Archives of Virology
Archives of Virology 58, 253--258 (1978)
© by Springer-Verlag 1978
Rapid Detection of IgG and IgM Antibodies for Cytomegalovirus by the Enzyme Linked Immunosorbent Assay (ELISA) Brief Report By R. CARPEL, F. DE CUYrER, and J. DE BRAEKELEER Department of Virology, Institut Pasteur du Brabant, Brussels, Belgium With I Figure Accepted June 6, 1978
Summary A simple solid phase enzyme immunoassay for the detection of immunoglobulin G and M to cytomegalovirus (CMV) is described. Using this test IgM antibodies to CMV were detected in 0.7 per cent of newborns and regularly after CMV infection in transplant patients, furthermore in these latter patients IgM production was prolonged for several months. For the determination of IgG the enzyme immunoassay was more sensitive than the complement fixation test (CF) and the antibody titres were 4 to 8 fold higher. Since the E L I S A test is rapid, specific and unexpensive it can become an acceptable routine diagnostic procedule.
The incidence of cytomegalovirus infection is increasing with the increase use of immunosuppressive chemotherapy and this virus is also an important cause of mental retardation (5). Specific immunoglobulin M (IgM) is indicative of current or recent infection. The detection of this class of immunoglobulin m a y therefore be important in recognising persistent CMV infection, for example in infants infected in utero or in immuno-compromised patients who experience a primary infection or reactivation by CMV. Several techniques are available for the detection of specific IgM, based on gradient eentrifugation followed b y immunofinorescence or radioimmunoassay (RIA). Although sensitive and reliable, these techniques need special equipment and are not feasible on a large scale, furthermore non-specific immunofluorescence is not uncommon. More recently enzyme linked immunosorbent assays (ELISA) have been described for measuring viral antigen and antibodies (1, 4, 11). 17 Arch.ViroL 58/3
]~. CAI~PEL, F. D$; CUYPER,
and J. DE BRAEI~ELEER:
We herein describe the results of a simple and rapid E L I S A test using commercial antigen and peroxidase labeled antibodies for the detection of specific IgG and IgM antibodies against CMV. CMV antigen prepared from the AD169 strain was purchased from Microbiological Associates (Bethesda, Maryland). The optimal antigen dilution to be used was determined by a checkerboard titration using a positive and negative serum diluted from 1 : 10 to 1 : 2000. The antigen dilution giving an absorbance of 1.0 at 490 nm with all the dilutions of the positive serum and less than 0.2 with the negative serum was used as working dilution. The antigen was usually used at a dilution ranging from 1 : 50 to 1 : 200. All the tests were also run in parallel using a control antigen prepared from W I 38 uninfected cells. The tests were performed in disposable, flat bottom Cook Microtiter plates M 129A (Dynatech Laboratories, Alexandria, Virginia, U.S.A.) and also, in parallel, in microeuvettes from Finnpipette Labsystem (Helsinki, Finland). Briefly, 200 ~1 of the antigen dilution, in carbonate bicarbonate buffet: p H 9.6, were distributed in the wells and incubated overnight at 4 ° C. The wells were then washed with phosphate-buffered saline (PBS) containing 0.05 per cent Tween 20 and fixed with a 10 per cent formaldehyde solution for 10 minutes. After a thorough wash the wells were filled with 10 per cent calf serum in PBS and incubated for 2 hours at 37 ° C. After a further -wash the plates or euvettes were stored at - - 7 0 ° C until used. The day of the test 200 ~] of two-fold dilutions of serum starting at a dilution of 1 : 50 were distributed in the wells or euvettes and incubated for 37 ° C. After the incubation the wells were washed with PBS, and ]00 B1 of a 1/2000 dilution of a commercial anti-human IgG or IgM antiserum labeled with horse radish peroxidase (Microbiological Associates, Bethesda, Mary[and) was added to the wells or euvettes and incubated at 20 ° C, for 2 hours for IgG and 4 hours for IgM. The wells we1~ again washed and 200 [~l of substrate was added. After 30 minutes the reaction was stopped by adding 50 ~l of 4 ~ HCI. The substrate consisted of a mixture of 0.4 per cent diphenylenediamine and 0.2 per cent urea peroxidase in phosphate-citrate buffer, pH 5.0. An orange to yellow color developed with positive samples whereas the wells or euvettes remained colorless for negative specimens. Since the end point was sometimes difficult to determine in the microplates, these were photographed to provide a permanent record, as shown on Figure 1. The results of the test performed in the Labsystem cuvettes were measured with a nine channel photometer (Finnpipette Analyzer System, F P 9 mode], Helsinki, Finland) ; as described by others we chose as end point the serum dilution that gave an absorbance at 4:90 nm twice the standard deviation above background absorbanee (6). The specificity of the commercial antisera -was tested by immnnodiffusion against normal serum. The anti-IgG and anti-IgM antisera gave only one line of precipitation corresponding to the specific immunoglobuiin. To further test the specificity of these antisera, normal human serum was placed on the top of a 10---40 per cent sucrose gradient and centrifuged for I5 hours at 37,000 rpm in a SW4i rotor. Twenty fractions were collected and dialyzed overnight. These fractions, diluted from 1 : 10 to 1 : i0,000, were tested by E L I S A as described above. Anti-IgG and anti-Ig~I antisera each gave only one peak of activity correspond-
ELISA for CMV IgG and IgM Antibodies
ing, respectively, to the serum fractions containing IgG or IgM immunoglobulin, thus confirming that the labeled antibodies were specific. Complement fixation tests (CF) were also done on all the sera by a mieromethod (10), with the same antigen used for the E L I S A test. Tests were carried out on serum specimens collected from 150 apparently healthy babies 1 to 2 days after birth; three urine and saliva specimens were also obtained from each infant, on the three successive days after birth. The urine and saliva specimens were examined by immunofluorescenee for the presence of CMV antigen, as described previously (2).
