IgM antibody detection of ppUL80a and ppUL32 by immunoblotting: An early parameter for recurrent cytomegalovirus infection in renal transplant recipients

The value of IgM detection for the early diagnosis of an active cytomegalovirus (CMV) infection in renal transplant recipients was evaluated prospectively. Sequential serum samples obtained from 22 allograft recipients with active CMV infection were tested for the presence of CMV-specific immunoglobulin M antibodies (IgM) by an enzyme-linked immunosorbent assay (ELISA) and a microparticle enzyme immunoassay (MEIA) and were compared with the Western-immunoblotting technique (IB). The time course of CMV IgM antibody detection was evaluated in relation to the shell vial assay (SVA), CMV disease, and immunosuppressive regimen. By IB, IgM antibodies against the capsid protein ppUL80a and the basic matrix phosphoprotein ppUL32 were detected in all 22 recipients with active CMV infection. Using the MEIA and the ELISA, the presence of CMV IgM antibodies was detected in 17 (77%) and ten (46%) of these 22 recipients, respectively. The SVA was the earliest parameter for detection of primary CMV infection in seven of nine (78%) recipients, in contrast to two of 13 (15%) patients with recurrent CMV infection (P < .05). The detection of IgM antibodies by IB was the earliest parameter for detection of recurrent CMV infection in seven out of 13 (54%) recipients in contrast to one out of nine (11%) patients with primary CMV infection (P < .05). During a primary CMV infection, the development of an abundant IgM antibody response was associated with recovery from CMV disease and the end of the viremic phase. © 1996 Wiley-Liss, Inc.     

Shell-vial culture and pp65 antigenemia assay in the detection of cytomegalovirus in the first blood sample of renal transplant recipients

The aim of the study was to compare the efficacy of pp65 antigenemia assay and the shell-vial culture (SVC; viremia) for the diagnosis of cytomegalovirus (CMV) infection in renal transplant recipients, comparing the results obtained in the first blood sample and the total number of blood samples analyzed in this group of patients. During the study period, 70 renal transplant recipients were studied: 44 (62.8%) with CMV infection. The method of sedimentation in a dextran solution for leukocyte extraction was used in the pp65 antigenemia assay. The MRC-5 shell-vial assay was used for CMV isolation from leukocytes (viremia). Eighty blood samples were examined from 70 renal transplant recipients: Of the 44 positive samples studied, in 77.5% of cases, both the antigenemia assay and the SVC were positive. In 16.2%, only the antigenemia assay was positive, and, in 6.2%, only the SVC was positive. In all blood samples studied, the antigenemia was present in 93.7% of cases, and the SVC was present in 83.7% (P = 0.04). If the results obtained in only the first blood sample taken for the diagnosis are studied, then we observe that the antigenemia assay was positive in 39 patients (88.6%), whereas the SVC was positive in 41 patients (93.1%), although the difference was not statistically significant (P = 0.39). It is concluded that the inoculation of all of the leukocytes extracted from blood samples in the SVC seems to produce a slight increase in the sensitivity of the cell culture and that the SVC becomes positive before the antigenemia for the detection of CMV in peripheral blood, especially in the first blood sample. J. Med. Virol. 55:240–242, 1998. © 1998 Wiley-Liss, Inc.     

Specific IgM class antibody production following infection with cytomegalovirus

Specific IgM class antibody production was studied in different groups of patients with characterized cytomegalovirus (CMV) infections using a radioimmunoassay (RIA). In pregnant women, IgM antibodies were detected only following primary infection and generally persisted less than 4 months. The demonstration of CMV-specific IgM during pregnancy is therefore diagnostic of recent primary CMV infection. In patients with symptomatic CMV infections, the appearance of IgM antibody was shown to be closely related to the onset of symptoms and coincided with production of complement fixing (CF) antibody. IgM antibodies were at maximum levels 3-4 weeks after presentation but generally declined to low or undetectable levels by 3-4 months. The significance of the results of testing for CMV-specific IgM in relation to clinical and other serological findings in these patients is discussed. IgM antibody production was also demonstrated in renal transplant patients with primary infections and in 6 of 21 recipients with secondary infections. In both groups the antibodies became detectable 3-6 weeks after transplantation but the titres were much higher following primary infection. IgM antibodies persisted throughout follow-up periods of up to 2 years after transplantation in some cases.     

