Clinical Biochemistry 48 (2015) 603–608
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum Jian-Wei Zhou a, La-Mei Lei b, Qian-Ni Liang a, Tian-Cai Liu a, Guan-Feng Lin a, Zhi-Ning Dong a, Rong-Liang Liang a, Zhen-Hua Chen a, Ying-Song Wu a,⁎ a b
Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, PR China Institute of Hydrobiology, Jinan University, Guangzhou 510515, Guangdong, PR China
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 13 January 2015 Accepted 18 January 2015 Available online 26 January 2015 Keywords: Rubella virus Cytomegalovirus IgM Dual-TRFIA
a b s t r a c t Objectives: This study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. Design and methods: Lanthanum elements labeled antibody and streptavidin–biotin system were used in the “capture sandwich” format simultaneously. Results: The working range of TRFIA for RV IgM was 2–80 AU/mL and for CMV IgM was 5–400 AU/mL. Intraand inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. Conclusion: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Rubella virus (RV), a single-stranded positive RNA virus, belongs to the Rubivirus genus of the Togaviridae family [1]. Rubella is an acute respiratory infectious disease caused by RV with mild or absent clinical features and is easily neglected. RV infection in pregnant women, especially during the first trimester [2], can cause intrauterine death, miscarriage, or premature birth [3]. When RV is transmitted to the fetus through the placenta, infection results fetal growth restriction or congenital rubella infection (CRI), which causes congenital rubella syndrome (CRS) in infants [4]. WHO estimated that at least 100,000 cases of CRS infants born annually worldwide [4]. Three characteristic manifestations of CRS, the most harmful symptom of rubella, include congenital cataracts, deafness and cardiovascular anomalies [2]. CRS is a huge burden on society and the economy, particularly in developing
Abbreviations: AU/mL, arbitrary units/milliliter; BSA, bovine serum albumin; CLIA, chemiluminescent immunoassays; CMV, cytomegalovirus; CRI, congenital rubella infection; CRS, congenital rubella syndrome; CV, coefficients of variation; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; HCMV, human cytomegalovirus; IgG, immunoglobulin G; IgM, immunoglobulin M; McAbs, monoclonal antibodies; PBS, phosphate buffered saline; RF, rheumatoid factor; RNA, ribonucleic acid; RV, rubella virus; SD, standard deviation; TRFIA, time-resolved fluoroimmunoassay. ⁎ Corresponding author. Fax: +86 20 37247604. E-mail address: [email protected] (Y.-S. Wu).
countries [5]. To reduce the incidence of CRS, it is important that screening and vaccination occur before childbirth. High positive rates of RV-specific IgM antibodies are present about 5–14 days after the rash appears. The levels then decrease and last for about 8–12 weeks [6]. Commercially available enzyme-linked immunosorbent assays (ELISA) [7] and chemiluminescent immunoassays (CLIA) [8] for IgG and IgM detection are generally used for clinical diagnosis. Positive levels of RV IgM strongly suggest that patients recently had an acute rubella virus infection [6]. Cytomegalovirus (CMV), a DNA virus, belongs to the β-herpes virus family [9]. Human cytomegalovirus (HCMV) is one of the most common causes of intrauterine infection, which can cause a variety of different infection syndromes [10]. CMV infection in pregnant women causes abortion, stillbirth, and premature delivery. About 10%–15% of neonates with congenital CMV infection show symptoms such as fetal growth restriction, microcephaly, hepatosplenomegaly and skin petechiae at birth, and 20%–30% of symptomatic infection neonates eventually die [11]. The early diagnosis and treatment of HCMV infection is importance clinically. IgM antibodies are present for several days after CMV primary infection and remain for about 3–6 months, suggesting a recently active CMV infection and virus replication [12]. To date, the methodologies used for serological investigation of CMV antibodies are the indirect ELISA [13] and CLIA [14]. Most CMV infections cause no symptoms, therefore the correlation between IgM responses and recent active CMV infection is
http://dx.doi.org/10.1016/j.clinbiochem.2015.01.009 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
604
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
of great significance in the clinic [15]. Pregnant women should be tested for CMV IgM antibodies as early as possible, as treatment should occur as early as possible as the early and accurate diagnosis of RV or CMV infection can reduce birth defects and improve the population quality. Time-resolved fluoroimmunoassay (TRFIA) using lanthanide chelates is widely used for clinical screening and diagnostics [16]. The main characteristics of TRFIA are long storage time, high sensitivity and specificity, good repeatability, a wide detection range and no radioactive contamination [17,18]. In this report, we developed a novel highly reproducible one-step dual-TRFIA, using Eu3 + and Sm3+ chelates as labels, to simultaneously measure the concentration of RV IgM and CMV IgM in human serum. Materials and methods Reagents and instrumentation Natural RV antigen was obtained from Microbix Biosystems Inc. (Mississauga, Ontario, Canada). CMV recombinant antigen (integrating the pUL80, pUL32, and ppUL83 genes), anti-RV monoclonal antibodies (McAbs) and anti-human IgM McAbs were obtained from Fapon Biotech Inc., (Shenzhen, China). Streptavidin was obtained from SigmaAldrich (St. Louis, MO, USA) and NHS-(N-hydroxysuccinimido)-biotin was from Roche (Mannheim, Germany). Eu3+ labeled kits and Sm3+ labeled kits were obtained from PerkinElmer (Turku, Finland). Transparent 96-well micro-titration strips were purchased from Thermo Labsystems (Milford, MA, USA). Victor™ 1420 time-resolved plate fluorometer and AUTODELFIA™ 1235 Automatic Immunoassay System were obtained from Perkin-Elmer Life and Analytical Sciences (Waltham, MA, USA). Enhancement solution and Wash solution were from Wallac (Turku, Finland). Bovine serum albumin (BSA) was obtained from Bovogen Biologicals Pty Ltd., (Victoria, Australia). RV IgM ELISA kits and CMV IgM ELISA kits were purchased from Serion (Würzburg, Germany). Rubella and CMV IgM CLIA kits were purchased from DiaSorin Liaison (Via Crescentino, Italy) and Abbott Architect (Sligo, Ireland). Serum panel PTC203 and PTR201 were purchased from SeraCare Life Sciences (Gaithersburg, MD, USA). Other chemicals and reagents used were of analytical grade. Coating with capture antibody 100 μl anti-human IgM McAbs, diluted to final concentration of 3 μg/mL with coating buffer (50 mmol/L, Tris, 0.9% NaCl, pH 7.4), was pipetted into each well. The plates were incubated at 4 °C overnight and then washed 3 times with washing buffer (25 mmol/L Tris, 0.9% NaCl, 0.06% Tween-20, pH 7.8). Then 200 μl of blocking buffer (50 mmol/L PBS, 0.9% NaCl, 0.1% BSA, 0.2% Tween-20, pH 7.4) was added to each well, and the wells maintained at 4 °C overnight. After removing the blocking buffer, the plates were vacuum dried and stored with a desiccant at 4 °C for short-term or at −20 °C for long-term preservation. Eu3+-labeled anti-RV McAbs and Sm3+-labeled streptavidin The buffer for anti-RV McAbs was exchanged for labeling buffer (100 mmol/L Na2CO3, pH 9.3) in a centrifuge tube containing a membrane before labeling. Then 0.2 mg DTTA-Eu (N1-[P-isothiocyanatobenzyl]-diethylene-triamine-N1,N2,N3-tetraacetate-Eu3+) was added to 1.0 mg of McAbs in 200 μl labeling buffer, then the mixture was stirred overnight at room temperature. The labeled antibody was separated from unreacted chelates and aggregated McAbs by gel filtration (Sephadex, G-50) using elution buffer (50 mmol/L Tris, pH 7.4 containing 0.9% NaCl and 0.2% BSA). The fluorescence of the Eu3 + labeled McAbs was measured at 615 nm. After adding BSA as a stabilizer, aliquots of the labeled antibody were stored at −20 °C. The streptavidin was labeled with Sm3 + chelates using the same procedure as for
