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Serodiagn. Immunother. Infect. Disease 1994; 6: 25-34
Evaluation of eight commercial enzyme-linked immunosorbent assays for detecting CMV-specif ic IgM antibodies in organ transplant recipients
J J Gray, T G Wreghitt
Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge, UK
Summary
Eight commercially available enzyme-linked immunosorbent assays (ELlSAs) for detect- ing cytomegalovirus (CMV) IgM antibodies were evaluated with 152 serum samples collected from 36 organ transplant recipients with evidence of CMV infection. A further 32 samples collected from patients with other virus infections and 11 sera containing rheumatoid factor were also included in the evaluation. The sensitivities of the assays ranged from 57.9-95.6% and the specificities from 80.0-100.0% when the results obtained with the samples collected from transplant recipients were assessed. When samples collected from patients with other infections and the rheumatoid factor positive samples were tested the specificities of the ELlSAs ranged from 68.3-95.5%.
Key words: ELBA, cytomegalovirus, IgM antibodies, organ transplants, rheumatoid factor
Introduction
Cytomegalovirus (CMV) infection in transplant recip- ients may be severe and life-threatening, particularly when acquired from the organ donor’. In heart and lung transplant recipients, who are more susceptible to CMV disease than other solid organ transplant recipi- ents, CMV infection may cause prolonged fever, hepatitis and pneumonitis, and donor-acquired infec- tion is often fatal if left untreated2,3. CMV, in common with all human herpesviruses. can establish latency after primary infection and reactivate periodically throughout the post-transplant period.
It is important to establish whether organ transplant recipients are experiencing CMV infection and although virus culture, detection of CMV antigens and
Received: 13 December 1993 Accepted: 16 December 1993 Correspondence and reprint requests to: JJ Gray, Clinical Microbiology and Public Health Laboratory. Addenbrooke’s Hospital, Cambridge CB2 2QU. UK 0 1994 Butterworth-Heinemann Ltd 0888-0786/941010025- 10
the histopathological examination of biopsy material are the principal diagnostic methods used4J, serology, particularly the detection of CMV-specific IgM antibodies, has a part to play. As primary CMV infec- tions are frequently more severe and treatment may require the modification of immunosuppressive therapy and the use of anti-viral drugs, it is helpful to measure CMV-specific IgM. In the majority of cases higher concentrations of specific IgM are produced in response to a primary infection than a reactivation of past infection or a reinfection with CMW.
A well-characterized panel of serial samples of serum, collected from organ recipients who had experi- enced either a primary CMV infection or a reactiva- tion of a past infection after transplant and had been tested previously by an ‘in-house’ IgM capture enzyme-linked immunosorbent assay (ELISA)6 were chosen for inclusion in this study. These were tested in eight commercially available ELISAs to determine the value of these assays in both detecting and distin- guishing between primary CMV infection and reacti- vation of past infection. Samples collected from patients with other virus infections or infections
26 Serodiagn. Immunother. Infect. Disease 1994; 6: No 1
common in transplant recipients and samples contain- ing rheumatoid factor (anti-globulin antibodies) were also included to identify and estimate the scale of non- specific reactivity in the eight commercial ELISAs.
Materials and methods
Serum samples
A total of 152 serum samples collected from 36 trans- plant recipients with evidence of CMV infection (25 with primary CMV infection and 11 experiencing a reactivation of past infection) was included in the study. Forty-three other samples, consisting of nine rubella IgM antibody-positive sera, 14 Paul Bunnell (heterophile antibody) positive sera, 11 rheumatoid factor positive sera, four hepatitis A virus IgM antibody-positive sera and five Toxoplasma gondii IgM antibody-positive sera were included in the evaluation.
