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Home , Immunofluorescence, Serology

Lab.med, 12: 363-367 (1988) Cytomegalovirus : Comparison of different with complement fixation and indirect for the detection of P. Hengster, M. P. Dierich Institute for Hygiene, University of Innsbruck

Summary: We have tested 5 commercially available ELISAs and compared them with immunofluorescence and comp- lement fixation on a panel of interesting sera. There were many discrepant results showing that new types of ELISA using monoclonal antibodies for IgG and anti-catching antibodies for IgM determination have a anti~ -catch higher sensitivity and specificity than all other tests. Results in detail are listened below.

Keywords: CM V - Serology — Quality of tests

Zusammenfassung: Wir haben 5 kommerziell erhältliche ELISAs ausgetestet und mit der Immun fluoreszenz und der Komplement- bindungsreaktion verglichen. Die einzelnen Tests ergaben sehr unterschiedliche Resultate. Neue Typen von ELISAs, die monoklonale Antikörper für die IgG-Bestimmung und Anti-Antikörper beschichtete Platten für Anti- -Antikörperdie IgM-Bestimmung verwenden, haben eine wesentlich höhere Sensitivität und Spezifität als alle anderen Tests. Die Ergebnisse werden im folgenden genau berichtet.

Schlüsselwörter: CM V — Serologie — Testqualität

determine IgG and IgM antibodies to cytomegalovirus, introduction and for comparison with complement fixation. The results are reported below. Efforts are undertaken to optimize the diagnostic pro- cedures. This has motivated us to evaluate the serological techniques, particularly with respect to new types of ELISAs, presently used to -ascertain a cytomegalovirus Material and Methods . The cytomegalovirus is an ubiquitous agent. samples:\Ne have tested 58 serum samples out of : Infection rates in developed countries reach about 50% our routine diagnosis selected of patients with typical Jn people of the age of 30 years. Routes of transmission Symptoms of CM V-, 5 sera of newborns, 5 sera are close or even intimate contact, diaplacentally in utero with previously serological diagnosed recent CMV infec- ' or with blood transfusions or blood products (1). Infec- tion, 10 sera expected to be clearly negative for CMV- * tions occur in normal hosts, often without Symptoms or antibodies, and another 5 sera "with positive rheumatoid with a variety of clinical Symptoms such äs infectious factor. Some sera of this panel present difficulties for the rnononucleosis like syndrome or , with an in- determination of CMV-antibodies, e.g. sera early in the creased prevalence in immuno compromised hosts with course of infection and sera of newborns contain only a /damage of transplanted orgäns or serious pneumonitis, Iow amount of anti-CMV-antibodies and sometimes only and in congenitally or perinatally infected newborns with IgM without IgG. infection in nearly all orgäns, especially the brain (2). « Sufficient CMV testing is absolutely necessary for an ex- Indirekt Immunofluorescence (IF): Immunofluorescence act diagnosis and for the prevention of infections with was used äs a reference method for the determination of blood and blood products äs a possible route of trans- anti-CMV-antibodies. CMV infected fibroblasts spotted mission (3). There are many tests available with various on slides (CAS GmbH Munich) were used äs Substrate. forms of and different test principle. The determi- 15 of a serum diluted 1:10 in PBS were added to the .. nation of IgG and IgM antibodies allows the diagnosis of slide and incubated for 30 min at 37°C. After washing for a recent primary infection but is of little value to diagnose 30 min in PBS FITC-conjugated rabbit anti human IgG « a reactivation with the (3—5). For this purpose the and IgM was added. After an incubation for 30 min at rise in titer is the only indirect indicator of an infection. 