Evaluation of a rapid enzyme immunoassay system for serologic diagnosis of Mycoplasma pneumoniae infection

A 5-min qualitative membrane enzyme-linked immunoassay (EIA) from Remel (Mycoplasma pneumoniae immunoglobulin G (IgG)/IgM Antibody Test System) was evaluated for its ability to detect IgG and IgG at levels indicating active or recent infection. Specimens from 131 patients were evaluated using an immunofluorescent antibody assay (IFA) to determine IgG and IgM titers and the membrane EIA. An enzymelinked immunosorbent assay (ELISA) performed by a reference laboratory was used for discrepancy resolution. There were 34 IgM positive specimens (titer ⩾ 1:16), 19 IgG positive specimens (titer ⩾ 1:64), and 78 negative specimens. Compared with IFA and/or ELISA, the membrane EIA was 97% sensitive for the detection of IgM and 79% sensitive for the detection of IgG. Of the 78 specimens called negative, 17 specimens had IgG titers (⩽1:32) or an ELISA result indicating prior exposure, and the membrane EIA called seven of 17 (41%) positive. For the detection of both IgG and IgM, the membrane EIA had a sensitivity of 91 %, specificity of 91%, and positive and negative predictive values of 87 and 93%, respectively. The Remel membrane EIA is a rapid and reliable assay for the diagnosis of active or recent M. pneumoniae respiratory tract infections.     

A repA-based ELISA for discriminating cattle vaccinated with Brucella suis 2 from those naturally infected with Brucella abortus and Brucella melitensis

The commonest ways of diagnosing brucellosis in animals include the Rose-Bengal plate agglutination test, the buffered plate agglutination test (BPA), the slide agglutination test, the complement fixation test, and the indirect enzyme linked immunosorbent assay (I-ELISA). However, these methods cannot discriminate the Brucella vaccine strain (Brucella suis strain 2; B. suis S2) from naturally acquired virulent strains. Of the six common Brucella species, Brucella melitensis, Brucella abortus, and B. suis are the commonest species occurring in China. To develop an ELISA assay that can differentiate between cows inoculated with B. suis S2 and naturally infected with B. abortus and B. melitensis, genomic sequences from six Brucella spp. (B. melitensis, B. abortus, B. suis, Brucella canis, Brucella neotomae and Brucella ovis) were compared using Basic Local Alignment Search Tool software. One particular gene, the repA-related gene, was found to be a marker that can differentiate B. suis from B. abortus and B. melitensis. The repA-related gene of B. suis was PCR amplified and subcloned into the pET-32a vector. Expressed repA-related protein was purified and used as an antigen. The repA-based ELISA was optimized and used as specific tests. In the present study, serum from animals inoculated with the B. suis S2 vaccine strain had positive repA-based ELISA results. In contrast, the test-positive reference sera against B. abortus and B. melitensis had negative repA-based ELISA results. The concordance rate between B. abortus antibody-negative (based on the repA-based ELISA) and the Brucella gene-positive (based on the ‘Bruce ladder’ multiplex PCR) was 100%. Therefore, the findings suggest that the repA-based ELISA is a useful tool for differentiating cows vaccinated with the B. suis S2 and naturally infected with B. abortus and B. melitensis.     

The value of ELISA vs. negative Coombs findings in the serodiagnosis of human brucellosis

This study was conducted to assess the value of ELISA findings in relation to negative findings in the Brucella-antihuman globulin (Coombs) test and in relation to the clinical condition of patients. One hundred and thirty three serum specimens, representing the same number of patients, submitted for serologic testing for brucellosis and showing negative Coombs, were tested by ELISA to determine their Brucella-IgG, -IgM and -IgA antibodies. Concordant negative results between Coombs and ELISA were found in 95 (71.4%) patients whose medical records also did not reveal suggestive clinical signs or symptoms of brucellosis. The elevated ELISA readings in the remaining 38 (28.6%) patients were distributed as follows: IgG + IgM + IgA in 1 patient, IgG + IgM in 8 patients, IgG alone in 24 patients and IgM alone in 5 patients. The clinical review of these patients indicated no current disease in 21 (15.8%), scanty evidence of brucellosis in 8 (6.0%) and suggestive or sufficient evidence in 9 (6.8%). Thus, ELISA is the test of choice to resort to in the case of clinical suspicion of brucellosis, even when the Coombs test shows negative findings.     