Antibody assays
CMV IgM antibody capture ELISA (‘in-house’ assay) This was performed as described previously6. In brief, the wells of a Falcon 3912 microtitre plate (Becton Dickinson) were coated overnight at 4°C with 100 ul of rabbit anti-human IgM (Dako) (diluted 1 : 2000 in 0.05 M carbonate/bicarbonate buffer pH 9.6). After washing the plate three times with 0.85% NaCl containing 0.8% Tween 20, 100 ul of test serum or controls (diluted 1 : 100 in phosphate buffered saline (PBS) containing 0.05% Tween 20) were added to each well and the plate incubated at 37” C for 3 h. The plate was washed as before and 100 pl CMV antigen (PHLS DMR Colindale, diluted 1 : 10 in PBS/Tween 20 containing 10% foetal calf serum) were added to each well. The plate was incubated overnight at 4” C, washed as before and 100 yl horseradish peroxidase (HRPO) conjugated anti-CMV IgG monoclonal antibody (DMR) were added to each well. The plate was incubated for 3 h at 37” C, washed three times as before and 100 ul of substrate solution (0-phenylenediamine 1 mg ml-’ and hydrogen peroxide 0.4 ul ml-’ in 0.05 M
citric acid/phosphate buffer pH 5.0) were added to all wells. After incubation at room temperature for 30 min the reaction was stopped by adding 25 ul of 3 M
sulphuric acid to each well and the optical density (OD) read at 492 nm wavelength. The amount of CMV-specific IgM in each sample was determined by reference to a standard curve, plotted from the results obtained with a reference positive control series, and expressed as arbitrary units of IgM (Figure 1). A sample was regarded as CMV IgM positive if 20.3 arbitrary units of IgM were detected.
Commercial CMV IgM ELISAs Eight commercial ELISAs for detecting CMV-specific IgM were evaluated. These were an indirect ELISA manufactured by Diamedix Corporation, Florida and
optical density 3r
011 I 100 33 10 33 10 0.33 01 0
arbitrary units
Figure 1. Standard curve constructed from the results of the reference positive control sera (100, 33, 10, 3.3, 1.0, 0.33, 0.1 and 0.0 arbitrary IgM units) tested in the ‘in-house’ assay.
‘in-house’ rabbII an1i+lu IQM HU IgM CMV *g anti-CMV-HRPO OPD
Mercia 1 and 2 lp--(a”--fo”
Sorin
Sigma
t
an,l-H” IQM HU IQM CM”-Ag-HRPO TMB
0
---cc-----l*+>~o ant,+” IQM
H” 1gt.A CMV AQ an,\-CM”-HRPO TMB
0
t ----+<-----l* + >---+O
bioelisa I---+--:
rabbit an,l-H” IQM H” IgM CM”-ag-HRPO TME COnlrOl AQ
EURO-PATH tf---xII*---io” ,
an,!-H" IgM H" IQM CM".AQ.HRPO TM8 CO",lOl AQ
0 Eurogenetics
*+~+/?---E 0
rat, 2‘“llVH” IQM CMV pig strep-HRPO HU IgM blolm-ant,-CM” TM8
0
Diamedix 1: F---‘o
Figure 2. Diagrammatic representation of the ELlSAs used to detect CMV-specific IgM antibodies. Hu: human; HRPO: horseradish peroxidase; OPD: orthophenylenediamine; TMB: tetramethyl benzidine; Strep-HRPO: streptavidin conjugated with HRPO; alk. phos.: alkaline phosphatase; pNP: p-nitrophenyl phosphate.
Bioelisa
Diamedix
Euro-genetics Euro-Path
Mercia version 1
Mercia version 2
Sigma
Sorin
Gray and Wreghitt: ELlSAs for detecting CMV-specific IgM antibodies in transplant recipients 27
Table 1. Quality control and test validation criteria
Assay Test validation criteria Result
Ratio OD neg cont/OD low pos cant ~0.8 Ratio OD high pos cont/OD low pos cant 21.2 OD calibrator 20.5 OD blank ~0.4 Negative control ~30 EU ml-l Positive control within range 78-132 EU ml-l None specified Mean OD cut-off control 0.150-0.450 OD neg cont/OD cut-off cant co.8 OD pos cont/OD cut-off cant >I.5 OD high positive control 20.8 Low pos cant mean >I.5 x mean neg control Low pos cant mean SO.5 x mean high pos cant
in this assay 20.153 10.630 OD high positive control 20.8 Low pos cant mean 21.5 X mean neg control Low pos cant mean 10.5 x mean high pos cant
in this assay 20.240 SO.698 OD negative control eO.200 OD positive control >0.400 OD pos cont/OD neg cant 22.0 CV of control replicate ~20.0%
Cut-off value >0.250 None specified
negative positive
0.27 7.90 0.987 0.081 1.28 EU ml-l
86.90 EU ml-l
0.208 0.5 8.9 1.261
0.274 1.369
0.356 0.139 0.667 4.8 3.1% 5.2% 0.403
seven IgM antibody-capture ELISAs: two manufac- tured and distributed by Mercia Diagnostics Ltd. (one assay marketed pre-1992 and no longer available and a second post-1992); one from Sigma Diagnostics, Dorset; one from Biokit S.A., Barcelona (distributed as Bioelisa by Biokit Ltd., Kent); one from Sorin Biomedica, Italy (distributed by Incstar Ltd., Berkshire); one from Euro-Path Ltd., Cornwall and one from Eurogenetics, Belgium. All assays were performed according to their manufacturers’ instruc- tions and the appropriate kit controls, cut-off controls, calibrators and reagent blanks were included as instructed in the manufacturers’ kit inserts. A diagram- matic representation of each of the commercial ELISAs is shown in Figure 2.
