Field evaluation of enzyme-linked immunosorbent assay for the serodiagnosis of tuberculosis

An enzyme-linked immunosorbent assay (ELISA) was evaluated as a serodiagnostic test for active tuberculosis in La Paz, Bolivia. ELISA was compared with sputum smear in 277 persons presenting to the Instituto de Torax and was used for screening in 1,458 military personnel. The test was performed under field conditions on 4-microliter samples of capillary blood obtained by finger prick. ELISA was found to have a sensitivity of 69% and a specificity of 88%. Sputum smear had a sensitivity of 79% and a specificity of 100%. ELISA was found to have undiminished sensitivity and specificity in patients who were sputum-negative, and the two tests could be combined to achieve a sensitivity of 92% and specificity of 88%. Positive and negative predictive values were highest for populations with tuberculosis prevalences in the range of that seen among patients presenting to the Instituto de Torax in Bolivia, but ELISA also led to the diagnosis of tuberculosis in 5 of 1,458 soldiers tested in the screening program.     

Multi-dot enzyme-linked immunosorbent assay for serodiagnosis of trematodiasis

A nitrocellulose membrane strip dotted with crude antigens of trematodes, such as Paragonimus miyazakii, P. westermani, Fasciola sp., Clonorchis sinensis and Schistosoma japonicum was applied for serodiagnosis of trematodiasis. With peroxidase conjugated anti-human IgG and 4-chloro-1-naphthol as a substrate, antibodies in trematodiasis patient sera were clearly detected on the strip as bluish purple spots. Densities of the spots correlated well with values obtained by standard micro-ELISA. Cross-reactions among the trematode antigens, which is occurred more frequently than by ELISA, were observed with this method. However, the darkest spot correlated with the infecting parasite in all cases. With the high reliability and advantage that crude antigens can be used, this simple method, multi-dot ELISA, is considered to be useful for serodiagnosis of trematodiasis.     

The serodiagnosis of tuberculosis by enzyme-linked immunosorbent assay with tuberculin purified protein derivative

Tuberculin purified protein derivative (PPD) is a relatively crude antigen prepared from Mycobacterium tuberculosis and has species nonspecificity in immunological reaction. It is, however, more readily available than more highly purified materials. Therefore, the detection of IgG antibody to PPD was done by enzymed-linked immunosorbent assay (ELISA) and its diagnostic useful was evaluated in this study. The patients with active tuberculosis had significantly high titer of IgG antibody to PPD compared with healthy persons and the patients without tuberculosis (P less than 0.001). An upper limit of normal set (=cut-off titer) at 2 standard deviations above mean of logarithmic titers in 220 healthy adult subjects would result in positive test reactions on the sera from 78 of 100 patients with active tuberculosis. Although 8 of 39 with atypical mycobacteriosis would be positive, 6 of 7 were distinguished almost with tuberculosis by detecting antibodies to PPD from M. intracellulare and M. kansasii concurrently. The antibody titer increased after chemotherapy would be gradually reduced under the cut-off titer when culture of mycobacteria turned to negative and markers of inflammation became negative. In false-negative cases, 4 were patients with hypo-gammaglobulinemia, 6 were with fresh tuberculosis before chemotherapy, 2 were with negative CRP in all clinical course and 4 were with bacilli needed over 7 weeks culture. From these results, this assay is helpful in the diagnosis of tuberculosis and a useful marker for judgment of clinical improvement, although detection of antibody has its limitations.     

