A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus

A recombinant baculovirus containing a cDNA clone encoding the nucleocapsid (NP) protein of Newcastle disease virus (strain Ulster 2C) has been used to infect insect cells (Spodoptera frugiperda). High levels of overexpressed NP protein were observed, comprising up to 40% of total cellular protein, which were subsequently shown to be antigenic. Nucleoprotein derived from the crude soluble lysate of infected insect cells has been used in an indirect ELISA to detect the presence of anti-NDV antibodies in a cohort of chicken sera. Data produced from these tests indicated a good correlation between ELISA titre and haemagglutination inhibition test data. The test was not affected by interference from background cellular proteins nor by cross-reactivity with non-NDV poultry pathogens. Additionally, the test did not generate false-positive readings.     

The use of monoclonal antibody for the immunodiagnosis of Fasciola gigantica infection in cattle

Antigens that were specific to Fasciola gigantica were obtained from the whole worm homogenate of the parasite by immunoaffinity chromatography in cyanogen bromide-activated sepharose 4B columns and used for the production of monoclonal antibodies. The F. gigantica-specific monoclonal antibody was labelled with horseradish peroxidase and used for the detection of circulating antigen by the direct ELISA method in the sera of cattle experimentally infected with the parasite. Circulating antigens were detectable in the sera of the animals as from the third week after infection while negative absorbance values were obtained 2 weeks after the termination of the infection by chemotherapy with oxyclozanide. This immunodiagnostic method offers an attractive alternative as a supplement to the conventional coprological diagnosis of fasciolosis.     

A longitudinal study of biological changes with age among the Brahmins of Calcutta

Peptides deduced from the central hydrophobic region (residues 158-189) of the G protein of bovine and ovine respiratory syncytial virus (RSV) and of human RSV subtypes A and B were synthesized. These peptides were used to develop ELISAs to measure specifically antibodies against these types and subtypes of RSV. We have evaluated the bovine RSV-G peptide in both an indirect ELISA and in a blocking ELISA. Specificity and sensitivity, relative to a routine diagnostic ELISA that detects antibodies against the RSV F-protein in bovine sera, were 98% and 92% respectively for the indirect peptide-based ELISA, and 98% and 98% for the blocking peptide-based ELISA. In paired serum samples, rises in antibody titer were detected more frequently with the indirect peptide-based ELISA than with the routine F-ELISA. Furthermore, the peptide-based G-ELISAs were able to differentiate between antibodies against BRSV and HRSV, and between those against BRSV and ORSV. In addition, the indirect peptide-based ELISA was selective for HRSV subtype A and B antibodies. This study shows that peptides, corresponding to the central hydrophobic region of the attachment protein G of several RSVs, can be used successfully as antigens in highly specific and sensitive immunoassays.     

Assessment of humoral immune response in mink (Mustela vison): antibody production and detection

A method for investigating the humoral immune response in mink (Mustela vison) was developed between October 1993 and March 1994. Protein A, 1:8000 dilution, had a high affinity for mink immunoglobulin, while anti-ferret (Mustela putorius) antibody, 1:200 dilution, had a weaker affinity. Four adult mink were immunized with a hapten, dinitrophenol (DNP), conjugated to a large carrier protein, keyhole limpet hemocyanin (KLH), and received two boosters at 3-week intervals. This provoked a strong T-lymphocyte dependent humoral immune response. An indirect enzyme linked immunosorbent assay (ELISA) was used to quantify the antibody produced. All mink had undetectable anti-DNP-KLH antibody in the pre-immune sera, with antibody levels increasing post-immunization, and peaking after the first or second booster.     

Isolation, characterization and comparison of antipeptide and antiprotein rabbit antibodies to the pi-isoform of glutathione S-transferase

The main linear epitopes of pi-glutathione transferase (pi-GST, EC 2.5.1.18), an enzyme related to cancer progression in a restricted number of tumours, were identified by testing in ELISA the reactivities of polyclonal anti-pi-GST rabbit sera against a panel of 51 overlapping decapeptides, covering the whole 216-residue sequence of the protein. Several major reactivity peaks were detected, each covering two or three adjacent peptides. The most active fragments were then reconstructed by conventional solid-phase synthesis, linked to Sepharose, and used as affinity ligands for isolating specific anti-pi-GST antibody subsets. A second group of antisera was then prepared in rabbits by using as immunogens some of the above described synthetic fragments, linked to a carrier protein, and antipeptide antibodies purified by affinity chromatography. An ELISA test was then performed, using as antigens a panel of peptides and different isoforms of GST, in order to establish whether antibodies isolated from total anti-pi-GST sera would display higher reactivity and specificity, as compared to traditional antipeptide antibodies. Binding data clearly confirm that the formers might be indeed better reagents for the detection and possibly quantitation of pi-GST.     

