Evaluation of an ultramicroELISA system for the diagnosis of rubella

An indirect Ultramicro ELISA assay, previously standardized in our laboratory, for detecting antibodies IgG to the rubella virus was assessed in comparison to the hemagglutination inhibition technique. This assessment allowed to determine its efficacy in the National System for Epidemiological Surveillance of this entity. One hundred and ninety serum pairs of clinically suspected cases of rubella were studied and a high percent of coincidence (99.4%), specificity (99.4%), and sensitivity (100%) was found between both techniques. In addition, 73 serum samples of blood donors were processed using an indirect microELISA system (Berhing) which was compared to the Ultramicro ELISA technique for rubella and it showed a 100% of sensitivity, specificity, and coincidence.     

Mercury-stimulated histamine uptake and binding in cultured astroglial and cerebral endothelial cells

BACKGROUND: The reference method for detecting specific Epstein-Barr virus (EBV) antibodies is indirect immunofluorescence (IF) with EBV-infected cells. The availability of protein purified from infected cells and more recently of recombinant polypeptides designed to contain immunodominant epitopes, has enabled the development of commercial enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of EBV infection. OBJECTIVE: Evaluation of ELISA-based EBV serodiagnosis in comparison with indirect immunofluorescence. STUDY DESIGN: We have first compared three commercial ELISA test systems with our in house indirect immunofluorescence assay for classifying correctly a set of serum samples into clinical categories (acute infection, past infection, interfering non-EBV infection, persistent infection). Additionally a prospective analysis with the best performing ELISA test (Enzygnost) was then carried out by running the ELISA test in parallel with the indirect immunofluorescence assay on 324 consecutive clinical samples sent to our laboratory for EBV serodiagnosis. RESULTS: For the serodiagnosis of past EBV infection and acute EBV infection all three commercial ELISAs performed well in comparison with indirect immunofluorescence. When testing samples positive for cytomegalovirus (CMV), Toxoplasma or herpes simplex IgM, interference in the IgM tests was observed with the three ELISAs. In some instances we could demonstrate that the positive IgM results were due to EBV reactivation. The observed discrepancies between ELISA and IF for the serodiagnosis of chronic EBV infection or EBV reactivation, point to the difficulty for the serodiagnosis of persistent EBV infection on single serum samples. According to our prospective study the EBV IgG determination was accurate. A positive IgM result was not always indicative of an acute infection. Positive IgM results due to EBV reactivation were observed. A positive EBV nuclear antigen (EBNA) IgG result in those samples precluded acute infection. CONCLUSIONS: 90-95% of samples could be classified correctly into clinical categories by a two parameter ELISA system detecting IgG and IgM against a standardized mixture of EBV antigens, allowing standardization and automation of EBV-specific serology. The absence of EBNA IgG was useful as a second line confirmatory assay for acute EBV infection.     

Detection of specific antibodies in saliva during dengue infection

Saliva was collected prospectively from patients presenting with suspected dengue infection 4 to 8 days after the onset of symptoms and assayed by a commercial dengue immunoglobulin M (IgM) and IgG capture enzyme-linked immunosorbent assay (ELISA) (PanBio Dengue Duo ELISA). Laboratory diagnosis was based on virus isolation and on hemagglutination inhibition (HAI) assay and an in-house IgM and IgG capture ELISA. With a positive result defined as either salivary IgM or IgG levels above the cutoff value, an overall sensitivity of 92% was obtained for both primary- and secondary-dengue patients (22 of 24), while no patients with non-flavivirus infections (n = 11) and no healthy laboratory donors (n = 17) showed elevation of salivary antidengue antibody (100% specificity). Salivary IgG levels correlated well with serum HAI titer (r = 0.78), and salivary IgG levels could be used to distinguish between primary- and secondary-dengue virus infections.     

Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays

The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.     

Bilateral fascia lata patch grafts in a patient with progressive scleromalacia perforans

An enzyme-linked immunosorbent assay (ELISA) test kit for the detection of feline leukemia virus (FeLV) antigen in saliva was evaluated in 150 cats. Saliva and blood samples from all cats were tested for FeLV using the saliva ELISA kit and a plasma ELISA kit, respectively. These results were compared with indirect immunofluorescent antibody (IFA) testing of blood smears also obtained from each cat. The proportion of cats that tested positive were 10%, 7%, and 8% for each test, respectively. Using the IFA test as the gold standard, the saliva FeLV test had a sensitivity of 91.7% and specificity of 97.1%, while the plasma ELISA test had a sensitivity of 91.7% and specificity of 100%.     

