Use of ELISA to measure antinuclear antibodies in children with juvenile rheumatoid arthritis.

OBJECTIVE: To compare a series of commercial ELISA tests with an indirect immunofluorescent antibody (IFA) test for the detection of antinuclear antibodies (ANA) in children with juvenile rheumatoid arthritis (JRA). METHODS: Sera from 178 patients with JRA (88 pauciarticular, 68 polyarticular, 22 systemic) were compared with 26 healthy pediatric subjects. Twenty-one samples from patients with systemic lupus erythematosus (SLE) were also tested. All samples were analyzed by IFA and by 3 commercial ELISA methods. Concordance of ELISA results with IFA results (selected standard) were used as a measure of performance. Sensitivity and specificity were calculated for each test and likelihood ratios (LR) were established for IFA and ELISA in pauciarticular and polyarticular JRA sera. The increment in pretest probability was then obtained for each test as an additional measure of test performance. RESULTS: IFA rendered positive results on 18-77% of the JRA sera depending upon the subset, 100% of SLE sera, and 15% of normal patient sera. Using IFA as the standard, correspondence with positive results among patients with JRA ranged from 0 to 74% for the 3 ELISA tests, while it ranged from 5 to 73% in IFA negative sera. IFA tests showed intermediate range likelihood ratios (0.3, 0.5, 3.5, and 5) and increments in pretest probability ranging from 25 to 45%. While one of the ELISA tests attained 50% of increment in pretest probability for the positive test, it showed 0% increment as a negative test. The other 2 ELISA tests incremented the pretest probability from 0 to 25%. CONCLUSION: Our findings indicate that in JRA, the lack of correspondence with the historic standard IFA precludes the use of ELISA tests for detection of ANA. In addition, IFA out-performs ELISA by a substantial degree when "clinical utility" analysis of test performance is utilized. Detection of ANA in children with JRA should either continue to rely on IFA or be based on a different set of antigens if an ELISA format is chosen.     

Evaluation of the micro enzyme-linked immunosorbent assay for antibodies to Trypanosoma cruzi

A micro enzyme-linked immunosorbent assay (ELISA) for antibodies to Trypanosoma cruzi was evaluated and the results obtained by ELISA were compared with those obtained by the complement fixation test (CF) and indirect fluorescent antibody test (IFA). Fifty sera collected from residents of the southeastern United States all had reciprocal ELISA titers less than or equal to 320. Similarly, serum samples from 17 patients with T. cruzi infection proven by xenodiagnosis had reciprocal ELISA titers of greater than or equal to 1,280. Specimens from 302 El Salvador Army recruits were tested by ELISA, IFA, and CF. Excellent correlation was observed between results obtained by the three serologic tests; 62.9% of the samples were negative by each of the three tests and 24.5% were positive by all. Overall, 29.5% of the sera were positive for antibodies to T. cruzi by ELISA, 29.5% by IFA, and 31.5% by CF. The data suggest that the micro ELISA is a promising serologic test for measuring antibodies to T. cruzi in individuals and in populations.     

Detection of specific IgG and IgM antibodies in the haemagglutination inhibition test and the enzyme-linked immunoassay for the diagnosis of rubella infection

A study has been carried out on the sera of 710 women who wished to know their state of rubella immunity using haemagglutination inhibition (HAI) and enzyme-linked immunoassay (ELISA) techniques. The majority of the women presented no symptoms. The ability of HAI to detect low antibody levels (1:8, 1:16) appears to be greater than that of Rubazyme ELISA IgG, employing the recommendations of the manufacturers. The correlation between HAI and IgG values above HAI titres of 1:16 is nearly 100%. In an additional study of 17 primary infections in pregnant women with definite rubella symptoms, the total titre of antibodies was determined by HAI and IgG and specific IgM with ELISA Rubazyme in successive samples. In four cases, IgM was clearly positive and no increase in either HAI or IgG antibodies could be demonstrated over three successive samples taken at an interval of 15 to 20 days. Therefore, we consider it necessary to determine IgM antibodies (evaluating these in the absence of the rheumatoid factor) in every doubtful case occurring in pregnant women, irrespective of whether clinical signs are present or not.     

Enzyme-linked immunosorbent assay (ELISA) for measles antibody. A comparison with haemagglutination inhibition, immunofluorescence and plaque neutralization tests.