Performance characteristics of the Euroimmun enzyme-linked immunosorbent assay kits for Brucella IgG and IgM

Brucella IgG and IgM ELISA kits manufactured by Euroimmun (Lubeck, Germany) were evaluated in a reference laboratory setting. Intraassay coefficient of variation (CV) values were ≤10% for positive sera and ≤12% for negative sera; interassay CVs were ≤12% for positive sera and ≤20% for negative sera. The tube agglutination test (TAT) was performed on 51 sera exhibiting various ELISA reactivity profiles. All 18 sera negative for both IgG and IgM by ELISA were TAT negative (titer <1:80), whereas 31 (94%) of 33 sera positive for IgG and/or IgM were TAT positive; the 2 discordant sera were IgG positive IgM negative by ELISA. None of 41 sera from healthy laboratory employees were ELISA IgG positive, whereas 1 (2%) of 41 was ELISA IgM positive. Similarly, 0 of 149 potentially cross-reactive sera (containing rheumatoid factor or antibodies to selected Gram-negative bacteria) was ELISA IgG positive, whereas 4 (3%) of 149 were ELISA IgM positive. These findings demonstrate the acceptable performance of the Euroimmun ELISAs for Brucella antibodies.     

Prevalence and incidence of Lyme borreliosis among Slovene forestry workers during the period of tick activity

Summary OBJECTIVES: To establish the prevalence and incidence of symptomatic and asymptomatic infection with Borrelia burgdorferi sensu lato during the period of tick activity, to compare the risk of infection with B. burgdorferi s.l. for forestry workers and indoor workers in Slovenia, and to compare the outcome of an in-house immunofluorescent assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA). METHODS: The study included 122 forestry workers; the control group consisted of 93 indoor workers. All participants were examined twice in 2002: before the beginning of tick activity (March) and at the end of tick activity (November). At each examination, principal demographic and epidemiological data were collected and a blood sample taken for serological analysis. Specific IgM and IgG antibodies against B. burgdorferi s.l. in the paired sera were determined with an in-house IFA and a commercially available ELISA flagellin test (DAKO). RESULTS: 9.8% of the forestry workers and 4.3% of the indoor workers tested positive for IgG with the IFA (p = 0.26); 23.8% of the forestry workers and 9.7% of the indoor workers tested positive for IgG with the ELISA (p = 0.02). During the study period the incidence of symptomatic Lyme borreliosis was 2.3% and the rate of IgG and/or IgM seroconversion of 10.2% was the same with both tests. CONCLUSIONS: The seroprevalence of antibodies against B. burgdorferi s.l. among the Slovene forestry workers was greater than among the indoor workers, but the difference between the two groups was not significant when the IFA was used. The incidence of Lyme borreliosis during the period of tick activity was lower than we expected, with a large proportion of seroconversions being asymptomatic.     

Analysis of antibodies to Aspergillus fumigatus antigens by class-specific enzyme-linked immunosorbent assay in patients with pulmonary aspergillosis

Antibodies to two commercial extracts of Aspergillus fumigatus were determined by a class-specific enzyme-linked immunosorbent assay (ELISA) in 203 serum samples from 139 patients with various pulmonary diseases. In 22 patients with aspergilloma immunoglobulin (Ig)M, IgA, and especially IgG antibodies were found, whereas in 50 patients with allergic alveolitis IgG antibody was most frequent, IgM occurring rarely. One patient with allergic bronchopulmonary aspergillosis demonstrated IgG and IgA antibodies. Of 20 cases with bronchial asthma, 10% reacted against A. fumigatus in immunodiffusion as well as in ELISA. Of 46 cases with carcinoma, tuberculosis, and miscellaneous pulmonary diseases, 17% were positive by immunodiffusion and 26% demonstrated antibodies usually IgG, by ELISA. Of 100 healthy blood donors, none had Aspergillus antibodies of the IgG class, whereas 3% were positive in the IgM and 3% in the IgA assay. The ELISA proved to be sensitive and useful in the follow-up of patients with aspergilloma after operation.     