Evaluation of serological methods for diagnosis of Puumala hantavirus infection (nephropathia epidemica)

Nephropathia epidemica (NE), Puumala (PUU) virus infection, is a febrile disease which is commonly associated with acute renal impairment. To differentiate NE from other acute febrile illnesses, a rapid and reliable serological diagnosis is important, and a number of different protocols have recently been introduced. In the present report we describe a comparative evaluation of six PUU virus immunoglobulin M (IgM) and seven IgG enzyme-linked immunosorbent assay (ELISA) protocols based on native, Escherichia coli-expressed, or baculovirus-expressed nucleocapsid protein (N). Neutralization and immunofluorescence assays were included for comparison. Equally high sensitivities and specificities were obtained with three mu-capture-based IgM ELISAs using native, baculovirus-expressed, and E. coli-expressed N antigens, respectively, and by an ELISA based on purified E. coli-expressed full-length N adsorbed to solid phase. The assays based on truncated amino-terminal N proteins, including a commercially available PUU virus IgM ELISA, all showed lower sensitivities. For detection of PUU virus-specific IgG, ELISAs based on monoclonal antibody-captured native or baculovirus-expressed N antigens showed optimal sensitivities and specificities, while the assays based on E. coli-expressed N did not detect all PUU virus IgG-positive serum samples. A commercially available PUU virus IgG ELISA based on E. coli-expressed amino-terminal N showed a significantly lower sensitivity than those of all other IgG assays.     

Salivary immunoglobulin G assay to diagnose Helicobacter pylori infection in children

An in-house enzyme-linked immunosorbent assay (ELISA) for measurement of Helicobacter pylori-specific immunoglobulin G (IgG) and IgA in saliva was evaluated by comparison with histopathologic (Giemsa staining) and biochemical (urease quick test) examination of gastric biopsy specimens obtained from 112 children referred for diagnostic gastroscopy. Serum H. pylori IgG was also measured in a subgroup of 50 children by the same ELISA. Salivary H. pylori IgG levels were significantly higher in H. pylori-positive (n = 57) than in H. pylori-negative (n = 55) children (P < 0.001). The sensitivity and specificity of the salivary IgG test were 93 and 82%, respectively; the positive and negative predictive values were 84 and 92%, respectively; and the accuracy was 87.5%. Salivary H. pylori IgA did not distinguish H. pylori-positive from H. pylori-negative children. The performance of serum H. pylori IgG was slightly (3 to 6%) better than that of salivary H. pylori IgG. The salivary IgG test can be considered a useful tool for the screening of H. pylori infection in children.     

Quantitation of intrathecal measles virus IgG antibody synthesis rate: subacute sclerosing panencephalitis and multiple sclerosis

A method for quantitating specific anti-viral antibodies in serum and cerebrospinal fluid (CSF) is established using enzyme-linked immunosorbent assay (ELISA). Quantitated antibody levels are used to determine intrathecal specific IgG synthesis rate for the particular antibody. Measles virus was used as a model for validating this quantitative technique: a mutated form of measles virus is a cause of subacute sclerosing panencephalitis (SSPE) and there is a possibility that measles virus is related to the cause of multiple sclerosis (MS). Matched serum and CSF samples were assayed. Concentration of anti-measles IgG was determined and intrathecal measles-specific IgG synthesis rate was calculated. For the SSPE samples, measles-specific IgG synthesis rate was elevated and comprised > 20% of the total intrathecal IgG synthesis rate; these results are consistent with the literature. The ELISA method can be performed routinely, providing a quick, simple, reproducible means of quantitating specific antibody concentrations, with sensitivity greater than 1 nanogram per milliliter. With this method, quantitation of IgG antibodies to any other viral antigen can be reliably and precisely determined.     

Emerging issues in sleep-disordered breathing

An amplified capture enzyme-linked immunosorbent assay (ELISA) has been developed by the use of the biotin-avidin detection system, for the measurement of canine plasma immunoglobulins (Ig) A, G and M. Test responses of dilutions of both the Ig standards and test plasma samples were consistently linear (r > 0.987) for the three Ig classes. The within-assay variation was 3.53 per cent for IgG, 5.84 per cent for IgM and 6.34 per cent for IgA. The analytical recoveries were 95 per cent for IgA, 97 per cent for IgG and 98 per cent for IgM. The lower detection limits of the assay were 38.4 ng ml-1 for IgG, 20.3 ng ml-1 for IgM and 41.2 ng ml-1 for IgA. The results indicate that this ELISA has a much higher sensitivity than the single radial immunodiffusion assay or the non-amplified ELISA for measurements of canine Igs, but has a comparable specificity and precision.     

