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PubMedDate not supplied

[Comparison of indirect immunofluorescence and enzyme immunoassay methods for the determination of antinuclear antibodies].

Antinuclear antibodies (ANA) are widely used for screening and monitoring of connective tissue diseases (CTD). Indirect immunofluorescence assay (IFA) is the standard method which is more often preferred for detecting these antibodies. Another method is enzyme immunoassay (ELISA) which includes extractable nuclear antigens (ENA). The aim of this study was to compare two different methods in view of their performances in the detection of ANA. A total of 27 sera from patients prediagnosed as different types of CTD, were screened by ANA-IFA (Zeus Scientific Inc, USA) and ELISA (Zeus Scientific Inc, ENA Profile-6, USA) methods. In addition, specific staining patterns of ANA on HEp-2 cells as a substrate, were enrolled with IFA. As a result, ANA positivity was detected in all of the 27 samples (100%) by IFA, and only in 7 (25.9%) by ELISA. The concordance rate between two different assays was estimated as 38.6%, and a statistically significant difference was found between the methods in the detection of ANA (x2=20, p<0.001). In conclusion, ANA-IFA method is still a reliable routine screening method for the laboratory diagnosis of CTD, while ENA Profile-6 ELISA may give false negative results, because of its limited antigen content, and should be supported with additional antigens.

Antibodies, Antinuclear↗
PubMed2004

[Experimental study on enzyme dot assay for detection of hemorrhagic fever with renal syndrome antigen].

OBJECTIVE: To establish a new and efficient method(IEDA) for detection of hemorrhagic fever with renal syndrome virus (HFRSV) antigen. METHODS: An immune enzyme dot assay (IEDA) with mixture of three sorts anti-HFRSV-IgG, which was obtained from rabbit vaccinated with EHFV R22, Chen and Hubei strain was employed to detect HFRSV antigen in serum and urine from epidemic hemorrhagic fever (EHF) patients, and compared with indirect immune fluorescence assay (I-IFA), 76 serum samples and 40 urine samples were detected in this study. RESULTS: The results showed that the total positive rate of HFRSV antigen detected by IEDA was 73.68% in serum and 65.00% in urine, while that detected by I-IFA was 75.00% and 70.00%, respectively. The positive rate in primary phase (within 5 days) of HFRSV antigen detected by IEDA was 94.34% in serum and 83.33% in urine, while that detected by I-IFA was 64.42% and 55.56%, respectively, there was significant difference in both serum and urine detections. Correlation study showed a high correlation in the result of IEDA and I-IFA. CONCLUSION: The results of this study suggested that the IEDA, as compared with I-IFA, was a more specific, sensitive, rapid and simple method with higher positive rate in primary phase. IEDA could be widely used for early diagnosis of HFRS in hospital at grassroots level.

Animals↗
PubMed1992

Enzyme linked immunosorbent assay & indirect fluorescence assay for rapid diagnosis of Bacteroides fragilis infections.

Antibodies for B. fragilis (NCTC 9343) were detected in sera of 121 patients and 37 controls using four methods viz., enzyme linked immunosorbent assay (ELISA), indirect fluorescence test (JFA), countercurrent immunoelectrophoresis (CIE) and indirect haemagglutination test (IHA). Of 121 patients, 57 were culture positive for B. fragilis, 38 positive for anaerobes other than B. fragilis and 26 were negative for anaerobes. In the B. fragilis culture positive group, antibodies to B. fragilis were positive in 82.5, 84.2, 85.90 and 91.2 per cent patients by CIE, ELISA, IHA and IFA respectively. In B. fragilis positive patients IFA was more sensitive than IHA, which for other groups IHA was found to be more sensitive than IFA. When all groups were taken together IHA was found more sensitive than IFA. ELISA and IFA tests are recommended for rapid serological diagnosis of B. fragilis infections, where facilities for these tests are not available, CIE and IHA could be done. Cross reactivity with other Gram negative anaerobic and aerobic bacteria should be kept in mind since seropositivity varied for B. fragilis (82-91%). In infections with microbes other than B. fragilis seropositivity varied between 23.7 to 63.2 per cent and in patients having cultures sterile or positive for other organisms seropositivity was 30.8 to 42.3 per cent. This nonspecificity could be due to other antigens that cross react between B. fragilis and other anaerobes and aerobes or the use of an antigen lacking high purity.

