The effects of destruction of carbohydrate moieties of Echinococcus granulosus antigen by sodium periodate treatment, and/or blocking phosphorylcholine specific antibodies by the addition of free reagent, were investigated to improve sensitivity and specificity of enzyme-linked immunoabsorbent assay (ELISA) for diagnosis of hydatid disease. Sera were collected, in Uruguay and Sweden, from patients with confirmed hydatidosis, non-hydatid tapeworm infections, other disorders, and healthy donors. ELISA performed with sodium periodate or free phosphorylcholine increased sensitivity, but decreased specificity. Western blot analysis showed reduced recognition of the 38 kDa antigen following both treatments, and of the 15.5 kDa antigen after periodate treatment. Addition of free phosphorylcholine enhanced the recognition of two antigens, 26 and 50 kDa, which may represent new cross-reacting epitopes.
Fibre optic immunosensor for the detection of the antibodies specific for Leishmania donovani in the sera of patients suffering from visceral leishmaniasis has been designed and fabricated. Technique invovles a two-step sandwich-type assay that gives quick analysis and is easy to use. Purified cell surface protein of L. donovani had been immobilized on tapered fibre optic core by covalent coupling. In the first step, treated fibres were incubated with the patient serum for 10 min and then with FITC-tagged goat anti-human IgG in the second step. FITC provided the specific fluorescent signal for detection. No accurate volumetric measurement of serum sample is desirable because of test fibre design and the whole procedure takes just 15 min. Fluorescent signal has been found to be sensitive and specific for Visceral leishmaniasis and correlates well with the conventional serodiagnostic procedures, like ELISA and IFAT.
Antigen derived from three major life-stages of human lymphatic filariid, Brugia malayi was fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and reactivity of filarial antibodies, present in sera of human bancroftian patients belonging to different categories of the disease, to immunoreactive molecules was evaluated by Western blotting and immune recognition. Antigen molecules of >180, ~180, ~116, ~66, 58, 33 and <20 kDa were strongly identified in blots especially by symptomatic and endemic normal sera. Although mf positive serum reacted with the majority of these molecules, the intensity of bands was poor. These functional molecules along with ~43 kDa were later isolated from a preparative SDS-polyacrylamide gel by elution. The diagnostic utility of these purified molecules was then assessed by enzyme linked immunosorbent assay (ELISA) for detecting the IgG antibodies in patients' sera of various categories. Tropical pulmonary eosinophilic subjects revealed highest reactivity with <20 and ~116 kDa molecules while mf positive ones reacted feebly with the majority of the molecules except ~43 and ~66 kDa. Sera of endemic normals revealed high IgG levels but antibodies to ~ 66 kDa were highest. Symptomatic patients showed moderate reactivity. Non-endemic sera neither reacted in blots nor in ELISA. The study demonstrates the usefulness of ~43 kDa in detecting IgG antibodies of mf positive asymptomatic patients. High IgG levels to ~66 kDa followed by ~116 kDa and <20 kDa in sera of endemic normals warrants their immunoprophylactic evaluation.
Considerable efforts are being made in the search for suitable candidates for an effective serogroup B meningococcal vaccine, but with limited success. We have identified a novel, high molecular weight antigen of approximately 200 kDa. The antigen was expressed during invasive disease as 100% of convalescent case sera contained antibody compared with approximately 50% of carriers. Many clinical isolates did not appear to express the 200 kDa antigen, but corresponding sera contained antibody, indicating in vivo expression. Antibody to this antigen cross-reacted with meningococci of diverse phenotypes. Further investigation of this antigen is required to determine its potential as a future vaccine component.