Use of an enzyme-linked immunosorbent assay technique in the differential diagnosis of active pulmonary tuberculosis in humans

Sera from patients with active pulmonary tuberculosis and pulmonary diseases frequently mimicking tuberculosis were assayed for immunoglobulin G antibody activity to purified protein derivative (PPD) by an enzyme-linked immunosorbent assay. A method of standardization was developed to limit assay variation. Patients with active pulmonary tuberculosis had a significantly greater mean level of antibody than had patients with atypical tuberculosis (P = 0.005), sarcoidosis (P = 0.0001), histoplasmosis (P = 0.004), blastomycosis (P = 0.008), or cryptococcosis (P = 0.017), patients who had received bacille Calmette-Guérin vaccination (P = 0.003) or who had a history of treated tuberculosis (P = 0.003), and PPD skin test-positive and skin test-negative control subjects (P = 0.001). This technique may have potential use as a rapid diagnostic aid in evaluating patients with suspected active pulmonary tuberculosis.     

A recombinant envelope protein-based enzyme-linked immunosorbent assay for West Nile virus serodiagnosis

Recombinant West Nile virus envelope (E) protein was examined in enzyme-linked immunosorbent assay (ELISA) to detect antibodies elicited during West Nile virus infection. Horses (nine of 10) and humans (six of six) with confirmed West Nile virus infection had IgG and/or IgM antibodies to the E protein. Antibodies to the recombinant West Nile virus membrane and nonstructural 1 proteins were not detected in any of these sera. An E protein-based ELISA may aid in the serological diagnosis of West Nile virus infection.     

Micro enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of New World leishmaniasis

Of 21 confirmed cases of New World leishmaniasis, 16 exhibited antibody to antigens of the promastigote of Leishmania braziliensis panamensis by the enzyme-linked immunosorbent assay (ELISA). Comparison of antibody titers obtained by ELISA with titers obtained by indirect immunofluorescence using an amastigote substrate confirmed that the sensitivities of the two techniques were within the same range (r = 0.80). Although sera from patients with New World leishmaniasis failed to react with antigens extracted from epimastigotes of Trypanosoma cruzi, sera from 39 cases of Chagas' disease were reactive with promastigotes of L. braziliensis panamensis. This apparent unidirectional cross-reactivity has been attributed to differences in potency of the antigenic stimulus presented in the two diseases.     

Enzyme-linked immunosorbent assay using Mycobacterium tuberculosis antigen 5 and PPD for the serodiagnosis of tuberculosis

Enzyme-linked immunosorbent assays (ELISA) of immunoglobulin G antibody to Mycobacterium tuberculosis antigen 5 and tuberculin purified protein derivative (PPD) were assessed for the serodiagnosis of tuberculosis in 41 patients with active tuberculosis, 19 patients with inactive tuberculosis, and 59 healthy control subjects. Patients with active tuberculosis were studied serially at monthly intervals following the initiation of therapy. When contrasted with our earlier studies of sera from patients in Bolivia and Argentina, serum titers in Cleveland patients with active tuberculosis were somewhat lower. Geometric mean titer in patients with active tuberculosis was 1:68 with antigen 5 and 1:46 with PPD. Titer was correlated with patient age, male sex, extent of tuberculosis, and history of prior tuberculosis. However, these associations were not statistically significant. During monthly follow-up for 16 months after the initiation of therapy, ELISA titers remained essentially stable. Thus, no convincing evidence was acquired to support the hypothesis that higher titers in sera from South American patients related to more chronic or more extensive disease. Receiver operating characteristics of ELISA with antigen 5 were better than those obtained using PPD and were similar to those reported by others for sputum smear. In a situation where tuberculosis screening is warranted, ELISA with antigen 5 might have a place if it recognizes a different population than does sputum smear.     

Specific serodiagnosis with adult Onchocerca volvulus antigen in an enzyme-linked immunosorbent assay

The cross-reactivity of the blood from onchocerciasis, loiasis, and dipetalonemiasis was tested by a micro-ELISA technique, utilizing adult Onchocerca volvulus antigen and blood samples taken on filter paper. The average ELISA values (OD at 500 nm) were as follows: 0.58 in persons with O. volvulus microfilariae (n = 81), 0.49 in microfilariae-negatives from the same endemic area (n = 39), 0.15 in dipetalonemiasis (n = 27), and 0.25 in loiasis (n = 12), while those of 65 Dipetalonema perstans-negative people were markedly low (average 0.14) and that of 22 Loa loa-negatives, 0.22, respectively. This ELISA could successfully differentiate onchocerciasis from dipetalonemiasis and loiasis.     

