Journal of Virological Methods, 14 (1986) 24s251 Elsevier

243

JVM 00529

The development of an M antibody capture ELISA for rubella IgM

Kathryn Bellamy ‘** S.A. Rousseau2+ and P.S. Gardner ‘3” ,

‘Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT; and ‘Public Health Laboratory, Luton and Dunstable

Hospital, Lewsey Road, Luton LlJ4 ODZ, U.K.

(Accepted 9 June 1986)

An M antibody capture enzyme-linked immunosorbent assay for rubella IgM was developed. The

enzyme label was prepared from a monoclonal antibody raised against rubella haemagglutinin (Tedder

et al., 1982). Paired sera from acute rubella infections and vaccines as well as sera from blood donors,

antenatal patients and patients whose sera contained rheumatoid factor and patients with acute non-

rubella infections were tested by this method.

ELISA, M antibody capture

Introduction

Until recent years the tests for rubella specific IgM involved a preliminary phys- ical separation of IgM and IgG (Pattison and Dane, 1975). In 1978 Diment and Pepys described a method which utilised an antibody to the F, fragment of IgM (anti p) attached to a solid phase to capture IgM. This allowed separation of IgM and IgG by a simple washing stage. In 1981 Mortimer et al. described an M an- tibody capture assay using I lZ5-labelled anti-rubella antibody; this was found to be a sensitive method for the detection of IgM in patients’ serum. Once IgM was cap- tured by the solid phase, rubella antigen and labelled antibody to rubella were in- cubated with the solid phase in succession. The number of counts is proportional to the amount of antibody in the sample.

An enzyme label may be substituted for the radio-label in which case, when the substrate is added to the solid phase, the intensity of colour is proportional to an- tibody.

The M antibody capture enzyme-linked immunosorbent assay overcomes a number of problems encountered when other methods are employed.

*Present address: Unilever Research Laboratory, Department of Immunology, Colworth House,

Sharnbrook, Bedfordshire MK44 lLQ, U.K.

0166-0934/86/$03.50 @ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

244

Firstly the inherent dangers of widespread use of radioactive isotopes is ob- viated. More importantly M antibody capture does not suffer from interference due to rheumatoid factor in sera.

This paper reports the development of an M antibody capture ELISA for use in the diagnosis of rubella infections.

Materials and Methods

Sera tested The sera examined for anti-rubella IgM included acute and convalescent phase

specimens from patients (36) with rubella-like illnesses, and post-immunisation specimens from women (24) given rubella vaccine. Other specimens tested in- cluded those from patients with recent infections of infectious mononucleosis (12), cytomegalovirus (4)) herpes simplex virus (1 l), respiratory syncytial virus (5)) in- fluenza A (5), toxoplasma gondii (2)) hepatitis A (I), parainfluenza (9), mumps (5) and mycoplasma pneumoniae (4) and sera from patients attending a rheuma- tology clinic (258).

Control sera An anti-rubella IgM positive control serum was prepared from the recalcified

plasma of a single donation from an adult attending the North London Transfu- sion Centre. The patient had had a recent rubella infection.

A negative control serum was prepared from pooled serum, negative for both IgG and IgM rubella antibody by single radial haemolysis, haemagglutination in- hibition and ELISA. The positive control was designated 100 units, dilutions of the positive serum were made in negative serum to give 30, 10, 3 and 1 arbitrary units. This method of standardisation was first described by Mortimer et al. (1981

a).

Preparation of horseradish peroxidase anti-rubella conjugate Monoclonal antibody prepared against rubella haemagglutinin (Tedder et al.,

1982) was used to prepare the enzyme label. Immunoglobulin was purified from ascitic fluid by chromatography, on DEAE 52 Cellulose (Whatman Ltd.) accord- ing to the method described by Tedder et al. (1982). The method uses 0.01, 0.03 and 0.06 M phosphate buffer to elute immunoglobulin from the cellulose. Each fraction was assayed for rubella haemagglutination-inhibition (HAI) activity and fractions containing the highest HA1 levels were pooled and the protein content estimated by the method of Lowry et al. (1951).

