Malignant hyperthermia during prolonged surgery for tumour resection

Patients with subacute cutaneous lupus erythematosus (SCLE) have circulating antibodies to Ro/SSA directed to two antigenically distinct ribonucleoproteins of 60 kDa and 52 kDa. Three laboratory tests may be used to detect anti-Ro/SSA antibodies: counterimmunoelectrophoresis (CIE), enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB). Their relative efficacy and clinical correlations were ascertained. We determined anti-Ro/SSA antibodies with CIE, with two different ELISA methods (ELISA 1 and 2) and with IB in 29 SCLE patients. Anti-52 kDa and -60 kDa Ro/SSA antibodies were also assayed with IB. In addition, we determined antinuclear antibodies with indirect immunofluorescence, anti-Sm, anti-RNP, anti-La/SSB and anti-Ro/SSA antibodies with CIE and ELISA, and anti-nDNA and cardiolipin antibodies using an ELISA method. CIE detected anti-Ro/SSA antibodies in 22 patients while ELISA 1 and 2 did so in 17 and 18 patients, respectively. In five patients, IB revealed a reactivity to 60 kDa polypeptides and in two, a reactivity to 52 kDa polypeptides. Of these seven patients, four had a myocardial infarction. Of these, two reacted to the 52 kDa antigen and two to the 60 kDa antigen. A combination of techniques was often needed to detect all specificities. ELISA proved to be very specific and sensitive. The IB technique detected a group of patients with myocardial infarction. A case-control study is needed to confirm the data of cardiac involvement.     

The autoantibody response to Ro/SSA in cutaneous lupus erythematosus

BACKGROUND AND DESIGN: Seventeen patients with subacute cutaneous lupus erythematosus (SCLE) were compared with 15 patients with discoid lupus erythematosus (DLE) to evaluate the relationship of 60- and 52-kd Ro/SSA autoantibodies to the clinical diagnosis and to evaluate assays for anti-Ro/SSA. RESULTS: All serum samples from patients with SCLE had precipitating anti-Ro/SSA antibodies in immunodiffusion, and all had high titer anti-60-kd Ro/SSA in enzyme-linked immunosorbent assay. Immunoblotting was inadequately sensitive for detecting anti-60-kd Ro/SSA. Fifteen patients with SCLE had anti-52-kd Ro/SSA (11 high titer, four low titer). Only one of the 15 patients with DLE had precipitating, high-titer anti-Ro/SSA. Nine other patients with DLE had low-titer anti-60-kd Ro/SSA, and four had low-titer anti-52-kd Ro-SSA. Low-titer anti-Ro/SSA did not confer an increased risk for photosensitivity in the DLE group. CONCLUSIONS: High-titer, precipitating antibodies to Ro/SSA are typical of SCLE and unusual in DLE. Low-titer, nonprecipitating antibodies to Ro/SSA are common in DLE and could be an indication of pathogenic factors shared with SCLE. However, low titers of anti-Ro/SSA do not confer a significant risk for SCLE skin lesions. For the purpose of clinical evaluation of skin disease, immunodiffusion assays for anti-Ro/SSA are cost-effective and informative.     

The effects of interferon-gamma on the central nervous system

A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.     

An enzyme-linked immunosorbent assay (ELISA) for monitoring guineapigs and rabbits for Bordetella bronchiseptica antibodies

An enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies specific to Bordetella bronchiseptica in guineapigs and rabbits was developed. In conventional and SPF colonies of guineapigs and rabbits, the ELISA was equally successful in detecting infected animals when compared to selective cultivation from the respiratory tract. The ELISA showed a sensitivity of 100% and a specificity of 90% in guineapigs. In rabbits the sensitivity and specificity of the ELISA were 97% and 91% respectively. In rabbit sera from infected colonies, ELISA activity showed a statistically significant correlation with titres obtained in the micro-agglutination test. Since serologically unrelated strains of the bacterium exist, the monitoring of animals for B. bronchiseptica infection by ELISA should be performed with various antigens.     

Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae. It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Cross-reactivity of anti-10kD heat shock protein antibodies in leprosy and tuberculosis patients

The response to recombinant 10-kD heat shock protein (HSP) of Mycobacterium leprae (rML10) was evaluated by indirect ELISA in sera from leprosy patients, household contacts, tuberculosis patients and healthy controls in a leprosy-endemic area in the North East of Argentina. Some technical parameters were analyzed: within-assay and between-assay variability, dose-response curves and detectability indexes (specificity and sensitivity) of ELISA applied to measure anti-10 kDa antibodies. High levels of these antibodies have already been reported in positive bacilloscopy patients; herein we have also demonstrated that tuberculosis patients sera cross-react with this M. leprae antigen. This test seems to have a low sensitivity and specificity for leprosy detection; it confirms that antibodies against highly conserved HSP antigens are important in the polyclonal response against mycobacterial epitopes in leprosy as well as in tuberculosis.     

Impact of ultraviolet irradiation on expression of SSA/Ro autoantigenic polypeptides in transformed human epidermal keratinocytes

SSA/Ro autoantibodies are frequently found in various autoimmune disorders including subacute cutaneous and neonatal lupus erythematosus. SSA/Ro patient sera precipitate a ribonucleoprotein complex consisting of multiple polypeptides and small RNA molecules (hY RNA). Such sera react in Western blot with at least four antigenically distinct proteins having molecular weights of 52-60 kD. Several laboratories have reported increased binding of anti-SSA/Ro patient serum to viable cultured human epidermal keratinocytes following UVB irradiation. However, it is currently unknown which SSA/Ro molecule(s) might be responsible for this increased antibody binding to UVB irradiated keratinocytes. To address this question, we studied the effect of UVB irradiation on the expression of three different polypeptide components of the SSA/Roautoantigen complex (60 kD SSA/Ro, 52 kD SSA/Ro, and 46 kD SSA/Ro (calreticulin) in A431 cells, a transformed human epidermal keratinocytes cell line. Total cellular and cell surface expression of each SSA/Ro antigenic polypeptide was examined by a whole cell ELISA and FACS using rabbit anti-synthetic peptide antisera as probes. Our results suggest that both total cellular and cell surface calreticulin, but not the 60 and 52 kD SSA/Ro polypeptides, is increased after 100 J/M2 of UVB irradiation, indicating that perturbed calreticulin expression may be primarily responsible for the UVB-induced increased binding of anti-SSA/Ro to keratinocytes. These results suggest that calreticulin could be a critical component of the SSA/Ro ribonucleoprotein complex that is involved in the pathogenesis of anti-SSA/Ro-associated photosensitive LE skin lesions.     

Oncologic surgery of the larynx after failure of radiotherapy]

An association between complete congenital heart block (CCHB) and anti-Ro/SSA antibody is well recognized but has never been reported in Thailand. We report here a 37-year-old female who was admitted because of massive epistaxis secondary to immune thrombocytopenia. She had given birth to a child with CCHB 2 years previously, when she was healthy. Antinuclear antibody and anti-Ro/SSA were positive in her sera, but were negative in her son. The relationship between anti-Ro/SSA antibody and outcome of mothers with infants with CCHB is reviewed.     

Differential effect of donor-specific blood transfusions after kidney, heart, pancreas, and skin transplantation in major histocompatibility complex-incompatible rats

The excretory-secretory (E-S) products of helminths are considered to comprise immunogenic molecules of high diagnostic value. In the present study, the serodiagnostic potential of the E-S products released in vitro by cultured female Onchocerca volvulus was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting using 190 serum samples from persons infected with O. volvulus and unexposed persons. The sensitivity of detection of anti-O. volvulus E-S antibodies was 94% for sera from patients with the generalized form of onchocerciasis and 100% for sera from patients with the chronic hyperreactive form (sowda). 95% of the sera from amicrofilaridermic persons, who subsequently became microfilaridermic within 2 years, reacted with O. volvulus E-S antigens and the donors were therefore regarded as having had a prepatent infection when first examined. These sera gave higher (P < 0.05) ELISA optical densities than sera from the same persons obtained when they had become patent, indicating a loss of antibody reactivity after emergence of microfilariae. The specificity of the E-S ELISA was 100% when sera of subjects infected with Wuchereria bancrofti were used, and at least 88% for Mansonella perstans sera. In Western blot analysis, the sera of persons with generalized onchocerciasis recognized 7 protein bands. Many E-S proteins were stained less intensely by the sera of subjects with generalized onchocerciasis than by the sera of sowda patients. Similar antigen bands were demonstrated using sera from the persons with prepatent infections.     

