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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.
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.
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.
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.
The rapid plasma reagin (RPR) card test manufactured by Beckman Instruments, Inc., was compared, qualitatively and quantitatively, with the Venereal Disease Research Laboratory (VDRL) slide test and the standard RPR 18-mm circle card tests for the serodiagnosis of syphilis. Sera from 638 individuals were used in this study. Two pilot lots and two production lots of antigen were submitted by Beckman Instruments, Inc., for evaluation. Qualitative agreement among the three RPR card tests was 98.1%; between the Beckman RPR card and the VDRL slide tests, 95.0%; and between the reference RPR card and the VDRL slide tests, 95.5%. The Beckman RPR card test was 95.3% specific, whereas the specificities of the reference RPR card and the VDRL slide tests were 98.8% and 96.1%, respectively. Sensitivities of the three nontreponemal tests were: Beckman RPR card test production lots, 94.7%; reference RPR card test, 96.8%; and VDRL slide test, 90.6%. Quantitative agreement +/- 1 dilution among the three RPR card tests was 93.0%, whereas quantitative agreement was approximately 40% when both RPR card tests were compared with the VDRL slide test. We found the Beckman RPR card test comparable to the standard RPR card tests. Therefore, the decision of which test to use for the serodiagnosis of syphilis is at the discretion of the user.
A home-made ELISA for serodiagnosis of HIV-infection was developed. It made use of the HIV viral lysate to coat on ELISA microplates. The purpose was to establish an ELISA for serodiagnosis of HIV-infection. The newly-developed ELISA, "H-ELISA", was applied to test 792 samples of HIV-positive serum as confirmed by Western blot. All 792 samples were positive by H-ELISA. It was also applied to test 540 samples of normal sera obtained from different laboratories in Srinagarind Hospital. A number 530 normal sera was negative, 8 samples were positive and confirmed by Western blot and 2 samples were false positive. It was concluded that the H-ELISA possessed 100% sensitivity with a false positive rate of 2/532(0.38%). The H-ELISA, the cost/test was less than 5 bahts, appears to be promising for substitution of imported commercial kits.
Western blot is used worldwide as a confirmatory assay after a positive or doubtful ELISA result in the serodiagnosis of HIV infections. Despite the use of this test some results may be not definitive due to the presence in Western blot of only few or a single band to HIV protein. This result has been defined as indeterminate. To resolve indeterminate results obtained with conventional Western blotting (WB) we used radio immune Western blotting (RIWB), a method capable of detecting antibodies to HIV-1 proteins with greatly increased sensitivity with respect to conventional WB. We used RIWB to perform a retrospective survey on 20 sera belonging to individuals undergoing HIV serodiagnosis (not blood donors) with positive or borderline EIA results, and indeterminate WB results who only later fully seroconverted. Fifteen of the sera tested by RIWB showed a positive result which clarified any doubtful indeterminate WB result; all bands present in conventional WB were enhanced in intensity using RIWB and new bands, absent in the WB assay, were highlighted using RIWB. RIWB may resolve most indeterminate WB results.
Demonstration of Mycobacterium tuberculosis in a smear or culture is the most reliable method for diagnosing tuberculosis (TB). In the last 10 years, several enzyme-linked immunosorbent assays (ELISAs) based on mycobacterial antigens (such as antigen 60, 38 kDa antigen, and antigen Kp90) have been used for the rapid diagnosis of TB. In this study, we report the isolation of an immunodominant lipopolysaccharide (LPS) antigen from M. tuberculosis H(37)Rv, which can be used for the serodiagnosis of TB. The LPS antigen was compared with three commercially available mycobacterium-specific antigens for the detection of TB. The antigens were evaluated using serum samples obtained from 59 Indian patients (19 patients with active pulmonary TB, 20 with extrapulmonary TB, and 20 with nontuberculous pulmonary disease) and 20 healthy adults. Antigen 60 IgG (sensitivity 89%, specificity 97%) and LPS (sensitivity 84%, specificity 97%) were more sensitive and specific than 38 kDa antigen IgG (sensitivity 79%, specificity 97%) and Kp90 IgA (sensitivity 82%, specificity 40%). These results indicate that the LPS antigen can be used as a sensitive tool for the serodiagnosis of TB and could be utilized to develop an ELISA for the screening of patients for TB.
A stabilized modification of the single radial complement fixation (SRCF) test in gel was developed for detecting various virus antibodies. The principle of the test is based on the use of a single-stage procedure with an agarose plate containing virus antigens and antibody-coated erythrocytes, and thin plastic film coated with complement. By filling the wells in the agar plate with a 1:4 diluted heat-inactivated sera and covering the agar surface with a complement film, a zone of unlysed cells surrounded by a hemolytic area appears after incubation overnight at 4 degrees C and then for 1-2 h at 37 degrees C, depending on the antibody titers. The SRCF antibody titer is calculated numerically from the square of the diameter of the unlysed cell zone. The stability of reagents could be significantly improved using thin complement film and several stabilizers. When this test was used for serodiagnosis of influenza, respiratory syncytial (RS), mumps, adeno virus type 3 and herpes simplex type 1 virus infections (using a total of 400 sera), excellent correlations were demonstrated for antibody titers between conventional complement fixation (CF) and SRCF titers. Furthermore, the expression of antibody titer as an SRCF unit with consecutive value, produced results sensitive to fluctuations in the antibody titers. The simplicity of the procedure, stability of the reagents, and excellent correlation with the conventional CF test might make this a useful test for routine serodiagnosis and seroepidemiological survey of various virus infections.
At present there are no accepted criteria to assess the usefulness of Western blot assays for the serodiagnosis of aspergilloma. An Aspergillus fumigatus cytosolic fraction complex (CFC) composed of four proteins (p90, p60, p40, and p37) has been identified. The usefulness of Western blotting with CFC antigens for the serodiagnosis of aspergilloma was evaluated in 25 patients with well-established diagnoses and in 94 controls. The most consistently reactive antigen was p90 (92% of patients with aspergilloma), followed by p40 (76%) and the entire CFC taken together (76%). With these data, interpretive criteria for positive and negative immunoblots were established, with p90 indicated as a helpful marker of aspergilloma.