The detection of cytomegalovirus (CMV)-specific IgM using peroxidase labelled antigen: comparison with an indirect enzyme-linked immunoassay

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 hundredand forty-sevenserumsamples wereobtainedfrom 54renaltransplant patients,sevenpatientswho had heterophilantibody negativemononucleosis, 23 with non-specificfebrile illnesses.10patientswho werepositive for anti-nuclearantibody and who had systemiclupuserythematosus(SLE). 58patientswith other diseases of non-viral aetiology and 195 healthy individuals. These were examined for the presenceof cytomegalovirus-specific IgM antibody by an IgM capture assayutilizing CMV-peroxidase labelled antigen and a commercially available indirect enzyme immunoassay(Enzygnost, Behring). The former assay detected 22 CMV-IgM positivespecimens whereasthe latter assaydetectedonly 16of these22(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 assayfollowing sucrosedensitygradient 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-labelledCMV antigenisa sensitiveand specificprocedurewhich shouldhave important applications in the diagnosis over the indirect immunoassay. Kic~word.s: cytomegalovirus.

of CMV

IgM. indirect

ELISA.

infections

with distinct advantages

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

0888-X)786/87/040267+07 $03.00/O

Cold

Unit.

267 (“ 1987 Academic

Press Limited

268

A. A. El-Mekki

et al.

detected by immunofluorescence (IF)‘, immunoperoxidase staining”‘, indirect radioimmunoassay (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 assaysand 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 sameantigen 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 sincehigh 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 virusZ4and 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.l4This 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 sampleswere 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)

13 41 10 7 23 58 195

Total

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 assaywas performed and evaluated as recommended by the manufacturer. The sametest 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 asdescribed by Schmitz et aLI4 The protein content was determined by a simple and sensitive method utilizing Coomassieblue2’. The antigen was labelled with activated peroxidase (POD) according to Schmitz et aZ.14Optimal CMV-POD working dilutions were determined by chequerboard 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 sealedand stored at 4°C for up to 4 weeks. Prior to use, plates were washedwith PBS Tween 20 and 100ul 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, 100ul 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 sucrosegradient (12.5-37.5%) and ultracentrifuged at 100,000g for 18h 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 specimensremained

270

A. A. El-Mekki et al. Table 2. Clinical data, CMV

Serial No. I 2 3 4 5 6 7 8 9 IO II 12 13 I4 15 16 17 18 19 20 21 22

Clinical category RT RT RT RT RT RT RT MN MN MN MN MN FI FI Fl FI FI FI FI RS RS RS

IgM, IgG titres and RF testing of positive specimens

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 ELISA

RF

NT NT NT NT NT

+ -

by indirect

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 diseasesof non-viral aetiology. Utilizing the capture assay, 22 of 347 (6.34%) specimenswere CMV-IgM positive. These included the 16 specimensalready 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 antinuclear 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 assaythan 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 peroxidaselabelled CMV antigen was more sensitive and specific than the indirect ELISA for

271

CMV detection by peroxidase labelled antigen

06 E c % * z 0 0

04

02

0-C

1

3

L

4

5-12 (overages)

Fractions

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

of sera that were negative control.

for CMV-IgM

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 demonstrated that one of the five sera containing anti-nuclear antibodies interfered with CMVIgM 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*28show that the IgM capture assay described in this study, does