Antibodies, Bacterial↗
PubMed1991

Comparison of four test kits for feline leukemia virus antigen.

The sensitivity and specificity of 4 commercial FeLV ELISA kits, using blood, were compared with results of virus isolation from blood and immunofluorescent antibody (IFA) testing on blood. Significant differences were not found among the 4 ELISA kits. Marked decrease in sensitivity of the ELISA kits was detected when virus isolation was used as the standard of positivity rather than the IFA test. Virus isolation was a more sensitive indicator of early infection, with marked discrepancy among results obtained by virus isolation, ELISA, and the IFA test. Results became progressively more concordant as infection became fully established. Cats FeLV-positive by virus isolation alone were more likely to eliminate viremia. All cats FeLV-positive by IFA testing remained persistently viremic. Virus isolation, ELISA, and IFA testing appear to differ in their prognostic value. The use of blood rather than serum for the ELISA resulted in several discordant results. Six cats were FeLV-positive by ELISA when blood was tested but were FeLV-negative when serum was tested. Positive ELISA results were obtained for 4 of these cats when serum was tested, using extended incubation to increase sensitivity. It is possible that blood may actually be more sensitive than serum for use of the ELISA method.

Animals↗
PubMed2005

[The importance of antinuclear (ABA) and anti-double stranded dna (anti-dsDNA) antibodies in the diagnosis of connective tissue diseases.].

Connective tissue diseases (CTD) are characterized by the presence of autoantibodies against several tissues. These autoantibodies occur against cell membrane, cell receptors, plasma proteins, and cytoplasmic and nuclear components. In laboratories, anti-nuclear antibodies (ANA) and anti-double stranded DNA (anti-dsDNA) antibodies are widely used in diagnosis of CTD. The aim of this study was to investigate the presence and accompaniment of ANA and anti-dsDNA antibodies in diagnosis of several CTD and also to study the prevalence of ANA and anti-dsDNA in a group of 88 patients with various types of CTD. ANA were detected by immunofluorescence (IFA) using HEp-2 cells (Zeus Scientific, Inc. USA) and anti-dsDNA antibodies using Crithidia luciliae (BioSystems, Spain) as substrates in immunofluorescence. ANA Western Blot (WB) Immunoassay (ImmuBlot, International Immuno-Diagnostics, USA) was also used along with the tests referred to previously. ANA was found in the sera of 84 (96.5%) patients while anti-dsDNA was detected in 7 (7.95%). Moreover different fluorescence patterns were also evaluated with ANA IFA in accordance with anti-dsDNA results. Mixed patterns in three and a homogeneous pattern in four anti-dsDNA positive patients' sera were determined on HEp-2 cell line by IFA. Seven sera which were ANA and anti-dsDNA positive with IFA were also found to be positive with WB and their ANA patterns with the specific ANA WB bands were also evaluated. It was observed that IFA results were in concordance with WB results. Our data indicated that the above findings should be controlled and evaluated with a more advanced method such as western blotting technique in order to confirm the presence of specific antibodies along with clinical outcome of the patients. As a result we think that ANA WB method is an appropriate technique in diagnosis of CTD as anti-dsDNA and ANA bands can be evaluated together with this method.

English Abstract↗
PubMed1991

[Detection of antibodies against Chlamydia in swine by an immunofluorescent test and an enzyme immunoassay].