Usefulness of whole-blood interferon-gamma assay and interferon-gamma enzyme-linked immunospot assay in the diagnosis of active pulmonary tuberculosis

PURPOSES: The aim of this study was to evaluate the usefulness of the whole-blood interferon-gamma assay (enzyme-linked immunosorbent assay [ELISA]) and interferon-gamma enzyme-linked immunospot assay (ELISPOT) based on early secretory antigenic target 6 and culture filtrate protein 10 in the diagnosis of active pulmonary tuberculosis (TB) in routine clinical practice. METHOD: We conducted a prospective study enrolling 144 participants with suspected pulmonary TB in a tertiary referral hospital in Seoul, South Korea, to investigate the diagnostic sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of these tests. Clinical assessment, tuberculin skin test (TST), whole-blood interferon-gamma ELISA (QuantiFERON-TB Gold [QFT-G]; Cellestis Ltd; Victoria, Australia), and an ELISPOT assay (T SPOT.TB; Oxford Immunotec; Oxford, UK) were performed. Test results were compared with the final confirmed diagnoses. RESULTS: Active pulmonary TB was diagnosed in 67 of 144 participants (47%). Sensitivities of QFT-G and T SPOT.TB for active pulmonary TB were 89% (95% confidence interval [CI], 79 to 96%) and 92% (95% CI, 83 to 97%), respectively; and specificities were 49% (95% CI, 37 to 61%) and 47% (95% CI, 36 to 59%). NPVs of QFT-G (84%; 95% CI, 69 to 93%) and T SPOT.TB (87%; 95% CI, 73 to 96%) were higher than that of TST (64%; 95% CI, 51 to 76%) [p = 0.001 and p < 0.001, respectively]. CONCLUSION: High NPVs of QFT-G and T SPOT.TB for the diagnosis of active TB suggest the supplementary role of these tests for the diagnostic exclusion of active TB, although the low PPV limits their usefulness in routine clinical practice in South Korea, where the prevalence of latent TB infection is considerable.     

Antibody-based enzyme-linked immunosorbent assay for determination of immune complexes in clinical tuberculosis

Immune complexes were isolated from sera of tuberculosis patients by precipitation with 2.5% polyethylene glycol. The precipitates were characterized by quantitative determination of different immunoglobulin classes by single radioimmunodiffusion, sodium dodecyl sulfate polyacrylamide gel electrophoresis for presence of serum components, Ouchterlony's double diffusion method for detection of complement components C3 and C4, and immuno-dot assay for detection of Mycobacterium tuberculosis antigens. The results showed that polyethylene glycol precipitates of patients' sera were indeed immune complexes, as they contained immunoglobulins, albumin, complement components, and mycobacterial antigens, whereas precipitates from control sera contained mainly albumin. The antibodies present in immune complexes were specific to M. tuberculosis antigen and showed no binding to Escherichia coli antigens. Immune complex levels, as determined by the ability to bind M. tuberculosis antigens in an enzyme-linked immunosorbent assay, were significantly higher in tuberculosis patients (n = 22) than in healthy control subjects (n = 18). Thus, immune complex level could be a useful parameter in the diagnosis of active tuberculosis.     