Evaluation of protein A and protein G as indicator system in an ELISA for detecting antibodies in mink to Pseudomonas aeruginosa

A modified, indirect enzyme-linked immunosorbent assay (ELISA) was developed and applied in the detection of mink antibodies to Pseudomonas aeruginosa. In this assay, peroxidase conjugated protein A and protein G were evaluated as indicator systems for detecting antigen-antibody complexes. It was found that protein A has a strong affinity for mink immunoglobulins. In contrast, protein G showed no such affinity. The affinity of protein A for mink immunoglobulins was further demonstrated by immunoprecipitation assays.     

ACP Broadsheet No 152: March 1998. Clinical implications of plasma homocysteine measurement in cardiovascular disease

Enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) are the most widely used serological tests for Maedi-Visna diagnostics. The purpose of the present study was to develop an indirect whole virus ELISA and an immunodiffusion test and compare their sensitivity. A total of 747 ovine serum specimens were analysed for antibodies against this ovine lentivirus. The number of positive results in the ELISA was 430 (57.56%). In the AGID test, a positive result was found in 380 samples (50.87%). In the group of discordant results 78 (10.4%) samples tested positive by the ELISA and negative by the AGID test and 28 sera (3.7%) were found to be positive by the AGID test and negative by the ELISA. The data in this report show the ELISA to be more sensitive than the AGID test, but accurate serological diagnostics should be based on a combination of the two tests.     

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.     

Nonsteroidal antiinflammatory drugs interact with horseradish peroxidase in an in vitro assay system for hydrogen peroxide scavenging

The established horseradish peroxidase/guaiacol in an in vitro assay system was used for investigation of the reactivity of nonsteroidal antiinflammatory drugs with hydrogen peroxide. Although the drugs rapidly seemed to react in the selected conditions, difficulties were encountered in attempts to quantify the reaction and an interaction with horseradish peroxidase was suspected. A more specific assay system based on the absolute specificity of the enzyme glutathione peroxidase for glutathione was subsequently used which demonstrated that none of the investigated nonsteroidal antiinflammatory drugs was able to scavenge hydrogen peroxide. An original procedure to further evidence the interaction was developed thereafter, based on the reaction of 5-aminosalicylic acid with similar hemoproteins. This led to the demonstration that nonsteroidal antiinflammatory drugs were substrates for horseradish peroxidase and explained their reactivity in the horseradish peroxidase/guaiacol assay system. The compound 5-aminosalicylic acid showed an unusual behaviour that was attributed to its ability to both scavenge hydrogen peroxide and interact with horseradish peroxidase. It was concluded that the lack of specificity of horseradish peroxidase for its donor substrate may lead to erroneous results in assays for hydrogen peroxide scavenging of some drugs. An alternative method is however available and a simple spectroscopic assay can evidence the interaction with horseradish peroxidase.     

Simple, speedy, sensitive, and specific serodiagnosis of pertussis by using a particle agglutination test

We developed a particle agglutination test (KPA) with poly(gamma-methyl L-glutamate) as the solid particle for measurement of pertussis toxin (PT) antibody. In this study, KPA was assessed as a means of serodiagnosing pertussis, and the results were compared with those of indirect enzyme-linked immunosorbent assay (indirect ELISA) and the microagglutination test. First, four serum samples were collected from each of 21 healthy children: before and 4 weeks after receiving three primary doses of acellular pertussis vaccines and before and 4 weeks after receiving a booster dose. In all 21 vaccinees, a significant rise in PT antibody titers was observed by KPA after each vaccination, and among all 84 serum samples collected, an excellent correlation was demonstrated between the values obtained by indirect ELISA and those obtained by KPA (r = 0.92). Second, paired serum samples were collected at intervals of approximately 2 weeks from 51 patients with culture-confirmed pertussis. A significant increase in titer (fourfold or more) was observed in 39 (76%) patients by KPA, 34 (67%) patients by indirect ELISA, and 23 (45%) patients by the microagglutination test. In acute- and convalescent-phase sera collected from 20 nonpertussis patients, there were no changes in titers by KPA. The KPA procedure was as simple as that of the microagglutination test, and the reaction time was only 2 h (or overnight). In this study, KPA was demonstrated to be a simple, speedy, sensitive, and specific serodiagnostic method for pertussis.     

