Serological diagnosis of brucellosis caused by Brucella canis

Blood serum samples from 2,328 dogs were tested to detect antibodies against Brucella canis with the agar gel immunodiffusion (AGID) and 2-mercaptoethanol slide agglutination test (ME-SAT) using Brucella ovis as the antigen. All blood serum samples were also evaluated for antibodies against Brucella abortus and Brucella melitensis using the Rose Bengal test. Twentyfive (1.07%) of the sera evaluated were considered positive with AGID test. Only 4 (16%) of these blood serum samples were positive when evaluated with ME-SAT. The 25 AGID positive samples and 25 AGID negative serum samples were also examined by: the complement fixation test (CFT) using B. ovis hot saline extract (HSE) as the antigen, indirect enzyme linked immunosorbent assay (ELISA) and immunoblotting (IB) using B. canis and B. ovis HSE antigens. Two positive canine sera from culture positive dogs and the serum of an experimentally RM6/66 B. canis-infected rabbit were employed as positive controls and one serum from a known uninfected dog as a negative control. ELISA with B. canis antigen gave 9 (18%) positive results (6 AGID-positive and 3 AGID-negative sera). ELISA performed with B. ovis antigen detected 15 (30%) positive samples (10 AGID-positive, 5 AGID-negative and 8 B. canis ELISA positive sera). IB analysis of known positive controls sera employing B. canis antigen detected bands with molecular weights of 94-80, 64-50, 35, 32-30, 28, 23, 20-18, 15-12 kDa. The same sera tested with B. ovis antigen revealed bands of 35, 32-30, 25, 23, 20-18, 15-12 kDa. No bands were observed with the negative control serum and the 50 canine tested sera.     

An agar--gel immunodiffusion test for detection of Brucella antibodies in human serum.

A comparison was made of results obtained with a Brucella agar--gel immunodiffusion (AGID) test and the standard tube-agglutination test on 612 human sera. Agreement between the tests was 97% when the titer was 1:160 or higher. Of 448 sera that showed no agglutination titer, 447 were negative with the AGID test. Results of the AGID test were also compared to those obtained with the 2-mercaptoethanol (2-ME) agglutination test on 148 sera that demonstrated a standard tube-agglutination titer of 1:20 or higher. All sera with a 2-ME-agglutination titer of 1:40 or higher were positive with the AGID test. Of 123 sera that showed no 2-ME-agglutination titer, 21 were positive with the AGID test. Two of these 21 sera were obtained from patients with bacteriologically proven brucellosis, and eight were from abattoir employees with suspected but not bacteriologically proven brucellosis.     

Seroepidemiology of Anaplasma marginale in white-tailed deer (Odocoileus virginianus) from Louisiana

Antibodies to Anaplasma marginale were detected by the indirect fluorescent antibody test (IFA) in six of 331 (2%) serum samples of white-tailed deer (Odocoileus virginianus) from Louisiana. None of the serum samples were positive using the A. marginale modified rapid card agglutination test. Of the six IFA positive sera retested by the complement fixation test four sera gave anticomplementary and two gave seropositive reactions. The low A. marginale reactor rate in this white-tailed deer population was probably a reflection of the lack of cohabitation between cattle and deer and the fact that the primary arthropod vectors in Louisiana are tabanids. The validity of the indirect fluorescent antibody test for A. marginale antibodies in white-tailed deer should be evaluated.     

Development of a western blotting assay to discriminate Brucella spp. and Yersinia enterocolitica O:9 infections in sheep

Outer membrane proteins (OMPs) of Rev-1 strain of Brucella melitensis were used in a Western blotting assay for the serological diagnosis of brucellosis in ovine sera. Fifty-four sheep sera were tested and divided into the following groups: Group A) n. 9 samples from one sheep that had been experimentally infected with Y. enterocolitica O:9; Group B) n. 10 samples collected from sheep infected with Brucella melitensis and 1 sample from a sheep vaccinated with the Rev 1 strain; Group C) n. 10 samples collected in "officially brucellosis-free" herds; Group D) n. 12 samples classified as "suspicious"; Group E) n. 12 samples classified as "positive". Antibodies were detected by routine tests performed for the diagnosis of brucellosis in serum samples of the sheep infected with Y. enterocolitica O:9 after the 2nd week post infection. In the WB assay, sera of group B recognised a 17 kDa protein, whereas sera of groups A, and D and 9 out of 12 of group E exhibited no reactivity to this protein. The results obtained encourage the use of the WB assay as a confirmatory test for the diagnosis of brucellosis.     