Eu3+-labeled anti-RV McAbs, except the optimized weight proportion of streptavidin and DTTA-Sm3+ was 2:1. Biotinylation of CMV antigen The CMV antigen was biotinylated to allow its detection by timeresolved fluorescence of Sm3+ labeled streptavidin. The CMV antigen was centrifuged and washed with PBS to remove protective agents such as NaN3. Then NHS-Biotin was then added at an optimized ratio of antigen and NHS-Biotin (1:25). The reaction mixture was stirred slightly for 1 h at room temperature. Then the labeled antigen was separated from unconjugated chelate by gel filtration with PBS and the protein peak at 280 nm was collected and filtered through a sterile 0.2 μm Minisart filter. After adding BSA as a protectant, aliquots of conjugates were stored at −20 °C for daily use. Preparation of antigen working solution RV antigen and biotinylated CMV antigen were diluted into a total concentration of 1.0 μg/mL and 0.2 μg/mL using assay buffer (50 mmol/L Tris, 0.9% NaCl, 0.5% BSA, pH 7.8). The mixture was used as the antigen working solution. Calibration The determination of RV IgM antibody activity was given in AU/mL and correlated to the RV IgM reference serum which was from Leipzig (Germany). The concentration of reference serum was defined as 30 U/mL detected by Serion RV IgM ELISA kit and was used as the gold standard of the laboratory. Then the serum was diluted serially and used to revise the RV IgM standard of dual-TRFIA, the ratio of theoretical value and the measured value was between 0.9 and 1.1. Similarly, CMV IgM antibody activity was given in AU/mL and correlated to the CMV IgM reference serum which was from the Paul-Ehrlich Institute, Langen. The concentration of reference serum was defined as 1800 U/mL detected by Serion CMV IgM ELISA kit and was used as the gold standard of the laboratory. Then the serum was diluted serially from 1:5 to 1:80 and used to revise the CMV IgM standard of dualTRFIA, the ratio of theoretical value and the measured value was between 0.9 and 1.1. Samples A total of 411 serum samples were obtained from The Guangzhou Second People's Hospital and The Liuzhou Maternal and Child Health Hospital, including 56 RV IgM positive serum samples, 355 RV IgM negative samples, 65 CMV IgM positive serum samples and 346 CMV IgM negative samples. All serums were confirmed by Diasorin Liaision RV and CMV IgM CLIA (The sample was defined as negative when the value is less than 20 UA/mL, positive when greater than 25 UA/mL and equivocal when between 20 and 25 UA/mL in RV IgM detection; The sample was defined as negative when the value is less than 18 U/mL, positive when greater than 22 U/mL and suspicious when between 18 and 22 U/mL in CMV IgM detection) (Table 1). Table 1 Samples and serum panels confirmed by Diasorin Liaision RV and CMV IgM CLIA. Sample type
Number of Diasorin Liaision sera rubella IgM
Diasorin Liaision CMV IgM
Positive Negative Positive Negative Blood donors Pregnant women Diagnostic/hospital patients Serum panel PTC203 Serum panel PTR201 Total
134 149 128 21 25 457
13 23 20 / 6 62
121 126 108 / 19 374
21 25 19 6 / 71
113 124 109 15 / 361
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
605
Fig. 1. Schematic of dual-TRFIA for RV IgM and CMV IgM detection in serum.
104 positive serum samples for Cross-reactivity studies, including 15 anti-nuclear antibody, 8 systemic lupus erythematosus, 26 rheumatoid factor, 9 herpes simplex virus IgM types 1 and 2, 5 toxoplasma gondii IgM, 7 human anti-mouse antibody, 16 high titer Rubella IgG (N250 IU/mL) and 18 high titer CMV IgG (N8 IU/mL). The serum panel PTC203 consisted of a set of 21 specimens with anti-CMV reactivity, including 6 positive samples and 15 negative samples (Table 1). Samples were collected between 1999 and 2011 and selected to demonstrate IgG and/or IgM reactivity. The serum panel PTR201 consisted of a set of 25 specimens with anti-Rubella IgG and/ or IgM antibody reactivity ranging from negative to strongly positive, including 6 positive samples and 19 negative samples (Table 1). Samples were collected in 1994 and 1995. All samples were measured and analyzed by RV/CMV dual-TRFIA. Then the unmatched samples were tested using the Abbott Architect CLIA (The sample was defined as negative when the value is less than 1.20 Index, positive when greater than 1.6 Index and equivocal when between 1.20 and 1.60 Index in RV IgM detection; The sample was defined as negative when the value is less than 0.85 Index, positive when greater than 1.0 Index and suspicious when between 0.85 and 1.0 Index in CMV IgM detection). All procedures were carried out in accordance with the manufacturer's instructions. DiaSorin Liaison RV/CMV IgM CLIAs used the chemiluminescence automatic detection system commonly used for clinical detection, consisting of magnetic particles coated with either RV antigen or CMV antigen and McAbs-N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) as a tracer. Abbott Architect RV and CMV IgM CLIAs were similar, except the murine acridinium labeled McAbs.
Assay protocol Eu 3 +-labeled Anti-RV McAbs and Sm3 +-labeled streptavidin (N200 ng/mL) were both added into the antigen working solution to form the analysis solution. The serum samples and standards were diluted with dilution buffer (50 mmol/L Tris, 0.9% NaCl, 1.5% BSA, 0.4% casein, pH 7.8) in a 1:101 dilution. In brief, 100 μl of serum sample or 100 μl of standard was pipetted into the microtiter wells coated with anti-human IgM McAbs. The plates were incubated for 1 h at room temperature with horizontal shaking. After washing 4 times, 100 μl analysis buffer was added. The plates were incubated at room temperature with shaking for 1 h. After washing 6 times, enhancement solution (100 μl/well) was added to each well. The plates were shaken for 5 min and fluorescence intensity readings at 615 nm (Eu3 +) and 645 nm (Sm3+) were measured with a Victor™ 1420 time-resolved plate fluorometer (Fig. 1). The same experiments were also performed using the AUTODELFIA™ 1235 automatic immunoassay system with software designed in our lab.
Results Dilution test Two positive samples containing different levels of RV IgM and CMV IgM with several dilutions from 1:2 to 1:32 were investigated in this study. As shown in Fig. 2, the dilution curves were both linear over the whole range of concentrations for both RV IgM and CMV IgM.
Fig. 2. Dilution linearity for RV IgM (A) and CMV IgM (B), based on 2 different degrees of positive serum samples.
606
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
8.00% and 3.26 to 9.64% and for CMV IgM 4.03 to 6.80% and 6.03 to 8.72%, respectively.
Table 2 Recovery test of RV IgM/CMV IgM TRFIA. Sample
S1 S2
RV-IgM
Recovery %
Original concentration (AU/mL)
Measured concentration (AU/mL)
16.7 23.1
15.9 21.3
CMV-IgM
S1 S2
Cross-reactivity 94 91 Recovery %
Original concentration (AU/mL)
Measured concentration (AU/mL)
22.3 33
22.9 34.7
105 107
Parallelism demonstrated agreement among the results obtained at different dilution levels of the samples.