Autoantibody profiles in canine ANA-positive sera investigated by immunoblot and ELISA

Canine systemic lupus erythematosus (SLE) has a similar disease expression as human SLE, but the serological characterisation of the canine disease is as yet incomplete. In the present study, we examined the specificity of antinuclear antibodies (ANA) in indirect immunofluorescence (IIF) positive canine sera. Sixty-four canine IIF ANA positive sera were characterised using HeLa cell nuclear extract immunoblots and recombinant U1-70K ELISA. We compared these results with a previously shown concordance between indirect immunofluorescence and immunodiffusion in canine SLE serological diagnosis. One canine serum reacting with Sm proteins was observed, and five canine sera presented anti-RNP autoantibodies against the antigens 70K, A, C, and/or B/B'. The autoantigen most frequently recognised was a 43 kDa nuclear protein, previously described as hnRNP G. This prominent canine autoantigen was missing in the commercially available extract designed for immunodiffusion testing of human sera. Other prominent canine autoantigens were found not to be identical with the principal human ones, thus making present human test systems deficient for the use in canine systemic connective disease diagnosis. The development of antigenic extract designed for canine autoimmune autoantigens is necessary in order to make immunodiffusion a useful method in canine diagnosis. The anti-RNP positive canine sera were examined in more detail and we found that the human major antigenic region of the most prominent RNP antigen, the U1-70K protein, also is targeted by canine autoantibodies. Thus, the response against the RNP antigen seems to be conserved between man and dog.     

Survey of antitrypanosome antibodies and studies of their specificity and autoimmunity

As a part of investigations to characterize trypanosome infections in Taiwan, sera collected from patients admitted to Veterans General Hospital, Taipei were tested for antitrypanosome antibodies. A Trypanosoma cruzi extract-based enzyme-linked immunosorbent assay (ELISA) was used to screen and titrate 1,297 patient sera. High antitrypanosome titers were detected in 166 (12.8%) of these sera. Retitration of random samples of the high titer (HT) sera indicated a 5.4% false positive. Thirteen donors with high antitrypanosome ELISA titers were followed up. Twelve of then remained high serum titers also showed high ELISA titers against an extract of Trypanosoma conorhini. Hemocultures conducted on freshly drawn blood specimens of the 13 subjects did not provide any evidence of trypanosome infections. Electrophoretic analyses of sera from HT and low titer (LT) patients suggested differences between serum proteins of the subjects in each of the groups. Atypical reactions were observed in immunodiffusion tests performed with HT and LT sera and trypanosome extracts, while western blot analyses revealed a complex pattern of binding by both sera. The qualitative and quantitative differences in these tests suggested interactions of T. cruzi antigens with donor antibodies against unrelated antigens and/or with autoantibodies. Subsequent analyses did not indicate any association between rheumatoid factor and the reactivities of the HT sera with the parasites. However, antinuclear antibodies were detected with an indirect fluorescent antibody test (IFAT) in 50% of the HT sera and 22% of the LT sera. No differences were found between the levels of antilaminin activity of the two groups. The IFAT employing T. cruzi epimastigotes was positive for 100% of the HT sera and 22% of the LT sera. The data indicate that the high seropositivity recognized in this study is due in part to the activities of cross-reacting antibodies and/or autoantibodies in the sample population.     

Detection of Clostridium perfringens type D epsilon antitoxin in serum of goats by competitive and indirect ELISA

Indirect and competitive ELISA techniques were developed and their ability to detect antibodies to Clostridium perfringens epsilon toxin in goat serum was compared. Different dilutions of a hyperimmune goat serum, in serum from a colostrum-deprived kid, were used as positive controls, while sera from eleven colostrum-deprived kids were used as negative controls. The epsilon toxin antibodies in the hyperimmune serum were also measured by mouse neutralisation test (MNT). The correlation coefficient between both the indirect ELISA technique and MNT was 0.99, while the same coefficient for the competitive ELISA was 0.98. Both the indirect and competitive ELISAs proved to be rapid, simple, sensitive and specific for detecting antibodies to C. perfringens epsilon toxin in serum of goats.     