An enzyme-linked immunosorbent assay (ELISA) for measles antibodies was compared with Plaque Neutralization (PRN), Haemagglutination inhibition (HI) and Fluorescent antibody (IFA) tests in 181 sera from vaccinated children and umbilical cord. Of 179 positive samples by the sensitive PRN, only two, with titers of 8, were negative by ELISA (copositivity of 98.9%). IFA and HI presented, respectively, copositivities of 93.3% and 82.7%. The ELISA presented a high sensitivity as well as a good reproducibility and represents an alternative for the time consuming PRN for detection of low measles antibodies.     

Serodiagnosis of Rocky Mountain spotted fever: comparison of IgM and IgG enzyme-linked immunosorbent assays and indirect fluorescent antibody test

To characterize IgM and IgG antibody responses in Rocky Mountain spotted fever (RMSF), a microtiter enzyme-linked immunosorbent assay (ELISA) using density gradient-purified Rickettsia rickettsii as antigen was developed. Sera of vaccinated individuals and patients with RMSF were tested by ELISA and by indirect fluorescent antibody (IFA) tests. Diagnostic agreement between ELISA and the IFA test was 76% and 52% for IgG and IgM antibody, respectively. Diagnostic agreement between the ELISA for IgG antibody and the IFA test for total immunoglobulins was 84%. The ELISAs for IgM and IgG antibody were as specific (100%) and as sensitive (100%) as the IFA test (83%-100%) in detecting antibody increases in paired sera from persons with RMSF and were superior to the IFA test in detecting seroconversions in vaccinees. The ELISA also detected antibodies in a single convalescent-phase serum with sensitivity and reliability. The ELISA for IgG antibody is appropriate for seroepidemiology and serodiagnosis since it permits measurement of antibody at a single dilution of serum up to a year after illness.     

Serum antibodies to rubella virus in healthy individuals with indirect immunofluorescent method]

To investigate sensitivity to rubella virus (RV) in healthy individuals, we examined levels of antibodies to RV in sera by an indirect immunofluorescence assay (IFA) and compared levels of antibodies by IFA with those by a hemagglutination inhibition (HI) assay. Of 114 healthy individuals, we detected antibodies to RV in serum specimens from 103 (90.3%) by IFA and in those from 109 (95.6%) by HI assay. The peak value of levels of antibodies by HI assay was 4 fold higher than that by IFA. When levels of antibodies by IFA were less than 32, levels of antibodies by HI assay ranged from < 8 to 1024. We did not detect anti-rubella antibodies of IgM class in all serum specimens and detected anti-rubella antibodies of IgA class in serum from only 1 individual by IFA. We detected antibodies to rubella in sera from 51 (94.4%) by IFA and in sera from 52 (96.3%) by HI assay of 54 individuals who reported having had rubella, and in sera from 23 (88.5%), by IFA and in sera from 26 (100%) by HI assay of 26 individuals reported having been vaccinated. Also, we detected anti-rubella antibodies in sera from 13 (76.5%) by IFA and in sera from 15 (88.2%) by HI of 17 individuals who reported having had neither rubella nor vaccination. In serum from 1 individual who reported having had rubella, we detected antibodies to rubella by IFA but not by HI assay. In serum specimens from 2 individuals who reported having had rubella vaccination, from 3 having had vaccination, from 2 having had neither rubella nor vaccination, we detected anti-rubella antibodies by HI assay but not by IFA. On the other hand, by both assays, we detected antibodies to RV in all sera of individuals who reported having had rubella and been vaccinated. The serodiagnosis, at least, by two methods is necessary to prevent individuals from rubella virus infection, because of following results: 1) influence of an inhibitor in serum specimens was thought to be variable. 2) The results measured by IFA were differed from those by HI assay in some individuals. 3) It is difficult in diagnosis of rubella from clinical symptoms alone. Also, it might be required to use vaccine to the individual who lacks detectable antibodies to rubella in serum by any method to prevent rubella infection.     

Microtiter enzyme-linked immunosorbent assay for rubella antibodies in human sera--analysis by parallel line assay method]

Antibodies against rubella virus in human sera were measured by ELISA and antibody titers were calculated by the parallel line assay method. The dose response regression curves of standard sera and test sera containing IgG antibody calculated by the parallel line assay method showed linearity, and were parallel to one another. However, the regression lines of sera positive for IgM antibody against rubella virus were not parallel to one another in the low dilution region, but parallel in the higher dilution region. A good correlation was observed between the rubella IgG antibody titers measured by the hemagglutination-inhibition (HI) test and those calculated by the parallel line assay method. The coefficient of the correlation was 0.781. Time-course studies of IgG or IgM antibody titers against rubella virus in rubella patients or in vaccines with MMR vaccine indicated that the ELISA was more precise and specific method than the HI test. Thus, the parallel line assay method using ELISA is considered to be a more useful method for the detection and quantification of antibodies to rubella than the conventional HI test.     

