA.

106

TABLE 4

Results of testing MACRIA and Rubazyme-M standard sera

MACRIA (AU) Rubazyme-M

MACRIA

Standards

Rubazyme-M

40 +

10 + +/-

3.3 _ -

I Negative

24, 23, 32, 28, 25 High positive

7.5, 10, 15, 5, 3.6 Low positive

<I, <l Negative

An experimental Rubazyme-M confirmatory neutralisation test used on sera giving

possible false positive results in Rubazyme-M, showed that the reactivity in sera 8 and

9 was indeed rubella-specific IgM and that in serum 5 was due to reactivity to cellular

components. Sera 2 and 3 clearly did not contain rubella-specific IgM, whilst serum 4

gave equivocal results.

Eleven sera containing heterophil antibody and EB virus-specific IgM were tested

by MACRIA and Rubazyme-M. Six of these sera were positive by MACRIA and four

gave results in the equivocal area (Table 3). One serum gave an equivocal result in

Rubazyme-M and one (serum 9) was positive.

When the MACRIA standard sera were tested in Rubazyme-M, the 3.3 AU stan-

dard was negative and the 10 AU standard gave an equivocal result in one of two tests

(Table 4). The Rubazyme-M low positive gave MACRIA results between 3.6 to 15 AU

(mean 8.2 AU).

DISCUSSION

This evaluation has demonstrated the problems that may occur when detecting

rubella-specific IgM, whichever technique is employed. When testing this group of

carefully selected sera we found a number of problems with each of the tests used.

By testing the MACRIA control standard sera it has been established that the

sensitivity of SDGF and GF is 3-5 AU. However, this sensitivity can only be achieved

when such tests are set up by meticulous and experienced laboratory workers. Results

obtained by serum fractionation may be variable. One serum in this comparison,

probably obtained some weeks after subclinical rubella, had been initially negative

when tested by SDGF, but when found positive by both MACRIA (7.5 AU) and

Rubazyme-M, was retested by SDGF and found to be positive. It was thus considered

to give concordant results. It may also be impossible to obtain a satisfactory result by

serum fractionation if sera have been heat inactivated or if inhibitors of HA are

present in the IgM fractions. This problem was apparent with sera 4,8 and 9 (Table 2).

107

MACRIA, which is only available in a limited number of laboratories, has now

been evaluated for more than a year. This technique may also give results which are

difficult to interpret. It has been found that sera from cases of infectious mononucleo-

sis may give false positive results (Morgan-Capner et al., 1983) but that IgM rheuma-

toid factor does not usually interfere (Mortimer et al., 1981). The low (3.4 U) false

positive result obtained with one serum containing a high level of IgM-RF in this

comparison (serum 10 in Table 2) may be due to the fact that the serum had been

frozen and thawed several times before it was tested by MACRIA. Two sera (Table 2,

sera 7 and 17) gave results in the MACRIA equivocal area, although they were positive

by Rubazyme-M and on at least one occasion by SDGF. We have no explanation for

the false positive (serum 15; 5.8 AU) result obtained with serum from an infant aged 6

wk with situs inversus and biliary atresia. In order to avoid reporting false positive

results, it is necessary to give careful consideration to all sera which give low positive

MACRIA results.

Rubazyme-M was generally found to be slightly less sensitive than MACRIA and

serum fractionation (Table 4 and sera 1 and 16 in Table 2). These results indicate that

Rubazyme-M detects at least five to ten MACRIA AU of rubella-specific IgM.

However, it is questionable whether a strict comparison of antibody levels obtained by

different assay systems is advisable. Of the four sera which gave false positive results

(Table 2, sera 2, 3, 4 and 5) serum 3 gave consistently negative results, serum 5 gave

variable results due to reactivity with the cellular components of the rubella antigen,

but sera 2 and 4 were consistently positive or equivocal. However, the Rubazyme-M

indices were low (1.108- 1.559). A second serum from patient 2 gave a similar positive

result (1.462). Neutralization failed to confirm that the reactivity with sera 2 and 4 was

due to rubella-specific IgM and they should therefore be considered false positive

results.

