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The Enzyme linked immunosorbent Assay (ELISA), indirect haemagglutination (IHA), and immunoblot techniques (IB) were used for the serodiagnosis of surgically confirmed cystic echinococcosis (CE) caused by the tapeworm Echinococcus granulosus. Antigens used for the detection of IgG or total antibodies included crude sheep hydatid fluid (CSHF), autoclaved antigen B (AAB), boiled antigen B (BAB), and homogenate protoscoleces antigen (HPA). The overall sensitivity of the ELISA and IHA tests used for the serodiagnosis of 57 surgically confirmed human cases was 91.2% and 68.4%, respectively. The sensitivity of both tests was comparable in groups whose sera were collected one week before surgery and up to one year after surgery at 95.8% and 87.5%, respectively. In contrast, the sensitivity of the ELISA was significantly higher than that of IHA for sera of patients collected after one year of surgery. There was a positive correlation (r = 0.61) between the titers of antibodies detected by the ELISA and IHA. Using the IB technique, antigen B fractions (8/12, 16, and 24 KDa) were detectable by sera of 68.4% using either CSHF or AAB, 49.1% using BAB and 22.8% using HPA as detecting antigens. The overall sensitivity of the three AgB fractions was identical or similar to that of the 8/12 KDa fraction alone, indicating that the detection of the latter fraction is sufficient for the serodiagnosis of CE infection in humans. In conclusion, the ELISA is the test of choice for the serodiagnosis of CE and the follow up of cases following surgery using CSHF as an antigen. The IB test is a confirmatory test when antigen B fractions of CSHF or AAB are detected.
Genes for flagellin A (FlaA) proteins from European borrelial strains of Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii were cloned and sequenced. An identity of 92 to 93% was observed in the flaA sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in Escherichia coli and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from B. garinii and B. afzelii than against rFlaA from B. burgdorferi sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from Yersinia enterocolitica or beta-hemolytic Streptococcus infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.
The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnosis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based on the Falcon Assay Screening Test system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen.
Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.
A definitive diagnosis of neosporosis in cattle implies the examination of the aborted fetus. However, in many instances fetal material is not available. Therefore, most diagnosis are based on serological tests. At the moment, there are no consensuses about the cut-off for serodiagnosis of neosporosis in cattle, for any test. The objective of the present study was to estimate the best cut-off for the Neospora agglutination test (NAT) for serodiagnosis in cattle. For that purpose, 246 serum samples from 4 groups of dairy cows (aborted Neospora positive; not aborted healthy; aborted other diseases and herds endemic neosporosis) were collected and antibodies anti-N. caninum were determined by NAT. Additionally, immunoblot (IB) was performed with sera from all cows of the endemic neosporosis group and the patterns of seroreactivity were contrasted with the NAT titers for this group of cows. Evaluation of the optimized sensitivity and specificity was calculated using Youden's J-statistics. The best Youden's J-statistic was obtained at 1:40 titer, presenting 100% of sensitivity and 90.4% of specificity with negative and positive predictive values of 100 and 75.0%, respectively. The comparison between NAT titers and the IB banding pattern support the results of the statistical analysis, i.e. titers of 1:40 and higher showed a complex pattern of bands, while titers lower than 1:40 did not precipitate any bands. These results indicate that 1:40 was an optimized NAT cut-off for serodiagnosis in cattle.
The study concerns 100 patients who are divided in two dissimilar groups. The first group is composed of 39 patients who have signs of possible Yersiniosis infection. In second group composed of 61 cirrhotics patients, the serodiagnosis was systematic. The rate of positive serology, comparing with healthy patients, is significantly high in both groups: 61,5 p. 100 (first group) 64 p. 100 (second group). Six cases in the first group were very likely caused by Yersinia infection and confirm the utility of the serodiagnosis in front of evocative symptomatology. But many positive serodiagnosis remain unexplained and the greatest care must be taken in appreciation of these results. Among cirrhotic patients, the positive serology is not correlated with evocative symptoms and would be the result of asymptomatic bacteriemias.
The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.