Utility of an immunocapture-agglutination test and an enzyme-linked immunosorbent assay test against cytosolic proteins from Brucella melitensis B115 in the diagnosis and follow-up of human acute brucellosis

The utility of an immunocapture-agglutination (Brucellacapt, Vircell SL, Granada, Spain) test and an enzyme-linked immunosorbent assay IgG, IgA, and IgM (ELISA-IgG, ELISA-IgA, ELISA-IgM) against cytosolic proteins from Brucella melitensis B115 (R) was compared with ELISA-IgG, ELISA-IgA, and ELISA-IgM against smooth lipopolysaccharide (S-LPS) from B. melitensis 16M (S), serum agglutination test (SAT), and Coombs test in the diagnosis and follow-up for 10 months of 51 patients with acute brucellosis. The sensitivities of ELISA tests against cytosolic proteins varied from 49.0 % for ELISA-IgG to 64.7% for ELISA-IgM and were lower than the sensitivities showed by ELISA S-LPS (from 88.2% to 92.2%), SAT (88.2%), Coombs (96.1%), and Brucellacapt (98.0%) tests. Specificity was over 93% in all cases. The evolutionary behavior of the SAT, Coombs, and Brucellacapt tests was similar. There was a decrease of between 20% and 40% in antibody titer in the 10th month of evolution after treatment. The evolutional curves of IgG, IgA, and IgM against cytosolic protein increased slightly till the eighth month. The specific IgM and IgA antibodies against protein fractions began to show a drop from the eighth month on, showing levels slightly lower than the initial sera values by the end of the 10th month. In this month, titers of specific IgG against proteins fractions remained higher than the titers showed by the initial sera.     

Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16 M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors.Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS/MS.A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines.Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays.     

Performance of an enzyme-linked immunosorbent-based serological assay for Entamoeba histolytica: Comparison with an indirect immunofluorescence assay using stored frozen samples

IntroductionEntamoeba histolytica infections are increasingly diagnosed as sexually transmitted infections in Japan. However, the stool ova–parasite examination (O&P) test has been the only approved diagnostic method used in Japan since production of the indirect immunofluorescence assay (IFA) serum antibody test was discontinued at the end of 2017. Herein, we assessed whether an enzyme-linked immunosorbent assay (ELISA)-based serological test could substitute for IFA.MethodsThis cross-sectional study used stored frozen serum samples from the Biobank of the National Center for Global Health and Medicine. A serological ELISA-based test was performed on these samples and their titers were compared with those previously measured by IFA based on the medical record data.ResultsSixty seven stored frozen serum samples with differing recorded IFA antibody titers (16 samples with titers < ×100, 13 samples × 100, 16 samples × 200, 11 samples × 400, and 11 samples ≥ × 800) were analyzed. The sensitivity and specificity values for ELISA vs. IFA were 92.2% [95% confidential interval: 81.5–96.9] and 87.5% [64.0–97.8], respectively. A strong correlation between the antibody titers was confirmed by a one-way ANOVA (R square 0.83, p value < 0.0001) for the two diagnostic methods.ConclusionThe ELISA and IFA antibody titers for E. histolytica were well correlated, and results from these methods were highly concordant. Introduction of an ELISA-based serological test for E. histolytica should be considered to improve E. histolytica infection diagnosis in Japan.     

Class- and subclass-specific antibody response to hemocyanin in nonmalignant paraproteinemia.