Chlamydial IgG-antibodies were detected in 130 of 2050 porcine sera (6.3%) using an indirect immunofluorescence assay (IFA). Antibody titers varied between 1:8 and 1:16 in 87 sera (67%) and reached titers from 1:32 to 1:128 in 43 sera (33%). An ELISA closely related to the IFA-technique was developed, which confirmed the IFA-positive and IFA-negative results in 93% and 86% respectively. A significant higher percentage of positive IFA-titers greater than or equal to 1:32 was obtained from sows with abortions and infertility as well as from store pigs with pneumonia than from healthy pigs (p less than 0.5).

Animals↗
PubMed1990

Detection of antibodies to cytomegalovirus by immunofluorescence and enzyme immunoassay.

Immunofluorescence assay (IFA) and enzyme immunoassay (EIA) for detection of immunoglobulin M (IgM) to cytomegalovirus (CMV) correlated in 88.9%. All but one differences were due to negativity in IFA and positivity in EIA. The agreement between determination of immunoglobulin G (IgG) to CMV by IFA and determination of total antibodies to CMV by EIA was 89.9%. All the differences were due to a mild positivity in IFA and negativity in EIA. The advantage of EIA is in its objective colorimetric evaluation of test results excluding a subjective underestimation or overestimation of the reaction. In addition, EIA gives the result of IgM detection already in 6 hours, while IFA as late as in 24 hours.

Antibodies, Viral↗
PubMed1977

Comparisons of the complement-fixation and indirect fluorescent antibody reactions in the detection of bovine babesiasis.

Complement-fixation (CF) and indirect fluorescent antibody (IFA) antigens were prepared from Babesia bigemina isolates obtained in Texas. These serologic procedures were evaluated on 130 serum samples sequentially collected from 5 B bigemina-infected mature cattle, beginning on the day of exposure and continuing for 175 day thereafter. Both tests were effective in detecting specific antibodies for the first 84 days of infection, with 57 of 60 (95%) serums tested being positive on the CF test and 57 of 57 (100%) tests being positive to the IFA test. During the interval from 98 to 175 days, 24 of 60 (40%) of the serums tested were positive with the CF test, and 53 of 56 (95%) were positive with the IFA test. During the first 84 days, a similar linear regression occurred in both CF and IFA serum titers, but after 98 days the IFA regression flattened out, whereas the CF titers decreased below the sensitivity threshold in 60% of the serums tested.

Animals↗
PubMed1986

Seroepidemiology of malaria in northern Thailand: II. Focal and temporal variations in endemicity.

Four population groups from regions of Northern Thailand were surveyed for the presence of antibodies to Plasmodium falciparum using the indirect immunofluorescent antibody (IFA) test. Each of the four populations was selected from areas known to represent different patterns of malaria transmission. Group 1 was from an area where there had been no malaria transmission for approximately 30 years. Individuals in this group below age 40 showed an extremely low prevalence of malaria antibodies. Group 2 was chosen from an area where low levels of transmission have continued despite more than 30 years of DDT spraying. In this group the age related pattern of malaria antibodies varied from village to village but in all villages there was a sharp increase in the prevalence of IFA positive tests in individuals over 30. The third group has had continuously high levels of transmission. Although there are differences in the age related prevalence of IFA positives when individual villages are compared, there is a greater prevalence at all ages than in the first two groups. The fourth group was selected from an area where transmission had recently resumed after freedom from indigenous cases for approximately six years. There was little difference in the prevalence of IFA positive individuals below the age of 25 but above that there was a steady increase in prevalence with age. The correlation of IFA antibody positives with known patterns of malaria transmission in these four areas demonstrates the usefulness of this serological technique in assessing malaria endemicity and the effectiveness of control measures as well as in the interpretation of other malaria statistics.

Adolescent↗
PubMed1978

Comparisons of the complement-fixation, indirect fluorescent antibody, and card agglutination tests for the diagnosis of bovine anaplasmosis.