Results obtained with four of the commercial ELISAs (Mercia version 1, Mercia version 2, Eurogenetics and Diamedix) which offered quantita- tive or semiquantitative estimations of CMV-specific IgM antibodies were compared with results, expressed in arbitrary units, obtained with the ‘in-house’ antibody capture ELISA.
Results
Quality control and test validation criteria
Some manufacturers stipulated certain performance characteristics for their assays. The results were only acceptable if the assay conformed to the criteria laid down. The test validation criteria and the results obtained are shown in Table 1.
In all the commercially available assays, the controls were within the limits set by each manufacturer and therefore the results were accepted.
Serum samples collected from transplant recipients
The results obtained from 38 samples collected from nine transplant recipients (four with primary CMV infection and five with a reactivation of past infection) and tested in all nine ELISAs are shown in Table 2. The results of this evaluation were communicated to the respective manufacturers.
Further tests were performed on 114 samples to evaluate the Mercia version 2 ELISA, which was a modification of the Mercia version 1 ELISA, as a consequence of our previous results.
The results obtained with 62 samples collected from a further 14 transplant recipients, 11 with primary CMV infection, and tested with both Mercia ELISAs and the ‘in-house’ ELISA are shown in Table 3. Although all three assays detected CMV infection in the transplant recipients the Mercia version 2 assay showed slightly reduced sensitivity when used to detect a reactivation of CMV infection in these patients. The results obtained with 52 samples collected from a further 13 transplant recipients, ten with primary CMV infection, and tested with the two Mercia ELISAs and the Bioelisa are shown in Table 4. There was better correlation between Bioelisa and Mercia version 2 than Bioelisa and Mercia version 1.
The sensitivity and specificity of each commercial assay, for detecting CMV IgM antibodies in this popula-
28 Serodiagn. Immunother. Infect. Disease 1994; 6: No 1
Table 2. Results obtained with 38 samples collected from nine organ transplant recipients and tested in all nine EtlSAs
CMV IgM ELISA
Patient CMV Days In- Mercia Mercia Sigma Sorin Bio- Euro- Euro Dia- Infect. post- house version version elisa Path gen. medix 7ry or TX assay 7 2 React.
React.
React.
lw
React.
React.
React.
15 34 40 45 85
168 573 615 702
22 45 94
271 364
2 43
336 36
402 14 34 45
136 231
14 119
4 22 37
114 43 98
197 21 34 41 59 62
- - + + + + + + + - + + + + - + + - + - -
+ +
+ - - + - - +
- - + + + + + + + - + + + + - + + - + - - - + +
- - - Equiv - - + Equiv -
- - + + f + + +
- - + + + + + + + - + + + + - + + - + - - - + + - + - - - - - + Equiv - - + + +
Equiv + + + + +
- - - - + - - - - - - - - - + + -
- - + + - - - - -
- + +
- Equiv + + + + + + + - + + + + - + f - + - - + + + -
- - - - - - - - - + + +
- - + + + + + + -
+ + + + - - + - + - - - + + - +
- - - - + - - - + + +
- - + + + + + + - - + + + +
- + - + - - - + + - +
- - - - Equiv -
- + + +
- - + + + + + + - - + + + + - + f - + - - - + + - +
- - - - Equiv Equiv - - + + +
- - - f + - - - Equiv
Equiv + - - - + + - - - - - - - - - - -
Iry = Primary infection; React. = Reactivation; TX = transplant.
tion of organ transplant recipients and defined as the ability to identify correctly both a positive and a negative sample, were calculated (Table 5)7. The ‘in-house’ ELISA was used as the ‘gold standard’ in this evaluation. Whether a patient experienced a primary CMV infection or a reactivation of past infection was determined by serological tests (complement fixation test’*, latex agglu- tination9 and CMV-specific IgG ELISA’O) pre- and post- transplant and virus culture and CMV antigen detection with samples collected after transplant.
