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PubMed1997

Serodiagnosis of alveolar echinococcosis: detection of antibody against EM18 in patients and rodents.

An international collaborative study on echinococcosis has been carried out for the establishment of a simple means for differential serodiagnosis of alveolar echinococcosis (AE) from other parasitic diseases including cystic echinococcosis (CE). The main candidate epitope is Em18 (previously undescribed epitope of a low molecular weight protein of 18.5 kDa). Evaluation of the usefulness of Em18 is introduced in this review paper. Serum samples showing antibody response against Em18 are exclusively from AE. The predominant IgG subclass recognizing Em18 is IgG4 or IgG1 or IgG4 + IgG1 but never IgG2. There are good correlations between (1) the antibody response against Em18 and the presence of active lesions and (2) the antibody response against Em18 and the Em2-ELISA values. Em18 is, therefore, expected to be reasonably reliable and useful for differentiation of active AE from inactive AE. A new ELISA system using a partially purified Em18 enriched fraction (PP-Em 18/16-ELISA) has been evaluated for serodiagnosis of AE compared with Em2plus-ELISA. A total of 194 serum samples were examined: 127 sera from AE (79) and CE (48) in China where both AE and CE are endemic, 21 sera from CE in Australia where CE only exists, 28 sera from cysticercosis (21), paragonimiasis (5) or sparganasis (2) in Korea where no indigenous AE nor CE exists and 11 normal sera. Antibody levels by PP-Em18/16-ELISA were much higher in AE than in CE and it was also true for commercially available Em2plus-ELISA. Some of CE from China showed exceptionally higher levels of antibody in comparison with those of CE from Australia. It is suggested that these strongly positive cases of CE from China may have been exposed to both species of Echinococcus. Although most of sera from paragonimiasis showed high antibody levels by Emplus-ELISA, they were negative by PP-Em18/16-ELISA. Therefore, PP-Em18/16-ELISA is expected to be more reliable for differentiation of AE from CE and others especially in Asian countries where paragonimiasis is still not rare. Antibody responses in rodents naturally infected with E. multilocularis: Serum samples from the wild vole, Clethrionomys rufocanus bedfordiae, infected with E. multilocularis showed similar antibody responses as in AE patients, whereas those from Norway rats, Rattus norvegicus, showed almost none. The latter rodents were simultaneously infected with Taenia taeniaeformis but showed no antibody response against T. taeniaeformis either. Therefore, we speculate that Norway rats may only be infected with E. multilocularis under some immunosuppressed conditions or genetic unresponsiveness. It is stressed that Em18 is highly specific to E. multilocularis, and antibody response against Em18 is reasonably reliable for differentiation of AE from other helminthic infections by Western blot and ELISA in humans and may be useful for detection of domestic animals contaminated with E. multilocularis in the endemic area.

Animals↗

PubMed1995

[The evaluation of antigens and antisera for their use in the serodiagnosis of aspergillosis].

Aspergillosis became an important opportunistic mycosis during the last years, with a great variety of clinical manifestations. To contribute to the replenishing of this mycosis serodiagnosis, biologic reactives (antigens and antisera) were prepared from strains of the species Aspergillus niger, Aspergillus flavus and Aspergillus terreus for their use in immunoprecipitation assays. The reactives were assessed by double immunospreading versus a reference commercial system; satisfactory results were obtained, and this guarantees the widening of the aspergillosis serodiagnosis in the Mycology Laboratory of the "Pedro Kourí" the Tropical Medicine Institute (IPK) with important imports savings.

Animals↗

PubMed1997

Advantage of dimeric peptide antigens in serodiagnosis of HIV-1 infection.

The reactivity of antibodies with dimeric and monomeric peptide antigens was compared by ELISA. A panel of highly purified synthetic peptides of HIV-1 representing defined regions, 598-609 and 524-533 (fusion domain) of gp41 and 306-320 of gp120, were used as antigens in the ELISA. These peptides were selected and synthesized taking into account the level of sequence conservation of various strains and hydrophilicity. The analysis included sera from 52 HIV-1 infected individuals and 53 HIV-1 negative controls. Both peptides from gp41 were found to be particularly immunoreactive with sera from HIV-1 infected individuals. The frequency of reactivity to the selected peptide from gp120 (V3 loop) in infected individuals was 82%. An interesting observation was that the dimeric peptide antigens had a detection rate more than 4-fold higher than the monomeric antigens. We found that lower levels of antibodies could be detected with dimeric antigens. The peptides reacted with few sera other than HIV-1 positive sera. These results implicate the potential dimeric peptide antigens to be utilized in the serodiagnosis of HIV-1 infection.

