Serodiagnosis and Immunotherap.v (1987) 1, 267-273

The detection of cytomegalovirus (CMV)-specific IgM using peroxidase lahelled antigen: comparison with an indirect

enzyme-linked immunoassay

A. A. El-Mekki*, W. Al-Nakibf’f, S. R. Yasin*t and 0. Strannegard*

*Department of Microbiology, Faculty of Medicine, University of Kuwait, and tMRC Common Cold Unit, Harvard Hospital, Coombe Road, Sulisbur~~. Wiltshire SP2 8B W,

U.K.

Three hundred and forty-seven serum samples were obtained from 54 renal transplant patients, seven patients who had heterophil antibody negative mononucleosis, 23 with non-specific febrile illnesses. 10 patients who were positive for anti-nuclear antibody and who had systemic lupus erythematosus (SLE). 58 patients with other diseases of non-viral aetiology and 195 healthy individuals. These were examined for the presence of cytomegalovirus-specific IgM antibody by an IgM capture assay utilizing CMV-peroxidase labelled antigen and a commercially available indirect enzyme immunoassay (Enzygnost, Behring). The former assay detected 22 CMV-IgM positive specimens whereas the latter assay detected only 16 of these 22 (73%) samples following rheumatoid factor (RF) removal. The CMV-binding activity detected in five of the sera which gave discordant results was shown to be located in the IgM fractions by the capture assay following sucrose density gradient fractionation. Thus there was no indication of false positive reactions in the IgM capture assay. In addition, the presence of anti-nuclear antibodies did not interfere with the enzyme immunoassay investigated.

These results. therefore, suggest that the CMV-IgM capture assay utilizing enzyme-labelled CMV antigen is a sensitive and specific procedure which should have important applications in the diagnosis of CMV infections with distinct advantages over the indirect immunoassay.

Kic~word.s: cytomegalovirus. IgM. indirect ELISA. capture ELISA.

Introduction

Cytomegalovirus (CMV)-specific IgM antibodies have been detected following primary infection in pregnancy’ ’ and in the sera of infants with congenital CMV infection’.O. In addition, these antibodies can be detected after infection in immunocompromised patients such as those receiving bone-marrow or renal transplantation”.‘. The detection of these antibodies, therefore, has been found to be useful in screening for evidence of CMV infection.

There are a number of assays for CMV-IgM detection. Thus CMV-IgM has been

$ To whom alI correspondence should he addressed. Dr W. Al-Nakib, MRC Common Cold Unit.

267 0888-X)786/87/040267+07 $03.00/O (“ 1987 Academic Press Limited

268 A. A. El-Mekki et al.

detected by immunofluorescence (IF)‘, immunoperoxidase staining”‘, indirect radioim- munoassay (RIA)“,“, indirect ELISA” and more recently by IgM capture procedures using “‘1-1abelled antibody7 or enzyme-labelled CMV antigen14.15. The specificity of most of the solid-phase indirect assays and the IF test have clearly been found to be affected by the presence of RF which is an IgM anti-IgG antibody in sera from normal and healthy individuals resulting in “false” IgM positive reaction’6. Moreover, the presence of specific IgG has also been shown to interfere with the detection of specific IgM by competing for the same antigen binding sites, thus affecting the sensitivity of the assay especially when the latter was present in small concentrations’““. The introduction of IgM capture assays, in which labelled immunoglobulins were used, partially resolved these limitations since high concentrations of specific IgG no longer interfered with IgM detection. However, as was demonstrated by Isaac & Payne2’, interference by RF, although reduced, was not totally eliminated. A modification of the IgM capture assay in which labelled antigens instead of labelled immunoglobulins were employed, resulted in a sensitive assay which was unaffected by the presence of either RF or high concentrations of IgG as has recently been demonstrated for CMV’4,‘5, rubella2’, Epstein-Barr virus 22 flavivirus*‘, varicella-zoster virusZ4 and measles”. ,

We were, therefore, very interested in developing an IgM capture assay for the detection of specific CMV-IgM using labelled antigens based on the method earlier described by Schmitz et a1.l4 This study compares the application of this procedure with that of a commercially available indirect assay for the detection of CMV-specific IgM in different categories of patients with or without evidence of a CMV infection.

Materials and methods

Human sera

A total of 347 serum samples were collected from different groups of patients (Table 1) and stored at - 20°C.