Evaluation of indirect fluorescent antibody test and enzyme-linked immunosorbent assay for diagnosis of hepatic amebiasis in Bangladesh

Serum samples of 31 amebic liver abscess (ALA) patients, 8 amebic hepatitis (AH) patients, and 60 controls were tested for anti-amebic IgG by enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody tests (IFAT). Sera of 29 (93.6%) ALA and 6 (75%) AH patients and 2 (3.3%) control subjects were positive by IFAT. Anti-amebic antibody titer above the cutoff point (= 0.168; x + 2 SD of control sera) was observed in sera of 27 (87%) ALA, 4 (50%) AH, and 1 (1.7%) control by ELISA. All the 8 pus samples were positive for anti-amebic antibodies by IFAT and ELISA. Sensitivity of ELISA was 87% for ALA, with a positive predictive value of 0.96, and 50% for AH cases, with a positive predictive value of 0.80. The sensitivity of IFAT was 93.6% for ALA, with a positive predictive value of 0.94, and 75% for AH, with a positive predictive value of 0.75. When pus samples were tested, the sensitivity was 100% for both tests. The specificity was 98.3% for ELISA and 96.7% for IFAT. Although not significant, IFAT was found more sensitive than ELISA (P>0.05).     

A dot-immunobinding assay (dot-Iba) for rapid diagnosis of pulmonary tuberculosis

IgG antibody to Mycobacterium tuberculosis from the sera of patients with 'definite' pulmonary tuberculosis (PT) was isolated and coupled with Cyanogen bromide-Sepharose 4B. Using an immunoabsorbent affinity chromatography, 14 kDa antigen was recovered from the culture filtrates of M. tuberculosis. With this mycobacterial antigen, a dot immunobinding assay (Dot-Iba) was developed for the detection of specific antibody to M. tuberculosis in the sera of patients with PT and controls. The assay gave positive results in all the 12 sputum-smear positive [acid fast bacilli (AFB)] patients with PT and gave negative results in the 50 sera from control groups. The Dot-Iba as described in this study, is simple, rapid and specific for laboratory diagnosis of PT.     

Naturally acquired IgM antibody response to the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax in Korea: use for serodiagnosis of vivax malaria

The antibody levels against the C-terminal region of the merozoite surface protein 1 of Plasmodium vivax (PvMSP1c) were measured in 276 patients with P. vivax malaria (patient group), 320 malaria-na?ve healthy individuals (control group 1), and 70 malaria-na?ve individuals with various disorders (control group 2) using the immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) and the direct sandwich ELISA. To evaluate the antibody response during relapse, 5 relapsed patients were tested using the IgM capture ELISA. The IgM antibodies were negative in 99.7% of control group 1 and in 100% of control group 2; they were positive in 90.6% of the patient group. The total antibody levels were positive in 88.4% of the patient group with the direct sandwich ELISA. The sera from the second malaria episode, i.e., relapsed patients, were 100% positive on the IgM capture ELISA. The results of this study suggest that the IgM capture ELISA may be a useful diagnostic method for P. vivax malaria for both primary infection and relapse.     

Brucellacapt、RBT、SAT、iELISA四种血清学检测方法对布鲁氏菌病检测价值的比较研究

目的:探讨布鲁氏菌病血清学试验(Brucellacapt)、虎红平板凝集试验(RBT)、试管凝集试验(SAT)、间接酶联免疫吸附试验(i ELISA)四种血清学检测方法对布鲁氏菌病检测价值的比较研究。方法:收集近两年110例布鲁氏菌病疑似病例人员的静脉血分离得到血清后进行Brucellacapt、RBT、SAT、i ELISA四种血清学检测,以卫生部制定的《布鲁氏菌病诊疗指南》中布鲁氏菌病确诊方法为诊断金标准,将检测结果与其确诊结果进行比较,评价比较各组血清检测方法对布鲁氏菌病的检测价值。结果:110例疑似人员中确诊为布鲁氏菌病阳性91例、阴性19例。Brucellacapt试验阳性89例、阴性21例;RBT试验阳性79例、阴性31例;SAT试验阳性71例、阴性39例;i ELISA检验阳性82例、阴性28例。Brucellacapt试验的灵敏度、符合率、Kappa值、ROC曲线下面积均最大,i ELISA试验、RBT试验、SAT试验依次减小;i ELISA试验的ROC曲线下面积最大,Brucellacapt试验次之,其次为RBT试验,SAT试验的ROC曲线下面积最小。结论:Brucellacapt、RBT、SAT、i ELISA四种血清学检测方法对于布鲁氏菌病检测均有一定的检测价值,对于常规普通患者可采用RBT试验、SAT试验进行检查,而对于疑似病例人员可采用灵敏度更高的Brucellacapt试验、i ELISA试验。     