AIDS Serodiagnosis↗

PubMed1982

Serodiagnosis of syphilis by an enzyme-linked immunosorbent assay for IgG antibodies against the Reiter treponeme flagellum.

An enzyme-linked immunosorbent assay for IgG antibodies against the flagellum (axial filament) of the Reiter treponeme (flagellum-ELISA) was developed and compared with the fluorescent treponemal antibody absorption (FTA-ABS) test and the Treponema pallidum immobilization (TPI) test with regard to diagnostic sensitivity and specificity. One serum from each of 827 individuals with and without syphilis was studied. In all diagnostic groups of syphilis there was no significant difference between the sensitivity of the FTA-ABS and the flagellum-ELISA, except in treated syphilis, where the FTA-ABS was more sensitive (P less than 0.01). In primary syphilis and in treated syphilis the sensitivity of the flagellum-ELISA was higher than the sensitivity of TPI (P less than 0.01 and P less than or equal to 0.05), respectively); in all other groups there was no significant difference between the sensitivity of TPI and flagellum-ELISA. The specificity of the flagellum-ELISA (99.0%) in 200 sera from blood donors without syphilis was not statistically different from the specificity of FTA-ABS (98.0%) and TPI (99.5%). The flagellum-ELISA seems to be well suited for routine serodiagnosis of syphilis and may replace other treponemal tests.

Antigens↗

PubMed1983

EDTA-treated plasma in the rapid plasma reagin card test and the toluidine red unheated serum test for serodiagnosis of syphilis.

Both the rapid plasma reagin (RPR) 18-mm circle card test and the toluidine red unheated serum test (TRUST) were designed to be more rapid than the Venereal Disease Research Laboratory slide test for the serodiagnosis of syphilis. The RPR card test, although originally designed as the teardrop card test for use with plasma, has received recognition from the Centers for Disease Control as a standard test for syphilis for use with serum samples only. In this study, 132 EDTA plasma-serum pairs were tested in both the RPR card test and TRUST immediately after the blood was drawn and the serum or plasma was separated from the cellular constituents of the blood. The plasma samples were further tested at 24-h intervals of storage at 25 and 4 degrees C for 72 h. Special attention was paid to any increase in roughness in nonreactive specimens. The greatest increase in roughness (16.7%) was seen with the TRUST after 72 h at 25 degrees C. An additional 174 plasma-serum pairs were tested after 18 h of storage at 25 degrees C. Comparisons of the results of the 306 serum specimens with their corresponding plasma pairs gave 99.0% qualitative agreement for both tests. Quantitative agreement +/- 1 dilution between serum-plasma pairs tested within 18 h was 92.1% (35 of 38) for the RPR card test and 94.1% (32 of 34) for the TRUST. Our results with plasma from blood drawn with EDTA as the anticoagulant were comparable with results with serum specimens for both tests when plasma specimens were tested within 18 to 24 h.

Antibodies↗

PubMed1989

Visuwell Reagin, a non-treponemal enzyme-linked immunosorbent assay for the serodiagnosis of syphilis.

A urease-based enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of reagin antibodies in serum. Visuwell Reagin (ADI Diagnostics Inc., Rexdale, Ontario, Canada) is a non-treponemal screening test for the serodiagnosis of syphilis which has the benefits of large batch testing, automatability, and objective interpretation of results. Unheated, undiluted sera are incubated in 96-well microtiter plates coated with a modified cardiolipin-lecithin-cholesterol antigen. Antibody bound to the plate is detected by an anti-human immunoglobulin G-urease conjugate. The procedure consists of three steps, with a total test time of 60 min. Visuwell Reagin ELISA was compared with the Venereal Disease Research Laboratory (VDRL) test and the reagin screening test (RST) with the following results. For ELISA versus the VDRL test, the sensitivities for untreated syphilis (n = 37) were 97.3% for both ELISA and the VDRL test, the confirmatory positive values (n = 79) were 84.8% for ELISA and 72.2% for the VDRL test, and the specificities for normal samples (n = 1,327) were 98.8% for ELISA and 99.5% for the VDRL test. For ELISA versus RST, the sensitivities for untreated syphilis (n = 57) were 94.7% for ELISA and 87.7% for RST, the confirmatory positive values (n = 26) were 96.2% for ELISA and 92.3% for RST, and the specificities for normal samples (n = 1,891) were 99.6% for ELISA and 99.3% for RST. The overall concordance values of ELISA with VDRL test and RST were 96.7 and 97.9%, respectively. The specificity of ELISA compared with that of RST may be underestimated, since confirmatory data were not available for all apparent false-positive samples. Visuwell Reagin had increased sensitivity and similar specificity compared with flocculation tests.