Immunofluorescence, enzyme-linked immunosorbent assay, particle agglutination and western blot for the detection of antibody to human immunodeficiency virus type 1

Immunofluorescence assay (IFA) has been applied for detection of antibody to human immunodeficiency virus type 1 (HIV-1). To compare the IFA with an enzyme-linked immunosorbent assay (ELISA) and particle agglutination (PA), we examined the antibody response to HIV-1 in 475 sera from AIDS, PGL and ARC patients as well as several risk groups and healthy persons by three methods. The positive results by any methods were confirmed by western blot (WB). The results by all methods were well correlated on the sera from 45 asymptomatic male homosexuals and 70 female prostitutes. There were some false positive results by ELISA in the sera from prisoners and healthy persons. Four sera from drug abusers were positive only by PA and IFA and were negative by ELISA. All were WB-inconclusive. Particle agglutination and IFA results were compared with western blot analysis on 208 ELISA-positive sera. All IFA-strongly positive sera (84%) were positive by western blot. The sera with weakly positive, negative and inconclusive results by IFA (16%) were possibly any of positive, inconclusive or negative by western blot. By PA, 200 of 208 (97%) sera were PA-positive and 1% of these sera were WB-inconclusive while the PA-negative sera were either negative or inconclusive by western blot. These results suggested that PA is a simple and sensitive method for screening of HIV-1 antibody while IFA could be a primary confirmatory test and western blot would then be used for confirming any IFA-negative or inconclusive results.     

Rapid detection of IgM and IgG antibodies to herpesviridae virus

In this study, the immunoconcepts EA indirect enzyme antibody technique (colorzyme) was used not only for detection of IgG antibodies but also for quantitative detection of IgM antibodies to Herpes Simplex virus (HSV), Cytomegalovirus (CMV) and Epstein Barr Virus (EBV) to diagnose recent iactivei infection. Reference reactive and negative antisera and randomly collected human sera were tested by complement fixation test (CFT) against HSV antigens and tested also by immunofluorescent (IF) and colorzyme Immunoconcepts EA tests. All sera that were negative to HSV, CMV and EBV antibodies by CFT were negative by IF and colorzyme EA tests. All antibody positive sera and reference positive antisera were also positive by IF and colorzyme EA tests with slight variation in antibody titres between CFT and colorzyme test results. Human sera which were negative or IgM positive to HSV, CMV and EBV by ELISA as well as negative and positive reference sera from different diagnostic kits were retested by IF and colorzyme EA for IgM antiviral reactivity results were concordance by the three rests. All incubations in colorzyme test were at room temperature and only an ordinary microscope used in IF test or plate washers and readers needed for ELISA test. The colorzyme immunoconcepts is a simple, rapid and sensitive for viral diagnosis and can be used in any private laboratory.     

Comparison of indirect immunofluorescence test for measles antibodies with haemagglutination inhibition and plaque neutralization tests

Indirect Immunofluorescence (IFA), Plaque Reduction Neutralization (PRN) and Haemagglutination Inhibition (HI) tests for measles antibodies were carried out in 197 sera obtained from umbilical cord and vaccinated children. The IFA was also applied to blood samples collected with filter paper. IFA results demonstrated that the test is relatively simple to perform, with good reproducibility for different antigen lots. Good correlation was obtained between IFA, PRN and HI antibody titers. Better correlation was demonstrated with IFA and PRN than with HI and PRN tests. Sensitivity of IFA in detecting antibody was less effective than PRN, however more effective than HI using rhesus monkey red blood cells. PRN antibody titers over 100 were detected by IFA but not by HI (9.7% with negative results). IFA may be of considerable practical use and able to substitute HI in seroepidemiological surveys and to evaluate vaccine efficacy. It also can be simplified by employing filter paper collected samples.     

吸毒人群尿液血液标本HIV-1抗体ELISA检测对比分析

目的　用酶联免疫吸附试验 (ELISA)检测吸毒人群尿液标本中艾滋病病毒 1型 (HIV 1 )抗体 ,与血清学检测结果进行比较。方法　采集某市劳教所吸毒者尿液、血液标本 354例 ,HIV 1抗体阳性吸毒者复检尿液标本1 9例 ,共计 373例。应用ELISA初筛试剂检测尿液及血液标本中HIV 1抗体 ,阳性者取其血液标本进一步用Genelabs试剂做蛋白印迹 (WB)试验确认。结果　尿液、血液标本中检测HIV 1抗体的特异性为 99 72 % ,尿试剂的假阳性率为 0 2 8% ;血液标本HIV 1抗体阳性者 ,尿液标本检测也呈阳性 ,灵敏度为 1 0 0 %。结论　尿液标本用ELISA方法检测HIV 1抗体与血液标本ELISA方法的检出率有高度的一致性。尿试剂适用于对高危人群进行尿液HIV 1抗体的筛查工作     

Rubella antibody assay by the immunoperoxidase technique: comparison with the hemagglutination inhibition test for determination of immune status.