Recovery test Treatment of RV IgM samples was based on the addition of 0.1 mL of standards consisting of RV IgM at two different concentrations (13.5 AU/mL and 19.9 AU/mL) to a 1 mL original sample aliquot containing 3.2 AU/mL endogenous RV IgM. After repeating experiments 3 times, mean values and recovery were calculated. The CMV IgM recovery experiment was similar to the above experiment. Results in Table 2 show that dual-TRFIA accurately measured the concentration of RV IgM and CMV IgM in serum samples, yielding a mean recovery of 92.5% for RV IgM and 106% for CMV IgM.
Standard curve, analytical sensitivity and precision The standard curves of RV IgM and of CMV IgM were linear over the concentration used. The measurement range of RV IgM was 2–80 AU/mL and that of CMV IgM was 5–400 AU/mL. No high dose hook effect was observed in the standard curve (Fig. 3). Sensitivity of RV IgM was 0.141 AU/mL and for CMV IgM it was 1.129 AU/mL. The limit of detection was defined by the concentration of RV IgM and CMV IgM corresponding to fluorescence of the zero calibrators plus 2 standard deviation (SD). As shown in Fig. 3, the precision (CV%) was less than 6% in the detection range for both in RV IgM and CMV-IgM, when each point was measured 10 times. The imprecision (intra-assay and inter-assay) for dual-label TRFIA was obtained by testing three concentrations of RV IgM and CMV IgM in serum (negative, weakly positive and strong positive), respectively. The intra- and inter-assay imprecision for RV IgM was from 3.46 to
Cross-reactivity studies were performed on 149 pregnant women and 104 specimens positive for anti-nuclear antibody, systemic lupus erythematosus, rheumatoid factor (RF), herpes simplex virus IgM types 1 and 2, toxoplasma gondii IgM, human anti-mouse antibody, high titer rubella IgG (N 250 IU/mL) and CMV IgG (N 8 IU/mL). With these specimens, the RV/CMV IgM dual-TRFIA and a commercially available diagnostic kit (DiaSorin Liaison CLIA) showed 100% agreement (253/253). Correlation between CLIA and TRFIA All 411 specimens and serum panel (PTC203 and PTR201) were tested by RV/CMV dual-TRFIA. The results of Diasorin Liaision RV IgM/CMV IgM and dual-TRFIA were shown in Tables 3. For dual-TRFIA, sample was defined as negative when the value was less than 1.8 AU/mL, positive when greater than 2.0 AU/mL and suspicious between 1.8 and 2.0 AU/mL in RV IgM detection; sample was defined as negative when the value is less than 8.0 AU/mL, positive when greater than 10.0 AU/mL and suspicious between 8.0 and 10.0 AU/mL in CMV IgM detection. The results obtained were analyzed by SPSS13.0 statistics software. Based on the above findings, we analyzed the sensitivity and specificity of the RV/CMV IgM dual-TRFIA assays. Equivocal values were regarded as positive when calculating the sensitivity and negative when calculating specificity. The sensitivity and specificity of RV IgM detection by dual-TRFIA was 93.5% (58/62) and 99.2% (371/374), with a kappa value of 0.953. For CMV IgM detection, the sensitivity and specificity were 90.1% (64/71) and 98.3% (355/361), with a kappa values of 0.917. No significant statistical difference between the RV/CMV IgM DiaSorin Liaison CLIA and dual-TRFIA assays. The unmatched samples were then tested using the Abbott Architect CLIA. For RV IgM detection, of the three unmatched equivocal samples in TRFIA, there were three positive in the Abbott Architect CLIA, and both methods found two unmatched negatives and two unmatched positive. For CMV IgM detection, of the seven unmatched equivocal samples in TRFIA, there were seven positive in the Abbott Architect CLIA, and both methods found two unmatched negatives and four unmatched positives (Table 3). Discussion The capture assay used to detect IgM antibodies in serum was previously shown to be feasible and effective [19]. Compared with the
Fig. 3. Standard curve of RV IgM (A) and CMV IgM (B) and the intra-assay CV% of each point.
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608 Table 3 Qualitative results of rubella IgM and CMV IgM. RV/CMV dual-TRFIA
Positive Equivocal Negative Total
Diasorin Liaision Rubella IgM
Diasorin Liaision CMV IgM
Positive
Negative
Total
Positive
Negative
Total
58 2 2 62
2 1 371 374
60 3 373 436
64 3 4 71
2 4 355 361
66 7 359 432
Note: Dual-TRFIA equivocal values were regarded as positive when calculating the measure of agreement (kappa value).
607
method for measurement of RV and CMV IgM antibodies. This method had a high sensitivity, specificity and good reproducibility. Additionally, this assay was suitable for the PE AUTODELFIA™ 1235 automatic system, which might allow the development of automated testing for RV and CMV IgM antibodies. Conflict of interest None. Acknowledgments
indirect method, it had a higher sensitivity and specificity and was not affected by RF or high concentrations of pathogen IgGs [20,21]. In this study, RF and related pathogen IgGs were tested and demonstrated no correlation between RV IgM and CMV IgM in clinical serum samples. The labels were thus independent of each other. To maintain maximum antigen activity, we used Eu3+-DTTA to label RV McAbs and directly diluted the RV natural antigen at an appropriate proportion. Before the serum reaction, Eu3+-RV McAbs were combined with specific antigen, which retained all epitopes and specificity of the natural antigen, to allow maximal detection of RV-specific IgM antibodies in serum. The choice of McAbs in this assay was of importance based on the principle that the activity between natural antigen and Eu3 +McAbs did not affect the subsequent specific reaction between the same antigen and IgM antibodies in serum. The study of CMV antigen was relatively comprehensive. Currently, the envelope protein ppUL83 is the most widely used protein in the clinic as the main antigen to detect IgM and IgG antibodies of CMV. Its antigenicity is high among numerous CMV proteins, and the corresponding antibody titers were very high during the acute stage of infection and early recovery [22,23]. Additionally, the CMV protein mainly induced IgM antibodies including pUL80 and pUL32 [24]. The CMV recombinant antigen used in this study contained the ppUL83, Pul80 and pUL32 genes, thus it harbored all antigen epitopes that specifically bind with CMV IgM antibody. This demonstrated that Eu3 + labeled streptavidin and time-resolved fluorometry is an appropriate indirect label because of its high affinity to biotin and low nonspecific binding properties. As a tetrameric structure with four binding sites for biotin, it can be used to amplify signals [25]. Feng-Bo developed a doubleantigen sandwich TRFIA to detect total anti-HCV antibodies based on biotin–streptavidin interactions and verified that this system was more specific and reliable for screening