The enzyme label was prepared according to the method of Nakane and Kawaoi (1974). Four mg of horseradish peroxidase (HRP) type VI (Sigma Chemical Co.) was dissolved in 1 ml 0.3 M sodium bicarbonate buffer pH 8.1 and gently mixed at room temperature for 1 h with 100 ~1 1% 2,4 dinitrofluorobenzene in absolute alcohol. The mixture was oxidised with 200 ~1 freshly prepared 0.1 M sodium per- iodate for 30 min, after which the mixture was dialysed against 1 mM sodium ace-

245

tate buffer pH 4.4 overnight at 4°C. The pH of this was raised to 9.5 with 0.2 M sodium bicarbonate buffer pH 9.5 immediately prior to the addition of an equal volume of rubella antibody (2 mg/ml) which had been dialysed at 4°C against 0.01 sodium carbonate buffer pH 9.5. The mixture was mixed gently at room temper- ature for 2 h. The conjugate formed was stabilised by the addition of 100 ~1 of 0.1 M sodium borohydride. The solution was allowed to stand for 2 h at 4°C and then concentrated, using a Minicon B15 concentrator (Amicon Ltd.). It was fraction- ated by downward flow on a 2.6 x 40 cm column of Sephacryl S300 (Pharmacia Ltd.) previously equilibrated with 0.1 M borate buffer pH 8.0 containing 0.4% sa- line, 0.1% BSA and 5% p(+)mannose. Fractions containing HRP-labelled IgG were pooled and concentrated using a Minicon B15 concentrator. The conjugate was mixed with an equal volume of 60% glycerol in borate buffer, and stored at 4°C.

ELBA for rubella specific IgM The test procedure adopted after investigating all parameters was as follows. (1) Nunc immuno I plates were coated with antibody to the Fc fragment of hu-

man IgM (anti k). The antiserum used was obtained from Northumbria Biologi- cals and was produced in swine. 100 ~1 of 1:lOOO anti lo, in 0.02 M Tris-HCl buffer pH 7.6 was added to each well. Plates were sealed and incubated at 20°C for 18 h. The plates were subsequently washed with 0.02 M Tris buffer and each well completely filled with a solution of 1% bovine serum albumin (BSA) (Armour Pharmaceuticals) in Tris buffer. After incubation at 20°C for 1 h the plates were aspirated if to be used immediately or partially aspirated leaving 50-100 ~1 of the BSA solution per well.

(2) 1:lOO dilutions of all sera, including controls were made in phosphate buff- ered saline containing 0.05% Tween 20 (PBST) and 100 l.~l of each added to du- plicate wells of the plate. After incubation at 37°C for 2 h, the plates were washed four times with PBST and aspirated to dryness.

(3) Rubella haemagglutinin diluted 1:20 in PBST was then added to each well (100 l.r.1 per well) and the plate incubated at 20°C for 18-20 h.

(4) The plates were again washed and then incubated at 37°C for 2 h with 100 ~1 per well of horseradish peroxidase labelled-monoclonal antibody to rubella. (The conjugate was diluted in PBST containing 1% human serum with no antibody to rubella and 1% normal swine serum - Tissue Culture Services.)

(5) Plates were washed four times with PBST and 100 ~1 per well enzyme sub- strate added (8 mg 2,2’-azino-bis(3-ethyl)benzthiazoline sulphonic acid (ABTS) in 10 ml 0.05 citric acid phosphate buffer pH 4.0 containing 100 ~1 20 volume hy- drogen peroxide).

(6) Incubation at 20°C for 30 min was followed by the addition of 50 )*l per well of a 2 mg/ml sodium fluoride solution.

(7) The absorbance at 405 nm was read on a Multiskan spectrophotometer (Flow Laboratories). Absorbance values of sera under test were compared with those obtained for the control sera.

246

TABLE 1

Sera from patients with clinical rubella.