Fig. 1. The microplates are simply placed on sensitized paper and transilluminated, the positive wells are therefore those that appear white on the picture Among the 150 children examined only 2 (0.7 per cent) excreted virus at birth and continued to excrete virus for 3 and 4 weeks, respectively, this incidence of CMV infection is similar to that obtained by virus isolation studies (3). The 2 babies excreting virus at birth were also the only 2 to have detectable CMV speeiiic IgM as detected b y the E L I S A test. CMV IgG antibodies on the other hand, were found in 56 of the newborns (37 per cent) with titres of 100 or higher. B y CF, only 46 (33 per cent) had detectable antibodies and the titres obtained by E L I S A test were generMly 4 to 8 fold higher than the CF antibody titre (Table 1). The correlation coefficient (r) for E L I S A test and CF test for IgG antibody was of 0.85, indicating a significant correlation. 17"
F. DE CUYPER,
'~nd J. DE BBAEKELEER:
Table 1. Correl~ttion between CF antibody levels and IgG antibody titres determined by the E L I S A in 150 newborns
Titre of ELISA 800 400 200 100 50
Titre of the CF test 16 32 64
We had also the opportunity to study 35 renal transplant patients. Sera had been collected from them at monthly intervals starting before transplantation, over a period of 1 to 2 years. Before transplantation, 22 of these patients were seronegative for IgG antibody by CF but only 14 of them were negative for IgG by ELISA and we consider therefore that only these latter cases were really seronegative. During the observation period i0 of these 14 patients underwent a primary infection recognized by ELISA test,,developing not only IgG antibody but also IgM antibody. The IgM antibody response lasted for 2 to 3 months for 6 of these patients but persisted for several months (6 to II) for the 4 remaining patients suggesting a persisting antigenic stimulation. Indeed, when virus excretion in the urine or saliva or both sites was examined we found that virus excretion occurred for 3 to 4 weeks in most patients but that it persisted for 4 to 6 months in those who showed a persisting IgM production. The clinical features associated with these infections was HBs Ag negative hepatitis in 2 eases, diffuse lung infiltration in 2 other cases, and no symptoms in the remaining 6 eases. Among the 21 patients who possessed IgG antibody as detected by the E L I S A test before the transplantation 4 had no detectable CF antibody suggesting again t h a t the E L I S A test is more sensitive than the CF. Five to six weeks after renal graft 9 of these 21 patients showed a 4 fold or greater rise in CMV-speeific IgG as detected b y the E L I S A test and 7 of these latter patients also developed IgM and excreted virus in the urine. The 12 remaining seropositive patients showed no change in their IgG antibody titres although 4 of them excreted virus and 4 excreted virus and developed CMV-specifie igM antibody. Reactivation or reinfection can therefore apparently be followed by an IgM response even in the absence of detectable IgG response. Since these patients are under immnnosuppressive chemotherapy (azathioprine 2 mg/kg) it is possible that the immunosuppression is less effective on the IgM production than on the I g G production. This finding is also compatible with the observations of others (9) who found that in patients under immnnosuppression repeated antigenic stimulation could produce a primary type of response with predominating IgM production. The observation of persisting IgM production in patients who were also long term exeretors of CMV is in agreement with other studies (7) showing that the production of IgM in transplant patients could be prolonged for years. To exclude the possibility of false positive Ig3/[ antibody results the neonatal and transplan~ sera found to be positive for CMV specific IgM were examined for rheumatoid factor (Pd~~) b y the I~A test (Hyland); all were negative for I~F.