anti-HCV antibodies than those indirect methods [26]. Because the recombinant antigen did not contain all the epitopes of the natural antigen, we selected biotin to bind to the antigen, which minimally affected antigen activity. Additionally, Sm3 + was used to label streptavidin to produce a streptavidin– biotin system with increased detection sensitivity and detection range while maintaining specific immune responses [27,28]. The dual-TRFIA system, based on the wavelength of lanthanum elements Eu3 + and Sm3 + at 615 nm and 645 nm, was used to measure Eu3 + and Sm3 + fluorescence intensity to simultaneously obtain the concentration of RV IgM and CMV IgM. The impact of background fluorescence values caused by Eu3+ to Sm3+ was deducted by the correction system in Victor™ 1420 or AUTODELFIA™ 1235. In the absence of an international standard, we established a quantitative analysis dual-TRFIA that could accurately determine the degree of RV IgM and CMV IgM in serum, based on a laboratory internal standard. Establishment of the quantitative determination method allowed the further study of changes in RV IgM and CMV IgM titers in serum and the occurrence, development and prognosis of pathogen infection [29–32]. Compared with commercial CLIAs, the RV IgM and CMV IgM dual-TRFIA developed in the current study had a higher sensitivity and specificity, suitable for clinical applications. Taken together, this study demonstrated the use of dual-TRFIA and anti-human IgM McAbs as captured antibodies to establish a quantitative
This work was supported by the National Science and Technology Major Project (grant number 2013ZX10004-803). References [1] Atreya CD, Mohan KV, Kulkarni S. Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level, 70; 2004 431–7. [2] De Santis M, Cavaliere AF, Straface G, Caruso A. Rubella infection in pregnancy, 21; 2006 390–8. [3] Chen M, Zhu Z, Liu D, Huang G, Huang F, Wu J, et al. Rubella epidemic caused by genotype 1E rubella viruses in Beijing, China, in 2007–2011, 10; 2013 122. [4] Robinson JL, Lee BE, Preiksaitis JK, Plitt S, Tipples GA. Prevention of congenital rubella syndrome—what makes sense in 2006?, 28; 2006 81–7. [5] Verma R, Khanna P, Chawla S. Rubella vaccine: new horizon in prevention of congenital rubella syndrome in the India, 8; 2012 831–3. [6] Banatvala JE, Brown DW. Rubella, 363; 2004 1127–37. [7] Hudson P, Morgan-Capner P. Evaluation of 15 commercial enzyme immunoassays for the detection of rubella-specific IgM, 5; 1996 21–6. [8] Portella G, Galli C. Multicentric evaluation of two chemiluminescent immunoassays for IgG and IgM antibodies towards rubella virus, 49; 2010 105–10. [9] Dimberg J, Hong TT, Skarstedt M, Lofgren S, Zar N, Matussek A. Detection of cytomegalovirus DNA in colorectal tissue from Swedish and Vietnamese patients with colorectal, cancer, 33; 2013 4947–50. [10] Griffiths PD. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation, 12; 2012 790–8. [11] Yinon Y, Farine D, Yudin MH. Screening, diagnosis, and management of cytomegalovirus infection in pregnancy, 65; 2010 736–43. [12] Adler SP. Screening for cytomegalovirus during pregnancy, 2011; 2011 1–9. [13] Revello MG, Gerna G. Diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant, 15; 2002 680–715. [14] Juhl D, Vockel A, Luhm J, Ziemann M, Hennig H, Gorg S. Comparison of the two fully automated anti-HCMV IgG assays: Abbott Architect CMV IgG assay and Biotest antiHCMV recombinant IgG ELISA, 23; 2013 187–94. [15] Schmitz H, von Deimling U, Flehmig B. Detection of IgM antibodies to cytomegalovirus (CMV) using an enzyme-labelled antigen (ELA), 50; 1980 59–68. [16] Lovgren T, Hemmila I, Pettersson K, Eskola JU, Bertoft E. Determination of hormones by time-resolved fluoroimmunoassay, 31; 1984 909–16. [17] Merio L, Pettersson K, Lovgren T. Monoclonal antibody-based dual-label timeresolved fluorometric assays in a simplified one-step format, 42; 1996 1513–7. [18] Wang K, Huang B, Zhang J, Zhou B, Gao L, Zhu L, et al. A novel and sensitive method for the detection of deoxynivalenol in food by time-resolved fluoroimmunoassay, 19; 2009 559–64. [19] Welch RJ, Chang GJ, Litwin CM. Comparison of a commercial dengue IgM capture ELISA with dengue antigen focus reduction microneutralization test and the centers for disease control dengue IgM capture-ELISA, 195; 2014 247–9. [20] Lakos G, Teodorescu M. IgM, but not IgA rheumatoid factor interferes with anticardiolipin and antibeta2 glycoprotein I measurements: a quantitative, analysis, 20; 2011 614–9. [21] Namekar M, Ellis EM, O'Connell M, Elm J, Gurary A, Park SY, et al. Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of dengue virus infection, 51; 2013 3102–6. [22] Wirgart BZ, Claesson K, Eriksson BM, Brundin M, Tufveson G, Totterman T, et al. Cytomegalovirus (CMV) DNA amplification from plasma compared with CMV pp65 antigen (ppUL83) detection in leukocytes for early diagnosis of symptomatic CMV infection in kidney transplant patients, 7; 1996 99–110. [23] Holtappels R, Podlech J, Grzimek NK, Thomas D, Pahl-Seibert MF, Reddehase MJ. Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65), 75; 2001 6584–600. [24] Maine GT, Stricker R, Schuler M, Spesard J, Brojanac S, Iriarte B, et al. Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer, 38; 2000 1476–81. [25] Suonpaa M, Markela E, Stahlberg T, Hemmila I. Europium-labelled streptavidin as a highly sensitive universal label. Indirect time-resolved immunofluorometry of FSH and TSH, 149; 1992 247–53. [26] Wu FB, Ouyan HQ, Tang XY, Zhou ZX. Double-antigen sandwich time-resolved immunofluorometric assay for the detection of anti-hepatitis C virus total antibodies with improved specificity and sensitivity, 57; 2008 947–53.
608
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
[27] Du L, Cheng S, Wang S. Determination of diethylstilbestrol based on biotin– streptavidin-amplified time-resolved fluoro-immunoassay, 27; 2012 28–33. [28] Dundas CM, Demonte D, Park S. Streptavidin–biotin technology: improvements and innovations in chemical and biological applications, 97; 2013 9343–53. [29] Sen MR, Shukla BN, Tuhina B. Prevalence of serum antibodies to TORCH infection in and around Varanasi, Northern India, 6; 2012 1483–5. [30] Sanz JC, Mosquera M, Ramos B, Ramirez R, de Ory F, Echevarria JE. Assessment of RNA amplification by multiplex RT-PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella, 118; 2010 203–9.
[31] Neirukh T, Qaisi A, Saleh N, Rmaileh AA, Zahriyeh EA, Qurei L, et al. Seroprevalence of cytomegalovirus among pregnant women and hospitalized children in Palestine, 13; 2013 528. [32] Rasmussen L, Kelsall D, Nelson R, Carney W, Hirsch M, Winston D, et al. Virusspecific IgG and IgM antibodies in normal and immunocompromised subjects infected with cytomegalovirus, 145; 1982 191–9.