IgM, IgG, and IgA class-specific, as well as IgG subclass-specific antibody titers against the primary immunogen HPH were measured with ELISA in 19 patients with nonmalignant paraproteinemia (eight with IgG1, two with IgG2, two with IgG4, four with IgM, and three with IgA) and in a simultaneously studied age- and sex-matched control group. After primary immunization only IgM and IgA anti-HPH titers were significantly lower in the patient group. Four patients with relatively high IgG or IgA serum paraprotein levels did not produce antibodies in some Ig classes or IgG subclasses, whereas all other patients and all controls developed antibody titers in all classes and IgG subclasses. Low or absent antibody titers did not occur preferentially in the Ig (sub)classes to which the paraproteins belonged. After secondary immunization the patients could not increase or maintain their antibody titers as well as the controls, and this was most clear in the IgM and IgA antibody class. A direct correlation between polyclonal serum IgM levels and IgM anti-HPH titers was present in the patients. Such a correlation was absent for IgA in the patients and for all classes in the controls. It is concluded that humoral immunosuppression as measured with a newly encountered antigen in patients with nonmalignant paraproteinemia is most clearly expressed in the IgM and IgA antibody class and that the paraprotein (sub)class is not preferentially involved.     

Value of a specific IgA assay on cord blood for the diagnosis of congenital toxoplasmosis

Congenital toxoplasmosis (CT) is a severe disease which must be recognized without delay to enable effective treatment. Detection of specific IgG and IgM antibodies in cord blood is not adequate for early diagnosis of CT. The diagnostic value of specific IgA detection in cord blood was assessed by testing cord blood samples from 41 infants with CT and 155 infants without CT. Each IgA assay was performed on serum diluted to 1 : 20 and 1 : 100. IgA values were compared with IgM values determined using the ISAgA^® test. Among the 41 CT infants, 38 had IgA positive response at the 1 : 20 dilution, 30 had IgA positive response at the 1 : 100 dilution, and 26 had IgM positive response. Among the 155 infants without CT, nine had IgA at the 1 : 20 dilution and two had IgA at the 1 : 100 dilution. Rate of true positives was higher for IgA (93%) than for IgM (63%). Diluting to 1 : 20 significantly improved sensitivity (93%) as compared with diluting to 1 : 100 (73%) and is therefore recommended for IgA cord blood assays with the reagent used in this study, even if it is associated with a lower specificity (94% instead of 99%). Our data suggest that IgA assays are helpful for the early diagnosis of CT.     

Identification of Brucella spp. isolates and discrimination from the vaccine strain Rev.1 by MALDI-TOF mass spectrometry

Brucellosis' surveillance and control programs require robust laboratory techniques that can reliably identify and biotype Brucella strains and discriminate between vaccine and field infection. In the recent years, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has revolutionized the routine identification of several microorganisms in clinical microbiology laboratories. Nevertheless, its application on Brucella spp. identification is limited since there are no reference spectra in the commercial databases, due to the microorganism's potential bioterrorist use.In this study, a custom MALDI-TOF MS reference library was constructed and its performance on identification at species level was evaluated using 75 Brucella spp. isolates. Furthermore, distinct peak biomarkers were detected for biovar assignment and discrimination from vaccine strain Rev.1. Analysis of mass peak profiles allowed Brucella accurate identification at genus and species level (100%) with no misidentifications. Despite the high intrageneric similarity, MALDI-TOF MS database succeeded in classifying at biovar level, 47 out of 62 B. melitensis bv. 3 isolates (75.81%), whereas all B. melitensis strains, except for one, were correctly discriminated from vaccine strain Rev.1.MALDI-TOF MS appeared to be a rapid, cost-effective and reliable method for the routine identification of brucellae which reduces time consumption in pathogen identification and could replace in the near future the current conventional and molecular techniques. Its ability to differentiate vaccine from field infection could facilitate brucellosis’ monitoring systems contributing in the effective control of the disease.     

Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.     

Serological response to plasmid-encoded antigens in children and adults with shigellosis

The immunological response to plasmid-encoded antigens of virulent Shigella was determined in Thai children less than 4 yr of age and in Thai adults by immunoblot analysis and ELISA. Forty-two percent (8/19) of Thai children and 4% (1/22) of Thai adults with shigellosis developed a greater than or equal to 4-fold rise in IgG antibody titer to water-extracted antigens of Shigella flexneri M90T by ELISA (p = 0.006). Two children and one lactating mother with shigellosis developed a 4-fold rise in serum IgA antibody titers to water-extracted antigens of M90T. The results of the ELISA were confirmed by immunoblot analysis in all of the 41 paired sera examined. Five patients developed IgA, and four developed IgM, antibodies as detected by immunoblot analysis, that were not detected by ELISA. The reciprocal log2 geometric mean titers of antibodies to plasmid-encoded antigens in acute sera was higher in Thai adults than Thai children: IgG 7,265 versus 1,659; IgM 879 versus 480; and IgA 662 versus 60 (p less than 0.001). Thai adults had high titers of antibodies to plasmid-encoded antigens in their acute sera, but were susceptible to Shigella infections, although they were historically less susceptible than Thai children.     