A novel fusion protein-based indirect enzyme-linked immunosorbent assay for the detection of bovine tuberculosis

Enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine tuberculosis has been widely explored over the years. Three Mycobacterium bovis-specific antigen genes, namely, mpb70, mpb83, and esat-6 were recombined in tandem by spliced overlap extension technology and expressed in Escherichia coli to obtain the fusion protein (rM70-83-E6). Western blot analysis showed that rM70-83-E6 can specifically react with bovine tuberculosis-positive sera but not those from cattle infected with other bovine diseases such as bovine paratuberculosis. An indirect ELISA (iELISA) method was established with rM70-83-E6 as the diagnostic antigen. The diagnostic criteria were determined using 150 serum samples from healthy cattle. Analyses of 85 serum samples from cattle with bovine tuberculosis and 100 serum samples from healthy cattle demonstrated that the sensitivity of the iELISA was 69.4% (59/85) and the specificity was 96.0% (96/100). Moreover, 46 out of 67 purified protein derivative (PPD) skin test-positive samples were also positive by iELISA, giving a positive coincidence of 68.7%, while all 50 PPD skin test-negative samples were negative by iELISA, giving a negative coincidence of 100%. The total coincidence between iELISA and the PPD skin test was 82.1%. This study demonstrated that iELISA using rM70-83-E6 antigen is simple, sensitive and easy to perform and can be used to analysis of a large number of samples for serodiagnosis of bovine tubercuiosis.     

Enzyme-linked immunosorbent assay for the serodiagnosis of Campylobacter pylori infection

Antibody activities to campylobacter pylori in serum were estimated by an enzyme-linked immunosorbent assay (ELISA) to crude antigens, prepared by sonication of whole organisms obtained from bacterial culture in 100 patients with chronic gastritis. Significantly raised serum IgG antibody activities to C. pylori was found in colonised patients with gastritis, especially in patients with active gastritis. High activities were also found beyond the age of 30. In 6 patients cleared of C. pylori with furazolidone and/or colloidal bismuth subcitrate (De-NoL), serial testing has shown a fall in activity to normal level by 1/2 year in 4 patients. The specificity and sensitivity of the sero-diagnostic assay was 85.3% and 97% respectively. The positive and negative predictive values were 92.8% and 93.5% respectively. The results indicate that such a serodiagnostic assay could be used to screen patients with C. pylori colonisation in epidemiological surveys.     

A competitive enzyme-linked immunosorbent assay for diminazene

Diminazene aceturate has remained a very important therapeutic drug for trypanosomosis in cattle, sheep and goats since its introduction into the market in 1955. Despite its continued use, the methods available for its detection in body fluids are lengthy and inefficient for routine monitoring of drug levels in treated animals. A competitive enzyme linked immunosorbent assay (ELISA) has now been developed and optimized for the detection of diminazene in bovine serum. In the assay, diminazene in the test samples and that in a newly developed diminazene-horseradish peroxidase conjugate compete for antibodies to diminazene raised in rabbits and immobilized on a microtitre plate. Tetramethylbenzidine-hydrogen peroxide (TMB/H(2)O(2)) is used as chromogen-substrate system. The assay has a detection limit of 0.8 ng/ml of serum with a high specificity for diminazene. Cross-reactivity with either homidium bromide and quinapyramine sulphate/chloride of 0.0004% is negligible while that with isometamidium chloride is 0.71%. The assay was able to detect diminazene levels in normal Boran steers for at least two weeks after intramuscular injection with the drug at a dose of 3.5 mg/kg bw. The assay will be useful in monitoring diminazene use, and development of resistance in trypanosomosis endemic areas.     

Serologic diagnosis of bone and joint tuberculosis by an enzyme-linked immunosorbent assay

Sera from patients receiving treatment for active bone and joint tuberculosis and sera from patients with inactive bone and joint tuberculosis were examined by an enzyme-linked immunosorbent assay for antibody to antigen 6, a homogeneous protein prepared from Mycobacterium tuberculosis strain H37Ra by immunosorbent affinity chromatography. Sera from 21 control subjects had a geometric mean titer of 1:6 with no difference between tuberculin purified protein derivative-positive and -negative patients. Sera from 20 patients with inactive disease had a geometric mean titer of 1:19. Fifteen patients receiving treatment for M. tuberculosis infection had a geometric mean titer of 1:179, which is significantly different from the geometric mean titers of both of the patients with inactive tuberculosis (P less than 0.001) and the control subjects (P less than 0.001). At a cut-off titer of 1:32, the sensitivity of the assay is 94% and the specificity for the control subjects and patients with inactive disease was 100%.     