A review of esthetic alternatives for the restoration of anterior teeth

Helicobacter pylori infection is an important cause of peptic ulcer disease and chronic gastritis. Infection with this bacterium stimulates the production of immunoglobulin (Ig) G antibody. Salivary IgG antibody tests to detect H pylori infection offer a convenient and noninvasive method of diagnosis. To evaluate an IgG salivary antibody kit, saliva was collected from 157 out-patients with dyspepsia referred for endoscopy to a tertiary centre. A salivary IgG ELISA antibody assay was performed using the Helisal Helicobacter pylori (IgG) assay kit, and at least four gastric biopsies were obtained. H pylori infection was confirmed by demonstration of the organism on Warthin-Starry silver stain (sensitivity 85%, specificity 55%). The prevalence of infection with H pylori was 30%. When the analysis was redone, excluding those treated with eradication therapy, the results were similar (sensitivity 86%, specificity 58%). The positive predictive value of the assay was 45% and the negative predictive value was 90%. Despite the ease of sampling, the assay used has limited diagnostic utility, lacking the predictive value to indicate which patients referred with dyspeptic symptoms to a tertiary care setting are infected with H pylori.     

Performance characteristics of an enzyme-linked immunosorbent assay for determining salivary immunoglobulin G response to Helicobacter pylori

We evaluated the salivary immunoglobulin G (IgG) immune response to Helicobacter pylori in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive H. pylori culture showed significantly higher titers of antibodies than subjects with no detectable H. pylori: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-H. pylori IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.     

Exposure to blood borne viruses and the hepatitis B vaccination status among healthcare workers in inner London

The performance of a commercially available assay for detection of hepatitis C virus (HCV) antibody in saliva samples was assessed. Samples of saliva were collected from 270 individuals whose HCV antibody status was determined by serum assay (161 HCV-positive, 109 HCV-negative). The saliva samples were tested for the presence of HCV antibodies using a modified protocol. The sensitivity was 94.4% (95% CI, 89.3-97.2%) and the specificity 99.1% (95% CI, 94.3-100%). Although the optical density in tests on HIV-positive individuals was lower than that among HIV-negative individuals, the HIV status had no significant influence on the results of the HCV assay in saliva. These findings suggest that tests on saliva can be useful in epidemiological studies for estimating the prevalence of HCV in populations that are difficult to reach.     

Regulation of thrombosis and hemostasis by antithrombin

An ELISA has been set up for quantifying mouse monoclonal antibodies in culture supernatant. The assay includes rabbit anti-mouse IgG antibodies chromatographycally purified. This preparation was used as coating and as conjugated antibodies in the ELISA. The assay can detect IgG1 with sensitivity of 0.2 ng/mL, IgG2a (0.85 ng/mL), IgG2b (0.13 ng/mL), and IgG3 (3.19 ng/mL) in culture supernatants. The effective working range was from subnanogram per mL quantities to 30 ng/mL by using a computer statistical program. Variation coefficient of ELISA was below 7%. Correlation estimates with a similar ELISA using commercial reagents were performed for each mouse antibody subclass. The assay was able to detect the four mouse monoclonal antibody subclasses in pure human serum as compared with the same ELISA using commercial antibodies. A 24-h pharmacokinetic profile of 1 patient treated with an IgG2a monoclonal antibody is presented.     

Determination of salivary and serum immunoglobulin concentrations in the cat

The concentrations of immunoglobulin(Ig)G, IgM, and IgA were determined in unstimulated saliva (n=14), stimulated saliva (n=6), and serum (n=14) from healthy adult cats. Analysis by single radial immunodiffusion (SRID) was compared with class-specific enzyme linked immunoassays (ELISA), and good correlation was demonstrated between the two techniques. Mean (s.d.) serum concentrations of 19.08 (5.38) mg/ml IgG, 2.04 (0.83) mg/ml IgM and 2.6 (2.16) mg/ml IgA were obtained by SRID. The immunoglobulin concentrations of the saliva samples frequently fell below the quantification limits for SRID, however, all samples could be quantified by ELISA making this the method of choice for the determination of salivary immunoglobulin concentrations. IgA was the predominant class of immunoglobulin secreted by the major feline salivary glands, and the concentration of each immunoglobulin class was greater in unstimulated versus stimulated saliva. Analysis of sequential unstimulated saliva samples collected each morning and evening over a 4-day period from four cats revealed the salivary immunoglobulin concentrations to be relatively constant.     