Contents lists available at ScienceDirect
Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem
Dual-labeled time-resolved immunofluorometric assay for the determination of IgM antibodies to rubella virus and cytomegalovirus in human serum Jian-Wei Zhou a, La-Mei Lei b, Qian-Ni Liang a, Tian-Cai Liu a, Guan-Feng Lin a, Zhi-Ning Dong a, Rong-Liang Liang a, Zhen-Hua Chen a, Ying-Song Wu a,⁎ a b
Institute of Antibody Engineering, School of Biotechnology, Southern Medical University, Guangzhou 510515, Guangdong, PR China Institute of Hydrobiology, Jinan University, Guangzhou 510515, Guangdong, PR China
a r t i c l e
i n f o
Article history: Received 23 September 2014 Received in revised form 13 January 2015 Accepted 18 January 2015 Available online 26 January 2015 Keywords: Rubella virus Cytomegalovirus IgM Dual-TRFIA
a b s t r a c t Objectives: This study established a novel time-resolved fluorescence immunoassay (TRFIA) that allows the simultaneous determination of rubella virus (RV) IgM and cytomegalovirus (CMV) IgM in human serum. Design and methods: Lanthanum elements labeled antibody and streptavidin–biotin system were used in the “capture sandwich” format simultaneously. Results: The working range of TRFIA for RV IgM was 2–80 AU/mL and for CMV IgM was 5–400 AU/mL. Intraand inter-assay coefficient of variation (CV) for RV IgM and CMV IgM were both less than 10% and recoveries were from 90% to 110%. No significant statistical difference in sensitivity or specificity was observed between dual-TRFIA and commercial chemiluminescent immunoassays (CLIA) in serum samples. Conclusion: The novel dual-TRFIA for RV IgM and CMV IgM detection might have valuable clinical application, with satisfactory sensitivity, specificity and accuracy. © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
Introduction Rubella virus (RV), a single-stranded positive RNA virus, belongs to the Rubivirus genus of the Togaviridae family [1]. Rubella is an acute respiratory infectious disease caused by RV with mild or absent clinical features and is easily neglected. RV infection in pregnant women, especially during the first trimester [2], can cause intrauterine death, miscarriage, or premature birth [3]. When RV is transmitted to the fetus through the placenta, infection results fetal growth restriction or congenital rubella infection (CRI), which causes congenital rubella syndrome (CRS) in infants [4]. WHO estimated that at least 100,000 cases of CRS infants born annually worldwide [4]. Three characteristic manifestations of CRS, the most harmful symptom of rubella, include congenital cataracts, deafness and cardiovascular anomalies [2]. CRS is a huge burden on society and the economy, particularly in developing
Abbreviations: AU/mL, arbitrary units/milliliter; BSA, bovine serum albumin; CLIA, chemiluminescent immunoassays; CMV, cytomegalovirus; CRI, congenital rubella infection; CRS, congenital rubella syndrome; CV, coefficients of variation; DNA, deoxyribonucleic acid; ELISA, enzyme-linked immunosorbent assay; HCMV, human cytomegalovirus; IgG, immunoglobulin G; IgM, immunoglobulin M; McAbs, monoclonal antibodies; PBS, phosphate buffered saline; RF, rheumatoid factor; RNA, ribonucleic acid; RV, rubella virus; SD, standard deviation; TRFIA, time-resolved fluoroimmunoassay. ⁎ Corresponding author. Fax: +86 20 37247604. E-mail address: [email protected] (Y.-S. Wu).
countries [5]. To reduce the incidence of CRS, it is important that screening and vaccination occur before childbirth. High positive rates of RV-specific IgM antibodies are present about 5–14 days after the rash appears. The levels then decrease and last for about 8–12 weeks [6]. Commercially available enzyme-linked immunosorbent assays (ELISA) [7] and chemiluminescent immunoassays (CLIA) [8] for IgG and IgM detection are generally used for clinical diagnosis. Positive levels of RV IgM strongly suggest that patients recently had an acute rubella virus infection [6]. Cytomegalovirus (CMV), a DNA virus, belongs to the β-herpes virus family [9]. Human cytomegalovirus (HCMV) is one of the most common causes of intrauterine infection, which can cause a variety of different infection syndromes [10]. CMV infection in pregnant women causes abortion, stillbirth, and premature delivery. About 10%–15% of neonates with congenital CMV infection show symptoms such as fetal growth restriction, microcephaly, hepatosplenomegaly and skin petechiae at birth, and 20%–30% of symptomatic infection neonates eventually die [11]. The early diagnosis and treatment of HCMV infection is importance clinically. IgM antibodies are present for several days after CMV primary infection and remain for about 3–6 months, suggesting a recently active CMV infection and virus replication [12]. To date, the methodologies used for serological investigation of CMV antibodies are the indirect ELISA [13] and CLIA [14]. Most CMV infections cause no symptoms, therefore the correlation between IgM responses and recent active CMV infection is
http://dx.doi.org/10.1016/j.clinbiochem.2015.01.009 0009-9120/© 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
604
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
of great significance in the clinic [15]. Pregnant women should be tested for CMV IgM antibodies as early as possible, as treatment should occur as early as possible as the early and accurate diagnosis of RV or CMV infection can reduce birth defects and improve the population quality. Time-resolved fluoroimmunoassay (TRFIA) using lanthanide chelates is widely used for clinical screening and diagnostics [16]. The main characteristics of TRFIA are long storage time, high sensitivity and specificity, good repeatability, a wide detection range and no radioactive contamination [17,18]. In this report, we developed a novel highly reproducible one-step dual-TRFIA, using Eu3 + and Sm3+ chelates as labels, to simultaneously measure the concentration of RV IgM and CMV IgM in human serum. Materials and methods Reagents and instrumentation Natural RV antigen was obtained from Microbix Biosystems Inc. (Mississauga, Ontario, Canada). CMV recombinant antigen (integrating the pUL80, pUL32, and ppUL83 genes), anti-RV monoclonal antibodies (McAbs) and anti-human IgM McAbs were obtained from Fapon Biotech Inc., (Shenzhen, China). Streptavidin was obtained from SigmaAldrich (St. Louis, MO, USA) and NHS-(N-hydroxysuccinimido)-biotin was from Roche (Mannheim, Germany). Eu3+ labeled kits and Sm3+ labeled kits were obtained from PerkinElmer (Turku, Finland). Transparent 96-well micro-titration strips were purchased from Thermo Labsystems (Milford, MA, USA). Victor™ 1420 time-resolved plate fluorometer and AUTODELFIA™ 1235 Automatic Immunoassay System were obtained from Perkin-Elmer Life and Analytical Sciences (Waltham, MA, USA). Enhancement solution and Wash solution were from Wallac (Turku, Finland). Bovine serum albumin (BSA) was obtained from Bovogen Biologicals Pty Ltd., (Victoria, Australia). RV IgM ELISA kits and CMV IgM ELISA kits were purchased from Serion (Würzburg, Germany). Rubella and CMV IgM CLIA kits were purchased from DiaSorin Liaison (Via Crescentino, Italy) and Abbott Architect (Sligo, Ireland). Serum panel PTC203 and PTR201 were purchased from SeraCare Life Sciences (Gaithersburg, MD, USA). Other chemicals and reagents used were of analytical grade. Coating with capture antibody 100 μl anti-human IgM McAbs, diluted to final concentration of 3 μg/mL with coating buffer (50 mmol/L, Tris, 0.9% NaCl, pH 7.4), was pipetted into each well. The plates were incubated at 4 °C overnight and then washed 3 times with washing buffer (25 mmol/L Tris, 0.9% NaCl, 0.06% Tween-20, pH 7.8). Then 200 μl of blocking buffer (50 mmol/L PBS, 0.9% NaCl, 0.1% BSA, 0.2% Tween-20, pH 7.4) was added to each well, and the wells maintained at 4 °C overnight. After removing the blocking buffer, the plates were vacuum dried and stored with a desiccant at 4 °C for short-term or at −20 °C for long-term preservation. Eu3+-labeled anti-RV McAbs and Sm3+-labeled streptavidin The buffer for anti-RV McAbs was exchanged for labeling buffer (100 mmol/L Na2CO3, pH 9.3) in a centrifuge tube containing a membrane before labeling. Then 0.2 mg DTTA-Eu (N1-[P-isothiocyanatobenzyl]-diethylene-triamine-N1,N2,N3-tetraacetate-Eu3+) was added to 1.0 mg of McAbs in 200 μl labeling buffer, then the mixture was stirred overnight at room temperature. The labeled antibody was separated from unreacted chelates and aggregated McAbs by gel filtration (Sephadex, G-50) using elution buffer (50 mmol/L Tris, pH 7.4 containing 0.9% NaCl and 0.2% BSA). The fluorescence of the Eu3 + labeled McAbs was measured at 615 nm. After adding BSA as a stabilizer, aliquots of the labeled antibody were stored at −20 °C. The streptavidin was labeled with Sm3 + chelates using the same procedure as for