Other serum samples
A total of 26 serum samples, four rubella IgM antibody-positive, 14 Paul Bunnell antibody-positive, five rheumatoid factor positive and three Toxoplasma
go&ii IgM antibody-positive, were tested in all eight commercial ELISAs. A further 17 samples, five rubella IgM antibody-positive, six rheumatoid factor positive, two T. gondii IgM antibody-positive and four hepatitis A virus IgM antibody-positive, were tested in the Mercia (Version 2), Sigma and Eurogenetics ELISAs (Table 6).
The specificity of each assay when used to test these ‘non-CMV’ samples was calculated and is shown in Table 6.
Quantitative and semiquantitative estimabn of CMV- specific IgM antibody
In the absence of any recognized international or national CMV antibody standards, CMV-specific IgM
Gray and Wreghitt: EllSAs for detecting CAN-specific /gM antibodies in transplant recipients 29
Table 3. Results obtained with 62 samples collected from transplant recipients and tested in the ‘in-house’ ELISA and the two Mercia ELlSAs for CMV IgM
CMV IgM ELlSA
Patienr CMV Infect. lry or React.
Days post-TX
In- house assay
Mercia version
7
Mercia version
2
- - +
- +
Equiv - -
+ - + + + - - -
+ + + + + +
-
+ + -
- + f t
f + +
10
11
12
13
Iv
lrv
React. 1 ry
1 rv
17 58
376 84
168 375
26 33 30 34 34 53
-164 43 47
121 -13
39 97
192 23
124 206 444
28 60
109 744
1474 -187
28 35 40 91
393 41 45 70
179 344
24 36 47 49 81
105 167 349
13 25 33 88
368 16 25 30 44 82
640 -1 69 95
303
+ + 14 1 w
+ +
+ +
+ + +
+ -t +
+ +
+ + +
+
+ + + + + +
+ + - 15 1 ry
+ +
16 1 v
React. React.
;quiv + - + + Equiv + - - Equiv + + + + +
iquiv +
17
18 React.
React. 19
20 React. Iv
21
22
1 v
+ +
+ + -
23 1 ry
Iry = Primary infection; React. = Reactivation; TX = transplant.
30 Serodiagn. Immunother. Infect. Disease 1994; 0: No 1
Table 4. Results obtained with 52 samples collected from 13 organ transplant recipients and tested in both Mercia and the Bioelisa CMV IgM assays
CMV IgM ELISA
Patient CMV Infect. lry or React.
Days post-TX
Mercia Mercia version version
1 2
Bioelisa
24
25
26
27
28
29
30
31
32
33
34
35
36
React.
Iv
1 ry
Iw
1 v
React.
React.
Iw
lw
Iw
1 w
17 58
127 183
32 38
175 1095
33 99
129 320
28 38 62 78
1197 47 61
181 30 89 95
209 65
117 394
30 37
108 153 226 289
23 56
1097 29 36 55 65 71 27
365 20 40 47
163 1550 1642 1651 1656 1814
+ + + - + + + Equiv + -I- + + + + -I- + + - - - + + + + - + Equiv - - + -I- - - Equiv - + + + - + + + +
+ + -t
+ + +
+ + + + - + + + + + + - Equiv -
+ + -
-
-
-
+
-I-
+ -
+
+
-I- -
+
+
+
+ -
+
+
+
f
+
-I-
+ -
+ +
-I- -
+
+
+ -
+
+
+ -
lry = Primary infection; React. = Reactivation; TX = transplant.