Enzyme-linked immunosorbent assay (ELISA) with crude extracts of adult Clonorchis sinensis has been reported to have a high degree of sensitivity with a moderate degree of specificity for the serodiagnosis of clonorchiasis. The cystatin capture ELISA was investigated for its usefulness for the serodiagnosis of human clonorchiasis. Cystatin bound specifically to cysteine proteinases in crude extracts of adult C. sinensis worms, and its binding capacity was not hindered competitively by the other proteinase inhibitors tested. The cystatin capture ELISA for clonorchiasis showed a higher degree of specificity than the conventional ELISA, which produced some cross-reactivities to sera from patients with cysticercosis, sparganosis, and opisthorchiasis. Immunoglobulin G antibodies to C. sinensis cysteine proteinases were produced in experimental rabbits at week 3, and their levels increased rapidly and remained at a plateau after 8 weeks of infection. Of the proteins from the C. sinensis crude extract captured with cystatin, seven proteins were reactive with the serum from patients with clonorchiasis. The cystatin capture ELISA is indicated to be a sensitive and highly specific immunodiagnostic assay for serodiagnosis of human clonorchiasis.
Previously we reported the development of a highly sensitive enzyme-linked immunosorbent assay specific for anti-tuberculous glycolipid (anti-TBGL) for the rapid serodiagnosis of tuberculosis. In this study, the usefulness of an anti-TBGL antibody assay kit for rapid serodiagnosis was evaluated in a controlled multicenter study. Antibody titers in sera from 318 patients with active pulmonary tuberculosis (216 positive for Mycobacterium tuberculosis in smear and/or culture tests and 102 smear and culture negative and clinically diagnosed), 58 patients with old tuberculosis, 177 patients with other respiratory diseases, 156 patients with nonrespiratory diseases, and 454 healthy subjects were examined. Sera from 256 younger healthy subjects from among the 454 healthy subjects were examined as a control. When the cutoff point of anti-TBGL antibody titer was determined as 2.0 U/ml, the sensitivity for active tuberculosis patients was 81.1% and the specificity was 95.7%. Sensitivity in patients with smear-negative and culture-negative active pulmonary tuberculosis was 73.5%. Even in patients with noncavitary minimally advanced lesions, the positivity rate (60.0%) and the antibody titer (4.6 +/- 9.4 U/ml) were significantly higher than those in the healthy group. These results indicate that this assay using anti-TBGL antibody is useful for the rapid serodiagnosis of active pulmonary tuberculosis.
Procedurally similar competitive enzyme-linked immunoassay (cELISA) methods were developed for the serodiagnosis of Babesia equi and Babesia caballi (piroplasmosis), Trypanosoma equiperdum (dourine), and Burkholderia mallei (glanders) infections in horses. Apparent test specificities for the B. equi, B. caballi, T. equiperdum, and B. mallei cELISAs were 99.2%, 99.5%, 98.9%, and 98.9%, respectively. Concordances and kappa values between the complement fixation (CF) and the cELISA procedures for the serodiagnosis of B. equi, B. caballi, T. equiperdum, and B. mallei infections in experimentally exposed horses were 76% and 0.55, 89% and 0.78, 97% and 0.95, and 70% and 0.44, respectively. The cELISA method may be a technically more reproducible, objective, and convenient approach for piroplasmosis, dourine, and glanders serodiagnosis in qualifying animals for international movement and disease eradication programs than the CF systems currently in use. Use of the cELISA method also obviated the problems associated with testing hemolyzed or anticomplementary sera.
Protein microarrays offer the possibility to circumvent most of the current limitations in the serodiagnosis of allergy, autoimmune, and infectious disease by allowing the simultaneous, multiparametric determination of specific subclasses of antibodies directed against many pathogenic antigens. Microarray immunoassays have been developed with these characteristics. A first-generation assay, for the serodiagnosis of infectious disease, allows the determination of IgG and IgM antibodies to various viral and bacterial antigens. In addition, a second-generation assay, designed for the serodiagnosis of allergic disease, permits the determination of IgE antibodies to various allergens implicated in allergic disease. Slides printed with antibody dilution curves and antigen are first incubated with serum samples and then subsequently with secondary antibodies. For detection of human IgG and IgM, fluorescently labeled secondary antibodies are employed. However, because of low-level concentrations of circulating IgE antibodies, a more sensitive protocol is required for human IgE detection. Here, fluorescence is delivered via the coupling of the secondary antibody to tyramide signal-amplification reagentry. Human IgG, IgM, or IgE bound to the printed antigens can then be revealed by confocal scanning microscopy and quantified with internal calibration curves. Generation of analytical and clinical data have demonstrated that the microarray test format provides equivalent performance to enzyme-linked immunosorbent assay (ELISA) tests and offers a significant advantage in convenience and cost when compared to traditional test formats.
The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.