Eu3+-labeled anti-RV McAbs, except the optimized weight proportion of streptavidin and DTTA-Sm3+ was 2:1. Biotinylation of CMV antigen The CMV antigen was biotinylated to allow its detection by timeresolved fluorescence of Sm3+ labeled streptavidin. The CMV antigen was centrifuged and washed with PBS to remove protective agents such as NaN3. Then NHS-Biotin was then added at an optimized ratio of antigen and NHS-Biotin (1:25). The reaction mixture was stirred slightly for 1 h at room temperature. Then the labeled antigen was separated from unconjugated chelate by gel filtration with PBS and the protein peak at 280 nm was collected and filtered through a sterile 0.2 μm Minisart filter. After adding BSA as a protectant, aliquots of conjugates were stored at −20 °C for daily use. Preparation of antigen working solution RV antigen and biotinylated CMV antigen were diluted into a total concentration of 1.0 μg/mL and 0.2 μg/mL using assay buffer (50 mmol/L Tris, 0.9% NaCl, 0.5% BSA, pH 7.8). The mixture was used as the antigen working solution. Calibration The determination of RV IgM antibody activity was given in AU/mL and correlated to the RV IgM reference serum which was from Leipzig (Germany). The concentration of reference serum was defined as 30 U/mL detected by Serion RV IgM ELISA kit and was used as the gold standard of the laboratory. Then the serum was diluted serially and used to revise the RV IgM standard of dual-TRFIA, the ratio of theoretical value and the measured value was between 0.9 and 1.1. Similarly, CMV IgM antibody activity was given in AU/mL and correlated to the CMV IgM reference serum which was from the Paul-Ehrlich Institute, Langen. The concentration of reference serum was defined as 1800 U/mL detected by Serion CMV IgM ELISA kit and was used as the gold standard of the laboratory. Then the serum was diluted serially from 1:5 to 1:80 and used to revise the CMV IgM standard of dualTRFIA, the ratio of theoretical value and the measured value was between 0.9 and 1.1. Samples A total of 411 serum samples were obtained from The Guangzhou Second People's Hospital and The Liuzhou Maternal and Child Health Hospital, including 56 RV IgM positive serum samples, 355 RV IgM negative samples, 65 CMV IgM positive serum samples and 346 CMV IgM negative samples. All serums were confirmed by Diasorin Liaision RV and CMV IgM CLIA (The sample was defined as negative when the value is less than 20 UA/mL, positive when greater than 25 UA/mL and equivocal when between 20 and 25 UA/mL in RV IgM detection; The sample was defined as negative when the value is less than 18 U/mL, positive when greater than 22 U/mL and suspicious when between 18 and 22 U/mL in CMV IgM detection) (Table 1). Table 1 Samples and serum panels confirmed by Diasorin Liaision RV and CMV IgM CLIA. Sample type
Number of Diasorin Liaision sera rubella IgM
Diasorin Liaision CMV IgM
Positive Negative Positive Negative Blood donors Pregnant women Diagnostic/hospital patients Serum panel PTC203 Serum panel PTR201 Total
134 149 128 21 25 457
13 23 20 / 6 62
121 126 108 / 19 374
21 25 19 6 / 71
113 124 109 15 / 361
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
605
Fig. 1. Schematic of dual-TRFIA for RV IgM and CMV IgM detection in serum.
104 positive serum samples for Cross-reactivity studies, including 15 anti-nuclear antibody, 8 systemic lupus erythematosus, 26 rheumatoid factor, 9 herpes simplex virus IgM types 1 and 2, 5 toxoplasma gondii IgM, 7 human anti-mouse antibody, 16 high titer Rubella IgG (N250 IU/mL) and 18 high titer CMV IgG (N8 IU/mL). The serum panel PTC203 consisted of a set of 21 specimens with anti-CMV reactivity, including 6 positive samples and 15 negative samples (Table 1). Samples were collected between 1999 and 2011 and selected to demonstrate IgG and/or IgM reactivity. The serum panel PTR201 consisted of a set of 25 specimens with anti-Rubella IgG and/ or IgM antibody reactivity ranging from negative to strongly positive, including 6 positive samples and 19 negative samples (Table 1). Samples were collected in 1994 and 1995. All samples were measured and analyzed by RV/CMV dual-TRFIA. Then the unmatched samples were tested using the Abbott Architect CLIA (The sample was defined as negative when the value is less than 1.20 Index, positive when greater than 1.6 Index and equivocal when between 1.20 and 1.60 Index in RV IgM detection; The sample was defined as negative when the value is less than 0.85 Index, positive when greater than 1.0 Index and suspicious when between 0.85 and 1.0 Index in CMV IgM detection). All procedures were carried out in accordance with the manufacturer's instructions. DiaSorin Liaison RV/CMV IgM CLIAs used the chemiluminescence automatic detection system commonly used for clinical detection, consisting of magnetic particles coated with either RV antigen or CMV antigen and McAbs-N-(4-Aminobutyl)-N-ethylisoluminol (ABEI) as a tracer. Abbott Architect RV and CMV IgM CLIAs were similar, except the murine acridinium labeled McAbs.
Assay protocol Eu 3 +-labeled Anti-RV McAbs and Sm3 +-labeled streptavidin (N200 ng/mL) were both added into the antigen working solution to form the analysis solution. The serum samples and standards were diluted with dilution buffer (50 mmol/L Tris, 0.9% NaCl, 1.5% BSA, 0.4% casein, pH 7.8) in a 1:101 dilution. In brief, 100 μl of serum sample or 100 μl of standard was pipetted into the microtiter wells coated with anti-human IgM McAbs. The plates were incubated for 1 h at room temperature with horizontal shaking. After washing 4 times, 100 μl analysis buffer was added. The plates were incubated at room temperature with shaking for 1 h. After washing 6 times, enhancement solution (100 μl/well) was added to each well. The plates were shaken for 5 min and fluorescence intensity readings at 615 nm (Eu3 +) and 645 nm (Sm3+) were measured with a Victor™ 1420 time-resolved plate fluorometer (Fig. 1). The same experiments were also performed using the AUTODELFIA™ 1235 automatic immunoassay system with software designed in our lab.
Results Dilution test Two positive samples containing different levels of RV IgM and CMV IgM with several dilutions from 1:2 to 1:32 were investigated in this study. As shown in Fig. 2, the dilution curves were both linear over the whole range of concentrations for both RV IgM and CMV IgM.
Fig. 2. Dilution linearity for RV IgM (A) and CMV IgM (B), based on 2 different degrees of positive serum samples.
606
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
8.00% and 3.26 to 9.64% and for CMV IgM 4.03 to 6.80% and 6.03 to 8.72%, respectively.