Sera 8 and 9 were obtained 6 wk apart from the same pregnant woman as discussed

earlier. A further serum taken in the post partum period was also positive in Rubazy-

me-M (index = 1.344) and by MACRIA (mean of 4 tests = 5.4 AU). These three sera

gave positive or equivocal results on at least three occasions; by Rubazyme-M

neutralization the reaction was shown to be due to rubella-specific IgM. It is therefore

possible that this woman had an IgM response persisting for at least a year as she had

been screened ‘immune’ by radial haemolysis a year prior to the first serum. Such

long-lasting IgM responses have been described previously (Al-Nakib et al., 1975;

Pattison et al., 1975).

Rubazyme-M is not currently recommended by Abbott Diagnostics for the diagno-

sis of congenital rubella by examination of cord blood and this advice is supported by

false negative results obtained with 2 of the 20 sera from neonates with evidence of

intrauterine infection. However, two sera collected at 26 days and 2 mth of age (sera 12

and 13) were also negative by Rubazyme-M. Serum 12 was also tested at Abbott

Laboratories where a negative result was again obtained. This serum was obtained at 2

mth from a baby who had failed to thrive, while serum 13 was from a baby with

108

splenomegaly, thrombocytopenic purpura and congenital heart defects from whom

rubella virus was isolated. Serum 11 was a cord blood from a baby who was small for

dates and had splenomegaly; it was also negative by SDGF, although a serum taken

13 days later was clearly positive. It is possible that early IgM antibodies such as in this

serum and in serum 1 are more readily detected by antibody-capture than by an

indirect type of assay. The second cord blood (serum 14) was initially negative by

SDGF and positive when retested recently; the mother of this baby had had serologi-

tally confirmed rubella in the fifteenth week of pregnancy.

The manufacturers of Rubazyme-M have previously shown that false positive

results are not obtained when sera containing RF and anti-nuclear-antibody were

tested. When we tested mixtures of sera in order to test the effect of high and low

concentrations of rubella IgG antibody, no false positive results were obtained.

False positive results obtained with sera containing heterophil antibody apparently

present less of a problem in Rubazyme-M than in MACRIA, due to the slightly lower

sensitivity of Rubazyme-M. However, one serum, strongly positive by MACRIA

(serum 9 in Table 3) also gave a positive result in Rubazyme-M. The apparent rubella-

specific IgM detected in sera from patients with infectious mononucleosis may result

from EBV stimulation of B-lymphocytes already committed by prior stimulation with

rubella virus (as seems likely with serum 9). Alternatively, the IgM antibodies detected

may be directed against cellular antigens which are expressed as a result of virus

infection and are detected in IgM assays since similar antigens may also be incorpora-

ted in the lipoprotein envelope of the rubella virus used as antigen (Morgan-Capner et

al., 1983).

Both our laboratories offer a reference service to other laboratories and have had

further experience of sera from patients with no history of recent rubella, giving

positive results in Rubazyme-M, which could not be confirmed in other tests. HOW-

ever, Abbott Diagnostics are continuously improving the formulation of Rubazyme-

M, and such problems appear to be becoming less frequent. It is essential that results

of tests for rubella-specific IgM should be used in conjunction with results of other

tests, such as HI and SRH, previous screening tests and as much accurate clinical data

as possible. When the result is difficult to interpret or results are not consistent with

the history, we would recommend that the test for rubella-specific IgM should be

repeated and that such sera should also be sent elsewhere for testing in a different

assay.

ACKNOWLEDGEMENTS

We are grateful to Abbott Diagnostics for supplying Rubazyme-M kits, to Dr.

Richard Tedder, Middlesex Hospital, for supplying 1251-labelled monoclonal antibody

to rubella, and to Susan Fox for typing the manuscript.

109

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Best, J.M., Banatvala. J.E. and Watson, D., 1969, Lancet 2, 65.

Morgan-Capner, P., Davies, E. and Pattison, J.R.. 1980, J. Clin. Pathol. 33, 1082.

Morgan-Capner. P., Tedder, R.S. and Mace, J.E., 1983, J. Hyg. 90, 407.

Mortimer, P.P., Tedder, R.S., Hambling, M.H., Shafi, M.S., Burkhardt, F. and Schilt, U., 1981, J. Hyg. 86.

139.

Pattison, J.R., 1980, Public Health Laboratory Service, Monograph Series No. 16, HMSO.

Pattison, J.R., Dane, D.S. and Mace, J.E., 1975, Lancet 1, 185.

Tedder, R.S., Yao. J.L. and Anderson, M.J., 1982, J. Hyg. 88, 335.