was quantified in the ‘in-house’ assay by extrapolation each sample was determined by dividing the stated EU from a standard curve, constructed from the results ml-’ value of the calibrator by the OD of the calibra- obtained with a pool of CMV-specific IgM positive tor and multiplying by the OD of the test sample. In serum diluted in negative serum, and the results were the Eurogenetics assay, the arbitrary concentration of expressed as arbitrary units of IgM (Figure 1). In the CMV-specific IgM antibodies (M units ml-l) was deter- Diamedix assay, the ELBA unit value (EU ml-r) of mined from a calibration curve constructed from the
Gray and Wreghitt: ELlSAs for detecting CMV-specific IgM antibodies in transplant recipients 31
Table 5. Sensitivity and specificity of the eight commercial CMV IgM ELlSAs when used to test samples collected from transplant recipients (Patient numbers I-9)
Bioelisa Diamedix Eurogenetics Euro-Path Mercia version 1 Mercia version 2 Sigma Sorin
Sensitivity Specificity % %
88.0 100.0 57.9 100.0 95.6 94.1 88.0 100.0
95.6 94.1
95.6 94.1 62.8 80.0 88.0 88.8
results of the 0, 100 and 400 M units ml-r controls. A semiquantitative estimation of CMV-specific antibody was made in both Mercia ELISAs by calculating the ratio of test samples OD : low positive control OD to give an antibody index (AI).
The results of the quantification of CMV-specific IgM obtained with those assays where this option was available are shown in Table 7. The ability of these assays to distinguish between primary CMV infection and a reactivation of past infection was demonstrated by calculating and comparing the mean of the maximum values detected in serum samples collected from each group (Table 8). The statistical significance of the differences between the means in each assay was calculated using the Student’s t test.
Discussion
The presence of CMV-specific IgM in serum samples collected from transplant recipients post-transplant is
an indication of recent CMV infection, either a primary infection, a CMV reinfection or a reactivation of a latent CMV infection.
Commercial assays for detecting CMV-specific IgM antibodies include indirect and IgM antibody-capture ELISAs. In indirect ELISAs for detecting IgM antibodies all classes of CMV-specific antibodies bind to CMV antigens attached to a solid phase. CMV- specific IgM is subsequently detected with an enzyme- labelled anti-human IgM antibody. These assays suffer from competition by CMV-specific IgG leading to false-negative results and interference from rheuma- toid factor which may produce false-positive reactions.
Although the sensitivity and specificity of indirect ELISAs can be improved by pre-treating the sample to remove competing IgG and rheumatoid factor, antibody-capture (u-chain specific) ELISAs for detect- ing virus-specific IgM antibodies have gained widespread acceptance as the need to pre-treat samples is avoided.
ELISAs based on the IgM antibody-capture prin- ciple involve the binding of human IgM by an anti- human IgM antibody (u-chain specific), either monoclonal or polyclonal, attached to the solid phase. A proportion of the total IgM is captured and any virus-specific IgM is subsequently detected with either an enzyme-labelled CMV antigen (Mercia, Bioelisa and Euro-Path) or a complex of antigen and enzyme- labelled specific antibody (Sorin, Sigma, Eurogenetics).
Although the majority of antibody-capture ELISAs are more sensitive and specific when used to detect IgM antibody, non-specific reactivity can be demon- strated with rheumatoid factor positive samples and samples containing heterophile antibodies (Paul Bunnell positive). In some commercial CMV IgM antibody-capture assays a control antigen consisting of uninfected cellular components (Bioelisa, Euro-Path),
Table 6. Specificity of the commercial ELlSAs calculated from the results obtained with sampfes containing rheumatoid factor and samples collected from patients with infections other than CMV
CMV IgM ELISA
Mercia version
1
Mercia version
2
Sigma Sorin Bio- elisa
Euro- Path
Euro- Dia- genetics medix
Rubella IgM reactive 1 2 1 0 1 2 1 0 positive total 4 9 9 5 5 5 9 5 Paul Bunnell reactive 8 2 10 6 0 0 0 11 positive total 14 14 14 14 14 14 14 14 RF positive reactive 0 0 8 0 0 0 0 1
total 6 11 11 8 6 5 11 8 T. gondii IgM reactive 1 0 1 0 1 1 1 0 positive total 5 5 5 5 5 Hepatitis A reactive
N: 0 0 NT NT N: 0 N:
IgM positive total 4 4 4 Total reactive IO 4 20 6 2 3 2
total 29 43 43 32 30 27 43 ii Specificity 74.3% 91.4% 68.3% 84.2% 93.7% 90.0% 95.5% 72.7%
NT = not tested.