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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 representsa considerable and important advantage over the indirect assay with regard to both sensitivity and specificity. In view of this and the easewith 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. References 1. Griffiths PD, StagnoS,PassRF, Smith RJ, Alford CA. Infection with cytomegalovirusduring pregnancy:specificIgM antibodiesasa marker of recent primary infection. J Infect Dis 1982; 145:647-53. 2. Kangro HO, Griffiths PD, Huber JJ, Heath RB. Specific IgM classantibody production following infection with cytomegalovirus.J Med Virol 1982;10: 203-12. 3. Stagno S, Tinker MK, Elrod C, Fuccillo DA, Cloud G, O’Beirne JO. Immunoglobulin M antibodiesdetectedby enzyme-linkedimmunosorbentassayand radio-immunoassayin the diagnosisof cytomegalovirus infections in pregnant women and newborn infants. J Clin Microbial 1985;21: 93&5. 4. Ahlfors K, Ivarsson S-A, JohnssonT, Svenberg L. Primary and secondary maternal cytomegalovirusinfections and their relation to congenital infection. Acta Paediatr Stand 1982;71: 10913. 5. Griffiths PD, StagnoS, PassRF, Smith RJ, Alford CA. Congenitalcytomegalovirusinfection: diagnostic and prognostic significanceof the detection of specific immunoglobulin M antibodiesin cord serum.Paediatrics1982;69: 544-9. 6. Al-Nakib W, DeverajanLV, Thornburn H et al. Prospectiveserologicaland clinical studies on infants born in Kuwait with an elevatedIgM in cord blood. Bull WHO 1985;63: 745-50. 7. SutherlandS, BriggsJD. The detectionof antibodiesto cytomegalovirusin the serumof renal transplant patientsby an IgM antibody capture assay.J Med Virol 1983;11: 147-59. 8. PassRF, Griffiths PD, August AM. Antibody responseto cytomegalovirus after renal transplantation:comparisonof patients with primary and recurrent infections. J Infect Dis 1983;147:40-6. 9. Hanshaw JB, Steinfeld HJ, White CJ. Fluorescent antibody test for cytomegalovirus macroglobulin.N Engl J Med 1968;279: 56670. 10. Gerna G, ChambersRW. Rapid detection of human cytomegalovirus and herpesvirus hominisIgM antibody by the immunoperoxidasetechnique.Intervirology 1977;8: 257-71. 11. Kangro HO. Evaluation of a radio-immunoassay for IgM-class antibodiesagainstcytomegalovirus. Br J Exp Path011980;61: 512-20. 12. Torfason EG, Kallander C, Halonen P. Solid-phaseradio-immunoassay of serumIgG, IgM and IgA antibodiesto cytomegalovirus.J Med Virol 1981;7: 85-96. 13. Schmitz H, Doerr HW, Kampa D, Vogt A. Solid-phaseenzymeimmunoassayfor immunoglobulin M antibodiesto cytomegalovirus.J Clin Microbial, 1977;5: 629-34. 14. Schmitz H, von Deimling V, Flehmig B. Detection of IgM antibodiesto cytomegalovirus (CMV) usingan enzyme-labelledantigen(ELA). J Gen Virol 1980;50: 59-68. 15. van Loon AM, HeessenFWA, van der Logt JTM, van der Veen J. Direct enzyme-linked immunosorbentassaythat usesperoxidase-labelled antigenfor determinationof immunoglobulin M antibody to cytomegalovirus.J Clin Microbial 1981;13: 416-22. 16. Kangro HO, Booth JC, Bakir TMF, Tryhorn Y, SutherlandS. Detection of IgM antibodies againstcytomegalovirus:comparisonof two radio-immunoassays, enzyme-linkedimmunosorbentassayand immunofluorescentantibody test, J Med Virol 1984;14: 73-80.

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17. Meurman OH, Ziola Br. IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies. J Clin Path01 1978; 3 1: 483-7. 18. Salonen E-M, Vahari A, Suni J, Wagner 0. Rheumatoid factor in acute viral infections: interference with determination of IgM, IgG and IgA antibodies in an enzyme immunoassay. J Infect Dis 1980; 142: 250-5. 19. Vejtorp M. The interference of IgM rheumatoid factor in enzyme-linked immunosorbent assays of rubella IgM and IgG antibodies. J Viral Meth 1980; I: 1-9. 20. Bonfanti C, Meurman OH, Halonen PE. Detection of specific immunoglobulin M antibody to rubella virus by use of an enzyme-labelled antigen. J Clin Microbial 1985; 21: 963-8. 21. Isaac M, Payne RA. Antibody class capture assay (ACCA) for rubella-specific IgM antibody. J Med Viral 1982; 10: 5564. 22. Schmitz H. Detection of immunoglobulin M antibody to Epstein-Barr virus by use of an enzyme-labelled antigen. J Clin Microbial 1982; 16: 361-6. 23. Schmitz H, Emmerich P. Detection of specific immunoglobulin M antibody to different flaviviruses by use of enzyme-labelled antigens. J Clin Microbial 1984; 19: 664-7. 24. Sundqvist V-A. Frequency and specificity of varicella-zoster virus IgM response. J Virol Meth 1982; 5: 219-27. 25. Salonen J, Vainionpaa R, Halonen P. Assay of measles virus IgM and IgG class antibodies by use of peroxidase-labelled antigens. Arch Virol 1986; 91: 933106. 26. Krishna RV, Meurman OH, Ziegler T, Krech VH. Solid-phase enzyme immunoassay for determination of antibodies to cytomegalovirus. J Clin microbial 1980; 12: 4651. 27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976; 72: 248-54. 28. Yolken R, Leister FJ. Enzyme immunoassays for measurement of cytomegalovirus immunoglobulin M antibody. J Clin Microbial 1981; 14: 427-32. 29. Wiellaard F, Scherders J. Hooijmans A, Dagelinckx C. Development and preliminary evaluation of two ELISAs for detection of anti-CMV IgG and IgM antibodies. J Viral Meth 1985: 10: 36339. (Manuscript

accepted

8th April

1987)