Eight commercially-available tests were compared for the diagnosis of Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). They consisted in four slide agglutination assays (one of them using horse erythrocytes and three of them latex beads coated with purified bovine erythrocyte antigen), in a classical Paul-Bunnell-Davidsohn (PBD) tube-test using sheep erythrocytes, and in three assays intended to detect EBV-specific IgM antibodies either by indirect immunofluourescence (one test) or by ELISA (two tests). Sixty serum specimens from patients with symptoms compatible with IM were selected on the following criteria: presence of EBV-specific IgM by at least 2 of the 3 specific assays and absence or very low titres (≤5) of antibodies to EBV nuclear antigen (EBNA). Using this panel of positive samples, the sensitivity of the 8 assays ranged from 55 to 100%. The EBV-specific IgM tests exhibited the higher sensitivities, especially the one using a combination of peptides directed to different viral antigens. Whereas the PBD tuve test was shown to be time-consuming and insensitive, even in young adults, the slide agglutination tests were easy to perform and exhibited sensitivities ranging from 70 to 80%. The latter tests represent a good alternative to EBV-specific serology for the rapid diagnosis of IM; however, in case of negativity, an EBV specific testing must be done, especially in children under 5 years.
The relative sensitivity of immediate early nuclear antigen detection (IENAD) and conventional cell culture for human cytomegalovirus (HCMV) and the role of the first test in surveillance for infection remain controversial. If all positive results were considered accurate, IENAD and culture gave similar sensitivities [207/284 (73%) vs. 192/284 (68%)] with urines, heparinised bloods, bronchoalveolar lavage fluids, and other clinical specimens. Nonetheless, IENAD could not replace culture, because only the latter identified 77 (27%) of 284 positive samples. The role of IENAD in surveillance for HCMV infection is discussed in the light of low positivity rates with surveillance specimens.
Leptospirosis is underdiagnosed owing to its non-specific presentation, especially in the early stages of the infection. In the present research, sera from 2368 healthy blood donors of a local blood bank were screened for the presence of leptospiral antibodies using IgM-Pk-ELISA, followed by the microscopic and macroscopic agglutination tests. Serum samples showing two positive and/or borderline results in these assays were confirmed by immunoblotting. The IgM-antibody profile was defined according to the following criteria: sera reactive to a diffuse band of molecular weight 14.8-22 kDa were considered leptospirosis positive, sera exhibiting no reactivity to this band were considered negative. Our results showed a prevalence of 1.01% positivity. There is evidence that transmission of Leptospira interrogans by blood transfusion is possible, but, to date there has been no documentation of transfusion-transmitted leptospirosis. Thus, according to the currently available information, there is no need for new bloodbanking procedures to identify possible carriers of L. interrogans.
Three functional assays on immunity, serum bactericidal activity (SBA), phagocytic killing (PK) and an index calculation of chemiluminescence induced in polymorphonuclear leukocytes (CL-index), were compared with an enzyme linked immunoassay (ELISA) of the concentration of the total Ig-fraction of anticapsular antibodies in 28 healthy adults immunized against Haemophilus influenzae type b (Hib) or Neisseria meningitidis serogroup A and C (Me A + C). The immunization evoked a serum response measurable with all assays and a high concentration of antibodies against McA, McC and Hib usually was associated with functional activity and vice versa. However, qualitative differences were found and five of the vaccines (18%) were non-responders in functional assays despite a significant seroconversion measured by ELISA. A functional test may prove valuable in bacterial vaccine trials as complementary to immunochemical calculations of anticapsular antibodies. For estimates of opsonic activity bactericidal serum activity interferes in the PK assay, which is demonstrated in the present study. As an alternative, the CL assay, with the index technique described, is a reliable, less laborious and effective test on opsonophagocytosis.
