Serodiagnosis and Immunotherapy in Infectious Disease最新文献

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.

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.

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.

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 (Cl_95%) = 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.

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.

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.

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.

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.