Ann. Virol. (Inst. Pasteur) 1982, 133 E, 67-72

R U B E L L A - S P E C I F I C IgM D E T E C T I O N BY E L I S A :

C O M P A R I S O N OF 3 S E R A P R E T R E A T M E N T S

by B. Fortier, J. C. Nicolas and F. Bricout

Laboraloire de Virologic, HOpilal Trousseau, 75571 Paris Cedex 12

SUMMARY

Rubella-specific IgM were studied in 236 human sera by ELISA and by sucrose gradient centrifugation techniques. The rheumatoid factor frequency made it necessary to treat the sera before app]ying the EI,ISA technique. Unfol%unately, the results obtained both after rheumatoid factor absorption by aggregated IgG and after IgG absorption by protein A of Staphylococcus aureus were unsatisfactory.

KEY-WORDS: IgM, Rubella, ELISA; Density gradient centrifugation, Rheumatoid factor, Protcin A.

INTRODUCTION

The detection of viral-specific IgM antibodies can be used as a method for the rapid diagnosis of some viral diseases. IgM antibodies are usually detected after separation of IgM and IgG by sucrose density gradient centri- fugation. The enzyme-linked immuno-sorbent assay (ELISA) has been proposed to replace such a t ime consuming separation [3, 9, 14, 17]. Contra- dictory results have been quoted, especially for rubella IgM dctection by this method, involving the presence of rheumatoid factor (RF) which is judged as an interfering element of variable importance [2, 9, 13, 18].

In this report, we compare the detection of rubella-specific IgM anti- bodics by a modified ELISA test and by sucrose density gradient centrifu- gation with subsequent haemagglutination inhibition.

MATERIALS AND METHODS Sera.

Among 236 sera, 208 were obtained from pregnant women and 28 from new- born infants; 16 of the latter were collected from infants whose mothers had been

Manuscrit re~u le 14 d6cembrc 1981, accept6 le 19 f6vrier 1982.

68 B. FORTIER, J .C . NICOLAS AND F. BRICOUT

proved to be infected by rubella virus during pregnancy. All the 236 sera contained rubella virus-specific antibodies by haemagglutination inhibition (HI).

Detection o/rubella-specific IgM.

Rubella-specific IgM was detected by density gradient eentrifugation (DGC) with subsequent HI t i tration of 19S and 7S fractions as described by Vesikari et al. [16]. We have considered the results of DGC as ~ reference results ~ for speci- fic IgM detection in the absence of other completely fool-proof detection methods.

Pretreatment of sera.

Before applying the ELISA technique, each serum was divided into three samples and each sample was treated systematical by in a different way. One sample was simply diluted (1/50) in PBS pH 7.2. A second sample was treated accor- ding to Vejtorp's method [13] for rheumatoid factor absorption (RF absorption): the serum was diluted (1/10) in PBS and one volume of this dilution was added to 4 volumes of a suspension of latex particles coated with aggregated human IgG (Latex RF, Behring Werke). After an incubation period of 1 h at 37 ~ C, the particles were sedimented by eentrifugation (2,000 y for 10 min) and the super- natant was collected. The third sample was treated for IgG absorption by the pro- tein A of Staphylococcus aureus strain Cowan I (S. aureus treatment): the serum was diluted (1 [10) in PBS and one volume of this dilution was added to 4 volumes of a suspension of S. aureus (10 % PBS), which was prepared according to Lind and Massa [7]. After an incubation period of 30 min at 22 o C, the cocci were sedi- mented by centrifugation (2,000 g for 15 min) and the supernatant was collected.

E L I S A test.

The ELISA test used for the detection of rubella-specific IgM was a modification of a technique described before [11. Rubella HI antigen and the corresponding control antigen (~Vellcome) were used for microplate sensitization [2]. For each serum, the three samples (without treatment, RF absorption, S. aureus t reatment) were diluted at 1/2 using PBS with Tween 20 (1%) and foetal calf serum (3 %). The final dilution of the sera was then 1/100. Anti-human IgM (~ chain)peroxidase- labelled antibodies (Insti tut Pasteur Production) were used for IgM detection. Each test, viral and control antigens, was done in duplicate. The optical density value of each serum (OD) at 492 nm for the viral antigen was corrected (ODe) after subtracting that of control antigen. A pool of negative human sera for rubella antibody by standard HI was used for determining the cut-off value. After repeti- tive titrations of this pool, it was agreed that the ODe limit for negative results should he 0.3.

R E S U L T S

Tab le I summar izes the resul ts of rubel la-specif ic IgM de tec t ion b y t h e E L I S A tes t us ing t h e 144 sera j udged as nega t ive a f t e r H I on f rac t ions col lected a f t e r DGC. In t he absence of t r e a t m e n t or a f t e r R F absorp t ion , 46 and 12 sera were r e spec t ive ly found to be rube l la IgM-posi t ive while no pos i t ive resul ts were obse rved a f t e r S. aureus t r e a t m e n t .

D G C = d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . E L I S A = e n z y m e - l i n k e d i m m u n o s o r b e n t

a s say .