IgA antibodies to tissue transglutaminase: An effective diagnostic test for celiac disease

OBJECTIVE: Tissue transglutaminase (tTG) is the main autoantigen recognized by endomysial antibodies. The aim of this study was to assess sensitivity, specificity, and predictive value of IgA and IgG antibodies to tTG in the diagnosis of celiac disease compared with endomysial antibodies. STUDY DESIGN: We established enzyme-linked immunosorbent assay procedures to measure IgA and IgG antibodies to tTG in sera from 48 untreated and 33 treated patients with celiac disease and from 63 patients with gastrointestinal disease who were in a control group. Sera from 10 patients with celiac disease were examined at various times after gluten was reintroduced into the patients' diet. RESULTS: Both IgA and IgG to tTG were significantly (P <.001) higher in serum of untreated patients with celiac disease versus those in the control group; IgA but not IgG was significantly (P <.001) higher in untreated versus treated patients with celiac disease. IgA and IgG antitissue tTG had a diagnostic sensitivity, specificity, and positive predictive value of 92% and 21%, 98% and 97%, and 98% and 83%, respectively. The concordance rate of IgA anti-tTG with IgA antiendomysial antibodies was 95%. In 5 of the 10 patients undergoing gluten challenge, IgA antiendomysium antibodies were detected earlier than IgA anti-tTG antibodies. CONCLUSIONS: tTG-based enzyme-linked immunosorbent assay is an effective diagnostic test, although immunofluorescent-based assays are more sensitive, particularly during gluten challenge.     

Neutralizing antibodies to Escherichia coli Vero cytotoxin 1 and antibodies to O157 lipopolysaccharide in healthy farm family members and urban residents

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to Escherichia coli O157 lipopolysaccharide (LPS) was developed with sera from 63 children with confirmed recent E. coli O157 infection and from 256 age-stratified urban controls. The median ELISA values for control and case sera were 0.05 (interquartile range, 0 to 0.20; mean +/- standard deviation [SD], 0.15 +/- 0.22) and 1.41 (interquartile range, 1.11 to 1.59; mean +/- SD, 1.41 +/- 0.53), respectively (P < 0.001). With a breakpoint of 0.59 (mean ELISA value of the control sera + 2 SDs), the assay had a sensitivity, specificity, and positive and negative predictive values of 95, 94, 80, and 98%, respectively, for recent E. coli O157 infection. The O157 LPS assay and Vero cytotoxin (VT) 1-neutralizing-antibody (NAb) assay were used to compare the relative frequencies of O157 LPS antibodies and VT1-NAbs in an age-stratified urban population from Toronto, Ontario, Canada, and in 216 healthy family members from dairy farm in southern Ontario. The frequency of O157 LPS antibodies was about threefold higher in dairy farm residents (12.5%) than in urban residents (4.7%) (P < 0.01). Similarly, the frequency of VT1-NAbs was about sixfold higher in dairy farm residents (42.0%) than in urban residents (7.7%) (P < 0.001). These findings are consistent with a greater level of exposure of dairy farm residents to VT-producing E. coli (VTEC) strains. The high rate of seropositivity to VT1 in farm residents probably reflects the booster effect of repeated VTEC exposures and argues against a sustained generalized immunosuppressive effect of VT1. Seroepidemiological studies may help in assessing the level of exposure of different populations to VTEC strains.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.     

Evaluation of a commercial immunodiagnostic kit incorporating lipoarabinomannan in the serodiagnosis of pulmonary tuberculosis in Ghana

We evaluated 'Mycodot', a commercially marketed immunodiagnostic test for tuberculosis which detects antibodies to lipoarabinomannan antigen. Serum was tested from 52 patients with newly diagnosed smear-positive pulmonary tuberculosis, of whom 20 were HIV-positive and 32 HIV-negative. Control sera were taken from 40 patients of whom 20 had acute non-tuberculous lobar pneumonia and 20 patients had no respiratory disease. The test was found to have a very high specificity of 97.5% (95% CI:92.5-100%). However, the sensitivity in HIV-negative patients was 56% (95% CI:39-73%), and was substantially lower at 25% (95% CI:6-44%) in HIV-positive patients. In conclusion: 'Mycodot' was found to be a highly specific and easily performed assay. However, the poor sensitivity, especially in HIV-infected patients, renders it unlikely to be useful either as a primary or adjunctive diagnostic test for tuberculosis, particularly in countries with a high prevalence of HIV. A larger trial of this assay in Ghana was not deemed necessary.     