Results of complement-fixation (CF), indirect fluorescent antibody (IFA), and card agglutination (CT) tests were statistically compared, using 380 serum samples obtained from 140 cattle which were disease-free or naturally or experimentally infected with Anaplasma marginale of Colombian origin. The IFA test was significantly the most sensitive for detection of amimals infected with anaplasmosis (97%); the CT test and the CF test were less so (84% and 79%, respectively). However, the most efficient test for identifying noninfected animals was the CF test (100%), and the CT and the IFA tests were less efficient (98% and 90%). A linear regression analysis performed on the average IFA and CF titers of 10 calves artificially infected with A marginale during a 20-week period showed significant regression coefficients for both tests. The regression line for the CF titers decreased below the sensitivity threshold at 14 weeks after calves were inoculated, whereas the regression line for the IFA titers continued above the sensitivity threshold 20 weeks after inoculation. The CT test also detected antibodies until the end of the observation period.

Agglutination Tests↗
PubMed1979

Evaluation of enzyme linked immunosorbent assay in the immunodiagnosis of schistosomiasis.

Eighty-six bilharzial patients divided into 5 clinical groups were studied. Enzyme linked immunosorbent assay (ELISA), indirect fluorescent antibody (IFA) and indirect haemagglutination (IHA) test were performed for all the patients. ELISA gave the most sensitive results (82.6% and 80.2% positivity rate in Egypt and Lille respectively), followed by IFA (79.1% positivity rate) and IHA (77.9% and 75.6% positivity rate in Egypt and Lille respectively). The humoral antibodies detected by all methods (ELISA, IFA and IHA) showed increasing values with the progress of the disease which is parallel to the antigenicity of the disease. Both the positivity rate and the mean value of antibody titres recorded by the 3 diagnostic techniques (ELISA, IFA and IHA) were significantly higher in mansoni than haematobium infection. This may be explained by species specificity of antibody response. The superiority of ELISA over IFA and IHA techniques was discussed.

Adolescent↗
PubMed1983

Comparison of ELISA and indirect immunofluorescence in the detection of IgG and IgM antitoxoplasma antibodies.

We have compared ELISA and IFA for the quantitation of IgG and IgM antitoxoplasma antibodies on whole sera and purified IgM fractions. In all the sera examined from adult patients the good correspondence of the two tests is expressed by a correlation coefficient of 0.77 (p = 0.001). However, we have observed that both IgM-ELISA and IgM-IFA performed on whole serum may lead either to false positive results due to presence of rheumatoid factors or to false negative results due to the competition with high level of IgG antibodies. The use of the simplified gel filtration method for separation of IgM fraction allowed the detection of IgM in 40 out of 75 adults patients by IgM IFA and in 39 by the same tests resulted negative if performed on whole serum. The separation of IgM fractions seems a necessary step for detecting IgM antibodies by IgM IFA in 12 of 14 newborns sera with clinical and serological evidence of congenital toxoplasmosis. The conventional IgM ELISA was constantly negative when performed on both whole sera or pure IgM IFA positive fractions of the same newborns.

Animals↗
PubMed1984

Characterization of tick antigens inducing host immune resistance. I. Immunization of guinea pigs with Amblyomma americanum-derived salivary gland extracts and identification of an important salivary gland protein antigen with guinea pig anti-tick antibodies.

Guinea pigs immunized by subcutaneous injection of an emulsion of incomplete Freund's adjuvant (IFA) containing tick salivary gland extract antigens (SGA) from partially fed female ticks expressed a significant level of tick rejection when challenged 17 days later. This level of tick rejection was similar to animals actively sensitized by tick feeding and challenged at the same time. SGA emulsified with complete Freund's adjuvant (CFA) or administered with saline was ineffective. However, ticks that fed on animals immunized with SGA+IFA or SGA+CFA expressed significant reductions in engorgement weight. SGA was active when prepared with or without protease inhibitors. The minimum effective immunizing dose of SGA was between 100 and 280 micrograms per animal. Extracts made from salivary gland-derived cement material (CA) from partially fed female ticks administered at 50 micrograms in IFA induced levels of tick rejection comparable to animals immunized with 280 micrograms of SGA+IFA. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS/PAGE) of 35S- and 125I-radiolabeled SGA and CA extracts immunoprecipitated by guinea pig anti-tick serum that transferred immune resistance demonstrated a unique protein of 20,000 m.w. Serum from animals immunized with SGA+IFA (successful immunization) recognized this same protein, whereas serum from animals immunized with SGA+CFA (unsuccessful immunization) did not. The results of this study suggest that a 20,000 m.w. protein derived from the tick salivary gland may be responsible for the induction and perhaps elicitation of host immune resistance responses to Amblyomma americanum ticks.