Table 2 Recovery test of RV IgM/CMV IgM TRFIA. Sample
S1 S2
RV-IgM
Recovery %
Original concentration (AU/mL)
Measured concentration (AU/mL)
16.7 23.1
15.9 21.3
CMV-IgM
S1 S2
Cross-reactivity 94 91 Recovery %
Original concentration (AU/mL)
Measured concentration (AU/mL)
22.3 33
22.9 34.7
105 107
Parallelism demonstrated agreement among the results obtained at different dilution levels of the samples.
Recovery test Treatment of RV IgM samples was based on the addition of 0.1 mL of standards consisting of RV IgM at two different concentrations (13.5 AU/mL and 19.9 AU/mL) to a 1 mL original sample aliquot containing 3.2 AU/mL endogenous RV IgM. After repeating experiments 3 times, mean values and recovery were calculated. The CMV IgM recovery experiment was similar to the above experiment. Results in Table 2 show that dual-TRFIA accurately measured the concentration of RV IgM and CMV IgM in serum samples, yielding a mean recovery of 92.5% for RV IgM and 106% for CMV IgM.
Standard curve, analytical sensitivity and precision The standard curves of RV IgM and of CMV IgM were linear over the concentration used. The measurement range of RV IgM was 2–80 AU/mL and that of CMV IgM was 5–400 AU/mL. No high dose hook effect was observed in the standard curve (Fig. 3). Sensitivity of RV IgM was 0.141 AU/mL and for CMV IgM it was 1.129 AU/mL. The limit of detection was defined by the concentration of RV IgM and CMV IgM corresponding to fluorescence of the zero calibrators plus 2 standard deviation (SD). As shown in Fig. 3, the precision (CV%) was less than 6% in the detection range for both in RV IgM and CMV-IgM, when each point was measured 10 times. The imprecision (intra-assay and inter-assay) for dual-label TRFIA was obtained by testing three concentrations of RV IgM and CMV IgM in serum (negative, weakly positive and strong positive), respectively. The intra- and inter-assay imprecision for RV IgM was from 3.46 to
Cross-reactivity studies were performed on 149 pregnant women and 104 specimens positive for anti-nuclear antibody, systemic lupus erythematosus, rheumatoid factor (RF), herpes simplex virus IgM types 1 and 2, toxoplasma gondii IgM, human anti-mouse antibody, high titer rubella IgG (N 250 IU/mL) and CMV IgG (N 8 IU/mL). With these specimens, the RV/CMV IgM dual-TRFIA and a commercially available diagnostic kit (DiaSorin Liaison CLIA) showed 100% agreement (253/253). Correlation between CLIA and TRFIA All 411 specimens and serum panel (PTC203 and PTR201) were tested by RV/CMV dual-TRFIA. The results of Diasorin Liaision RV IgM/CMV IgM and dual-TRFIA were shown in Tables 3. For dual-TRFIA, sample was defined as negative when the value was less than 1.8 AU/mL, positive when greater than 2.0 AU/mL and suspicious between 1.8 and 2.0 AU/mL in RV IgM detection; sample was defined as negative when the value is less than 8.0 AU/mL, positive when greater than 10.0 AU/mL and suspicious between 8.0 and 10.0 AU/mL in CMV IgM detection. The results obtained were analyzed by SPSS13.0 statistics software. Based on the above findings, we analyzed the sensitivity and specificity of the RV/CMV IgM dual-TRFIA assays. Equivocal values were regarded as positive when calculating the sensitivity and negative when calculating specificity. The sensitivity and specificity of RV IgM detection by dual-TRFIA was 93.5% (58/62) and 99.2% (371/374), with a kappa value of 0.953. For CMV IgM detection, the sensitivity and specificity were 90.1% (64/71) and 98.3% (355/361), with a kappa values of 0.917. No significant statistical difference between the RV/CMV IgM DiaSorin Liaison CLIA and dual-TRFIA assays. The unmatched samples were then tested using the Abbott Architect CLIA. For RV IgM detection, of the three unmatched equivocal samples in TRFIA, there were three positive in the Abbott Architect CLIA, and both methods found two unmatched negatives and two unmatched positive. For CMV IgM detection, of the seven unmatched equivocal samples in TRFIA, there were seven positive in the Abbott Architect CLIA, and both methods found two unmatched negatives and four unmatched positives (Table 3). Discussion The capture assay used to detect IgM antibodies in serum was previously shown to be feasible and effective [19]. Compared with the
Fig. 3. Standard curve of RV IgM (A) and CMV IgM (B) and the intra-assay CV% of each point.
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608 Table 3 Qualitative results of rubella IgM and CMV IgM. RV/CMV dual-TRFIA
Positive Equivocal Negative Total
Diasorin Liaision Rubella IgM
Diasorin Liaision CMV IgM
Positive
Negative
Total
Positive
Negative
Total
58 2 2 62
2 1 371 374
60 3 373 436
64 3 4 71
2 4 355 361
66 7 359 432
Note: Dual-TRFIA equivocal values were regarded as positive when calculating the measure of agreement (kappa value).
607
method for measurement of RV and CMV IgM antibodies. This method had a high sensitivity, specificity and good reproducibility. Additionally, this assay was suitable for the PE AUTODELFIA™ 1235 automatic system, which might allow the development of automated testing for RV and CMV IgM antibodies. Conflict of interest None. Acknowledgments
indirect method, it had a higher sensitivity and specificity and was not affected by RF or high concentrations of pathogen IgGs [20,21]. In this study, RF and related pathogen IgGs were tested and demonstrated no correlation between RV IgM and CMV IgM in clinical serum samples. The labels were thus independent of each other. To maintain maximum antigen activity, we used Eu3+-DTTA to label RV McAbs and directly diluted the RV natural antigen at an appropriate proportion. Before the serum reaction, Eu3+-RV McAbs were combined with specific antigen, which retained all epitopes and specificity of the natural antigen, to allow maximal detection of RV-specific IgM antibodies in serum. The choice of McAbs in this assay was of importance based on the principle that the activity between natural antigen and Eu3 +McAbs did not affect the subsequent specific reaction between the same antigen and IgM antibodies in serum. The study of CMV antigen was relatively comprehensive. Currently, the envelope protein ppUL83 is the most widely used protein in the clinic as the main antigen to detect IgM and IgG antibodies of CMV. Its antigenicity is high among numerous CMV proteins, and the corresponding antibody titers were very high during the acute stage of infection and early recovery [22,23]. Additionally, the CMV protein mainly induced IgM antibodies including pUL80 and pUL32 [24]. The CMV recombinant antigen used in this study contained the ppUL83, Pul80 and pUL32 genes, thus it harbored all antigen epitopes that specifically bind with CMV IgM antibody. This demonstrated that Eu3 + labeled streptavidin and time-resolved fluorometry is an appropriate indirect label because of its high affinity to biotin and low nonspecific binding properties. As a tetrameric structure with four binding sites for biotin, it can be used to amplify signals [25]. Feng-Bo developed a doubleantigen sandwich TRFIA to detect total anti-HCV antibodies based on biotin–streptavidin interactions and verified that this system was more specific and reliable for screening anti-HCV