Serodiagnosis of cutaneous leishmaniasis: assessment of an enzyme-linked immunosorbent assay using a peptide sequence from gene B protein

An enzyme-linked immunosorbent assay (ELISA) using a 28 amino acid sequence of the repetitive element of gene B protein (GBP) from Leishmania major was developed for serodiagnosis of cutaneous leishmaniasis (CL). The assay was compared to ELISAs using crude amastigote and promastigote antigens from L. donovani and the major surface glycoprotein (Gp63) from either L. donovani or L. major as a solid-phase ligand. The sensitivity of the assays was tested in 33 patients suffering from CL caused by L. major. The sensitivity of the GBP peptide (GBPP) ELISA was 82%. This was higher than in the assays using crude amastigote (67%) or promastigote (67%) antigens, but the difference was not statistically significant. The sensitivity in the assays using Gp63 from L. donovani (52%) or L. major (39%) was significantly lower than in the assay using GBPP (P = 0.019 and P < 0.001, respectively). Plasma samples from healthy Sudanese individuals living in an area endemic for malaria but free of leish-maniasis were negative in all the assays. Significantly higher levels of antibodies were found in the patients who had suffered from the disease for more than eight weeks than in patients with a shorter clinical history (GBPP ELISA; P = 0.038; amastigote ELISA; P = 0.004; and promastigote ELISA; P = 0.017). In the former group, the sensitivities of the five ELISAs were 100% (GBPP), 87% (amastigote), 93% (promastigote), 67% (L. donovani), and 53% (L. major), respectively.     

Clinical features and evolution of antinuclear antibody positive individuals in a rheumatology outpatient clinic

OBJECTIVE: To identify individuals with antinuclear antibodies (ANA) not fulfilling criteria for systemic lupus erythematosus (SLE) or other connective tissue diseases (CTD); to describe their clinical and serological features, to identify factors indicating evolution to SLE. METHODS: A case-control study, based on retrospective evaluation of clinical files. The cases were ANA positive individuals (n = 50) examined in a medical outpatient setting, for symptoms compatible with SLE, but not fulfilling SLE classification criteria. Two patients with SLE were matched to each case in terms of age at initial symptom onset and sex. Thyroid autoimmunity was assessed by detecting antithyroid antibodies. Fisher's exact test and conditional logistic regression were used to evaluate the predictive ability of initial findings for SLE development. RESULTS: ANA positive individuals suspected of having a CTD present a wide variety of symptoms and findings, usually at the 4th to 5th decade of life. Antibodies to Sm and U1RNP were absent; anti-Ro(SSA) and anti-La(SSB) occurred in 6%, while anti-dsDNA occurred in less than 10% of the cases. Arthritis, butterfly and discoid rash, Raynaud's phenomenon, and anti-Ro/SSA antibodies are the initial findings indicating evolution to SLE. Hematological abnormalities such as leukopenia and thrombocytopenia as well as constitutional symptoms such as easy fatigue and arthralgias are not associated with evolution to SLE. Antithyroid antibodies were detected in 16% of the cases and 2.3% of controls. CONCLUSION: ANA may connote a form of systemic autoimmunity that is expressed as a wide variety of complaints, even in the absence of a definite diagnosis of CTD. Arthritis, rash, Raynaud's phenomenon, and anti-Ro/SSA antibodies indicate evolution to SLE. Autoimmune thyroid disease occurs in ANA positive individuals not fulfilling SLE classification criteria rather than in patients with SLE.     

Characterization of two corneal epithelium-derived antigens associated with vasculitis