37 °C and a further washing step the slides were examined H/Ve have tested serum samples with different ELISA sys- with a Zeiss Standard Lab 16 with an H PO f tems and immuno- äs reference method to 50 lamp at a magnification of 400 (6). Lab.med. 12: 363 (1988) 363 Abbott ELISA: The Abbott ELISA uses CM V antigen- second step the conjugate is added, and in a third step coated beads to determine total IgG, l g M, and IgA anti- the Substrate reaction finishes the procedure. bodies against CMV. The test was performed according to the included instructions. Results with this type of Behring ELISA: Behring provides a plate coated with ELISA can be compared with the CF. The serum dilution antigen and with control antigen beside. The test for IgG and IgM is performed in the same manner s in the Virion used in the test is 1:21. test. The serum dilution used in the test is 1:40. For Medac ELISA: Medac supplies two different ELISA Sys- interpreting the test the optical density value of the tems for the detection of IgG and IgM antibodies. For unifected control is subtracted from the infected D value the detection of IgG antibodies the plate is coated with and a difference of 0.200 or more is expected to indicate . In a first step the patient serum in a a positive reaction. dilution of 1:32 is added at the same time with an enzyme-labeled CMV antigen. If the patient serum con- Complement fixation (CF): CF is a widely used Standard tains antibodies against the CMV antigen the antibodies method to determine antibodies against different viral form immune complexes and bind with their Fc poition . We used the antigen supplied by VIRION to the rheumatoid factor. After a washing step the sub- Germany. The test is performed s described by Kolmer. Sera were considered to contain anti CMV antibodies if strate is added and the reaction can be read immediately. a serum dilution of 1:10 resulted in a positive reaction. For the IgM determination the plate is coated with anti- IgM- s catching antibody. In a first step serum Calculation of sensitivity, specificity, false negative, false is added to the plate in a dilution of 1:100 and IgM positive, negative and positive predictive value: antibodies with divergent specificities become bound to Correct results based on criteria the catching antibodies. By washing all unbound anti- bodies are eliminated. In the next step the enzyme-labeled antigen is added. After a second washing step the sub- strate is added and after 30 min the reaction can be Comparative A B stopped and read with a photometer (7). assay results C D Organon ELISA: Organon supplies two different ELISAs, named Vironostica. For the determination of IgG anti- Percent sensitivity = A/(A+C) χ 100 bodies the plate is coated with a monoclonal anti-CMV Percent specificity = D/(B + D) χ 100 Positive predictive value = A/(A+B) χ 100 antibody. In a first step the CMV antigen is incubated for Negative predictive value = D/(C+D) χ 100 1 h at 37 °C. After washing in a second step the 1:100 False positive rate = B/(B + D) χ 100 diluted serum sample is incubated for 1 h at 37°C. After False negative rate = C/(A+C) χ 100 washing in a third step the enzyme labeled rabbit anti human IgG conjugate, and after a further washing step the Substrate solution is added. Results The principle of the IgM test is the same s in the Medac The Immunofluorescence was used s Standard method IgM test. for the detection of CMV antibodies. A serum was con- Virion ELISA: Virion provides a lyophilized antigen. This sidered positive for IgG if the Immunofluorescence and if is reconstituted with 1 ml of distilled water, diluted 1:100 at least two ELISAs gave a positive reaction. These criteria in coating puffer, 200 μΐ of this dilution is pipeted per were chosen to increase the specificity of a terminal result, well, and incubated overnight at room temperature in a which with Immunofluorescence only might be seriously moist chamber. Each serum is tested in a dilution of 1:100 influenced by a false positive result in this test. For IgM the results were based on the Immunofluorescence. In for IgG and IgM and a Standard serum runs in each test. case of 3 rheumatoid factor (RF) positive sera the deter- This Standard serum has a optical density value for IgG and IgM and every sample has to be corrected with re- mination was based on the concordance of the results of spect to this value. Optical densities of gre ter than 0.250 the Medac and Organon ELISA because the RF should are expected to indicate clearly positive reactions. The not effect this tests. principle of the ELISA for IgG and IgM is the name. In a Table 1 lists the different reaction patterns in all tests first step the patient serum reacts with the antigen, in a obtained with sera for the IgG determination. 35 sera

Table 1: IgG determination results No. of sera Diagnosis1 IF Medac Organon Virion Behring Abbott CF