32 Serodiagn. Immunother. Infect. Disease 1994; 6: No 1
Table 7. Quantitative determination of CMV IgM antibody and differentiation between primary infection and reactivation of past infection
Days post transplant
‘in-house’ arbitrary
units
Diamedix EU ml-1
Eurogenetics M units ml-’
Mercia version I
Al
Mercia version 2
Al
15 Iry 0.0 9.3 0 0.4 0.3 34 0.1 10.4 0 0.7 0.4 40 1.0 27.1 728 3.3 1.5 45 10.0 76.3 809 3.8 4.7 85 100.0 214.9 793 5.6 5.2
168 10.0 14.7 742 3.0 3.1 573 1.0 7.6 772 2.0 1.2 615 0.3 9.1 793 1.9 1.1 702 0.3 32.4 768 1.3 1.1
22 Iry 0.0 0.0 0 0.4 0.3 45 33.0 39.7 830 5.3 8.5 94 3.3 113.9 779 3.4 3.8
271 1.0 28.6 613 2.2 2.3 364 3.3 28.2 556 2.2 2.5
21 Iry 0.0 0.0 0 0.3 0.4 34 0.0 0.0 0 0.4 0.4 41 3.3 8.5 677 2.7 5.5 59 10.0 24.0 741 2.4 4.6 62 10.0 24.5 741 2.7 4.7 14 Iry 0.0 0.0 0 0.4 0.4 34 0.1 0.0 0 0.4 0.3 45 0.0 7.0 7 0.8 0.5
136 3.3 9.1 850 2.5 4.5 231 33.0 4.0 744 3.5 3.1
2 React. 0.0 0.0 0 0.3 0.3 43 0.3 65.7 240 2.3 1.2
336 10.0 99.7 753 5.0 2.7 36 React. 0.0 0.0 0 0.3 0.3
402 1.0 8.0 323 1.7 1.8 14 React. 0.0 0.0 4 0.5 0.4
119 0.3 0.0 179 0.8 1.9 4 React. 0.0 0.0 0 0.4 0.4
22 0.0 0.0 19 0.6 0.4 37 3.3 0.4 89 1.1 0.5
114 0.1 0.0 5 0.5 0.4 43 React. 0.0 0.0 0 0.3 0.3 98 0.3 6.9 128 1.7 1.2
197 0.1 0.0 131 1.0 0.9
A sample was positive if in the ‘in-house’ assay there was 20.3 AU, Diamedix; k-40 EU ml- ‘, Eurogenetics; 2150 M units ml-’ and in both Mercia ELlSAs; >I.1 Al; Iry = Primary infection; React. = Reactivation.
or specific blocking reagents (Eurogenetics) are added to minimize non-specific reactivity.
In this study the poor sensitivity of the indirect ELISA (Diamedix) (57.9%) (Table 5) probably reflects competition by CMV-specific IgG. This is highlighted by the inability of this assay to detect CMV-specific IgM in samples collected from four out of five patients with a reactivation of past infection and in whom CMV IgG was already present. IgM was detectable for a much shorter time with this assay in those patients with a primary CMV infection where a CMV-specific IgM response was detected (see Table 2).
The results of the initial evaluation (Table 2) indicate that all the IgM antibody-capture ELISAs were more sensitive or more able to identify correctly the positive samples than the indirect ELISA. The sensitivities ranged from 62.8% (Sigma) through
88.0% (Sorin, Bioelisa and Euro-Path) to 95.6% (Mercia version 1, Mercia version 2, Eurogenetics) (Table 5). Although the Mercia versions 1 and 2 assays showed comparable sensitivities (95.6%) in the initial evaluation (Table 2) they had sensitivities of 97.5% and 92.0% respectively when the results of all samples were evaluated (Tables 2-4). The wide range of sensi- tivities seen in this study may be due to the use, in two of the assays, of a monoclonal anti-human IgM of low affinity when compared to a poiycional serum or an inability to optimize the assay. The two assays which used a monoclonal antibody to capture IgM were Sigma (sensitivity: 62.8%) and Sorin (sensitivity: 88.0%).