Detection of IgM antibodies against cytomegalovirus: comparison of two radioimmunoassays, enzyme-linked immunosorbent assay and immunofluorescent antibody test

The sensitivity and specificity of direct antibody radioimmunoassay (RIA), M-antibody capture RIA (MACRIA), enzyme-linked immunosorbent assay (ELISA), and the immunofluorescent antibody (IFA) test for the detection of CMV-specific IgM was compared using 40 sera selected from different groups of patients. RIA, MACRIA, and ELISA gave concordant results with thirty-two sera but discordant results with eight sera, of which three were cord sera from congenitally infected babies, three were from immunocompromised patients with recurrent CMV infections, and two were from patients with lymphadenopathy and Paul-Bunnell-positive mononucleosis, respectively. RIA, MACRIA, and ELISA were of similar sensitivity with sera from adult patients, but ELISA was apparently less sensitive than RIA and MACRIA for the detection of CMV IgM in cord serum. By comparison IFA was significantly less sensitive than the other three tests. Rheumatoid factor is reactive in RIA, ELISA, and IFA but can efficiently be removed by absorption with latex-IgG beads or cross-linked human IgG.     

A comparison of antibody capture radio- and enzyme immunoassays with immunofluorescence for detecting IgM antibody in infants with congenital rubella

IgM antibody capture radioimmunoassay (MACRIA) and enzyme immunoassay (MACEIA) were compared with immunofluorescence (IF) for detecting specific IgM antibody in 99 sera from 76 infants with confirmed congenital rubella, and 61 sera from a comparative group of 59 infants who had miscellaneous abnormalities but in whom congenital rubella was not confirmed.All of 35 specimens collected from confirmed cases within 12 weeks of birth were positive by all three methods and all but one of 17 specimens collected after the age of 18 months were uniformly negative. At intermediate ages discrepancies occurred in 18 specimens, of which eight were positive and 10 negative by IF. Three of these 18 specimens were negative by both antibody capture procedures but showed weak fluorescence; the other 15 were negative by MACEIA, but positive by MACRIA which appears to be the more sensitive of the antibody capture methods. Sera from five infants in the comparative group were clearly positive by all three methods. These five infants were probably congenitally infected with rubella. Sera from the other 54 infants were negative, except for one that gave a weakly positive result by MACRIA alone.Antibody capture procedures offer several advantages over previous methods for detecting IgM antibody. Although MACRIA was found to be slightly more sensitive than MACEIA, the greater stability of the enzyme label, together with the possibility of both visual and quantitative assessment, could make MACEIA the method of choice for detecting rubella-specific IgM.     

An enzyme labelled nuclear antigen immunoassay for detection of cytomegalovirus IgM antibodies in human serum: specific and non-specific reactions

A mu-capture enzyme linked immunosorbent assay was developed for detection of IgM antibody to cytomegalovirus (CMV). Virus-specific IgM was detected using horseradish peroxidase labelled nuclear CMV antigen (CMV-ELA). False-positive reactions caused by Paul-Bunnell-Davidsohn (PBD) positive sera and antinuclear antibody (ANA) positive sera were identified in a combination assay employing enzyme labelled nuclear control antigen (CO-ELA) in parallel to the CMV-ELA. Four of five PBD positive and 30 of 31 ANA positive sera reactive with the CMV-ELA were identified as false positive reactions in the combined ELA-assay. The reactivity in PBD-positive sera could not be explained by antigenic cross reactivity between CMV and Epstein-Barr virus, and the results further suggested that different cell specified components of the CMV-ELA were responsible for the reactivity of PBD-positive as compared to ANA-positive sera. One of 314 healthy blood donors, 12 of 12 patients with primary CMV infection, and 11 of 15 patients with secondary CMV infection had detectable CMV IgM antibodies. Comparison of different CMV-ELAs revealed that pronounced differences in specificity as well as sensitivity may exist.     