Animals↗
PubMed1995

Evidence for a high rate of false-positive results with the indirect fluorescent antibody test for Ehrlichia risticii antibody in horses.

OBJECTIVE: The original objective was to determine seroprevalence of Ehrlichia risticii antibody among horses in California. On the basis of the unexpected results of the survey, an investigation into the accuracy and reproducibility of results of the indirect fluorescent antibody (IFA) test for E risticii was carried out. DESIGN: Prospective, seroprevalence study. ANIMALS: Healthy horses (n = 655) and horses with clinical signs of equine monocytic ehrlichiosis (EME; n = 514) from various regions of California. PROCEDURE: The IFA test was performed. Results were compared with results of an ELISA and with results of western immunoblot analysis. RESULTS: Overall, 104 of 655 (15.9%) healthy horses had evidence of an antibody response. However, 84 of 514 (16.3%) horses with clinical signs of EME also had positive test results, and of the 8 seropositive diseased horses for which paired (acute and convalescent) samples had been submitted, only 1 had a rise in antibody titers between the acute and convalescent samples. Comparison of results for the IFA test, ELISA, and western immunoblot analysis revealed a high rate of false-positive results for the IFA test. Subsequent studies suggested that routine vaccination of horses with non-E risticii vaccines may have contributed to the false-positive reactions. CLINICAL IMPLICATIONS: The data failed to provide conclusive evidence of E risticii infection among California horses. Owing to the high percentage of false-positive test results, caution is advised when using the IFA test to diagnose EME in horses or to determine the necessity for E risticii vaccination.

Animals↗
PubMedDate not supplied

[Comparison of instructions to authors of Mexican medical journals and the Vancouver requirements].

OBJECTIVE: To find out how much information of the Vancouver norms (VANNOR) are given to prospective authors in the instructions for authors (IFA) in some Mexican medical journals (MMJ). DESIGN: Prospective, cross sectional, descriptive, observational. MEASUREMENTS: To score the IFA in the MMJ published between 1989 and 1991. The scoring was done on the basis of presence in the IFA of the VANNOR characteristics grouped in categories (characteristics and presentation of the manuscript, title page, summary and keywords, text order and contents, references, tables, illustrations, and other instructions). A total score of 60 characteristics (grouped in nine categories) was obtained for each journal. Statistical differences as a function of VANNOR affiliation were investigated by means of Student's t test, chi-square, and exact Fisher tests using a 0.05 alpha level (two-tailed). RESULTS: 50 MMJ were included (21 affiliated to VANNOR). No statistical difference was found for VANNOR affiliation and registration in bibliographic data bases. But statistically significant higher scores were found in the affiliated journals in 12 of 60 characteristics (three of nine categories) and in the total score. CONCLUSIONS: Independently of VANNOR affiliation, there were deficiencies in the IFA of all journals, particularly in instructions regarding the manuscript's contents. Possibly, some inadequacies in manuscripts might be prevented with IFA that comply more thoroughly with the VANNOR requirements.

Mexico↗
PubMedDate not supplied

Countercurrent immunoelectrophoresis, a technique for the diagnosis of bovine babesiosis.