Early diagnosis and treatment for an immunocompromised patient with a deep-seated mycosis are very important for the prognosis of the patient. Several methods of serodiagnosis for deep-seated mycosis have been developed and are clinically available. Detection of (1,3)-beta-D-glucan from serum samples of a patient can be evaluated with high sensitivity and specificity. Antigens detection test for fungi is another useful method for this serodiagnosis. A novel sandwich ELISA to detect galactomannan has higher sensitivity than the latex agglutination test. The cryptococcus capsular antigen detection technique is also very useful for patients with cryptococcosis. Thus, serodiagnosis for deep-seated mycosis appears to be a useful tool for treatment of immunocompromised patients.
A successful modification of the indirect hemagglutination test to demonstrate antibodies for serodiagnosis of amebic liver abscess has been described in the present study. In the modified test, the protein A-IMA, Staphylococcus aureus (Cowan's strain I) bearing protein A (SAPA) cells were used to enhance hemagglutination of sensitized red cells. Use of SAPA cells markedly enhanced sensitivity of the test and greatly increased the titers obtained with most of the sera. At a diagnostic antibody titer of 1:128 and above, the protein A-IHA could detect 72 (100%) of amebic liver abscess (ALA) cases. Amongst the controls, no false positive reaction was observed in non-amebic liver disease controls. However 1(2%) of sera demonstrated false positive reactions from healthy controls. The protein A-IHA was highly sensitive when compared with that of the indirect hemagglutination (IHA) for serodiagnosis of amebic liver abscess. The novel immunoassay is simple, inexpensive and requires little technical skill. It has the potential for wide application in serodiagnosis of amebic liver abscess.
Serodiagnosis in current practice by the method ELISA. Utilization of commercial kits. I. - Rubella. Comparative study with hemagglutination inhibition test. A comparative study of the hemagglutination inhibition test and the method ELISA for the serodiagnosis of rubella in current practice has been achieved on 1 000 sera using commercial products in kits. The conditions of utilization, the principal factors of the reaction and the expression of results in ELISA titres are defined. The results show a higher sensibility and a higher precision for ELISA and an uncertain interpretation concerning the low titres for IHA. The use of ELISA for the serodiagnosis of rubella in routine with the kit Rubelisa does not present any particular difficulty and raises the quality of the results.
The excretory-secretory antigens collected from the axenic culture medium and conventional somatic antigen prepared from the whole trophozoites of E. histolytica were compared for their efficacy in serodiagnosis of amoebiasis. A total of 280 sera collected from different clinically proven cases of amoebiasis and healthy subjects were analysed against both the antigens in ELISA. Both antigens showed a 100 per cent correlation in amoebic liver abscess cases, patients infected with enteropathogens other than amoeba and healthy subjects. However, excretory-secretory antigens showed slightly higher detection rate in patients suffering from acute amoebic dysentery and asymptomatic cyst passers groups. These results clearly suggested the use of excretory-secretory antigens by ELISA for serodiagnosis of amoebiasis due to its better or equal sensitivity, specificity and easier preparation compare to conventional antigen. The use of excretory-secretory antigen in serodiagnosis will not only help in performing more tests utilizing the same chemicals, but also save the cost, time and troubles for importing the foreign chemicals required for cultivation of E. histolytica.
The general applicability of the new peptide immobilization strategy in which the peptide of interest is N-terminally extended with an acetyl-thio-acetyl group or (poly)-Lys extension during synthesis, has been demonstrated in epitope-mapping experiments and serodiagnosis. Ala-scanning experiments and minimal epitope determination showed that the antigenicity of Ata-extended peptides derived from the human chorionic gonadotropin (hCG) and hepatitis B virus (HBV) amino acid sequence, was superior to the free parent peptides. Further, it could be shown that the choice of the epitope-mapping procedure (peptide in solution or immobilized on a solid support) may lead to a different perception of which residues constitute the epitope. In addition, a time-consuming conjugation process could be circumvented since the ELISA reactivity of BSA-conjugates was comparable to that of Ata-extended peptides. In the serodiagnosis using sera from various HIV-positive individuals, the lysyl-peptide showed a signal/noise ratio 10 times higher than the parent peptide, indicating that sensitivity increased as a result of this N-terminal lysyl tail. In all experiments we observed that antibody detection could be performed at roughly 10 times lower amounts of peptide when N-terminally linked to an Ata-group or lysyl-extension compared to the free parent peptide or the BSA-conjugated equivalent.