Using the polymerase chain reaction (PCR), and DNA extracted from a lesion of a syphilis patient, we isolated the gene encoding for the mature Treponema pallidum TmpA membrane protein, and expressed this antigen in the cytoplasm of E. coli as a soluble fusion protein. The antigen, purified in one step by immobilized metal ion affinity chromatography (IMAC), was used to develop a visual immunoassay and an ELISA. When these systems were compared with the non-treponemal Venereal Disease Research Laboratories (VDRL) test we found very low discrepancy (0.9% and 0.83%, respectively) in a study of more than 1000 samples. The reactivity pattern of our sera panel in the TmpA visual and ELISA tests indicated that anti-TmpA antibodies are elicited and/or disappear concomitantly with anticardiolipin antibodies. Finally, we obtained a good correlation between the TmpA assays and the Treponema pallidum Haemogglutination Assay (TPHA) in classifying VDRL weakly reactive samples, suggesting their potential use in the confirmation algorithm of syphilis.
Sera from 192 children participating in studies of a pertussis toxoid vaccine were analyzed by enzyme-linked immunosorbent assay (ELISA) for pertussis toxin IgG antibodies and by the Chinese hamster ovary cell assay for pertussis toxin neutralizing antibodies. A significant linear correlation was found between titers obtained by ELISA and the neutralization assay (r = 0.84, P < 0.0001). The study shows that these two assays give similar information for groups of sera, although an antibody titer obtained by one method could generally not be used to predict a titer for the other method with accuracy in an individual serum. We propose that ELISA alone can be used in studies of IgG antibody response to pertussis toxin after vaccination with pertussis toxoid.
Efficient control of measles apparently requires reimmunization of susceptibles. The classic techniques used for estimation of measles susceptibility, neutralization and hemeagglutination inhibition, are neither convenient nor exactly comparable when carried out in different laboratories. We have compared a one-dilution commercial enzyme-linked immunosorbant assay (ELISA) to a classic neutralization test with respect to its ability to designate susceptibles as well as its ability to indicate post-reimmunization rises in measles antibody levels. A group of controls not deemed to require reimmunization was tested in parallel. The results indicate that the simple and standardized commercial assay may be able to substitute for the classic assays, which can only be carried out in specialized laboratories, when simple screening for persons with low levels of antibodies to measles or when evaluations of responses to (re)immunizations are required.
Recombinant Escherichia coli LamB proteins containing meningococcal PorA epitopes were used for the analysis of the human antibody response to specific PorA epitopes. Sera from meningococcal cases, carriers and controls were examined by an ELISA-inhibition assay. Antibody to the serosubtype P1.16 epitope was detected with similar frequency regardless of whether recombinant E. coli LamB protein containing P1.16 or SDS-PAGE purified meningococcal class 1 protein (serosubtype P1.16) was used as the antigen.
Context: Neisseria gonorrhoeae is a Gram-negative diplococcus, an obligate human pathogen, and the etiologic agent of the sexually transmitted infection (STI), gonorrhoea. culture is the standard procedure for diagnosis, which may be supported by nucleic acid tests and microscopy. Aims: To determine the best possible method of diagnosis for Gonococcus infection in resource-limited settings. Settings and Design: The meta-analyses were designed to determine the difference in diagnosis between Culture and nucleic acid amplification tests (NAATs) and also between the different Amplification Tests and widely available Roche COBAS AMPLICOR test. Subjects and Methods: Databases searched were Pubmed, Medline, Google Scholar and Cochrane reviews. Risk ratio (RR) with 95% confidence intervals was estimated for the dichotomous outcomes. The random-effect model was applied for all the studies in the analysis. Statistical Analysis Used: The meta-analysis was computed in RevMan Version 5.3, Copenhagen, Denmark. Results: In the first analysis, NAATs significantly improved the chances of detection in comparison to the standard culture and final RR was 1.24 (1.05–2.51), which put the diamond on the right of no-effect axis, indicating more positives by NAATs. In the second analysis, AMPLICOR had the more positive results, which may have indicated better detection rate, as well as less specificity and final RR was 0.809 (0.737–0.888), which put the diamond on the left of the non-effect axis, indicating more positives by AMPLICOR. Conclusions: In a resource-limited scenario like India, the syndromic management of STIs are considered to be the norm. A positive diagnosis is only given if the tests are confirmed by Culture, as it is still considered to be the gold standard of diagnosis. However, in many cases, due to suboptimal transportation and lack of proper handling, culture in unable to grow even if the patient is infected. In such cases, Nucleic Acid Tests should be able to detect an infection.