Determination of specific cytomegalovirus IgM antibodies using infected air dried cells and isolated nuclei by immunoperoxidase assay

A simple immunoperoxidase assay (IPA), adapted for detection of serum IgM antibodies to cytomegalovirus (CMV) is described. The antigen consisted of CMV infected human embryonic fibroblasts or isolated nuclei. The sera were absorbed with aggregated gamma-globulins prior to testing. Rabbit anti-human IgM peroxidase conjugate was used to detect IgM bound to viral antigen. In parallel the enzyme linked immunosorbent assay (ELISA) technique was used to determine IgG and IgM antibodies to CMV, respectively. All patients with acute CMV infections who were tested had CMV-specific IgM antibodies by IPA, both whole cell and nuclei antigen. The maximal IgM titers were higher by ELISA than by IPA but in 3 of the CMV patients IgM was detected earlier by IPA (with both types of antigens) than by ELISA. In 3 of 5 transplant patients with recurrent CMV infection IgM was demonstrated by immunoperoxidase techniques, while by ELISA IgM was demonstrated in only 2 of them. No cross reactivity with other herpes viruses was observed. The described IPA is simple, rapid and has the potential for widespread use in routine laboratories.     

Human cytomegalovirus glycoprotein B genotypes in renal transplant recipients

Based on sequence variation of the glycoprotein B (gB) gene, human cytomegalovirus (HCMV) strains can be classified into four gB genotypes. In a previous study of bone marrow transplant recipients, infection with the gB type 1 correlated with a more favorable clinical outcome than infection with the gB types 2, 3, or 4. The gB type was determined in 60 renal transplant and in 47 bone marrow transplant recipients using PCR and restriction analysis. All HCMV variants in patient specimens could be assigned to one of the four previously described gB types. Two or more specimens obtained from 39 patients were analysed; in 31 of these patients the gB type was the same in all samples. The gB type did not correlate with the clinical outcome or the level of viremia in renal transplant recipients. © 1996 Wiley-Liss, Inc.     

Detection of measles,mumps, and rubella antibodies in saliva using antibody capture radioimmunoassay

Antibody capture radioimmunoassays were developed for detecting virus specific IgM (MAC-RIA) and IgG (GACRIA) to measles, mumps, and rubella and used to investigate saliva as an alternative specimen to serum for diagnosis. Saliva was collected from 63 patients with measles, 19 with mumps, and 150 with rubella, which were all clinically diagnosed and serologically confirmed. Virus specific IgM was detected in 92% of measles, 75% of mumps, and 100% of rubella saliva samples collected during the first week of illness. Between 1 and 5 weeks after onset virus specific IgM was detected in 100% of saliva specimens. After the 5th week the proportion of reactive specimens declined. The specificity of the MACRIA tests was established by testing saliva samples collected from blood donors for measles (88), mumps (88), and rubella IgM (91). All of the saliva specimens tested for measles and rubella specific IgM were unreactive, 1/88 specimens tested for mumps specific IgM contained significant reactivity. Saliva specimens collected from acute cases of MMR were tested in all 3 MACRIAs. A small proportion of saliva samples contained detectable IgM of more than one virus infection. Rubella and measles specific IgG was detected in the saliva of all cases from the 4th or 5th day of illness, respectively. Detection of mumps specific IgG was less successful. We have demonstrated that virus specific IgM can be reliably detected in saliva samples collected from acute cases of measles, mumps, and rubella and identified 1–5 weeks after onset of illness as the optimum time for collection of samples. © 1993 Wiley-Liss, Inc.     

抗人巨细胞病毒单克隆抗体的建立及初步临床应用

应用人巨细胞病毒AD169株(HcMV-AD169)免疫BALB/c小鼠,取脾细胞与Sp2/0小鼠骨髓瘤细胞融合,获得两株(1F9 2H10)分泌抗HCMV单克隆抗体(McAb)的杂交瘤细胞系。经鉴定,两株McAb的Ig亚类为IgG1,腹水效价间接ELISA法为10~(-5)和10~(-6)。两株McAb仅与HCMV反应,而与其他疱疹病毒无反应,2H10有中和病毒作用而1F9则无。用HCMV-McAb建立抗体捕获ELISA法测定150例孕妇血清中HCMV-IgM抗体,阴阳性总符合率与间接ELISA法相比较,为99.3％(149/150)。文中尚对HCMV-McAb用途作了讨论。     

Solid-phase radioimmunoassay of serum IgG, IgM, and IgA antibodies to cytomegalovirus