Anticardiolipin Antibodies in Patients with Type 2 Diabetes Mellitus

Background: There is controversy about an increased prevalence of antiphospholipid antibodies in diabetic patients. The possible implications are little known.Methods: We prospectively studied all consecutive outpatients with type 2 diabetes mellitus (DM) attended to in an Internal Medicine office. IgM and IgG anticardiolipin antibodies (ACA) were determined by standardized enzyme-linked immunoassay.Results: Fifty-six patients were included. Only one patient (1.8%) had a titer of IgM ACA higher than 15 MPL units and no patient had a titer of IgG ACA higher than 15 GPL units. Six patients (10.7%) had low IgM ACA titers (4–15 MPL units) and 18 patients (32.1%) had low IgG ACA titers (4–15 GPL units). There were no differences in the frequencies of a low IgM or IgG ACA titer or in the means of IgM and IgG ACA titers in patients with complicated and uncomplicated DM, with and without cardiovascular disease, with and without nephropathy, or with and without retinopathy.Conclusions: Moderate to high ACA titers must be exceptional in patients with type 2 DM. Low ACA titers may occur in patients with type 2 DM. These low titers do not seem to be associated with complicated DM, cardiovascular disease, nephropathy or retinopathy.     

Evaluation of IgG ELISA using N-lauroyl-sarcosine–soluble proteins of Bartonella henselae for highly specific serodiagnosis of cat scratch disease

Conventional IgG-ELISA methods for diagnosing cat scratch disease (CSD) caused by Bartonella hensela are still poor in sensitivity and specificity, which generally employ bacterial whole-cell proteins or N-lauroyl-sarcosine–insoluble proteins as the antigen. By Western blot analysis, we found that sarcosine-soluble fraction of proteins (SSP) showed highly specific reaction to immunofluorescence assay (IFA)–positive sera obtained from CSD patients compared with the above antigens. Clinical utility of the new ELISA employing SSP was evaluated using sera from 118 patients with clinically suspected CSD (sera positive by IFA: titers ≥ 1:256, n = 46; negative: titers < 128, n = 72) and 88 sera from healthy individuals. Sensitivity and specificity of distinguishing IFA-positive patients from healthy individuals were 95.7% and 97.7%, respectively. Fifteen discordant results were observed (13 ELISA(+)/IFA(−); 2 ELISA(−)/IFA(+)). However, all 15 sera reacted with SSP by Western blot analysis, indicating superiority of the new ELISA over IFA. The ELISA employing SSP greatly improved the accuracy of diagnosing CSD.     

重组汉坦病毒核蛋白抗原用于免疫滴金技术检测肾综合征出血热抗体的研究

目的　探索应用重组汉坦病毒核蛋白 (rNP)的免疫滴金法 (CGIDA)对肾综合征出血热 (hemorrhagicfeverrenalsyndrome ,HFRS)的诊断价值。 方法　制备纯化汉坦病毒核蛋白抗原 ,构建了HTN型汉坦病毒的核心区域 (334个碱基 ) ,克隆至原核表达载体 pGEX 4T 1中进行原核表达及纯化。在相同的CGIDA系统中 ,比较研究rNP与天然汉坦病毒核蛋白 (NP)的抗原功能。并与酶联免疫吸附试验 (ELISA)和间接免疫荧光法 (IFA)对比检测。结果　用rNP和天然NP平行检测HFRSIgM符合率达 89.7% ,HFRSIgG符合率达92 .3% ;CGIDA法检测HFRSIgM的敏感性为 75 .0 % ,特异性为 10 0 % ;CGIDA法检测HFRSIgG的敏感性为 83.1% ,特异性为10 0 %。结论　重组核蛋白的CGIDA对HFRS的早期诊断具有较好的应用价值。     