Antibodies↗

PubMed1975

Application of the enzyme-linked immunosorbent assay (ELISA) in the serodiagnosis of syphilis.

The enzyme-linked immunosorbent assay (ELISA) technique, using an ultrasonicate of Treponema pallidum as antigen, has been evaluated as a serological test for syphilis. It is concluded that the test is simple, reliable, and relatively quick and that its sensitivity in all stages of syphilis is equal to the FTAABS test. Because its specificity is probably also high, ELISA might be used in future as a first-line screening test for the serodiagnosis of syphilis.

Adsorption↗

PubMed1992

Advantages of branched peptides in serodiagnosis. Detection of HIV-specific antibodies and the use of glycine spacers to increase sensitivity.

The reactivities of antibodies with branched and monomeric peptides were compared in ELISA assays. We found that lower amounts of antibodies could be detected with branched peptides than with monomeric peptides. This was observed with a monoclonal antibody and with antibodies in the sera of various HIV-positive individuals. To investigate the physical aspects of branched peptides important for the observed increase in sensitivity, glycine spacers of different lengths were introduced between the branched lysine core and the epitope reacting with the monoclonal antibody. The effect of the number of glycine residues, both on the sensitivity of antibody detection and on the amount of branched peptide needed to produce a given signal, was studied and the optimum was found at 4-5 residues. We discuss the basis for these findings and conclude that the routine use of branched peptides for serodiagnosis will give both greater sensitivity and appreciable cost savings.

AIDS Serodiagnosis↗

PubMed1991

[The use of the indirect hemagglutination reaction for the serodiagnosis of HIV infection].

An assay system, based on the passive hemagglutination test and permitting the serodiagnosis of HIV infection with correct results in more than 99% of cases, has been developed. Three kinds of freeze-dried erythrocyte diagnostica (with shelf life exceeding 6 months), possessing high serological activity and sensitized with recombinant gene-engineering polypeptides, have been obtained. The proposed assay system is highly promising for mass examination of sera for the presence of antibodies to HIV due to the simplicity of assay techniques, the possibility of storing the diagnostica within a wide range of temperatures (4 degrees-30 degrees C) and obtaining results in a short time (3 hours).

AIDS Serodiagnosis↗

PubMedDate not supplied

[Detection and quantitative determination of antigen to the human immunodeficiency virus during development of a test-system for the serodiagnosis of AIDS].

One of the variants of IFA using a conjugate on the basis of anti-HIV-IgG was used for control of the content of human immunodeficiency virus (HIV) antigen at different stages of production of a test system for serodiagnosis of AIDS. This method permits HIV antigen quantitation in virus lysates, its nonpurified concentrates, and in native culture fluid in titres from 2000 to 60,000 and from less than 27 to greater than 729, respectively. In most cases, high titres of antigen in the liquid fraction of the culture corresponded to high values of the antigen-containing cells on the basis of immunofluorescence data and signs of intensive HIV production in electron microscopic culture control. The authors developed a method allowing to determine the antigen titre in the study material using only one dilution (1:9) of it in IFA technique.

AIDS Serodiagnosis↗

PubMedDate not supplied

[Extraction of the soluble protein fraction from Treponema Reiter for use as antigen in syphilis serodiagnosis].

The present paper reports on two methods for extraction of the protein antigen from Treponema Reiter and shows the chemical and immunochemical characteristics of the extracts. Serologic testing of 1810 sera from syphilitic patients in various stages of the disease, from healthy subjects and from subjects with a false positive reaction to lipoid antigens, showed the value of the Reiter antigen to be equal to the cardiolipin antigens and those prepared from pathogenic Treponema. It is recommended to associate this antigen, in view of its qualities, to the cardiolipin tests used in the serodiagnosis of syphilis, especially in the laboratories that do not currently apply other treponema tests.