The specificities or the ability of the ELISAs to identify correctly the negative samples when used to test samples collected from transplant recipients
Gray and Wreghitt: ELlSAs for detecting CMV-specific IgM antibodies in transplant recipients 33
Table 8. The mean of the maximum values of CMV IgM antibody obtained from patients with primary CMV infection and CMV reactivation
Mean of maximum values
Primary Reactivation infection
Statistical significance
between means (Student’s
t test)
‘In-house’ Diamedix Eurogenetics Mercia version 1 Mercia version 2
44.0 AU 91.8 EU ml-’
807.0 M units ml-’
4.2 Al
5.9 Al
3.0 AU 23.0 EU ml-l
295.0 M units ml-’
1.8 Al
1.6 Al
P <0.05 P<O.l P<O.Ol
P<O.l
P <0.002
ranged from 80.0% (Sigma) to 100% (Diamedix, Bioelisa and Euro-Path) (Table 5) but when samples collected from patients with other infections were tested the specificity ranged from 68.3% (Sigma) to 95.5% (Eurogenetics) (Table 6). The three assays which showed 100.0% specificity with transplant recip- ient samples had specificities of 72.7% (Diamedix), 93.7% (Bioelisa) and 90.0% (Euro-Path) with the non- transplant samples collected from patients with no evidence of CMV infection. The Mercia versions 1 and 2 assays both had specificities of 94.1% with transplant recipient samples but had specificities of 74.3% and 91.4% respectively with the non-transplant samples.
Non-specific reactivity, although at an acceptable level in four of the commercial assays (Mercia version 2, Bioelisa, Euro-path and Eurogenetics), was widespread and found with rubella IgM antibody- positive sera, Paul Bunnell (heterophile antibody) positive sera, RF positive sera and T. gondii IgM antibody-positive sera. There was an unacceptably high incidence of non-specific reactivity with the Paul Bunnell positive sera in the Mercia version 1 (57.1% of samples tested). Sigma (71.4%), Sorin (42.8%) and Diamedix (78.5%) ELISAs (Table 6). Although it has been suggested that non-specific reactions caused by Paul Bunnell positive samples may be due to polyclonal stimulation of B-lymphocytes by Epstein Barr virus (EBV). it is unlikely in this study for two reasons. The false-positive reactivity was not consistent throughout ali the assays and in the assays which included either an uninfected control antigen (Bioelisa, Euro-Path and Mercia version 2) or specific blocking reagents (Eurogenetics) false-positive reactivity was not detected. Non-specific reactivity associated with the presence of rheumatoid factor was only detected in the Sigma (72.7% of samples tested) and Diamedix (12.5%) ELISAs. The low incidence of RF associated non-specific reactivity in the Diamedix ELISA, which is an indirect ELISA, may be due to the presence of an immunoabsorbent in the sample diluent which has been shown to neutralize the activity of up to 348 III ml-’ of lgM rheumatoid factor (see kit insert).
Only two of the four commercial ELISAs with the ability to quantify CMV-specific IgM (Eurogenetics
and Mercia version 2) were able to distinguish between primary CMV infection and a reactivation of past infection. Although a difference was detected between the means of the maximum values of antibody in primary infection and reactivation with the Mercia version 1 ELISA, it was not statistically significant, Also, in the Diamedix ELISA the difference was not significant and the mean maximum value (23.0 EU ml-‘) of samples collected from patients with a reacti- vation of past infection was in the assay negative range (~30 EU ml-l) (Table 8).
We have thus shown that there is considerable varia- tion in the performance of commercial ELISAs for detecting CMV-specific IgM antibody. In general, antibody-capture assays are preferable to indirect assays and those which include control antigen or blocking reagents minimize the non-specific reactivity detected in serum samples from some patient categories, particularly those experiencing infection with another virus (EBV). In choosing a commercial assay for detecting CMV-specific IgM antibody, care must be taken to assess the assay performance in the patient populations to be tested.
References
Wreghitt TG, Hakim M, Gray JJ. Kucia S. Wallwork J, English TAH. Cytomegalovirus infections in heart and heart and lung transplant recipients. J C/k fathol 3988; 41: 660-7 Hutter JA, Scott J, Wreghitt T, Higgenbottam T. Wallwork J. The importance of cytomegalovirus infection in heart-lung transplant recipients. Chest 1989: 95: 627 Wreghitt TG. Cytomegalovirus infections in heart and heart-lung transplant recipients. J Antimicroh Chemother 1989; 23: 49-60 Wreghitt TG, Smyth RL, Scott JP, Higgenbottam T, Gray JJ, Stewart S, Wallwork J. Value of culture of biopsy material in diagnosis of viral infections in heart-lung transplant recipients. Transplant froc 1990; 22: 1809-10 Griffiths PD, Panjwani DD, Stirk PR. Ball MG, Ganczakowski H, Blacklock HA, Prentice HG. Rapid detection of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancer 1984: ii: 1242-S
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