A radioimmunoassay (RIA) using polystyrene beads as the solid phase for cytomegalovirus (CMV) antigen and iodinated immunosorbent purified anti-human IgG, IgM, and IgA as indicator antibodies was developed for the detection of immunoglobulin class-specific antibodies to CMV. An antigen prepared from extracellular virus was essential for reliable results, and a preparation ultracentrifuged and sonicated twice was better than a crude antigen. The optimal antigen gave low cpm values with a negative reference serum, resulting in cpm ratios of 10 or higher between early convalescent phase serum and negative reference serum. Of six patients with an increase in CMV CF titres, all six had an increase in RIA IgG titres, four had an increase in IgA titres, and all had IgM antibodies. The IgG titres were high, up to 1/64,000. In a group of 17 infants negative in CMV CF test, 14 had CMV IgG antibodies in RIA test, indicating mainly low levels of maternal antibodies. In six of seven patients with CMV isolations from urine specimens, an increase in IgG or IgA titres or the presence of IgM antibodies was found, and only one of these patients had an increase in CMV CF titre. The specificity of the developed CMV RIA test was further demonstrated by detecting no significant increase in RIA titres in serum specimens of patients with primary herpes simplex infection, chickenpox, herpes zoster, or infectious mononucleosis.     

Antibody response to the immediate early protein of cytomegalovirus in renal transplant recipients.

The antibody response to the immediate early protein (IEP) of cytomegalovirus (CMV) was examined in seven renal transplant recipients during CMV reactivation and associated clinical abnormalities. Three cases with antibody increase to the IEP in the week after CMV isolation had no CMV-induced disease. The four patients without antibody increase exhibited laboratory abnormalities and three of them had renal dysfunction. In this small number of patients, the antibody response to the IEP in the week after isolation of CMV was associated with absence of abnormal clinical laboratory findings including renal function, and the prognosis was good for graft function in renal transplant recipients with CMV reactivation.     

Human cytomegalovirus specific IgA antibodies detected by immunoperoxidase assay in serum of patients with cytomegalovirus infections

Cytomegalovirus (CMV)-specific IgA antibodies were determined by an immunoperoxidase assay in sequential serum samples of 10 patients with CMV infection in order to evaluate the feasibility of the use of this technique for diagnosis. In parallel, IgM and IgG antibodies to CMV were studied by enzyme-linked immunosorbent assay (ELISA) and by the immunoperoxidase assay, respectively. CMV IgA antibodies were detected in all 10 CMV patients studied. Specific IgM was detected earlier than IgA in only one of these ten patients. No CMV-specific IgA antibodies (titer less than 2) were detected in 45 medical students. Neither were they found in paired sera of 5 patients with herpes simplex infection, 5 patients with varicella, 6 patients with zoster and 2 patients with Epstein-Barr virus infection. The potential application of the indirect immunoperoxidase IgA assay for serodiagnosis of CMV infections is discussed.     

Detection of anti-epithelial cell antibodies in association with pediatric renal transplant failure using a novel microcytotoxicity assay

We have developed a microcytotoxicity assay allowing sera to be screened for anti-epithelial cell cytotoxic antibodies. Cells from the epithelial cell line A549 were cultured overnight in Terasaki trays prior to the addition of the sera to be screened. Using this assay, 63 pediatric recipients of 78 renal transplants have been studied retrospectively. Seventeen transplants carried out in 13 patients were found to be associated with the production of antibodies reactive only against epithelial cells (AEC). Eleven of these transplants failed as compared with 19 failures out of 52 transplants not associated with AEC production (Fisher's p = 0.04). We conclude that transplantation in the face of pre-existing AEC should be approached with caution.     

Comparison of the neutralizing and ELISA antibody titres to human cytomegalovirus (HCMV) in human sera and in gamma globulin preparations

Administration of anti-cytomegalovirus gamma globulin has been reported to prevent acute human cytomegalovirus (HCMV) infection in immunocompromised patients. Most likely the gamma globulin acts by its neutralizing activity. However, the antibody titer to HCMV in hyperimmune gamma globulin is usually detected by an enzyme immunoassay (ELISA). We, therefore, compared the ELISA titres with the neutralizing antibody titres in the sera of blood donors and in hyperimmune gamma globulins. A poor correlation between both titres was found. Gamma globulin preparations with identical ELISA titres clearly differed in the neutralizing titre. If neutralization is the important mechanism by which the hyperimmune gamma globulins work, our results would favour a routine testing of the neutralizing antibody titre in HCMV hyperimmune gamma globulins.     

