Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

The respective advantages of complement fixation and ELISA for routine diagnosis of cytomegalovirus infection

The results of CF and ELISA tests for cytomegalovirus performed on 270 sera of hospitalized patients show a positive correlation. As a general rule, ELISA is more sensitive than CF, except for a few sera collected from patients with immunological disorders. When two sequential sera are available, the CF remains a reliable and inexpensive method. But when only one serum can be obtained, the probability of an active CMV infection can be estimated on the IgM/IgG ratio. In 26% of the patients, this ratio was greater than or equal to 1. The ELISA is twice as expensive as the CF test. To reduce its cost, a simple method for preparing ELISA antigen from commercially-obtained CF antigens is described.     

Immunological diagnosis of cytomegalovirus infections. Application to blood transfusion centers

The Blood Transfusion Centers (B.T.C.) are mainly concerned with the selection of CMV infection free blood donors, the screening of the anti CMV antibody high titre plasma donors and the evaluation of specific anti CMV Immunoglobulin preparations. Various serological methods could be used but they are of different value depending the purposes of the B.T.C. The neutralization test (Nt), with the addition of complement is specific and detects the protecting AB against the glycoproteins of the viral envelope. The complement fixation test (CF) using extracts of CMV infected cells as antigen largely varies in its sensitivity according to the quality of the antigen. In any case, the CF test is not sensitive enough to detect a latent CMV infection in a certain percentage of the non immunosuppressed adults, but could be used for the selection of anti CMV antibody high titres carriers. Three sensitive methods: passive haemagglutination, indirect immunofluorescence and indirect ELISA tests, might be used for the detection of latent CMV infections. They detect various AB against various internal and external components of the CMV. They are submitted to various sources of errors. The sensitivity of the indirect IF test is mainly restricted by the quality of the antigen preparation, its specificity by the presence of anti cells antibodies in the sera, the Fc receptors in the antigens and the specificity of the conjugates. The indirect ELISA which is submitted to the same causes of errors is a highly sensitive test, easy to perform, reagents are available, and automatic processors have been developed. When compared with the previous techniques, the ELISA test is suitable for the screening of CMV free donors, when it is performed with an highly sensitive antigen. It could be also used for the screening of high antibody titre carriers: its correlation with the CF test is quite good (r = 0,82). When comparatively applied to the titration of Immunoglobulins preparations, made from plasmas or placentas, for either IM or IV administration, the Elisa test gives AB titres different from those obtained with Nt and indirect IF. The calibration of a standard Immunoglobulin reagent is urgently needed and double blind clinical assays of the protective effect of various preparations of specific anti CMV Immunoglobulins have to be promptly designed.     

Specific antibodies in children excreting cytomegalovirus

Acute-and convalescent-phase sera from 22 children were examined by ELISA in comparison with a routine complement fixation (CF) test for detection of anti-CMV antibodies. All these subjects were excreting CMV from urine and/or saliva. The results showed that ELISA is more sensitive than CF test. Particularly ten children showed, by ELISA, anti-CMV antibody titers more agreeing with clinical-virological features. Generally, in other subjects the results of the two serological tests were similar. Three cases showed discordances both between the two methods and between serological data and clinical virological findings.     

Comparison of 4 serological tests--complement fixation, neutralization, fluorescent antibody to membrane antigen and immune adherence hemagglutination--for assay of antibody to varicella-zoster (V-Z) virus.

Four tests for antibody to varicella-zoster (V-Z) virus were compared; these were tests of complement fixation (CF), neutralization (NT), fluorescent antibody to membrane antigen (FAMA) and immune adherence hemagglutination (IAHA). Fifty-two sera from patients with varicella and zoster and from recipients of live varicella vaccine were examined by the 4 tests. The CF test was least sensitive, but the antibody titers by the NT, FAMA and IAHA tests were roughly comparable. The IAHA test was the simplest and fastest to perform, and appeared suitable for routine serological assay to V-Z virus. The correlation between the IAHA antibody titer and susceptibility of individuals to clinical varicella was investigated retrospectively using sera obtained during 2 outbreaks of varicella in an institution for children, where all the unvaccinated children had developed varicella symptoms. Most of the 25 pre-exposure sera from unvaccinated children examined by the IAHA test had tiers of less than 1:2. In contrast, all the 23 sera from vaccinated children who did not develop varicella had detectable antibody titers of 1:2 to 1:64. These results indicate that the IAHA titer reflects the susceptibility or resistance of individuals to clinical varicella.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Herpesvirus papio 2: alternative antigen for use in monkey B virus diagnostic assays