Rapid dot sputum and serum assay in pulmonary tuberculosis

A rapid direct sputum (Sp.) and/or antibody assay, based on immunoblotting and enzyme immunoassay is described. The test can detect mycobacterial antigens or antibodies in clinical specimens from pulmonary tuberculosis (TB) patients. In this study, 87 sputa, 87 sera and 40 paired sputa and sera were utilized from smear-positive and smear-negative, culture-positive patients; 59 sputa, 37 sera and 22 paired sputa and sera from nontuberculosis respiratory disease patients and 68 sera from healthy controls. The antigen detection in sputum by dot assay has 86.1% sensitivity on active tuberculosis patients, 92.9% specificity, 91.6% positive predictive value (PPV), 88.2% negative predictive value (NPV) and 10.3% error. The antibody assay has 83.6% sensitivity, 95.4% specificity, 94.4% positive predictive value, 85.6% negative predictive value and 11% error. The test performed on paired sputum and serum (Sr.) samples has a sensitivity of 93.3%, which rose to 96.1% on smear-positive and culture-positive patients, but the specificity decreased to 83% in sputum, whereas in serum it was 92%. The results of the assay, combined with clinical and radiological data, could form the basis for starting an earlier course of treatment for tuberculosis.     

Enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola cathepsin L1 for the diagnosis of human fasciolosis caused by Fasciola hepatica/gigantica hybrid type

Recombinant Fasciola cathepsin L-1 (rCatL1) was evaluated in enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of human fasciolosis in Japan. Quality characteristics of the test were accessed by receiver operating characteristic (ROC) analysis, with sera from fasciolosis patients (n = 10), patients with no evidence of parasitic infections (n = 29), and patients with other helminth infections (n = 119). Both the sensitivity and specificity of the test achieved 100% with the control samples. To test the performance of the assay in an authentic situation, 311 serum samples, which had been sent to our laboratory for the diagnosis of parasitic infections from January 2018 to February 2019, were re-assessed using the rCatL1 ELISA. In this case, the sensitivity of the rCatL1 ELISA was 100%, giving positive results to all fasciolosis sera (n = 7), and the specificity was 99.0%, in which three of the 304 non-fasciolosis samples were judged positive. Careful re-examination of the laboratory data and medical imaging of these three patients revealed that one of the patients, who had been diagnosed as having larva migrans syndrome, was judged to be infected with Fasciola, in addition to ascarid nematodes. Thus the true specificity of the assay in the authentic reached 99.3% (302/304). As the rCatL1 ELISA exhibited a highly significant positive likelihood ratio (152.0) and negative likelihood ratio (0.0), calculated from the 311 sample data, this rCatL1 ELISA can be used for routine screening and definitive diagnosis test for fasciolosis in reference laboratories.     

Detection of antibodies in serum and cerebrospinal fluid to Toxoplasma gondii by indirect latex agglutination test and enzyme-linked immunosorbent assay.

Sensitivity of anti-Toxoplasma antibody (IgG) test by enzyme-linked immunosorbent assay (ELISA) was evaluated in comparison with indirect latex agglutination (ILA) using 2,016 paired human samples of serum and cerebrospinal fluid (CSF). The samples were collected from neurologic patients in Korea with mass lesions in central nervous system (CNS) as revealed by imaging diagnosis (CT/MRI). When the sera were screened for anti-Toxoplasma antibody by ILA, 76 cases(3.8%) were positive (1:32 or higher titers). In the paired samples of CSF, no positive reactions were observed. When ELISA was performed using PBS extract of Percoll purified tachyzoites as antigen, cut-off absorbance was determined as 0.40 for serum and 0.27 for CSF tests. The antibody positive rates by ELISA were 7.0% in serum and 5.6% in CSF. Of them, 40 cases (2.0%) showed positive reactions in both serum and CSF. The antibody positive rates were higher in groups older than 40 years. The rates were higher in male (4.7% by ILA, 8.3% by ELISA) than in female (2.2% by ILA, 5.0% by ELISA). The rates in CSF showed no such sex difference. ELISA showed twice higher positive rates when serum was tested, and was sensitive enough to detect specific antibodies in CSF. Etiologic relations between positive antibody tests and CNS lesions remained unknown.     