35 ·+ +_ ' _+ _+ _+ _+ ·+ + 10 — — 1 + _j_ _f_ · _j_ _ 4. + + 1 + •f + + — · — -j- 4. 1 + + + + + ' — _ 4 2 + + - + · nd 4 . 4 1 + + + + " + — 4 — 1 + + · + + * + nd _|_ _ 1 + + '+ + ' - - nd — — 1 + nd — — 1 - ' + - nd _ — 1 - +. - - - + — — 2 + _· - _ — — nd = not determined. 1 Diagnosis: positive = IF + and two IgG specific ELISAs + ; negative = IF - or IF + and less then two IgG specific ELISAs +. 364 Lab.med. 12:364(1988) : Results of the differem feste for IgG Table 4: Results of the differem tests for IgM Numbers in parentheses indicate numbers of false positive and false negative reactions, respectively, of the total amount indicat- Test Test result Sample ed dilution positive negative Test Test result Sample •1:1.. ·*.:».. Immuno- positive negative fluorescence 17(3) 41 (0) 1:20 Medac IgM 15(1) 43(0) 1:100 Immuno- Organon IgM 14(1) 44(1) 1:100 fluorescence 46 (3) 12 (0) :20 Virion 17(6) 41 (8) 1:100 Medac IgG 42(1) 16 (2) :32 Behring 1 11 (6) 10(2) 1:40 Organon IgG 44 (1 ) 13 (0) :100 Abbott total lg (0) 1:21 Virion 38(0) 20 (5) :100 Complement (1) 1:10 Behring 17(1) 4 (1) :40 f ixation 1 Abbott total Ig1 41 (0) 17 (2) 1:21 Complement 40(0) 18 (3) 1:10 1 These tests do not distinguish between IgG and IgM. fixation1

1 These tests do not distinguish between IgG and IgM.

reacted uniformly positive and 10 uniformly negative. 13 the false positive and negative results are listed and the sera revealed discrepancies between the various lest, positive and negative predictive values are calculated for Given the criteria for a positive diagnosis the IF showed IgG (table 5) and for IgM (table 6). 3 false positive results. The results with the Abbott ELISA and the complement fixation (CF) are indicated for com- It is the general experience that the detection of IgM parison, since these tests do not distinguish between IgG antibodies in the sera due to the Iow titers äs in sera of and IgM antibodies, while the other tests are IgG specific. newborns represents a particular problem. Table 7 dem- onstrates that in the IF test two sera are IgM positive. To facilitate the overview of the false positive and false These two sera are not detected by two of the four ELISAs. negative reactions for each test System the results are In addition the CF is negative in one case. This clearly grouped äs in table 2. In addition the serum dilutions used shows the problems arising by testing sera of newborns according to the recommendations of the manufacturer only by CF or ELISAs. are indicated. Table 3 lists the different reaction patterns in all tests obtained with sera for the IgM determination. Rheuma- Discussion toid factor positive sera are marked by "R F". 6 sera reacted uniformly positive, 28 uniformly negative. 25 sera showed As for other infectious serological methods are discrepancies between the various test results. The widely used for routine diagnosis of recent CMV infection Abbott ELISA and the CF results are listed for comparison. or to determine the immune Status. We have tested 4 These tests detect IgM and IgG. IgG and IgM specific commercially available ELISAs and compared them with immunofluorescence. In addition As for the IgG determination also for the IgM results the the results obtained with a false positive and false negative results are listed detecting IgG and IgM class antibodies and the Abbott (table 4). ELISA detecting IgG, IgM and IgA were evaluated. The Based on the IgG and IgM values displayed in table 1 purpose of the evaluation of these test Systems was to through 4 the sensitivity and the specificity are analyzed, compare more recent commercially available reagents to

N Table 3: IgM determination results No. of sera Diagnosis1 IF Medac Organon Virion Behring Abbott CF

6 + +. + + + + " + + 28 _ — _ · — — — — 1 + 44- 4- — 4- 4 4 1 RF 4 .4 — — ·" "-' + ~t~ 2 + + 4- H- — "~ + "^ 1 + — — — -f 4- + + 1 4 — — + -r- — -f 4- 3 + — — — — 4 44 1 RF 4 -l- — — 44 44 1 + — — — -l· 4 44 3 4 -l- -|- 4 — nd 44 1 RF 4 -f — — . 4 nd 4 4 1 + .4. + _ — nd 44 4 4 _ _ - · 4 nd 4 4 1 — 4 — 4 — + — 1 + + + - 'nd .4 2 - · - - 4 . nd

nd - not determined; RF « Rheumatoid facior RF + 1 Diagnosis: positive - IF -h (except when «F +; in this case for a positive diagnosis the results of Medac and Organon ELISA-lgM both have to be positive, because these test are not influenced by the rheumatoid factor); negative « IF - or IF + but RF also + and Medac and Organon ELISA negative. Lab.med. 1*2: 365 (1988) 365