The indirect immunofluorescence (IIF) test was standardized using antigenic particles of Cysticercus longicollis (Cl), ORF strain, for the detection of antibodies in cerebrospinal fluid (CSF) in human neurocysticercosis (NC). ELISA with a total saline extract of Cysticercus cellulosae (Cc) and IIF with Cc particles were performed comparatively. The IIF-Cl test presented 84.8% positivity (95% confidence interval (Cl95%) = 74.2%–95.4%) in 46 CSF samples from patients with NC, 100% negativity in 45 CSF samples from the control group, and 92.3% agreement with ELISA and 98.9% agreement with the IIF-Cc test. No difference was observed between the homologous and heterologous antigens in the IIF test, suggesting that particulate Cl antigens may be employed alternatively in the laboratory diagnosis of NC by the determination of antibodies in the CSF.
This study was conducted to assess the value of ELISA findings in relation to negative findings in the Brucella-antihuman globulin (Coombs) test and in relation to the clinical condition of patients. One hundred and thirty three serum specimens, representing the same number of patients, submitted for serologic testing for brucellosis and showing negative Coombs, were tested by ELISA to determine their Brucella-IgG, -IgM and -IgA antibodies. Concordant negative results between Coombs and ELISA were found in 95 (71.4%) patients whose medical records also did not reveal suggestive clinical signs or symptoms of brucellosis. The elevated ELISA readings in the remaining 38 (28.6%) patients were distributed as follows: IgG + IgM + IgA in 1 patient, IgG + IgM in 8 patients, IgG alone in 24 patients and IgM alone in 5 patients. The clinical review of these patients indicated no current disease in 21 (15.8%), scanty evidence of brucellosis in 8 (6.0%) and suggestive or sufficient evidence in 9 (6.8%). Thus, ELISA is the test of choice to resort to in the case of clinical suspicion of brucellosis, even when the Coombs test shows negative findings.
An indirect fluorescent antibody (IFA) test using Leishmania donovani promastigote and amastigote antigens for diagnosis of kala-azar was evaluated in a kala-azar endemic area of Bihar, India. The test was found to be highly sensitive and specific using the promastigote antigen. It was positive in about 99% of parasitologically proven cases (n = 105), 89% of clinically suspected cases (n = 70) and all the sodium antimony gluconate (SAG) resistant cases (n = 80). None of the non-endemic control subjects (n = 40) or subjects infected with tuberculosis (n = 20), leprosy (n = 28), malaria (n = 20) and amoebiasis (n = 20) showed positive response. Only about 3% of the control subjects (n = 70) of the endemic area showed false positive response. Similar results were obtained with the amastigote antigen.
Of 135 serum samples from 135 patients suspected of hepatitis C virus (HCV) infection, 67 were detected by Abbott IMX antibody assay, 89 by Murex anti-HCV (version III), and 47 by Roche Amplicor polymerase chain reaction (PCR). Furthermore, 44 of the 62 positive serum samples by both Abbott and Murex antibody assays, 2 of the 27 positive samples by Murex antibody assay only, none of the 5 positive samples by Abbott antibody assay only, and one of the 43 negative samples by both Abbott and Murex antibody assays had measurable HCV RNA by Roche Amplicor PCR, suggesting active hepatitis C viremia. Whereas Abbott and Murex antibody assays were in agreement with each other in 103 of the 135 serum samples tested, they showed discrepancy with regard to the other 32. Despite generating a small percentage of false positives, Abbott and Murez antibody assays are useful in monitoring serum antibody levels of the past or continuing hepatitis C virus infection. Abbott IMX appears to be more specific than Murex anti-HCV (version III). The use of Roche Amplicor PCR provides a means of revealing active hepatitis C viremia, and helping clarify antibody indeterminate serum samples.