Serum levels of autoantibodies against monomeric C-reactive protein are correlated with disease activity in systemic lupus erythematosus

This study was performed to investigate the relation between IgG autoantibodies against human C-reactive protein (anti-CRP) and disease activity measures in serial serum samples from 10 patients with systemic lupus erythematosus (SLE), of whom four had active kidney involvement during the study period. The presence of anti-CRP was analysed by enzyme-linked immunosorbent assay. The cut-off for positive anti-CRP test was set at the 95th centile of 100 healthy blood donor sera. Specificity of the anti-CRP antibody binding was evaluated by preincubating patient sera with either native or monomeric CRP. Disease activity was determined by the SLE disease activity index (SLEDAI), serum levels of CRP, anti-DNA antibodies, complement components and blood cell counts. Of 50 serum samples, 20 (40%) contained antibodies reactive with monomeric CRP, and 7 of 10 patients were positive on at least one occasion during the study. All patients with active lupus nephritis were positive for anti-CRP at flare. Frequent correlations between anti-CRP levels and disease activity measures were observed in anti-CRP-positive individuals. Accumulated anti-CRP data from all patients were positively correlated with SLEDAI scores and anti-DNA antibody levels, whereas significant inverse relationships were noted for complement factors C1q, C3 and C4, and for lymphocyte counts. This study confirms the high prevalence of anti-CRP autoantibodies in SLE and that the antibody levels are correlated with clinical and laboratory disease activity measures. This indicates that anti-CRP antibodies might have biological functions of pathogenetic interest in SLE. Further prospective clinical studies and experimental studies on effects mediated by anti-CRP antibodies are warranted.     

Detection of antibodies to Staphylococcus aureus Toxic Shock Syndrome Toxin-1 using a competitive agglutination inhibition assay

AIMS: To develop a competitive agglutination inhibition assay (CAIA) for the detection of anti-Toxic Shock Syndrome Toxin-1 (TSST-1) antibody in serum samples using a commercially available reverse passive agglutination assay (RPLA) kit. METHODS AND RESULTS: TSST-1 toxin and sera were incubated together, so that anti-toxin IgG would complex with the toxin. Latex particles sensitized with rabbit IgG anti-TSST-1 were added to test for un-complexed toxin. The sensitivity and specificity of the CAIA assay was determined relative to positive and negative ELISA results. The sensitivity (proportion of positive ELISA sera which tested positive by CAIA) was 66% whilst the specificity (proportion of ELISA negative sera which tested negative by CAIA) was 75%. Seven sera (14%) were negative by ELISA but positive for CAIA and 12 (18.8%) were positive for ELISA but negative for CAIA, suggesting some interference with the assays. Statistical analysis showed no significant difference between the methods in terms of the numbers of individuals testing positive (chi2, P = 0.04). CONCLUSIONS: The CAIA assay allowed detection of anti-TSST-1 within 18 h and was simple to read visually. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is a useful test for individual serum samples and a preliminary investigation for medical screening of suspected toxic shock syndrome and is applicable in situations where antibody assays are not routinely used for anti-TSST-1 and also where sophisticated equipment (e.g. microtitre plate reader) is not available.     

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).     

Seroepidemiology of enteric fever and brucellosis in a developing country

The seroepidemiology of infection due to S. typhi and Brucella spp. in a sub-tropical, developing country of the Middle East was investigated in 130 children of age less than 1 to 15 years, 117 young adults (16-30 years) and in a further 40 adults (greater than 30 years) using an agglutination test to detect antibodies to of S. typhi, B. abortus and B. melitensis. Only 4.5% of the children's and adult's sera respectively showed the presence of antibodies to both O and H antigens of S. typhi with reciprocal titers greater than or equal to 80. Prevalence rates of 11.6%, 11.3% respectively in children and adults of antibodies to B. abortus with reciprocal titers greater than or equal to 80 and similarly 13.1%, 12.4% to B. melitensis were found. The usefulness of a single Widal test to diagnose enteric fever in a non-endemic area and the agglutination test in the diagnosis of brucellosis in an endemic area was investigated. Of 244 patients with suspected enteric fever, the sera of 100 showed reciprocal antibody titers 80 to both O and H antigens and included in these were 8 patients with concurrent culture positivity. Among 138 children and adults with suspected brucellosis, 29 culture proven cases where serologically confirmed and a further 90 were detected serologically. The sera of all the culture proven cases exhibited antibody reciprocal titer greater than or equal to 640. A similar antibody response was noted in seventy-two of the culture negative cases diagnosed serologically for brucellosis. In culture proven, cases B. melitensis was the isolate. Comparative evaluation indicated the potential usefulness of the Rose Bengal Antigen Slide Agglutination Test (Brucelloside Test) as a screening test in the serodiagnosis of human brucellosis.     