Application of the enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human African trypanosomiasis (sleeping sickness).

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human African trypanosomiasis (sleeping sickness) is described. A crude extract of a Trypanosoma brucei suspension which was purified from all blood components was used as antigen. In rabbits experimentally infected with T. brucei or T. rhodesiense both homologous (anti-T. brucei) and heterologous (anti-T. rhodesiense) Trypanosoma antibodies could be detected with ELISA using T. brucei as antigen. The sensitivity of ELISA was comparable with that of the immunofluorescence (IF) technique. Sera of patients with sleeping sickness were examined with ELISA and IF. It proved possible to discriminate between groups of individuals with and without trypanosomiasis. Cross reactions were only observed with serum from a patient in which antibodies to Leishmania were detected. No cross reactions were observed in sera from patients with malaria, toxoplasmosis, schistosomiasis, or echinococcosis. ELISA represents a good alternative to IF in the serology of African trypanosomiasis, and may be particularly suitable for mass screening purposes.     

结核杆菌特异性酶联免疫斑点技术在HIV感染人群中的应用

我国艾滋病病毒(HIV)感染人群中结核病的发病率高,造成了严重的疾病负担。提高潜伏结核和活动性结核的诊断准确性,对控制HIV病人中结核病的治疗和控制至关重要。该文总结了近年来出现、发展并完善的一种新的结核诊断技术,即结核杆菌特异性酶联免疫斑点技术,及其在HIV人群中的相关研究结果,并着重对该技术在潜伏结核和活动性结核的诊断、治疗效果监测以及结核相关性免疫重建炎症反应综合征中的应用进行了评价。     

Dot enzyme immunosorbent assay for the serodiagnosis of typhoid fever.

A nitrocellulose membrane strip dotted with a specific 50 kDa outer membrane protein of Salmonella typhi was applied for the serodiagnosis of typhoid fever. Using horseradish peroxidase conjugated IgM and IgG antibodies with 4-chloronaphthol as substrate, antibodies in typhoid patients were clearly visualised as bluish purple dots while sera from patients with non-typhoid fevers gave negative results. The detection of specific IgM and IgG antibodies in typhoid patients suggest either recent or current infection. Combined with the high specificity, reliability and rapidity of the test, the dot EIA technique provides a simple and useful method for the serodiagnosis of typhoid using a single serum specimen.     

An enzyme-linked immunosorbent assay for platelet compatibility testing

The selection of platelet donors for patients who are refractory to random donor platelets often presents a difficult clinical problem. We describe an enzyme-linked immunosorbent assay (ELISA) for evaluating alloantibodies in refractory patients. Platelets from prospective donors are immobilized on microtiter plates and, after incubation with test serum and washing, platelet-bound IgG is detected with enzyme- linked anti-human IgG. Platelets from 46 prospective donors were tested. Twenty-two were judged compatible (reciprocal of the antibody titer less than 16) and, of these, 15 were used as platelet donors; each gave a measurable platelet increment after transfusion. The magnitude of the response was roughly proportional to the assay results. Platelets from donors giving antibody titers less than 4 resulted in platelet increments at 1 hr ranging from 4,890 to 22,200 (median 12,600), while platelets from donors giving titers of 8 or 16 resulted in lesser increments (550-4548). Conversely, 5 of the 24 patients found incompatible by the assay (titer greater than 16) gave no platelet increment, and in 3 instances, the recipient developed fever and chills after the transfusion. The assay is sensitive, simple, and adaptable to the clinical laboratory. Platelets from volunteer donor panels can be plated and stored for up to 6 mo.