.Journal o,f Virological Methods, 3 (I 98 1) 45--49

Fkevier/North-Holland Biomedical Press

45

RAPID DETECTION OF RUBELLA-SPECIFIC IgM ANTIBODIES BY THE USE OF

MICROIMMUNOBEADS (MIB-IgM)

R. BRAUN' , H.W. DOERR** and C. HORING’

‘Ernst-Rodenwaldt-Institut, Kohlenz; and ‘Institu t fir Medizinische Virologie der Universittit, Im

Neuenheimer Feld, 324, O-6900 Heidelberg, (F.R.G.)

(Accepted 24 February 1981)

A simple haemagglutination (HA) titre reduction assay for the detection of rubella-specific IgM

antibodies has been developed. Serum IgM is rapidly isolated by immune adsorption to heavy chain-

specific anti-human IgrM antibodies covalentty coupled to polyacrylamide microimmunobeads. The

immob~ized immunocomplexes are incubated with rubella virus HA antigen. Subsequently. the

particles are centrifuged from the solution, and the reduction of the HA titre is determined. This

procedure has proven to be as reliable as haemagglutination inhibition (HAI) tests carried out with

IgM fractions separated on a sucrose density gradient.

INTRODUCTION

Solid phase immunosorption of serum IgM has been proven to enable rapid serologica

diagnosis of recent cases of rubella, hepatitis and other viral diseases in patients (Doerr,

1979; Doerr et al., 1980; Krech and Wilhelm, 1979; Gerlich et al., 1980; Roggendorf

et al., 1980). However, since only simple adsorption procedures are used, high initial

dilutions have been usually found necessary to avoid non-specific reactions (Roggendorf

et al., 1980). Otherwise, the test results might have to be confirmed by the mercapto-

ethanol reduction method (Doerr et al., 1980). A solid phase carrier, to which heavy

chain-specific anti-human IgM antibodies have been futed covalently, promises an im-

provement of test specificity. In the following, we introduce such a test system using

polyac~lamide micro~munobeads, which are commercially available.

MATERIALS AND METHODS

Lyophilized microimmunobeads (anti-human IgM) were purchased from Bio-Rad,

Munich (F.R.G.). They are hydroph~ic polyacrylamide particles, S-10 m in diameter,

to which heavy chain-specific rabbit anti-human IgM antibodies have been coupled

covalently via a peptide bound. Once rehydrated the beads can be stored for at least one

* To whom requests for reprints should be addressed.

o166-0934i8liOOOn-0000/$02.5o 0 E:lsevier/North-llolland Biomedical Press

46

month at 4°C. They are stable in suspension (10 mg/l ml of 0.2 M PBS, pH 7.2) for several

hours.

The microimmunobeads have been recommended for different immunoassays (Langone,

1978) and B-cell labelling (Ammann et al., 1978). Test procedure: 20 ~1 of undiluted

serum sample is added to 0.2 ml bead suspension, thoroughly mixed and incubated for

1 h at +37”C. Subsequently, the mixture is diluted with 2 ml PBS-Tween 20 (0.5% in

0.2 M buffer solution, pH 7.2) and the particles are pelleted (7000 g, 1 min). This washing

procedure is repeated twice and the last pellet is resuspended in 0.2 ml rubella haemag-

glutination antigen solution (obtained from Behringwerke, Marburg, F.R.G., diluted

1 : 4 in Hepes salt albumin gelatin buffer (HSAG), pH 6.2, corresponding to 64-128

haemagglutination (HA) units). After a second incubation period of 1 h at +37”C, the

particles are centrifuged from the solution, and the rubella HA titre is determined with

trypsinized human 0-erythrocytes in the usual microplate system (U.S. Department of

Health, Education, and Welfare, 1970) three times in parallel. Twofold dilution steps are

used. A serum specimen of a person with a high anamnestic rubella antibody titre in the

haemagglutination inhibition (HAI) test originating from a previous infection (1 : 512,

no specific IgM detectable after Ig differentiation by density gradient centrifugation)

served as control.

The exclusion of IgG contamination was checked by two methods.

1. Resuspension of the bead pellet after the washing procedures in 0.2 ml peroxidase-

labelled anti-human IgG solution (obtained from Dako, Copenhagen/Denmark; diluted

in PBS 1 : 200). After an incubation period of 1 h at +37”C and three further washing

steps, the enzyme reaction was started by the addition of the adequate substrate solution,

as previously described (Doerr et al., 1980).

2. Resuspension of the bead pellet, as given above, in 0.2 ml serum sample with a

high antibody titre to the blood group Rh+ (D) (1 : 2 8 192) instead of the rubella antigen

solution. After the analogous incubation and particle sedimentation, an eventual anti-

body reduction was easily checked by a simple HA test.

The serum IgM isolation of control experiments was carried out by sucrose density

gradient centrifugation with 2-mercaptoethanol treatment of the IgM fractions, as des-

cribed elsewhere (Caul et al., 1978). The IgM fractions were investigated for specific

activity in the CDC standard rubella HA1 test (U.S. Department of Health, Education,

and Welfare, 1970). Absence of IgG contamination was demonstrated by Laser nephelom-

etry.