Detection of cross-reactive antibody to BLV p24 in sera of human patients infected with HTLV

For detection of antibody to bovine leukemia virus (BLV) major core protein of p24 and cross-reactive antibody in human patients infected with human T cell leukemia virus type I (HTLV-I), monoclonal antibody, D432 against BLV p24 was used by competitive binding enzyme-linked immunoadsorbed assay (ELISA). In sera from cattle with enzootic bovine leukosis (EBL) which were positive for BLV antibodies by immunodiffusion test, 109 out of 112 (97.3%) were positive for BLV p24 antibody by competitive binding ELISA. By using the same procedures, 21 samples from adult T cell leukemia (ATL) patients and healthy carriers with HTLV-I were tested for cross-reactive antibody to BLV p24. All 21 samples were positive for HTLV-I antibodies by immunofluorescence test and/or ELISA. By competitive binding ELISA using non-treated BLV antigens, none of these 21 samples inhibited the binding of the D432. When the BLV antigen was treated by several different denaturation procedures, several HTLV-I positive samples showed the inhibition of the D432 binding and the most effective treatment was by 2-mercaptoethanol (2-ME). Sixteen out of 21 samples showed the presence of cross-reactive antibody against 2-ME-treated BLV antigens. The cross-reactivity of human sample to BLV p24 antigen was further confirmed by Western blotting of the 2-ME-treated BLV antigens. None of the 28 samples from leukemia patients other than ATL which were negative for HTLV-I antibodies showed inhibition of the D432 by the competitive binding ELISA.     

Anti-galactocerebroside antibodiesin human cerebrospinal fluids determined by enzyme-linked immunosorbent assay (ELISA)

The standard ELISA technique was improved for the detection of antigalactocerebroside antibody in biological fluid. Mouse monoclonal antigalactocerebroside antibody was used to demonstrate specificity and sensitivity of the technique. After optimization of the assay, the usefulness of this measurement for the evaluation of patients with multiple sclerosis was assessed. The presence of antigalactocerebroside antibodies in the cerebrospinal fluid of 20 patients with multiple sclerosis, 10 with other neurological diseases and 10 normal individuals was determined. All the CSF samples from normal individuals were negative. In patients with multiple sclerosis 14 of the 20 samples had elevated levels of antigalactocerebroside antibody, whereas with other neurological diseases 5 out of 10 were positive. Antigalactocerebroside levels were lower in samples from patients during an acute relapse than in those from more chronic cases. These results indicate that the presence of anti-galactocerebroside antibody in cerebrospinal fluid is not specific to MS but may reflect previous damage to myelin.Abbreviations and trivial names used ELISA Enzyme-Linked Immunosorbent Assay - CSF cerebrospinal fluid; galacto- or glucocerebroside, ceramide-1-0-beta-galactoside or-glucoside     

A Comparative Examination of Swine Sera for Antibody To Aujeszky Virus with the Conventional and a Modified Virus-Serum Neutralization Test and a Modified Direct Complement Fixation Test

Sera of pigs from élite breeding herds, of boars and sows collected at slaughter-houses, and of pigs from herds known to be infected, were examined for antibody to Aujeszky virus. The conventional and a modified virus-neutralizing antibody (VNA) test and a modified direct complement fixation (CF) test were employed. In simultaneous titrations of positive sera the modified VNA test gave titers approx. 4 log2 units above the titers obtained by the conventional test. The conventional VNA test was found insufficiently sensitive. Unspecific neutralization in the modified VNA test was infrequent in serum dilution 1/2 and rare in dilution 1/4. The GF tests on sera of slaughter sows and animals from known infected herds showed a remarkable consistency with the VNA tests. Inconsistent results were obtained with but few sera. Abt. 5 % of the sera could not be examined because of complement fixation with control antigen.     

Evaluation of circulating immune complexes in lymphomas and leukemias using two different assays

Summary Circulating immune complexes (CICs) have been detected in the sera of patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease, chronic myeloid leukemia, and acute lymphoblastic leukemia by using C1q-binding and L1210-binding assays. Both assays gave broadly similar patterns of reactivity in terms of frequency and magnitude, though there are some differences. Significantly elevated CIC levels were observed in all pathologic groups. However, sera from NHL patients with an unfavorable prognosis consistently exhibited the highest frequency of positive values and mean CIC levels in both these assays.The two tests showed concordance in 66.6% of the NHL patients' sera and were significantly correlated. Of the sera from NHL patients 12.7% were positive in the C1q-binding assay only and 15.9% in the L1210-binding assay only. Both the assays gave positive results in some patients, and a degree of overlap indicates the presence of different types of CIC in cancer patients' sera. The combined use of two methods for detecting CICs may be useful for evaluation of the activity, the extent, and the prognosis of the malignant disease.