Patient

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21 22 23 24 25 26

27 28

Days after onset of rash ELISA arbitrary units HA1 titre

2 l-3 < 10 15 >40 > 1280 1 Cl < 10

14 >40 320 5 <l < 10

27 >40 320 2 <l < 10

15 >40 320 3 3-10 < 10

14 >40 > 1280 2 l-3 < IO

17 240 320 1 it < 10

10 >40 160 2 <l < 10

17 >40 320 3 3-10 < 10

10 >40 >1280 2 >40 80

17 >40 320 3 <l < 10

10 >40 320 2 il < 10

10 >40 640 13 >40 160 39 10 160

3 <l i 10 20 >40 160

3 >40 >1280 16 >40 >1280 3 >40 20

24 >40 320 2 >40 40

18 >40 640 2 10 < 10

16 >40 160 1 ‘Cl < 10

20 >40 160 2 3-10 20

12 >40 160 6 >40 1024 1 >40 512 8 >40 1024 6 >40 1024 7 >40 1024 1 10-30 < 16

10 >40 256 11 >40 512 7 >40 1024

247

Patient Days after onset of rash ELISA arbitrary units HA1 titre

29 30 31

32 33

34

35 36

12 >40 1024 5 >40 1024 5 >40 256

15 >40 256 4 >40 256 3 >40 128

17 >40 512 0 >30 16

16 >40 1024 10 >40 512 1 >3 < 16 9 >40 1024

Results

The results of the assay on samples from 36 patients are shown in Table 1. The sera were collected between 1 and 39 days after onset of illness, with one sample actually taken on the day of onset. It was possible to detect rubella specific IgM in six specimens, where no rubella antibody was detectable by HAI. IgM was de- tected in 13 out of 23 samples taken within 3 days of onset of rash, and the re- maining patients had detectable IgM when a second sample was tested. IgM was detected in samples up to 39 days after onset of rash, although it is possible that it would be detected for longer if suitable specimens were available.

Serum samples from patients with non-rubella infections were examined and the results are shown in Table 2. Of the 84 sera tested 80 gave a negative result and four gave very low reactivity of between 1 and 3 units which would also be con- sidered negative. Three of these sera were from cases of EBV infections all of which contained heterophile antibodies. A cross-reactivity between EBV sera and the M

TABLE 2

MACELISA arbitrary units for non-rubella infections.

Infectious agent Number tested MACELISA arbitrary units <l l-3 >3

Mycoplasma pneumoniae Cytomegalovirus Herpessimplex Varicella zoster Respiratory syncytial Influenza A Epstein-Barr virus Toxoplasma gondii Hepatitis A Parainfluenza Mumps

4 4 4 4 _

11 10 1 _

26 26 5 5 5 5 _

12 9 3 2 2 _

1 1 _

9 9 _

5 5

Totals 84 80 4 0

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TABLE 3

Comparison of sucrose density gradient centrifugation and HA1 and ELISA for IgM detection.

Days HAI titre MACELISA after onset

Arbitrary units

Fraction 2 Fraction 2 Fraction 9 Fraction 9 Fraction 2 Fraction 9

+2 ME control +2 ME control

Vaccinee 0 2 >2 2 16 <l <l

19 <2 8 8 16 30-100 3-10

76 2 8 32 16 l&30 3-10

Natural Infection 6 <2 64 32 32 >lOO 10

10 (2 64 32 32 >lOO l-3 120 4 4 32 32 1 1

antibody rubella immunoassay had previously been found in radioimmunoassay (Morgan-Capner et al., 1983). In order to ensure that no cross-reactivity occurred with sera containing high levels of rheumatoid factor (Rf), 258 sera were tested. These had previously been tested for Rf by a sheep cell agglutination test (SCAT) (sheep cells sensitised with rabbit amboceptor). Only six sera reacted at all in the assay, they gave results between 1 and 3 arbitrary units which was considered neg- ative. The SCAT titres of these sera which reacted ranged from >1:1024 to ~1.8.

Two small series of specimens were tested by ELISA and sucrose density gra- dient centrifugation followed by haemagglutination-inhibition (HAI) in the pres- ence and absence of 2-mercaptoethanol (Caul et al., 1974). The HA1 employed an overnight incubation with cells before reading the agglutination patterns. The two series consisted of three sera from two patients, one of whom had had clinical ru- bella, and the other had received rubella vaccine. The results are shown in Table 3. IgM was detected by both methods, the presence of total IgG and IgM in each fraction was determined by single radial immunodiffusion. Fraction 2 in each case contained IgM but not IgG whereas fraction 9 contained IgG but no IgM.

Paired sera from (24) patients who were vaccinated against rubella (the type of vaccine was not known) were assayed by M antibody capture ELISA, single radial haemolysis (Kurtz et al., 1980) and also Rubazyme M and G enzyme immunoas- says (Abbott Laboratories). The Rubazyme assays are both indirect assays not an- tibody capture assays and they were carried out according to the manufacturers’ instructions. The results are shown in Table 4. A Rubazyme G index of greater than 1 .O is considered positive and Rubazyme M indices of less than 0.910 are con- sidered negative, between 0.910-1.090 equivocal and greater than 1.090 are con- sidered positive. Sera which had less than 15 IU/ml by SRH are considered non- immune and offered vaccination. All the pre-vaccination samples were negative by SRH but some were positive by Rubazyme G. The post-vaccination specimens from five pairs did not contain IgM by the M antibody capture assay or the Rubazyme M. Of these five patients, three were found to be positive by Rubazyme G in the pre-vaccine specimens and the remaining two pre-vaccine samples gave equivocal results by Rubazyme G.

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TABLE 4

Serological tests on sera from vaccines.