The rubella immune status of 128 young women was determined with two assays for rubella antibody, the immunoperoxidase technique and the hemagglutination inhibition test. Comparison of the results of these techniques showed agreement in 125 cases (110 positive and 15 negative). Positive results were obtained for one woman only by the immunoperoxidase assay and for two women only by the hemagglutination inhibition test; all three of these women had a minimal titer of rubella antibody of 1:16. The sensitivity of the immunoperoxidase test was comparable to that of the hemagglutination inhibition test, and the specificity of the former test was verified. The peroxidase-labeled antibody technique is easier to perform than the hemagglutination inhibition test and is less time-consuming since sera do not require prior treatment and results can be obtained in as little as 90 min.     

Evaluation of a crude soluble antigen from in-vitro culture of Plasmodium falciparum for ELISA

The present study was to evaluate the soluble antigen prepared from the in-vitro cultured P. falciparum (FCR3) as an alternative source of antigen to those obtained from in-vivo models for enzyme linked immunosorbent assay (ELISA) in serology of malaria. Results obtained on known positive and negative reference sera revealed good correlation between the ELISA and the indirect fluorescent antibody (IFA) technique (rs = 0.797; p less than 0.001). However such close correlation was not observed on six local sera from patients whose peripheral blood smear showed ring stage of P. falciparum (rs = 0.43; p greater than 0.05), though all the six sera were positive by the IFA and ELISA tests. Test was repeated to establish its reproducibility. The results indicate that antigen prepared from in vitro culture and stored in liquid nitrogen was found sensitive in ELISA for serology of malaria.     

Enzyme-linked immunosorbent assay and indirect immunofluorescence assay for Lyme disease

The sensitivity and specificity of an indirect immunofluorescence assay (IFA) and ELISA for Lyme disease were estimated. Sera from patients with Lyme disease, patients with other infections, and healthy individuals were examined. Significant cross-reactivity occurred only with sera from patients with syphilis, yaws, and pinta . All tested sera from patients with Lyme disease, however, gave negative results in the rapid reagin screening test and the microhemagglutination assay for antibodies to Treponema pallidum confirmatory for syphilis. When sera from patients with treponemal diseases were excluded from the analysis, the IFA and ELISA were highly specific, having 97% and 100% reliability, respectively. Sensitivity of both tests varied with the stage of disease but was 100% for both tests during complicated Lyme disease. The results indicate that both tests are highly specific and sensitive for complicated Lyme disease but relatively insensitive for patients with erythema chronicum migrans alone.     

Serodiagnosis of canine visceral leishmaniasis in Portugal: comparison of three methods.

Sera collected in Portugal from 43 dogs were screened for specific antibodies to Leishmania donovani antigens. Three different techniques were compared: an indirect immunofluorescence assay (IFA), a direct enzyme-linked immunosorbent assay (ELISA) and a competitive-ELISA (C-ELISA) using two species-specific monoclonal antibodies, D2 and D13. By IFA, 22 of the sera examined showed positive reactions, compared with 26 by ELISA or 27 by C-ELISA. There was no direct correlation observed between the serum titre by IFA and the strength of the reaction in ELISA or inhibition in C-ELISA. However, a good correlation was observed between sera identified as positive (95.5%) by all three techniques. Western blotting on leishmanial membranes showed that common antigens with Mr of 26,000 and 70-84,000 were recognized by all infected dog sera, regardless of the serum titre. In large scale studies, ELISAs are preferred to IFA for the rapid diagnosis of canine visceral leishmaniasis because of their greater simplicity.     

Antibodies against human immunodeficiency virus in generalized lupus erythematosus

After studying a bisexual male with a clinical picture suggestive of AIDS a positive ELISA test for antibodies against the Human Immunodeficiency Virus (HIV), but negative results on indirect immunofluorescence testing, as well, as absence of core and envelope HIV antibodies by ELISA, and who later turned out to have Systemic Lupus Erythematosus (SLE) which become asymptomatic on corticosteroid therapy, we decided to test 70 patients with SLE for HIV antibodies. Four of them (5.6%) were positive by ELISA, but on a repeated test only 2 (2.8%) remained positive, and their sera was tested by indirect immunofluorescence. They were negative, as was the ELISA test for core and envelope HIV antibodies. We conclude that the possibility of more such cases, of SLE mimicking AIDS, should be kept in mind, including the occurrence of false positive ELISA tests in such patients.     