BACKGROUND AND PURPOSE: Serologic testing for antibody to monkey B virus (BV) in macaque sera is problematic due to the biohazardous nature of BV and BV antigens. Herpesvirus papio 2 (HVP2), a herpesvirus of baboons, is more closely related genetically and antigenically to BV than is human herpes simplex virus 1 (HSV1). The potential for use of HVP2 relative to HSV1 as an alternative test antigen for detection of anti-BV antibody in macaque sera was assessed. METHODS: Standard ELISA formats were developed, using BV-, HVP2-, and HSV1-infected cell extracts. Performance of the HVP2 and HSV1 tests was assessed relative to that of the BV test. RESULTS: Using the BV antigen ELISA, 349 sera from 7 macaque species were tested, and results were classified as positive (253), negative (94), or suspect (2). The ELISA using HVP2 antigen detected 98.0% of BV-positive sera (248 of 253), whereas the HSV1-based ELISA detected only 96.0% (243 of 253). All three ELISAs identified the same two samples as suspect, and the HSV1 ELISA identified three additional BV-positive sera as suspect. CONCLUSIONS: The HVP2 antigen-based ELISA was equal in sensitivity and specificity to the BV antigen-based ELISA and was superior to the HSV1 ELISA for detection of BV-positive macaque sera. In addition, the HVP2 ELISA has greater laboratory safety, compared with BV antigen use for ELISA testing.     

应用转化细胞分泌的乙型肝炎病毒e抗原检测人血清中的乙型肝炎病毒e抗体

应用乙型肝炎病毒核心抗原基因转化的小鼠L细胞分泌的乙型肝炎病毒e抗原,采用ELISA法与Abbott公司抗-HBe EIA诊断盒平行比较,检测了31份抗-HBe阳性和19份抗-HBe阴性的人血清,结果完全相符。经多次重复试验,本法的OD490nm值的误差不超过8％。OD490nm值与血清稀释度之间呈直线关系。细胞培养液不经纯化即可应用,一般做1:4稀释。细胞分泌的抗原无感染性,价格低廉,不会因结合人血清蛋白而产生非特异性反应。因此比一般诊断盒中所用的人血清HBeAg有很大的优越性。     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Measurement of antibody to Jo-1 by ELISA and comparison to enzyme inhibitory activity

Antibody to the Jo-1 antigen (histidyl-tRNA synthetase) is found almost exclusively in myositis patients, usually those with adult PM, but has been found in only 30% of that group by immunodiffusion or other techniques thus far reported. We have reexamined the prevalence of antibody to Jo-1 in sera from 130 patients and 82 controls by using the sensitive ELISA technique. The ELISA used affinity-purified, enzymatically active bovine Jo-1 antigen. A wide range of antibody level by ELISA was found among 24 immunodiffusion positive sera. Six myositis and two control sera had apparent specific antibody detectable only by ELISA. Overall, however, the antibody continued to show high myositis specificity with predominance in adult PM (35.8% in that group). Because the antibody inhibits enzymatic activity of the synthetase antigen, we also studied the quantitative inhibitory activity of these sera to compare with the antibody activity as determined by ELISA. Twenty-four immunodiffusion-positive sera, 29 immunodiffusion-negative sera, and 15 normal sera were tested at 1/50 dilution in the reaction mixture. There was background inhibition by all normal sera tested that averaged 30.5%. All but one immunodiffusion negative myositis sera (a high binder by ELISA) inhibited less than 50% of the average with normal serum. Twenty-three of 24 immunodiffusion positive sera inhibited greater than 80% of this normal average; the other inhibited 66%. The serum dilution giving 50% inhibition was highly correlated (R = 0.83) with the ELISA activity. Thus, inhibition of histidyl-tRNA synthetase activity is a relatively accurate measure of Jo-1 antibody. This method should be applicable to measuring antibody to other aminoacyl-tRNA synthetases.     

Serological Diagnosis of Human Melioidosis with Indirect Hemagglutination and Complement Fixation Tests

An indirect hemagglutination (IHA) test and a complement fixation (CF) test were evaluated from test results on sera from 212 human melioidosis patients of which 119 were culturally proved cases. Significant antibody titers (IHA titers of 1:40 or greater and CF titers of 1:4 or greater) were demonstrated with either test in all except five patients. IHA and CF titers ranged as high as 1:20,480 and 1:1,024, respectively. Antibodies were usually demonstrated by both tests 1 week after onset of disease. Transient seronegative reactions during the course of disease were seen in sera of approximately 19% of the patients with either IHA and CF but rarely with both tests. High titers in either test were obtained by the third week of disease and reached maximum levels in 4 to 5 months. Titers usually were detectable for 9 or more months. Antibodies were detected by IHA and CF tests in 80 to 100% of the sera obtained at various time intervals from 9 months to 2 or more years after disease onset. Antibody persistence occurred in patients who had a short disease course, as well as in patients with prolonged, complicated infections. The IHA test had excellent specificity when evaluated with normal human sera and diverse antimicrobial sera from hyperimmunized rabbits and human patients. The CF antigen appeared to contain common antigens with some but not all types of Pseudomonas aeruginosa. The specificity of the CF antigen could be enhanced without appreciable effect on its sensitivity by use of a titer of 1:8 in lieu of 1:4 as a criterion for a significant reaction. Either test could be used advantageously for the laboratory diagnosis of melioidosis.     

Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

The serodiagnosis of human hydatid disease: 2. Additional studies on selected sera using indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS).

Sixty-one serum samples selected on the basis of reactivity in the complement fixation (CF) and latex agglutination (LA) test, were further examined for sensitivity and specificity by indirect haemagglutination (IHA), enzyme linked immunosorbent assay (ELISA) and defined antigen substrate spheres (DASS). Twenty sera from healthy Europeans and 48 samples from patients with either schistosomiasis or trichinosis were also tested. Comparable levels of sensitivity were found between the CF and LA positive sera and IHA, ELISA and DASS. Of the CF positive LA negative group of sera, many were positive by DASS but only a few reacted in IHA and ELISA. Some cross reactivity was also observed in the schistosomiasis sera tested by IHA and ELISA.     

The effect on kaolin adsorption of serum on the virus neutralization and enzyme-linked immunosorbent assays of antibody to bovine herpesvirus 1

Two procedures have been used for measuring antibody titres to bovine herpes virus 1 (BHV1): the serum neutralization (SN) test and enzyme-linked immunosorbent assay (ELISA). One hundred and thirty-two sera selected for their low SN titres were tested both unadsorbed and after adsorption with kaolin to determine the effect of kaolin on the titres. With ELISA, the titres of unadsorbed and kaolin adsorbed were not significantly different but with the SN test many treated sera, originally with weak positive titres, became negative after kaolin adsorption. Thus, if the ELISA results are specific for BHV1 antibody then the SN test findings suggest that treatment of sera with kaolin, rather than removing a viral inhibitor, removes a substance from the serum which potentiates SN antibody. This in turn indicates that low SN titres (reciprocal of titre less than or equal to 4, for instance) are probably specific for BHV1 SN antibody whether or not they are abolished by kaolin treatment of the serum.     

Cross-reactive antibodies to mycoplasmas found in human sera by the enzyme-linked immunosorbent assay (ELISA)

Antibodies against Mycoplasma pneumoniae in patients' sera with M. pneumoniae infection were measured by the complement fixation (CF) test and enzyme-linked immunosorbent assay (ELISA). Many patients' sera cross-reacted with heterologous mycoplasmal ELISA antigens such as M. hominis, M. hyorhinis, M. orale, M. pulmonis and M. salivarium. The sera with high CF (CF greater than or equal to 40) titers gave significantly higher ELISA values to M. hyorhinis (P less than 0.001) and M. pulmonis (P less than 0.001), which are not parasitic for humans, than those with low CF (CF less than 20) titer. Human normal immunoglobulin G (human normal IgG) containing 98% or more IgG, prepared from pooled plasma of at least 500 normal human donors, showed ELISA reactions with all mycoplasmal strains used. The nonspecific adsorption of human normal IgG on the surface of plate wells and on medium components which might contaminate mycoplasmal ELISA antigens could be disregarded. These results suggest that cross-reactive antibodies to mycoplasmas exist in human sera, and they affect the results of ELISA for serodiagnosis of M. pneumoniae infection.     

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Attempts to demonstrate specific antibody responses in the vitreous body of the eye

Postmortem serum and vitreous humor specimens obtained from 31 autopsied human bodies were assayed for specific antibody responses to adenoviruses, RS virus and Mycoplasma pneumoniae using the complement-fixation (CF) test and the ELISA procedure (in 23 of the bodies examined). The antibody responses as measured by the CF test were negative in all vitreous body samples tested, with the ELISA five specimens gave a positive reaction at a titre 1 : 40 and one at 1 : 80. These positive antibody titres turned out to invariably coincide with the high-titre antibody levels in the serum. Implicitly, at high-titre levels in the serum these antibodies tend to penetrate in the vitreous body of the eye.     

Sensitive Matrix Gel Diffusion Test for the Detection of Australia Antigen

A matrix gel diffusion (MGD) procedure with a sensitivity comparable to the complement fixation test (CF) has been developed for detecting Australia antigen in serum. The test utilizes a thin layer of agar (0.1 mm) with an applied plastic matrix. Reactants are introduced directly onto the surface of the agar through wells in the plastic matrix. End points obtained by CF with a panel of 11 sera varied from 1:8 to 1:512. When these sera were tested by MGD, end points for detection of antigen were within one dilution of that obtained by CF.