The countercurrent immunoelectrophoresis (CIE) test was performed as a diagnostic tool for bovine babesiosis using a partially purified soluble antigen obtained from a saline extract of B. bovis-infected bovine erythrocytes by 60% ammonium sulphate precipitation (Bb-ag), and it was compared with the indirect fluorescent antibody test (IFA). Antibodies against B. bovis were detected in all 3 experimentally infected calves at the 8th post-infection day by IFA while antibodies to Bb-ag at the 10th postinfection day by CIE. These antibodies persisted until the end of the observation period (90th day post-infection). 43 serum samples from 116 bovines coming from a Babesia-enzootic area were positive (antibody titers > 1:30) by IFA test. 36 from the 43 (94%) IFA -positive bovine serum samples had antibodies to B. bovis soluble antigen (Bb-ag) detected by CIE. In contrast, the 20 serum samples from a Babesia-free area were all negative by both these methods. No cross-reactivity was observed with the soluble antigen obtained from normal red blood cells in the 3 experimentally infected calves nor in the bovines coming from a Babesia-enzootic area. CIE employing Bb-ag was as specific as IFA test but slightly less sensitive (100 vs. 94%) and detected antibodies to B.bovis early during the infection (8 vs. 10 days post-infection). Since CIE is easy to perform, does not require expensive reagents, material or equipment, and one can analyze up to 50 serum samples at once in a short time it is recommended as a useful test for the diagnosis of bovine babesiosis in developing countries.

Animals↗
PubMedDate not supplied

Comparative evaluation of in vitro and in vivo assays for the detection of reticuloendotheliosis virus as a contaminant in a live virus vaccine of poultry.

Indirect immunofluorescence (IFA), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) were used for detection of reticuloendotheliosis virus (REV) as a contaminant in a live virus fowl pox (FP) vaccine of poultry. A FP vaccine known to be contaminated with REV was tested by in vitro and in vivo assays in chicken embryo fibroblasts (CEFs) and day-old specific-pathogen-free (SPF) chicks, respectively. Using in vitro assays, IFA and PCR were more sensitive than ELISA in detection of REV in CEFs inoculated with REV-contaminated FP vaccine. However, when the vaccine was tested by in vivo assays using SPF chickens, the sensitivity of ELISA was comparable with that of IFA and PCR. Antibody to REV was not detected in SPF chickens within 4 wk postinoculation with REV-contaminated FP vaccine at hatch. Filtration of vaccine to eliminate vaccine virus from the inoculum before testing in CEFs resulted in a significant reduction in the frequency of REV detection by PCR or IFA. The data suggest that the sensitivity of IFA, PCR, and ELISA depends on the concentration of REV in the vaccine and that in vivo assays of vaccines for contamination with REV should include a test for virus because a negative antibody test may be misleading.

Animals↗
PubMed1997

[Isolation of Chlamydia trachomatis and immune response in different populations].

We studied the presence of C. trachomatis-specific IgG and IgM in adults and newborns, respectively, and attempted isolation of the bacteria in cell culture. The determination of antibodies was carried out by an IFA on C. trachomatis infected (L2 434/Bu serotype) McCoy cells, cultured in 24-well plastic plates. We found C. trachomatis-specific IgG in 27% of women with clinical symptoms, in 40% of women being attended for periodic gynecological control, in 60% of infertile women and in 10% of pregnant women. A proportion comparison test revealed the presence of specific IgG as highly significative for the group of infertile women as compared to the group of pregnant women (p < 0.0001). We divided the patients into four groups, in relation to the results of the tests for specific IgG and C. trachomatis isolation. Seven out of 10 had positive isolation and negative IFA, 5 out of 8 had positive isolation and negative IFA. Twenty five out of 28 pregnant women had negative isolation and positive IFA, finally, 63 out of 76 had both tests negative. Statistical analysis using the McNemar proportion-comparison test suggests that IgG's presence is highly significant in pregnant women with respect to other groups (p < 0.001). Our results suggest that the demonstration of IgG is not enough for diagnostic purposes, except in infertile women with a previous history of infection with C. trachomatis. We isolated C. trachomatis in 20% of the newborns tested and 10% were also positive for IgM IFA. The diagnosis was improved by combining both techniques. These results show the importance of the detection of C. trachomatis in youngsters to avoid infertility and in pregnant women to prevent newborn infections and the possibility of premature births and low weight babies.

Adult↗