Serum samples of patients with and without suspect of having a recent rubella infection

were collected in the routine diagnostic service of our institutes. 110 specimens of 70

patients were investigated.

RESULTS

In preliminary experiments, each lot of microimmunobeads used for serum IgM im-

munosorption was examined for IgG contamination, as described above. No colour

47

reaction was visible by the use of peroxidase-labeled anti-human IgG (r) antibodies until

45 min after the addition of the enzyme substrate. When a serum specimen, presenting

a high anti-D antibody level was used, no titre reduction could be detected in HA tests

with human erythrocytes of the blood group Rht (D). These antibodies belong to the

IgG or IgA class exclusively.

Table 1 presents an evaluation of the MIB-IgM tests carried out with patients’ serum

samples. In all 49 serum samples containing virus-specific IgM antibodies, as confirmed

by ultracentrifugation, a titre reduction by the MIB-IgM test was seen. As shown in lines

6 and 7 of Table 1, we also detected a titre reduction by the MIB-IgM test in serum speci-

mens obtained from eight patients immediately at the onset of rubella symptoms (lymph-

nodes palpable, exanthema), although no antibody titres in their 19 S fractions were

detectable. All those eight patients, however, developed specific IgM antibodies and a

titre rise of at least fourfold in a second serum obtained 7-10 days later, whereas serum

samples without titre reduction in the MIB-IgM test produced no titre rise in subsequent

serum specimens and showed no IgM titres in the 19 S fractions of all serum samples.

Forty-three persons with only anamnestic or without any rubella antibodies revealed no

evidence for specific IgM in both methods (Table 1, lines 8 and 9).

All virus titrations were performed three times in parallel. These results demonstrate

the absence of non-specific rubella antigen inhibitors. Furthermore, we looked for the

interference caused by rheumatoid factor IgM directed to human IgG. Ten serum speci-

mens without specific IgM but IgG antibodies to rubella haemagglutin~ (1 : 64-1 : 1024),

which produced positive test results ( 1 : 20-I : 640) in the RF Latex a~lutination test

(Doerr et al., 1980), were found to be completely negative in the MIB-IgM test. Qual-

TABLE 1

Comparison between titres of the 19 S fraction and titre reduction steps in the MIB-IgM test for the

detection of specific antibodies in serum specimens originating from patients with and without clinical

suspect of rebella

Sera

fnf

19 S fraction Titre reduction

in HA1 steps in HA (log, 1

Total serum

in HAI

3 128 5-6 512

13 64 3-6 12882048

13 32 3-5 128- 1024

17 16 2 --4 32- 256

3 8 2-4 128- 256

6a <8 l-4 8- 256

2a <8 l-3 <8

21 <8 0 8- 512

22 <8 0 <8

a In these cases, specific antibodies in the 19 S fraction and an at least fourfold titre rise were found

in subsequent serum samples obtained 7-10 days later without exception.

Tttre of 19 S fraction

Fig. 1. Comparison between titrz reduction assay and IgM titres derived by uItracenWugation.

itatively seen, we found a complete agreement between the new technique and the

established method for detecting rubelIa IgM. For quantitative evaluation, we plotted

the semm-specific amounts of HA reduction (based on a log, d&&ion row) into a dia-

gram, comparing them to the results seen in HA1 tests carried out with the isolated IgM

fraction (Fig. 1). A good correlation was found between the two methods, but, what is

more important, always a positive result, even if the HAI revealed only low titre values

in the isolated IgM fractions.

DISC‘USSION

The determination of IgM antibodies to rubella haemagglutinin by the help of the

titre reduction assay is easy to handle and shows a very good correlation to data obtained

by the classical density gradient centrifugation method. A preabsorption of the serum

specimens is not necessary, because non-specific factors (RF, ~-lipoproteins) do not

cause false positive results. Also the 2-mercaptoethanol treatment can be omitted. The

covalent fixation of the anti-IgM antibodies to the solid phase carrier enables stronger

washing procedures and assures a higher degree of reproducibility in the IgM isolation.

Furthermore, by the determination of the residual HA titre a considerable improvement

of test sensitivity is reached. Virus neutralization requires more antibodies than virus

fixation to antibodies on a carrier. Corresponding to that, in a normal HA1 no haemag-

49

glutination inhibition of the usual 2-4 HA units of rubella can be reached after IgM

immunosorption, using microimmunobeads in the concentration specified. This may

be the reason for the use of only one HA unit in a test system previously introduced

(Krech and Wilhelm, 1979) which may be critical. In fact, based on information given

by the producer (Bio-Rad, Munich), only the IgM of 2 fl of serum can be isolated via

0.2 ml beads suspension (10 mg/l ml), when a quantitative IgM concentration of 120 mg/

100 ml serum is assumed.

The new test consumes less time than any other method and requires no expensive

technical equipment. It may be applicable for many haemagglutinating antigens and with

some variations for the detection of neutralizing IgM antibodies, and also in radioim-

munoassay and enzyme-linked immunosorbent assay tests.

ACKNOWLEDGEMENT

The authors wish to thank Mrs. B. Maly for her excellent technical assistance.

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