Antigens↗

PubMedDate not supplied

[Passive hemagglutination test in the serodiagnosis of syphilis].

Passive haemagglutination test with pathogenic Treponema pallidum antigen in the serodiagnosis of syphilis, carried out on 752 sera (518 syphilitic patients and 234 subjects without evidence of syphilis) showed a greater general sensitivity as compared the cardiolipin tests and complement fixation test with Reiter protein antigen, and lower, to immunofluorescence (FTA--ABS). Its specificity is relatively lower than that of the cardiolopin and treponemal tests. Associated with the cardiolipin test it may be preferably used for the detection of syphilis, and moreover, passive haemagglutination may confirm the diagnosis in case of weak positive results with the FTA--ABS test. The latter is, however, essential for problem cases.

Antigens, Bacterial↗

PubMed1997

The use of radioimmune western blotting to evaluate indeterminate western blotting results in blood donor HIV serodiagnosis.

The serodiagnosis of HIV infection is sometimes puzzling due to questionable "indeterminate" results obtained by western blotting (wb), the confirmatory test used world-wide after a positive or borderline ELISA result. These situations cause anxiety in the person being tested and determine additional laboratory costs. We showed that radioimmune western blottting (riwb) an improved and sensitive modification of conventional wb, was able to resolve most "indeterminate" results in individuals who later fully seroconverted. We report in this paper the use of riwb in sera of 20 uninfected blood donors with an indeterminate wb continuing for at least 6 years, to verify the serological status in indeterminate not at risk individuals. Thirteen out of the 20 were indeterminate with conventional wb due to the presence of antibodies to p 24, two to p 17, two to p 55, two to p 51 and p 55 and one to p 24 and p 51 antibodies. The presence of all these bands was confirmed using riwb; moreover, in comparison with wb, the intensity of all bands was clearly enhanced. Despite the use of this highly sensitive method no new bands were found with riwb in indeterminate sera; negative results were obtained when sera borderline or positive with ELISA but negative with wb, were subjected to riwb.

AIDS Serodiagnosis↗

PubMed1996

Enzyme-linked immunosorbent assay using recombinant antigens for serodiagnosis of Japanese encephalitis.

Recombinant Japanese encephalitis (JE) virus proteins were evaluated as antigens for serodiagnosis of JE using an enzyme-linked immunosorbent assay (ELISA). The premembrane/membrane (prM/M) and envelope (E) proteins of JE virus were expressed in HeLa cells infected with a recombinant vaccinia virus that encodes the JE virus prM and E genes and were released from cells in a particulate form. The particulate antigens were partially purified from culture fluid from the infected cells by precipitation of particles with polyethylene glycol and then dissociated from the particles with 0.1% Triton X-100. This antigen preparation was used to evaluate one preimmune and two postvaccination sera from 20 volunteers given three inoculations of the commercial JE vaccine (Biken vaccine) by a conventional ELISA. The results from this assay correlated with neutralization data. The results of an IgM capture ELISA carried out with the recombinant antigen also correlated with the results of an existing IgM capture ELISA performed with JE virus-infected mouse brain, when tested with 29 serum and 13 cerebrospinal fluid samples from JE patients. These results indicated that recombinant JE virus antigens are useful for ELISA as an antigenically equivalent, highly productive, and safe alternative to authentic JE virus antigens.

Animals↗

PubMed2003

Serodiagnosis of human plague by a combination of immunomagnetic separation and flow cytometry.

BACKGROUND: Plague is a severe, highly communicable bacterial disease caused by Yersinia pestis. It is still endemic in more than 20 countries worldwide. Although known as a devastating disease for centuries, laboratory confirmation of clinical suspected cases is still problematic. No standardized and internationally approved test system is commercially available. The aim of this study was the introduction and evaluation of a combination of immunomagnetic separation and flow cytometry for the serodiagnosis of human plague. METHODS: Paramagnetic polystyrene beads were coated with purified F1 capsular antigen (F1 CA) and reacted with sera from plague patients, from 26 laboratory personnel vaccinated against plague and from 102 healthy blood donors (HBD). After incubation with fluorescein isothiocyanate-conjugated anti-human rabbit IgG, particle-associated fluorescence was detected by flow cytometry. RESULTS: Anti-F1 CA antibodies could be demonstrated in all patients with bacteriologically confirmed plague and in 22 sera (84.6%) from vaccinees. Only one serum in the HBD group showed a weakly positive reaction. The total assay time was less than 2 h. CONCLUSIONS: Compared with a recently published combination of an anti-F1 CA enzyme-linked immunosorbent assay (ELISA) and immunoblot, the new assay showed the same sensitivity as the ELISA and almost the same specificity (99.0 versus 100%) as the immunoblot. Allowing a rapid, reliable, and quantitative analysis, immunomagnetic separation combined with flow cytometry might replace both conventional immunoassays.