Antigenicity of a synthetic peptide from glucosyltransferases of Streptococcus mutans in humans

Human salivary immunoglobulin A (IgA) and serum IgG antibodies to the Streptococcus mutans glucosyltransferases (Gtfs) and to a synthetic peptide of 19 amino acids from a conserved region in the Gtfs (residues 435 to 453) were determined in young adults by enzyme-linked immunosorbent assay. Varying levels of antibody to Gtfs were detected in saliva or serum, with significantly higher levels of antibody to GtfD than to GtfB/C or GtfC. Anti-Gtf IgA levels in saliva did not correlate with those of IgG in serum. Caries-free (CF) volunteers exhibited significantly higher salivary IgA antibody levels to the peptide and to GtfB/C or GtfC than did the caries-active (CA) subjects. Preincubation of CF saliva and serum with the peptide inhibited the antibodies to the Gtfs in a dose-dependent manner, whereas preincubation of the samples from the CA group resulted in only partial inhibition. Our results indicated that this 19-amino-acid peptide includes one of the major B-cell epitopes of Gtfs and that CF individuals have higher titers of antibodies than CA subjects.     

Evaluation of three immunoassays for detection of Toxoplasma-specific immunoglobulin G and M

A time-resolved immunofluorometric assay of the IgG specific for Toxoplasma gondii has been compared with two commercial methods, one automated enzyme linked fluorescent assay (VIDAS) and one automated colorimetric enzyme immunoassay (BEIA). The coefficients of variation were 3.4% and 7.4% within-assay (n = 12), and 9.2% and 8.1% between-assay (n = 10) for two sera at low and high concentrations of specific IgG. The regression lines obtained with 96 samples were y = 1.04x + 2.1 and y = 0.98x - 1.1. We also compared a time-resolved capture fluoroimmunoassay for Toxoplasma-specific IgM with a commercial immunoenzymatic assay (BEIA TOXO-M). The sensitivity and specificity were 100%, calculated from assays of 78 samples.     

New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.     

PAICA: a method for characterizing platelet-associated antibodies--its application to the study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3

In idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA ("Platelet-Associated IgG Characterization Assay"), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient's own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA ("Monoclonal Antibody-specific Immobilization of Platelet Antigens"). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP IIb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP IIb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.     

Chlamydia trachomatis antibody detection and diagnosis of reactive arthritis

OBJECTIVE: To investigate whether determining the presence of serum or synovial fluid (SF) IgG and IgA of anti-Chlamydia antibodies with two recent commercially available enzyme-linked immunosorbent assays (ELISA) using synthetic peptides or recombinant antigen could be helpful to detect possible Chlamydia trachomatis (CT)-involved disease in rheumatological patients without evidence of urogenital CT infection. METHODS: The prevalence of such antibodies was determined in samples from patients with well-defined disease, i.e. CT sexually acquired arthritis and from patients with other inflammatory arthropathies unrelated to CT. RESULTS: When considering IgG and/or IgA anti-MOMP or anti-LPS antibodies, a sensitivity of 100% was obtained for serum and SF samples, but with a low specificity. A sensitivity and a specificity equal or close to 80% were observed for the SF IgG anti-MOMP antibodies. CONCLUSION: Clinically, the most appropriate determination was the SF IgG anti-MOMP antibodies. This commercially available ELISA test could be useful for the diagnosis of probable CT reactive arthritis.     

Usefulness of combined color Doppler/contrast in providing complete delineation of left ventricular cavity

Although detection of disease-induced hepatitis A virus (HAV)-specific antibodies in saliva has been successfully utilized in a few epidemiological studies, available assays fail to detect lower salivary anti-HAV levels associated with vaccine-induced immunity. We present a new capture enzyme immunoassay which employs a three-layer antibody recognition system. Evaluation of paired saliva-serum specimens from 1,025 international travellers, 134 other volunteers, and 91 hepatitis A vaccine recipients demonstrated 99.6% (95% confidence interval, 98.4 to 99.9) specificity and 98.7% (95% confidence interval, 97.7 to 99.4) sensitivity of this salivary assay in differentiating between immune and susceptible individuals, compared with serum-based methods. We conclude that this assay is sufficiently sensitive for reliable detection of both vaccine- and infection-induced HAV-specific immunoglobulin G in saliva, even when corresponding anti-HAV levels in serum are very low (< 1 IU/ml).