Indirect enzyme immunoassay

Commercially available indirect CMV-IgM ELISA was purchased from Behring, AG (Enzygnost). Briefly, sera were diluted 1: 20 and tested in duplicate wells that had been

Table 1. Groups of sera tested

Clinical condition No. of sera tested

Renal transplant patients (a) Suspected CMV cases (b) Follow up cases

Systemic lupus (anti-nuclear antibody-positive) Mononucleosis (Paul-Bunnel negative) Non-specific febrile illnesses Other diseases of non-viral aetiology Healthy individuals (routine screening)

Total

13 41 10 7

23 58

195

347

CMV detection by peroxidase labelled antigen 269

coated with CMV antigens or uninfected tissue culture fluids (control antigen). Rabbit anti-human IgM alkaline phosphatase conjugate was also obtained from Behring (Behring OSDI 04/05). The assay was performed and evaluated as recommended by the manufacturer. The same test was also used to detect and measure CMV-specific IgG. All sera found positive on initial screening were then tested for the presence of RF by a Rheuma-Wellco test (AD04). Specimens containing RF were absorbed with heat aggregated IgG as described by Krishna et ~1.~~ and were retested for the presence of CMV-IgM by the indirect ELISA. Reference CMV-IgM positive and negative sera were titrated (1: 20 to 1: 1280) and included in each test.

Capture enzyme immunoassay

Preparation of CMV-peroxidase conjugate: CMV (strain AD169) was propagated in human embryonic lung (HEL) fibroblasts in Roux bottles at an m.o.i. of approximately 0.5. When 9&100% of the cell layer showed cytopathic effect (CPE), usually 1 week after inoculation, cultures were harvested and CMV antigen prepared as described by Schmitz et aLI4 The protein content was determined by a simple and sensitive method utilizing Coomassie blue2’. The antigen was labelled with activated peroxidase (POD) according to Schmitz et aZ.14 Optimal CMV-POD working dilutions were determined by chequer- board titrations against reference CMV-IgM positive sera and a pool of negative sera containing HSV I specific IgM and heterophil antibodies.

Sera testing

Nunc immunoplates (No. 439454) were coated (100 ul well-‘) with a highly purified rabbit IgG anti-human IgM (u chain specific) produced for laser nephelometry total IgM determination (Behring, OSAT 14/14) at a concentration of 10 ugml-’ IgG in bicarbonate buffer, pH 9-6 for 2 h at 37°C in a water bath. Plates were then sealed and stored at 4°C for up to 4 weeks. Prior to use, plates were washed with PBS Tween 20 and 100 ul of test and control sera (at a 1: 40 dilution) were added in triplicate and incubated with the capture antibody for 60 min at 37°C. After washing, 100 ul of POD-labelled CMV antigen was added and the mixture incubated for 90min at 37°C. Plates were washed in PBS Tween 20 and the bound enzyme activity detected by the addition of substrate (hydrogen peroxide, U-phenylenediamine) for 20 min at room temperature in the dark. The plates were then read at 492 nm in a Titertek Multiscan. A serum sample was considered positive for CMV-IgM if its optical density (OD) at 492 nm was greater than twice the OD value (at 492 nm) of the negative serum pool. All CMV-IgM positive sera (at 1: 40) were then titrated (1: 20 to 1: 2560) and retested in the capture assay.

Also, five of the sera, as well as IgM negative control sera, were also layered on a discontinuous sucrose gradient (12.5-37.5%) and ultracentrifuged at 100,000 g for 18 h in an SW50.1 rotor (Beckman). Fractions (0.4ml) were collected, 12 fractions per gradient, dialysed overnight in phosphate-buffered saline and retested for CMV-specific IgM and specific IgG by the capture and indirect assays, respectively.

Results

Of the 347 sera tested, 33 specimens (9.51%) were initially positive for CMV-IgM by the indirect assay. However, after RF had been absorbed out, only 16 specimens remained

270 A. A. El-Mekki et al.

Table 2. Clinical data, CMV IgM, IgG titres and RF testing of positive specimens

Serial No. Clinical category

I RT 2 RT 3 RT 4 RT 5 RT 6 RT 7 RT 8 MN 9 MN

IO MN II MN 12 MN 13 FI I4 FI 15 Fl 16 FI 17 FI 18 FI 19 FI 20 RS 21 RS 22 RS

CMV-IgM titres indirect/capture

1:40/1:120 I :40/I :80 I :20/I :20 1:40/1:320 1:20/1:80 1:40/1:160

---/ I :40 1:80/1:320 1:80/1:640 1:40/1:320

--/I:80 -1 I :40

1:160/1:1280 1:80/ I:80

-/I:40 1:80/1:160 I :40/ I:40

--/I:80 1:80/1:640 1:40/l :80 1:40/I :640

--/I :40

CMV-IgG titres by indirect ELISA RF

NT - NT + NT - NT - NT - NT -

I:5120 - -

NT -

NT - I:5120 - I:1280 -

NT + NT -

I:1280 - NT -

NT - I:1280 -

NT - NT - NT - NT -

RF: Rheumatoid factor. NT: not tested, FI: febrile illnesses, RT: renal transplant patients, MN: mononucleosis, RS: routine screening (healthy).