.J" those which are longer known to find out the reliability of ficity äs the CF and can be used for blood donor screening. the test results, in comparison with HIV methods, where The differentiation between acute or passed infection is highly sensitive and highly specific methods are used. not possible with only one sample in this test. Testing a limited amount of sera, including some of par- With the indirect immunofluorescence, which is often ticular interest, we found many divergent results äs shown used äs a reference method because of its sensitivity, false in table 1 for IgG and table 3 for IgM. For IgG determi- positive results occur which for IgM determination are nation the Organon and the Virion use the same serum caused by rheumatoid factor. For the IgG determination dilution (1:100), the Behring and the Medac test applies the serum dilution of 1:10 may be responsible for the a 1:32 and a 1:40 dilution. The results äs seen in table 5 false positive results. It has to be known that in human are much better with the Organon and the Medac test. fibroblasts the CM V is able to induce IgG-Fc-receptors The CF has a higher specificity and sensitivity äs some which leads to binding of different not CMV specific IgG ELISAs. The 1:20 dilution in the IF seems to be too class antibodies with their Fc portion and not with the concentrated leading to false positive results which mark- antigen binding site (8). For this reason the Fc receptor edly diminishes the specificity of the test. has to be blocked or infected nuclei should be used äs There are two main problems for the IgM determination, Substrate. The IF is time consuming when many samples first, the competition of high level IgG with IgM for the are tested and needs expertise in interpreting fluorescence binding site leading to false negative IgM results/and patterns. second, the false positive results caused by the rheuma- toid factor. For the IgM determination (tables4, 6) the The CF is a very well known Standard method to deter- rate of false positive and false negative results in the mine antibodies against many and therefore es- Behring and Virion ELISA is even higher äs for the IgG tablished in many laboratories. With a serum dilution be- determination, which is not only correlated to the low 1:10 unspecific reactions occur but even with this mentioned problems. Also the dilution with 1:100 is the dilution Iow positive sera might be missed äs they occur same except in the Behring with 1:40. A pretreatment early in the course of infection or in newborns with Iow of all sera for the IgM determination with A for level IgM antibodies. As shown in table 7 the CF is not example to remove the IgG antibodies is very worksome able to detect antibodies in two cases one of them being and also expansive and for this reason rarely performed. definitely infected with the CMV. Also antibodies that do not fix complement are not detected. New types of The ELISA of Abbott recognizes IgG, IgM and in addition ELISAs which for IgG determination use monoclonal IgA. With the sample dilution of 1:21 this test shows a antibodies like Organon or measure immune complexes slightly increased sensitivity and about the same speci- with enzyme labeled CMV antigen äs Medac and for IgM

Table 5: Characteristics of IgG Tests Test Sensi- Speci- False False Predictive value tivity ficity positive negative % % % % positive negative % % Immunofluorescence 100 80 20 0 93 100 Medac IgG 95 93 7 5 98 88 Organon IgG 100 98 2 0 100 98 Virion 88 100 0 12 100 75 Behring 94 75 25 6 94 75 Abbott total Ig 95 100 (0) .5 (100) 88 Complement fixation 93 100 (0) 7 (100) 83 () These numbers in parenthesis have no significance, because the Abbott ELISA and CF can be positive due to IgM without having IgG antibodies detectable.

Table 6: Characteristics of IgM tests Test Sensi- Speci- False False. Predictive value % % % % positive negative % % . Immunofluorescence 100 93 7 0 92 100 Medac IgM 100 98 2 0 93 100 Organon IgM 92 98 2 7 92 98 Virion 43 75 25 57 - 35 80 Behring 71 57 43 29 45 80

Table 7: Results with 2 sera of newborns Nr. IF Medac Organon Virion Behring Abbott CF IgM IgM IgM IgM IgM total 6188 + + -H' - - + + ·· ^ 17 + + + — nd + -