ELISA for CMV IgG and IgM Antibodies
The specificity of all positive IgG and IgM E L I S A results was further checked b y fractionating the positive sera on sucrose density gradients and testing the fractions for IgM and igG by E L I S A ; peak activity was found in the relevant IgG and IgM containing fractions indicating a good specificity. E L I S A methods for the detection of viral antibodies are progressing rapidly. SCHMITZ et al. (8) recently described an E L I S A test for the detection of specific CMV IgM antibody and found a good correlation between immunofluorescence techniques and E L I S A tests if in this latter tests a nuclear antigen was used. We here described a simple and rapid test using a commercial antigen which can be performed in microtiter plates without special equipment. If large numbers of samples have to be tested a more automated system such a the Labsystem can be used. With the Labsystem cuvettes and photometer analyzer the results we obtained were comparable to those obtained in microtiter plates. 2~evertheless the Labsystem offers the possibility to handle a larger number of samples and to obtain ~n aeurate measure of the titre which is directly printed by the calculator. Furthermore with this system only 3 serum dilutions are needed saving time and material. Although SOH~aZTZet al. (8) have described a non specific reaction when using commercial antigen instead of purified nuclear antigen we found no false positive results when the starting dilution of the test serum was of 1 : 50. For lower dilutions non-specific reactions could be observed and we might therefore have missed low I g ~ antibody titres, nevertheless similar non-specific binding was observed when nuclear CMV antigen was used as described b y S C ~ T Z et al. (8). The nonspecific binding to the plastic could be partly prevented by treating the wells with calf serum which also binds to the IgG receptors t h a t could be present in the commercial antigen, preventing another factor of false positive test. As in other tests non-specific reaction could be caused b y R F and all positive sera were therefore tested for R F as well as for antinuclear antibodies which might also give rise to false positive results. This latter non-specific reaction can be avoided if tests are run in parallel with a control antigen prepared from uninfected cells as we did in our tests. The data presented indicate that E L I S A is rapid, specific and unexpensive. This test can therefore become an acceptable routine diagnostic procedure. Nevertheless to provide comparable results between different laboratories standardization of the test will be needed.
Referenees 1. Blsx~I, F., GA]~LI, R.: ELISA, a rapid diagnostic method. Lancet 1, 696--697 (1977). 2. CARPEL, R,., :HESTERMANS, 0., TOVSSAIN~, CH.,VEREERSTRAETEN, P., VAN BEERS, D., DE BRAE~ELEER, J., SCHOUTENS, E. : Cytomegalovirus Infection and graft survival in renal graft recipients. Arch. Virol. 56, 149--156 (1978). 3. ELEK, S., STER~r, H. : Development of a vaccine against mental retardation caused by cytomegalovirus infection in utero. Lancet 1, 1--5 (1974). 4. GE~RA, G., MeCLo~TD, C., CHA~BERS, R.: Immunoperoxidase techniques for detection of antibodies to human cytomegalovirus. J. clin. Microbiol. 3, 364--372 (1976).
R. CAPrEL et al. : E L I S A for CMV I g G a n d I g M A n t i b o d i e s
5. HANSHAW~ J . B., SC~:tEIIqER, A. P., MOXLEY, A. W., CTAEV, L., ABEL, V., SCHEINER, B. : School failure a n d d e a f n e s s a f t e r s i l e n t c o n g e n i t a l c y t o m e g a l o v i r u s infection. N e w E n g l . J . Med. 295, 4 6 8 - - 4 7 0 (1976). 6. LEINIKKI, F., P£SSILX, S.: Solid p h a s e a n t i b o d y a s s a y b e y r o a n s of e n z y m e conj u g a t e d t o a n t i - i r n m u n o g l o b u l i n . J . clin. P a t h o l . 29, 11 t 6 - - 1 1 2 0 (1976). 7. NAGING~O~', J . : C y t o m e g a l o v i r u s a n t i b o d y p r o d u c t i o n in rena.1 t r a n s p l a n t p a t i e n t s . J . H y g . (Carnb.) 69, 6 4 5 - - 6 6 0 (1971). 8. S c h l I T Z , H., DeEply, H . W., KAMPA, D., VOGT, A. : Solid-phase i m m u n o a s s a y for i m m u n o g l o b u l i n M a n t i b o d i e s t o c y t o m e g a l o v i r u s . J . clin. Mierobiol. 5, 6 2 9 - - 6 3 4 (1977). 9. ROWLEY, M. J., MACKAY, I. R., MoKExZlE, I. F. C.: A n t i b o d y p r o d u c t i o n i n i m m u n o s u p p r e s s e d r e c i p i e n t s of r e n a l allografts. L a n c e t 2, 7 0 8 - - 7 1 0 (1969). 10. SEVER, J . C. : A p p l i c a t i o n of a m i c r o t e e h n i q u e to v i r a l serological i n v e s t i g a t i o n s . J . I m m u n o l . 88, 3 2 0 - - 3 2 9 (1962). 11. VOLLER, A., BARLEY:T, A., BIDWEL, D., CLARK, M., ADA~IS, A.: T h e d e t e c t i o n of viruses b y 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 s a y ( E L I S A ) . J . gen. Virol. 33, 165 to 167 (1976).
Authors' address: Dr. ig. CAPPEL, Department of Virology, Institut Pasteur du Brabant, 28 Rue du gemorqueur, 1040 Brussels, Belgium. R.eceived M a r c h 30, 1978
Herausgeber, Eigentfimer und Verleger: Springer-Verlag, l~ISIkerbastei5, A-1011 Wien. Ffir den Textteil verantwortlich: Dr. Wilhelm Schwabl, M0tkerbastei 5, A-1011 Wien. Fiir den Anzeigenteil verantwortlich: Mag. Bruno Schweder, ~Slkerbastei 5, A-1011 ~Vien.Druck: R. Spies & Co., Straufiengasse 16,A-1050 Wien. Priutcd in Austria.