antibodies than those indirect methods [26]. Because the recombinant antigen did not contain all the epitopes of the natural antigen, we selected biotin to bind to the antigen, which minimally affected antigen activity. Additionally, Sm3 + was used to label streptavidin to produce a streptavidin– biotin system with increased detection sensitivity and detection range while maintaining specific immune responses [27,28]. The dual-TRFIA system, based on the wavelength of lanthanum elements Eu3 + and Sm3 + at 615 nm and 645 nm, was used to measure Eu3 + and Sm3 + fluorescence intensity to simultaneously obtain the concentration of RV IgM and CMV IgM. The impact of background fluorescence values caused by Eu3+ to Sm3+ was deducted by the correction system in Victor™ 1420 or AUTODELFIA™ 1235. In the absence of an international standard, we established a quantitative analysis dual-TRFIA that could accurately determine the degree of RV IgM and CMV IgM in serum, based on a laboratory internal standard. Establishment of the quantitative determination method allowed the further study of changes in RV IgM and CMV IgM titers in serum and the occurrence, development and prognosis of pathogen infection [29–32]. Compared with commercial CLIAs, the RV IgM and CMV IgM dual-TRFIA developed in the current study had a higher sensitivity and specificity, suitable for clinical applications. Taken together, this study demonstrated the use of dual-TRFIA and anti-human IgM McAbs as captured antibodies to establish a quantitative
This work was supported by the National Science and Technology Major Project (grant number 2013ZX10004-803). References [1] Atreya CD, Mohan KV, Kulkarni S. Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level, 70; 2004 431–7. [2] De Santis M, Cavaliere AF, Straface G, Caruso A. Rubella infection in pregnancy, 21; 2006 390–8. [3] Chen M, Zhu Z, Liu D, Huang G, Huang F, Wu J, et al. Rubella epidemic caused by genotype 1E rubella viruses in Beijing, China, in 2007–2011, 10; 2013 122. [4] Robinson JL, Lee BE, Preiksaitis JK, Plitt S, Tipples GA. Prevention of congenital rubella syndrome—what makes sense in 2006?, 28; 2006 81–7. [5] Verma R, Khanna P, Chawla S. Rubella vaccine: new horizon in prevention of congenital rubella syndrome in the India, 8; 2012 831–3. [6] Banatvala JE, Brown DW. Rubella, 363; 2004 1127–37. [7] Hudson P, Morgan-Capner P. Evaluation of 15 commercial enzyme immunoassays for the detection of rubella-specific IgM, 5; 1996 21–6. [8] Portella G, Galli C. Multicentric evaluation of two chemiluminescent immunoassays for IgG and IgM antibodies towards rubella virus, 49; 2010 105–10. [9] Dimberg J, Hong TT, Skarstedt M, Lofgren S, Zar N, Matussek A. Detection of cytomegalovirus DNA in colorectal tissue from Swedish and Vietnamese patients with colorectal, cancer, 33; 2013 4947–50. [10] Griffiths PD. Burden of disease associated with human cytomegalovirus and prospects for elimination by universal immunisation, 12; 2012 790–8. [11] Yinon Y, Farine D, Yudin MH. Screening, diagnosis, and management of cytomegalovirus infection in pregnancy, 65; 2010 736–43. [12] Adler SP. Screening for cytomegalovirus during pregnancy, 2011; 2011 1–9. [13] Revello MG, Gerna G. Diagnosis and management of human cytomegalovirus infection in the mother, fetus, and newborn infant, 15; 2002 680–715. [14] Juhl D, Vockel A, Luhm J, Ziemann M, Hennig H, Gorg S. Comparison of the two fully automated anti-HCMV IgG assays: Abbott Architect CMV IgG assay and Biotest antiHCMV recombinant IgG ELISA, 23; 2013 187–94. [15] Schmitz H, von Deimling U, Flehmig B. Detection of IgM antibodies to cytomegalovirus (CMV) using an enzyme-labelled antigen (ELA), 50; 1980 59–68. [16] Lovgren T, Hemmila I, Pettersson K, Eskola JU, Bertoft E. Determination of hormones by time-resolved fluoroimmunoassay, 31; 1984 909–16. [17] Merio L, Pettersson K, Lovgren T. Monoclonal antibody-based dual-label timeresolved fluorometric assays in a simplified one-step format, 42; 1996 1513–7. [18] Wang K, Huang B, Zhang J, Zhou B, Gao L, Zhu L, et al. A novel and sensitive method for the detection of deoxynivalenol in food by time-resolved fluoroimmunoassay, 19; 2009 559–64. [19] Welch RJ, Chang GJ, Litwin CM. Comparison of a commercial dengue IgM capture ELISA with dengue antigen focus reduction microneutralization test and the centers for disease control dengue IgM capture-ELISA, 195; 2014 247–9. [20] Lakos G, Teodorescu M. IgM, but not IgA rheumatoid factor interferes with anticardiolipin and antibeta2 glycoprotein I measurements: a quantitative, analysis, 20; 2011 614–9. [21] Namekar M, Ellis EM, O'Connell M, Elm J, Gurary A, Park SY, et al. Evaluation of a new commercially available immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of dengue virus infection, 51; 2013 3102–6. [22] Wirgart BZ, Claesson K, Eriksson BM, Brundin M, Tufveson G, Totterman T, et al. Cytomegalovirus (CMV) DNA amplification from plasma compared with CMV pp65 antigen (ppUL83) detection in leukocytes for early diagnosis of symptomatic CMV infection in kidney transplant patients, 7; 1996 99–110. [23] Holtappels R, Podlech J, Grzimek NK, Thomas D, Pahl-Seibert MF, Reddehase MJ. Experimental preemptive immunotherapy of murine cytomegalovirus disease with CD8 T-cell lines specific for ppM83 and pM84, the two homologs of human cytomegalovirus tegument protein ppUL83 (pp65), 75; 2001 6584–600. [24] Maine GT, Stricker R, Schuler M, Spesard J, Brojanac S, Iriarte B, et al. Development and clinical evaluation of a recombinant-antigen-based cytomegalovirus immunoglobulin M automated immunoassay using the Abbott AxSYM analyzer, 38; 2000 1476–81. [25] Suonpaa M, Markela E, Stahlberg T, Hemmila I. Europium-labelled streptavidin as a highly sensitive universal label. Indirect time-resolved immunofluorometry of FSH and TSH, 149; 1992 247–53. [26] Wu FB, Ouyan HQ, Tang XY, Zhou ZX. Double-antigen sandwich time-resolved immunofluorometric assay for the detection of anti-hepatitis C virus total antibodies with improved specificity and sensitivity, 57; 2008 947–53.
608
J.-W. Zhou et al. / Clinical Biochemistry 48 (2015) 603–608
[27] Du L, Cheng S, Wang S. Determination of diethylstilbestrol based on biotin– streptavidin-amplified time-resolved fluoro-immunoassay, 27; 2012 28–33. [28] Dundas CM, Demonte D, Park S. Streptavidin–biotin technology: improvements and innovations in chemical and biological applications, 97; 2013 9343–53. [29] Sen MR, Shukla BN, Tuhina B. Prevalence of serum antibodies to TORCH infection in and around Varanasi, Northern India, 6; 2012 1483–5. [30] Sanz JC, Mosquera M, Ramos B, Ramirez R, de Ory F, Echevarria JE. Assessment of RNA amplification by multiplex RT-PCR and IgM detection by indirect and capture ELISAs for the diagnosis of measles and rubella, 118; 2010 203–9.
[31] Neirukh T, Qaisi A, Saleh N, Rmaileh AA, Zahriyeh EA, Qurei L, et al. Seroprevalence of cytomegalovirus among pregnant women and hospitalized children in Palestine, 13; 2013 528. [32] Rasmussen L, Kelsall D, Nelson R, Carney W, Hirsch M, Winston D, et al. Virusspecific IgG and IgM antibodies in normal and immunocompromised subjects infected with cytomegalovirus, 145; 1982 191–9.