Name Radial Abbott ELISA Weeks post- haemolysis Rubazyme G Rubazyme M arbitrary units vaccine

MK

JMc

LP

IR

PA

SP

JE

PH

BU

GT

AM

SW

PH

JG

JH

PC

SL

so

W

RA

CI

SL

AK

HA

+ _ + _ +

+ _ + _ +

+ _ +

+

+

+ _ +

+ faint

+

+ _ +

+

0.450 1.723 0.43 2.004 0.37 1.96 0.436 1.606 0.546 1.91 0.41 0.882

0.665

0.708

0.397

1.17

0.443

1.898

1.146

0.57

1.812 0.832

0.323 0.65 1.90 0.295 1.946 0.568 1.691 0.596 1.679 0.452 1.74 0.458 1.936 0.552 1.578 0.612 1.323 0.34 1.44 1.183 2.173 0.972 1.765 0.918 1.637 1.17 2.75 1.55 1.83

0.59

0.949

0.552

0.918

0.74

1.773

1.308

0.648

0.84

0.427

0.553

0.211

0.395

0.37

< 1 7.5

< 1 2.5

i 1 3.0

< 1 31.0

< 1 5.5

< 1 >lOO < 1

20.5 < 1

3.0 < 1

20.5 < 1

3.5 < 1

5.5 < 1

27.5 < 1

8.5 < 1

18.5 < 1

23.5

>lOO < 1

15.5

5.5 < 1

23.5 < 1 < 1 < 1 < 1 < 1

1 < 1 < 1 < 1 < 1

9

18

22.5

6

7

4

6.5

8.5

6.5

8.5

12.5

6

7

6

6

6.5

6

6

6

8

6

6

8

6

250

A comparison of the results obtained in the M antibody capture assay with the Rubazyme M results gave rise to a number of discrepancies. Nine specimens taken between 6.0 and 12.5 wk (mean 7.4 wk) after vaccination gave positive results in the M antibody assay but negative results in the Rubazyme M assay. One other sample which was positive by the M antibody assay gave an equivocal result (0.918) in the Rubazyme M assay. The seroconversion of all these patients was confirmed by the SRH and Rubazyme G assay.

Discussion

The method we have described for detecting rubella specific IgM was found to be both sensitive and reliable. All except one of the M antibody capture assay re- sults on sera from patients with clinical rubella were confirmed by using HAI titres. The one unconfirmed result can be explained by the time at which the specimens were taken 13 and 39 days after the onset of rash. It was possible to detect IgM

as early as the day of onset of rash and up to 39 days after onset. It is probable that IgM detection is possible for a much longer period of time but suitable spec- imens were not available.

IgM was detected in three samples before HA1 antibody was detected and in all samples once the HA1 titre had reached 1:20. In theory therefore a diagnosis could be made on a single serum sample, in practise however it is extremely unlikely that the results of a single assay would be sufficient, especially if the patient were preg- nant. In 1985 Kurtz and Anderson found cross-reactions between IgM specific tests for parvovirus and rubella. They suggested that sera which give equivocal results when tested for rubella specific IgM, should be tested for parvovirus specific IgM. This may be helpful in the investigation of pregnant women with rubelliform rash illness as there are at present no clinical grounds for terminating pregnancies com- plicated by human parvovirus infections.

Tests on sera from non-rubella infections, although no parvovirus infections were tested and those sera with rheumatoid factor did not reveal significant non-specific reactivity. Previous reports (Duemeyer et al., 1979) suggested that Rf sera react with enzyme labelled IgG unless the IgG has been pepsin digested to remove the Rf binding site. This does not appear to be a problem in this assay but will require further investigation.

It would also appear that this method was suitable for detecting the response to rubella vaccination. The results also support those of Cubie and Edmond (198.5), in that the M antibody capture method was more sensitive than Rubazyme M. And finally the absence of IgM in post-vaccine samples from four women whose pre- vaccine sera were ‘positive’ by Rubazyme G, but negative by SRH supports the findings of Mortimer et al. (1981 b) and their query as to whether some women are vaccinated unnecessarily.

251

Acknowledgements

We are grateful to Dr. R. S. Tedder, Middlesex Hospital Medical School, for providing the monoclonal rubella antibody; Dr. Lee, Robert Jones and Agnes Hunt, Ortopaedic Hospital, Oswestry for the rheumatoid factor sera; Dr. P. Mor- timer, Virus Reference Laboratory, Colindale for paired sera and the Public Health Laboratories at Leeds, Poole, and Liverpool for additional sera; also Mrs. Ann Scott for technical assistance.

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