CMV-IgM positive including two sera (Nos 2, 13; Table 2) that were both RF and CMV-IgM positive. The 17 “false” positive IgM sera containing RF were from two healthy individuals, three patients with non-specific febrile illnesses, five with SLE and seven patients with diseases of non-viral aetiology.

Utilizing the capture assay, 22 of 347 (6.34%) specimens were CMV-IgM positive. These included the 16 specimens already detected by the indirect assay following RF absorption and six sera that were negative by the indirect assay. None of the sera that became CMV-IgM negative following RF absorption, including those containing anti- nuclear antibodies, reacted in the capture assay. However, five of these sera were fractionated on sucrose density gradient and the fractions retested by the capture ELISA. As shown in Fig. 1, CMV-specific antibody was detected in the IgM fractions with peak OD values around fraction 3 which were totally free of contaminating IgG or IgA. CMV-IgG titres of these sera are shown in Table 2. All the sera tested had relatively high CMV-IgG titres (1:1280 to 1: 5120). Generally, CMV-IgM titres were higher (two- to eight-fold) in the capture assay than those obtained in the indirect assay. Three sera (Nos, 3, 14 and 17), however. had the same titres in both assays.

Discussion

The data presented in this study show that the IgM capture assay using peroxidase- labelled CMV antigen was more sensitive and specific than the indirect ELISA for

CMV detection by peroxidase labelled antigen 271

06

E c % * 04 z 0 0

02

0-C L 1 3

Fractions

4 5-12 (overages)

Figure 1. IgM GiptUre assay of sucrose density gradient fractions of sera that were negative for CMV-IgM by the indirect assay. 0 Test sera; q negative control; n positive control.

CMV-IgM detection in sera from different groups of patients. Thus not only RF interfered with the IgM detection in the indirect assay, but also specific IgM titres were generally lower in the latter test than in the IgM capture ELBA. Furthermore, the indirect assay failed to detect CMV-IgM in six sera that were positive by the capture assay. Indeed, five of these sera were fractionated on sucrose density gradient and found to contain CMV-specific antibody activity in the IgM fractions. Peak IgM titres were detected in fraction 3, while peak CMV-IgG titres were detected in fractions 8-l 1 (only peak titres of fractions are presented in Table 2). The discrepancy in the rate of detection of CMV-IgM by the two methods may be accounted for by the fact that the two test systems by their design quantitate specific IgM in totally different ways. Thus, in the indirect assay, competition for the same antigenic binding sites by the relatively high titres of CMV-specific IgG may have interfered with the binding of CMV-specific IgM which were present at low titres in these sera (Nos 7, 11, 12, 15 and 18; Table 2). In contrast, the CMV-IgM capture assay selectively binds the IgM fractions of these sera thereby eliminating interference by CMV-IgG which do not bind and are removed following washing. This important feature of the assay clearly improves CMV-IgM detection especially if present at lower concentrations.

In this study, and as shown by other workers’4.28,29, RF did not interfere with IgM detection by the capture assays. Thus, none of the sera that were CMV-IgM positive by the indirect ELISA and became CMV-IgM negative following RF absorption, were positive by the IgM capture ELBA.

In a recent study, Wiellaard et ~1.~’ developed an IgM capture assay similar to that described by Yolken & Leister 28 but utilizing unlabelled CMV antigen. They demon- strated that one of the five sera containing anti-nuclear antibodies interfered with CMV- IgM detection by showing a weak positive reaction. In this study, we did not observe such cross-reactions when ten documented sera containing anti-nuclear antibodies were tested by the capture assay. It is therefore possible that the use of CMV-labelled antigen in the CMV-IgM capture assays may decrease or avoid the occurrence of “false” positives due to anti-nuclear antibodies. In addition, the present data and in agreement with those of others’4~‘5*28 show that the IgM capture assay described in this study, does

272 A. A. El-Mekki et al.

not detect “false” specific IgM due to other herpes viruses since sera known to contain herpes simplex IgM tested in the CMV assay did not give rise to false positive results (data not presented).

Therefore, in our opinion, this capture assay represents a considerable and important advantage over the indirect assay with regard to both sensitivity and specificity. In view of this and the ease with which CMV antigen could be labelled with peroxidases, this assay, we feel. should seriously be considered as the method of choice for the detection of CMV-IgM.

Acknowledgement

This work was supported by Kuwait University, Grant No. MI 011.

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(Manuscript accepted 8th April 1987)