Clinical reliability of IgG, IgA, and IgM antibodies in detecting Epstein-Barr virus at different stages of infection with a commercial nonrecombinant polyantigenic ELISA

We studied the diagnostic reliability of a modification of the Enzygnost EBV test (Behringwerke, Germany) for the detection of IgG, IgA, and IgM antibodies (Abs) in the diagnosis of Epstein-Barr virus (EBV) disease. One hundred and twenty-three serum samples were studied: 14 asymptomatic subjects without EBV infection, 48 patients with primary infection, 46 subjects with past EBV infection (11 patients with other acute infections), 8 patients without EBV infection but with other viral infection, and 7 patients with probable acute clonal stimulation of B lymphocytes caused by different microorganisms. Enzygnost EBV is based on an ELISA test with a pool of viral antigens. In our series the reliability of IgM for the diagnosis of recent primary EBV infection was: sensitivity 100%, specificity 95%, positive predictive value 90.5%, and negative predictive value 100%. The IgG detection with Enzygnost was: sensitivity 98%, specificity 100%, positive predictive value 100%, and negative predictive value 91.7%. Only two subjects had positive IgA. The Enzygnost test is an efficient method for the diagnosis of EBV infection although a few IgM false positives can occur.     

A cellular basis of immunity in experimental Brucella infection

Brucella suis, Brucella abortus, and Brucella melitensis were shown by microscopic and cultural procedures to multiply extensively within normal rat, mouse, and guinea pig monocytes maintained in vitro in cell cultures for 3 days. Intracellular growth of brucellae had no observable toxic effects on most monocytes, although many of the cells became completely engorged with brucellae within 3 days. Non-smooth brucellae and strain 19 multiplied slowly within normal monocytes. In contrast, "immune" monocytes) i.e. those derived from animals previously infected with smooth brucellae, greatly restricted the intracellular growth of smooth and non-smooth brucellae and strain 19. Growth of smooth Brucella, within either normal or "immune" monocytes, was not influenced by addition of Brucella antiserum to the culture medium. Desensitization of immunized guinea pigs did not diminish the refractory state of their monocytes. Cellular resistance did not develop when animals were vaccinated with heat-killed brucellae, though these animals did produce agglutinating antibody. Similarly, vaccination of animals with living, rough B. suis failed to induce a refractory state in their monocytes, even though the vaccinated animals developed delayed hypersensitivity to smooth Brucella antigen. In vivo studies of Brucella survival in the spleens of normal and vaccinated mice (treated with streptomycin to prevent extracellular survival) gave strong support to the in vitro demonstrations of acquired "cellular immunity." Some implications of these results are discussed.     

Recombinant OMP28 antigen-based indirect ELISA for serodiagnosis of bovine brucellosis

Brucellosis is a zoonosis of both public health and economic importance in many developing countries including India. Early detection and segregation of the infected animals are important in order to control the disease. Serodiagnostic tests for brucellosis is mainly based on detection of antibodies developed against lipopolysaccharide (LPS) component of cell. In this study we evaluated a protein antigen, 28 kDa outer membrane protein (OMP28), of Brucella melitensis as an alternative to LPS. Recombinant OMP28 was produced in Escherichia coli system. The efficacy of purified OMP28 was studied in an indirect enzyme-linked immunosorbent assay (ELISA) for diagnosis of brucellosis in field sera collected from different regions of country. Using known negative and known positive serum samples it was found that OMP28 is immunoreactive to Brucella infected cattle, sheep, goat and dog sera. Three hundred and eighty two cattle sera were screened by OMP28 antigen-based ELISA and the results were compared to rose Bengal plate agglutination Test (RBPT). Recombinant OMP28 antigen-based ELISA has shown sensitivity of 88.7%, specificity of 93.8% and accuracy of 92.9%. It was concluded that recombinant B. melitensis OMP28 could be used as a protein antigen for diagnosis of brucellosis in domestic animals.