PURPOSE: In a previous investigation into corneal autoimmunity, it was demonstrated that a putative autoantigen, a protein of 66 kDa, present in bovine corneal epithelium, binds circulating autoantibodies in approximately 60% of patients with Wegener's granulomatosis (WG). The aim of the present study was to characterize and identify the 66-kDa protein. METHODS: A purification protocol was established for the 66-kDa protein using standard chromatography techniques. During the purification procedure it became clear that the 66-kDa protein detected in patients' sera was in fact two proteins, both running at 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, that eluted in different fractions on DE-52 chromatography columns. These two proteins have been labeled bovine corneal epithelial antigen-A and -B (BCEA-A and BCEA-B). Further investigations of antibody binding have demonstrated that patients' sera bind to either one or the other of these proteins with no cross-reactivity between them. Separated BCEA-A and BCEA-B protein extracts were immunoblotted with 27 WG patients' sera, 10 Churg-Strauss syndrome (CSS) patients' sera, 31 rheumatoid arthritis (RA) patients' sera, and 40 healthy control subjects' sera from the blood bank. RESULTS: Forty-six percent of WG patients' sera had antibodies to one of the 66-kDa antigens, whereas none of the healthy control subjects' sera had 66-kDa antibodies (P < 10(-5)). In the WG group, 31% were positive to BCEA-A (versus controls, P = 0.0023), and 15% were positive to BCEA-B. WG patients with peripheral ulcerative keratitis (PUK) had a significant association with anti-BCEA-A antibodies when compared with healthy control subjects (50%, P < 10(-6)). However, in the RA group with no eye disease there was an association with BCEA-A (25%, P = 0.011) but not in the RA group with PUK. The frequency of anti-BCEA-B antibodies was significantly increased in patients with CSS (60%, P < 10(-7)). CONCLUSIONS: In summary, it has been shown that vasculitis patients have antibodies to two 66-kDa corneal antigens and that autoantibodies to these antigens are mutually exclusive. It has also been shown that antibodies to BCEA-B are associated with CSS, whereas BCEA-A antibodies are associated with WG and RA.     

The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle

A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.     

Combinations of three immunological assays for detecting anti-Toxoplasma IgG in the sera of patients infected with Toxoplasma gondii

Enzyme-linked immunosorbent assay (ELISA), Dot-ELISA and Dot-immunogold silver staining (Dot-IGSS) were simultaneously used to detect the specific IgG against Toxoplasma gondii in 65 patients infected with the protozoa. The positive rates were 86.51%, 92.51% and 98.64%, respectively. When ELISA and Dot-ELISA results were put together, the positive rate increased to 95.38%. When Dot-IGSS results were combined with those of ELISA or Dot-ELISA, the positive rate was raised to 100%. The difference in positive rate between ELISA and Dot-IGSS was significant (x2 = 6.93, p < 0.01), but no statistically significant differences were found between ELISA and Dot-ELISA or between Dot-ELISA and Dot-IGSS. Paired comparison of the reacting intensities of the sera in the 3 assays showed the correlations were highly significant (p < 0.001), with r = 0.608 between Dot-IGSS and Dot-ELISA, r = 0.8194 between Dot-IGSS and ELISA and r = 0.517 between Dot-ELISA and ELISA. Hence combination of different serological assays may increase their sensitivity and specificity for detecting the anti-Toxoplasma antibodies.     

Antigens for serological diagnosis of ovine footrot

An antigen extracted from Dichelobacter nodosus with potassium thiocyanate (KSCN) is currently used in enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of ovine footrot, but the test lacks specificity in mature sheep. Other antigens were therefore evaluated for use in this test. Structural components of the cell envelope of D. nodosus including outer membrane, cytoplasmic membrane, lipopolysaccharide and pilus and extracellular proteases were purified from cultured D. nodosus while recombinant membrane proteins, protease and pilus antigens were also evaluated. Many antigenic components of D. nodosus participated in reactions in ELISA that were not specific for infection with D. nodosus and apart from pilus, none of the antigens resulted in improved specificity of the ELISA. Using a positive-negative cut-off to yield sensitivity of 70%, ELISA using pili from cultured D. nodosus serogroup A had a specificity of 98.3% compared with 89.7% for the ELISA with KSCN-extract as antigen (P < 0.001). Recombinant pili morphogenetically expressed in Pseudomonas aeruginosa were unsuitable for use in ELISA due to copurification of Pseudomonas antigens to which apparently healthy sheep directed antibodies. The application of ELISA with D. nodosus pilus as antigen in footrot control programs is discussed.     

Analysis of conserved ambisense sequences within GB virus C

No analysis has been done of the ambisense of GB virus C (GBV-C). When the anti-genomes of 16 reported sequences of GBV-C were analyzed, nucleotide codons 1758 and 1402 within the anti-genome were conserved initiation and stop codons, respectively. Nucleotide sequences were also determined within the same region of 22 GBV-C strains. The anti-genomes of 38 sequences were translated and a consensus sequence was determined. In accordance with the consensus sequence, overlapping peptides were synthesized and used for the detection of anti-synthetic peptide antibodies by ELISA. The positivity of antibodies among sera with GBV-C RNA was significantly higher than among sera without GBV-C RNA (66.7% vs. 15.6%), regardless of the simultaneous presence of hepatitis B surface antigen or antibodies to hepatitis C virus (P < .05). These results indicated that a novel protein associated with GBV-C might be expressed from the ambisense of this virus.