366 Lab.med. 12:366(1988) determination use a catching antibody have markedly im- an elimination for the IgM determination is performed. proved the serological methods in their sensitivity and Given a fourfould rise in titre in the CF this classical specificity for the diagnoses of CMV infections. These method is also satisfactory, but some problems arise with tests have very Iow background and a very clear cut-off Iow titre sera of newborns and it needs two samples with value which may be due to the high grade of purity for 14 days between to make the correct diagnosis. A single the antigen used in the test. Especially for the IgM deter- high titre in all CMV tests never allows the diagnosis. mination there is no competition of high level IgG with ELISA with antigen coated plates should not be used any the IgM antibödies for the antigen binding site and there is longer, because of their lack in sensitivity and with respect no influence of rheumatoid factor which otherwise often to IgM also due to their lack of specificity, which is not causes false positive IgM results by binding to the IgG only correlated to the R F. bound to the antigen.. ELISAs with the antigen coated plates lack in sensitivity and also specificity especially for For blood donor screening a highly sensitive test for IgG the IgM determination. It seems that for routine diagnosis antibödies or for IgG -f IgM antibödies will be the best of CMV infection the ELISA principle with commercially to find out any CMV positive person; available antigen or antigen coated plate. should not longer be used. When these tests are performed not References: strictly according to the included instructions, but prop- 1. CHANG, N. T.. HUANG, J., GHRAYEB. J., McKINNEY, S., CHANDA, P. K., erly reset, and red from experienced persons they give CHANG. T. W.. PUTNEY, S., SARNGADHARAN, M. G., WONG-STAAL. F., GALLO, R. C.: An HTLV-III peptide produced by recombinant DNA is immunoreactive with better results äs demonstrated here. sera from patients with AIDS. Nature 315,151-154 (1985). 2. SCHILT, U.. SIDIROPOULUS. D.: Virusisolierung im Fruchtwasser bei Infektionen One could argue that these results are subjective because in der Schwangerschaft. Gynäk. Rdsch. 21.166-170 (1981). different results might be obtained in different laborato- 3. HENI, N.. HEISSMEYER, H., SCHMITZ, H.: Klinik und Diagnostik derZytomegalie- Hepatitis. Dtsch. med. Wschr. 44,1610-1612 (1976). ries, but all these tests are similar and were performed 4. ALFORD, A., BRITT, Jr., BRITT. J.: Cytomegalovirus. In: FIELDS, B. N. (eds.) with great care strictly related to the included instructions Virotogy. Raven Press, New York, pp.629-660 (1985). 5. KELLER, R., PEITCHEL. R.. GOLDMANN, J., GOLDMAN, M.: An IgG-Fc receptor and required controls were used. The results obtained are induced in Cytomegalovirus-infected human fibroblasts. J. Immunol. 116, 772—777 of value since in routine laboratories normally compara- (1976). 6. STAGNO,S.,TINKER. M. K., ELROD, C.. FUCCILLO, D. A., CLOUD,G.,0'BEIRNE, tive testing is not performed, It seems that quality control A. J.: Immunglobulin M antibödies detected by enzyme-linked immunosorbant assay and purity of antigen and supplied materials differs mark- and in the diagnosis of Cytomegalovirus infections in pregnant edly from Company to Company. With the results listened women and newborn infants. J. Clin. Microbiol. 21, 930-935 (1985). 7. SCHMITZ, H., DEIMLING, VON U., FLEHMING, B.: Detection of IgM amtibodies in tables 1 —6 the question about the clinical relevance to Cytomegalovirus (CMV) using an enzyme-labelled antigen (ELA). J. gen. Virol. 50, of results by many laboratories arises. in future it might 59-68(1980). 8. LUTH AR D, Th.: Transfusionsbedingte Zytomegalievirusinfektionen. In: LUTHARD, :be possible to make further improvements of the CMV Th. (Hrsg.) Transfusionsbedingte Zytomegalievirusinfektionen. Steinkopff Verlag diagnostics with the use of recombinant used äs Darmstadt, Darmstadt (1984). antigen. Acknowledgemen t Conclusions We thank Ms. Maria Kristof for excellent technical assistance. « For determination of antibödies directed against CMV we recommend an ELISA based on a principle äs applied by To whom correspondence should be addressed Medac and Organon; because these tests are easy to Dr. Paul Hengster perform and highly specific and sensitive. Alternatively Institute for Hygiene the IF äs a sensitive method can be taken in account, Fritz-Pregl-Straße 3 especially when a titration for the IgG determination and A-6010 Innsbruck t

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