Diagnosis of perinatally acquired HIV-1 infection using an IgA ELISA test

The clinical utility of the detection of anti-HIV-1 IgA antibodies using a modified commercial ELISA (EIA) test for the early diagnosis of perinatally acquired HIV-1 infection was evaluated. One hundred and seventeen sera were obtained from 86 infants born to HIV-1-infected mothers and tested for HIV IgA antibodies by an ELISA test (third generation) after removal of IgG with recombinant protein G. Infants were classified according to the Center for Disease Control and Prevention's (CDC) classification system after 15 months of age; 46 were classified as HIV-infected children and 40 as uninfected. HIV-IgA antibodies were detected in 53 of 64 serum samples from all infected children. No significant differences were observed in IgA detection among symptomatic or asymptomatic infected children. However, when analyzed by age a significant difference was observed in IgA detection when children who were over 6 months of age were compared with the younger group (Fisher exact test, p = 0.0000053). All 53 samples from 40 noninfected children were IgA-negative. Statistical analysis was assessed comparing IgA results with HIV infection status as the gold standard. Sensitivity (95%) and specificity (100%), positive predictive value (100%), and negative predictive value (94%) of IgA antibody determination were analyzed taking into account only one sample per child and only children older than 6 months. Positive likelihood ratio was 95.9% and negative likelihood ratio was 94%. Test efficiency was 97%. The detection of IgA HIV antibodies using EIA is an effective method for early diagnosis of HIV-infected infants in comparison with conventional IgG HIV antibody tests. It is a simple and inexpensive method that could be used in both developed and developing countries.     

Comparison of various serological test results using antigens from different strains of Plasmodium falciparum

Suitability of different strains of Plasmodium falciparum grown continuously in vitro was compared using the indirect haemagglutination (IHA) the indirect immunofluorescent (IFA) tests and ELISA. In the tests employing soluble antigens (IHA and ELISA), there was a significant higher mean log titer of the same sera tested against different strains. Ranking of the strains in term of sensitivity for the detection of malaria antibody in people in the endemic area were G-112 = SO = CC greater than SU greater than PS in the IHA test and G-112 = SO greater than CC greater than SU greater than PS in the ELISA. The difference in the mean log titers appear to relate neither to the geographical location not the isoenzyme markers tested. There was also an apparent correlation between the results of the IHA and the IFA test but not between these two tests and ELISA.     

COMPARISON OF ELISA AND lEA FOR ESTIMATION OF ANTIBODY LEVELS OF CATTLE TO THEILERIA ANNULATA VACCINE

Bovine tropical Theileriosis caused by Theileria annulata is an economically important disease of cattle. An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in vaccinated and unvaccinated cattle, using cellular schizont as antigen and its results were compared with immunofluorescent assay (IFA). For this test 126 sera collected (105 vaccinated, 31 not vaccinated) from cows and assayed with ELISA which among them 104 sera were positive and 32 sera were negative. Same sample assayed with IFA in which 99 were positive sera and 37 were negative sera. Thereby the sensitivity and specificity of this ELISA on comparsion with lEA were 95.5% and 66.6% respectively. This study revealed that ELISA could be successfully used for both differentiating vaccinated and not vaccinated cattle and obtaining the titer of vaccinated cattle.     

Fundamental studies on serological diagnosis of amoebiasis. 1. Application of amoebic antigen for CF test to ELISA

We studied the establishment of enzyme-linked immunosorbent assay (ELISA) using amoebic antigen for complement fixation (CF) test. Optimal dilution for ELISA of sera from patients was 1:100, and that of CF-antigen was 1:400. The upper limit of the 99% critical range of the reaction of negative sera was 0.068 (cut-off level). Absorbance of sera from patients diluted 1:100 to antigen and antibody titers of ELISA were strongly correlated, so it was possible to estimate antibody titers from absorbance of serum diluted 1:100. ELISA and CF test were done to compare sensitivity of the tests using 63 sera from patients with invasive amoebic disease. The sensitivity of ELISA compared well with CF test (62 sera were positive by ELISA and 61 by CF test). Only one sample was both positive by ELISA and negative by CF test. This sample had low ELISA titers, so this discrepancy was mainly due to the sensitivity of CF test in detecting lower levels of antibody. These results suggested that the amoebic antigen for CF test can be applicable to ELISA, and this method was so sensitive and specific.