Production and characterisation of a human monoclonal antibody to cytomegalovirus and its use in an early nuclear fluorescence assay

A human monoclonal antibody to cytomegalovirus (CMV) was produced by transforming peripheral blood mononuclear cells of a patient with recent CMV infection. It is directed against a late antigen located in the nucleus of CMV infected fibroblasts at 24-72 hours postinfection and immuneprecipitates 65K and 48K proteins from 35S-labelled CMV infected cells. Results of its use in an early nuclear fluorescence assay for rapid diagnosis are presented.     

Selective detection of IgM-antibody against core antigen of the hepatitis B virus by a modified enzyme immune assay.

IgM antibody against core antigen of the hepatitis B virus (anti-HBc IgM) was selectively determined by a new enzyme immunoassay (EIA). Microtiter plates were coated with anti-human micro chain immunoglobulin. On addition of serum IgM is bound by a factor of about 4,000 more than IgG. After removing the sample, HBcAg is added to the IgM-coated surface. Binding takes place if the IgM contained anti-HBc and was demonstrated by the aid of a conjugate made from anti-HBc IgG and horse radish peroxidase. Quantitation may be achieved without testing a dilution series. The assay was not disturbed by a large excess of anti-HBc IgG in the sample and rheumatoid factor did not produce false-positive results, provided the sample was diluted in an excess of aggregated IgG. The diagnostic relevance of the assay was demonstrated in selected cases of acute hepatitis B. Rapid diagnosis of acute hepatitis B infection is therefore now possible in those cases whihc are HBsAg-negative but anti-HBc-positive.     

Detection of immunoglobulin G antibodies to cytomegalovirus antigens by antibody capture enzyme-linked immunosorbent assay

An antibody capture enzyme-linked immunosorbent assay (ELISA) that uses horseradish-peroxidase-labeled antigen for the detection of immunoglobulin G (IgG) antibodies to cytomegalovirus (CMV) is described. A microtiter plate was coated with anti-human IgG and consecutively incubated with serum specimens, enzyme-labeled CMV antigen made from CMV-infected cell nuclei, and substrate. The CMV IgG antibody content was determined spectrophotometrically and expressed as absorbance. Furthermore, to reveal any nonspecific reactions, all sera were tested against an enzyme-labeled control antigen made from uninfected cell nuclei. The problem with nonspecific reactions was small and was circumvented by the addition of unlabeled control antigen to the conjugates. For epidemiological studies the test was not as sensitive as other serological tests. On the other hand, the IgG antibody capture ELISA was highly sensitive for detecting the serological antibody response in patients with primary and recurrent CMV infections. Thus, one positive serum remained positive at a serum dilution of 1:10(7). The specificity of the test was shown by a blocking experiment and by testing 126 complement fixation-positive sera, of which 97% were positive. There was a rather good correlation between the complement fixation test and the IgG antibody capture ELISA (rs = 0.79, P less than 0.001). The test is especially useful when tests for CMV antibodies of the IgM, IgA, and IgE classes are run by similar antibody capture ELISAs, since the same procedure and conjugate are used.     

Early diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA-DNA hybridisation assay

One hundred forty-eight urine specimens were collected from 47 renal transplant and dialysis patients and screened for the detection of cytomegalovirus (CMV). Diagnosis of CMV infection was suggested in 17 out of 47 patients (36.2%) by more than one of the five methods used. DNA hybridisation assay (DNA HA) using 32P-labelled probe detected CMV DNA in 15 (31.9%) of 47 patients, whereas virus isolation on conventional tube cell cultures (CTC), immunofluorescence incorporating monoclonal antibodies on centrifugation vial cultures (IF), complement fixation test (CFT), and electron microscopy (EM) yielded positive results in only nine (19.2%), 12 (25.2%), 11 (23.4%), and one (2.1%) of 47 patients, respectively. The significance of these results obtained by DNA HA lies not only in the apparent increase in number of patients diagnosed, but also in both early and rapid detection of CMV DNA. More importantly, the DNA HA is highly specific in that it correlates accurately with clinical and laboratory data characteristic of CMV disease. In respect of clinically manifest CMV disease, the specificity of DNA HA, CTC, IF, CFT, and EM was 87.5, 43.7, 56.3, 43.7, and 6.3%, respectively. These advantages of DNA HA make it the test of choice for early diagnosis of CMV infections in immunosuppressed patients.