H I ~ h a e m a g g l u t i n a t i o n inh ib i t i on . R F ~ r h e u m a t o i d f ac to r .

RUBELLA ANTI-IgM AND ELISA 69

TABLE I. - - False positive results of IgM detection by the ELISA technique in 144 sera found to be rubella-specific IgM negative after DGC: the results are given according to the treatment performed.

Without IgG absorption treatment RF absorption (S . aureus treatment)

46/144 12/144 0/144

TABLE II, - - False negative results of IgM detection by the ELISA technique in 92 sera found to be rubella-specific IgM positive after DGC: the results are given according to the treatment performed.

Without IgG absorption treatment RF absorption (S . aureus treatment)

11/92 21/92 15/92

Table II summarizes the results of rubella-specific IgM detection by the ELISA test using the 92 sera found to be positive after DGC. In the absence of treatment, and after RF absorption or S. aureus treatment, negative results were observed respectively in 11, 25 and 15 cases.

DISCUSSION

Among the 144 sera which were rubella IgM-negative after DGC, 46 (32 %) gave positive results when no t reatment was performed before the ELISA test. If we agree with the well-known safety use of the first method (RF absorption), these results are probably enhanced by the fixa- tion of RF with immunoglobulins already fixed on the antigenic sites as proposed by Salonen et al. [11]. Only 4 of these 46 sera contained RF after detection by an agglutination test (Latex l~F, Behring). The sensitivity of such a test is known to be very low [13].

The presence of RF was observed very differently according to the authors [2, 11, 15]. This difference must be explained by the method of detection, by agglutination Champsaur et al. [2] or by the ELISA test, Vejtorp et al. [15]. These differences could also be explained by differences in the physiological and pathological status of the populations studied. In fact, the RF is more frequent in new-born infants [10], in pregnant women [8] or during the course of some viral diseases such as rubella [11, 12].

This high frequency of RF interaction with IgM detection by the ELISA test made it necessary to pretreat the sera.

After RF absorption, 12 of the 144 sera (8 %) were considered as rubella IgM-positive by the ELISA test. These sera were also rubella IgM-positive without treatment. Similar results were found by Leinikki et al. [6] when they used RF absorption or protein A absorption before the ELISA test.

70 B. FORTIER, J.C. NICOLAS AND F. BRICOUT

However, in our case the protein A treatment gave a good correlation with the DGC. No positive results were found following this treatment.

Among the 92 sera which were rubella IgM-positive after DGC, negative results were found in 11, 15 and 21 cases respectively when no t reatment was performed, or after RF absorption or S. aureus treatment.

Competition between IgG and IgM versus the antigenic sites could explain this apparent lack of sensitivity as it was postulated before [6]. Indeed, after S. a u r e u s t reatment of the 11 sera found to be IgM negative without treatment, 3 remained negative and the remaining 8 could be considered as positive. This was probably due to the absorption of IgG by S. a u r e u s protein A.

However, such competition could not explain the failure of detection of specific IgM by the ELISA test after S. a u r e u s t reatment for 15 sera which were rubella IgM-positive after DGC. This lack of sensitivity after S. a u r e u s t reatment could be explained by an incomplete absorption of IgG by the protein A as already proposed by Leinikki et al. [6] or by an absorption of some IgM molecules on protein A as observed by Grangeot- Keros et al. [4] or Harboe and Folling [5].

In addition to the 11 sera found negative without treatment, 10 other sera were found negative after RF absorption. These results strongly suggest the lack of sensitivity of the ELISA technique for IgM detection. In fact, among the 81 sera found to be IgM positive without any treatment, some could be considered as non-specifically positive when RF was used. This can be assumed by the enhancement of the false negative results after RF absorption.

In conclusion, it is obvious that specific IgM detection requires pretreat- ment of the serum before performing an ELISA test. Treatment with RF or with S. a u r e u s protein A is not satisfactory and it seems extremely hazardous to use such an ELISA test for rubella-specific IgM detection in pregnant women.

RESUME

DI~TECTION IMMUNO-ENZYMATIQUE DES IgM SPI~CIFIQUES DE LA RUBt~OLE :

COMPARAISON DE 3 PRI~TRAITEMENTS DES St~RUMS

Les IgM sp6cifiques de la rub6ole de 236 s6rums humains out 6t6 reeher- ch6es par technique immunoenzymatique ainsi que par ultracentrifugation en gradient de saccharose. La fr6quence du facteur rhumatoYde a oblig6 h traiter ]es s6rums avant la technique immunoenzymatique. Cependant des r6sultats non satisfaisants ont 6t6 obtenus rant aprbs absorption du facteur rhumatoide par des IgG agr6g6es qu'apr~s absorption des IgG par la prot~ine A de S taphy lococcus aureus .

MOTS-CL~S : IgM, Rub~ole, ELISA ; Ultracentrifugation en gradient de densitY, Facteur rhumato~de, Prot~ine A.

RUBELLA ANTI-IgM AND ELISA 71

ACKNOWLEDGMENTS

This work was supported by a grant of the (( Fondation pour la Recherche Mddicale )).

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