Antibodies↗

PubMed1992

Improved serodiagnosis of non-A, non-B hepatitis by an assay detecting antibody to hepatitis C virus core antigen.

We examined sequential serum samples from 12 patients with well-characterized posttransfusion non-A, non-B hepatitis who had an acute, resolving self-limited type of clinical course for the presence of antibody to the hepatitis C virus nucleocapsid (core) protein (p22) expressed by a recombinant baculovirus. These sera were simultaneously examined for antibody to the hepatitis C virus nonstructural protein (C100-3) that is presently used for blood screening worldwide. In three patients, both anti-p22 and anti-C100-3 antibodies were detected, but anti-p22 was detected much earlier. In four patients, only anti-p22 was detected. Two other patients were considered to be hepatitis C virus carriers who had been already infected with hepatitis C virus. In one patient, only anti-C100-3 was detected, and it was transient. In two patients, neither antibody was detected. Anti-p22 was detected in at least one of eight samples of transfused blood. Of the nine samples of donated blood that were positive for anti-p22, only four were positive for anti-C100-3. This new assay detecting the antibody to the p22 protein is thus useful for the serodiagnosis of non-A, non-B hepatitis in the acute phase and for blood screening.

Antigens, Viral↗

PubMed1991

A novel IgM-indirect hemagglutination test for the serodiagnosis of acute toxoplasmosis.

A new indirect hemagglutination (M-Toxo-IHA) test was standardized for the serodiagnosis of acute toxoplasmosis. The reagent for this test consisted of stabilized human red cells coated with a Toxoplasma gondii heat-stable alkaline-solubilized extract which reacted predominantly with IgM antibodies found in serum samples from patients with a recent infection. Almost no reactivity was verified with serum samples from chronic or ancient toxoplasmosis. The reagent preparation was simple and obviated prior fractionation of parasite components reactive with IgM antibodies. Rheumatoid factor or high levels of IgG antibodies did not interfere in the M-Toxo-IHA test. A total of 358 serum samples was assessed for the evaluation of this test, comprising sera from patients with recent and ancient toxoplasmosis, from non-related diseases, as well as from clinically healthy individuals with no previous T. gondii infections. Serodiagnostic performance of the M-Toxo-IHA test in terms of sensitivity, specificity, efficiency, and positive and negative predictive values was verified, and the values obtained were 1.000, 0.985, 0.989, 0.952, and 1.000, respectively, in relation to IgM-immunofluorescence or IgM-capture ELISA. Six different batches of reagent, successively produced for the current study, provided reproducible results and were stable for at least 22 months at 4 degrees C.

Acute Disease↗

PubMed1991

Evaluation of direct agglutination test (DAT) and ELISA for serodiagnosis of visceral leishmaniasis in India.

The direct agglutination test (DAT) and the enzyme-linked immunosorbent assay (ELISA) were used for serodiagnosis of parasitologically confirmed Indian visceral leishmaniasis (VL) cases. All the sera of VL cases were positive by both the methods. DAT titres of VL cases were greater than or equal to 1:3,200, and ELISA values were greater than or equal to 0.55 1:400 dilution. In the control group, sera of widely prevalent diseases of India, such as leprosy, tuberculosis, malaria, and liver cirrhosis, were included. Both tests could discriminate between VL and other patients of the control group. The sera of post-kala-azar dermal leishmaniasis (PKDL) patients gave OD values of greater than 0:55 and had DAT titres of 1:1,600. Both tests are sensitive and specific for the diagnosis of VL cases. DAT, being simpler and more economical, will be suitable for diagnosis and epidemiological studies for VL under rural conditions of India.

Agglutination Tests↗