Separation-free electrochemical immunosensor for rapid determination of atrazine

A separation-free electrochemical immunoassay method for the detection of the pesticide atrazine is described. The method developed is a competitive ELISA incorporating disposable screen printed horseradish peroxidase modified electrodes as the detector element in conjunction with single-use atrazine immuno-membranes. Screen printed carbon electrodes were prepared using carbon ink incorporating horseradish peroxidase. A monoclonal antibody for atrazine was immobilised onto Biodyne C membranes which were, in turn, placed over the electrode surface. The assay was based on competition for available binding sites between free atrazine and an atrazine-glucose oxidase conjugate prepared 'in-house'. In the presence of glucose, H2O2 formed by the conjugate was reduced by enzyme-channelling via the HRP electrode. The HRP was in turn re-reduced by a direct electron transfer mechanism at a potential of +50 mV Vs Ag/AgCl. Any H2O2 formed in the bulk solution by unbound atrazine-GOD conjugate was scavenged by excess catalase thus removing the requirement for a washing step. The performance of the method was compared with a commercial immunoassay kit for atrazine.     

Immunodiagnostic studies on Onchocerca volvulus and Mansonella perstans infections using a recombinant 33 kDa O. volvulus protein (Ov33)

A total detergent-soluble extract of adult female Onchocerca volvulus (OvAg) and a recombinant O. volvulus protein (Ov33) linked to glutathione-S-transferase (GST) were compared with regard to their serodiagnostic suitability for differentiating between O. volvulus and Mansonella perstans infections in a region endemic for both filarial worms in western Uganda. Using OvAg in an enzyme-linked immunosorbent assay (ELISA), 98.8% sensitivity was obtained examining 84 O. volvulus microfilariae (mf) carriers living in the hyperendemic area. However, 5 of 18 (28%) sera from M. perstans mf carriers without O. volvulus mf, from another area hypoendemic for O. volvulus, cross-reacted with OvAg. Using the recombinant antigen Ov33-GST in an ELISA and Western blot assay, sensitivity for O. volvulus remained high (97.2% and 98.8% respectively) while none of 90 sera from M. perstans mf carriers reacted positively. Both antigens were used to examine a batch of sera from 260 persons living in the onchocerciasis hyperendemic area who did not have mf in their skin snips (9.5% of 2728 persons examined); 116 of these sera (44.6%) were positive in the OvAg ELISA, compared to 85 (32.7%) and 69 (26.5%) which were positive in Ov33-GST ELISA and Ov33-GST Western blot, respectively. Reaction with GST alone was minimal. The recombinant antigen Ov33 efficiently differentiates between O. volvulus and M. perstans infections, and is sensitive when used to detect patent and prepatent or low-level O. volvulus infections.     

Effects of phencyclidine on immediate early gene expression in the brain

An enzyme-linked immunosorbent assay (ELISA) was developed for sensitive and specific determination of pravastatin (PS) sodium, a HMG-CoA reductase inhibitor. Preparation of immunogens to obtain antisera was carried out using chemically modified PS; beta-alanine derivative of PS (for ELISA-1) and 5-deoxy- PS (for ELISA-2) were linked to bovine serum albumin via its terminal carboxylic acid by the N-succinimidyl ester method, to avoid intramolecular lactonization of PS. Enzyme-labeled antigens were prepared similarly by coupling with horseradish peroxidase, and were used by homogeneous combination of antisera. The enzymic activity was determined using a microtiter plate coated with second antibody and tetramethylbenzidine as a chromogenic substrate. Both of the ELISA systems enabled the determination of PS in a range of 5 to 500 pg/well, with an IC50 of 36 to 130 pg/well. Cross-reactivties with main metabolites in plasma, which differed from PS in decaline moiety, were less than a few percent. When ELISA-1 was applied to the determination of PS in human plasma directly after dilution with the ELISA buffer, the detection limit and the intra-assay coefficient (5 ng/ml of PS) were 500 pg/ml and 4.5%, respectively. Further, ELISA-1 was validated by gas chromatography-mass spectrometry with the determination of PS in human plasma after oral administration at a dose of 10 mg/body.     

Annexin V autoantibodies in rheumatoid arthritis

OBJECTIVE: To investigate the occurrence of anti-annexin V autoantibodies in sera of patients with rheumatoid arthritis to assess involvement with the disease and any relation to glucocorticoid treatment. METHODS: Anti-annexin V antibodies were measured by an enzyme linked immunosorbent assay (ELISA) which used the purified human recombinant protein as antigen. RESULTS: Concentrations of anti-annexin V autoantibodies, predominantly of the IgG class, were significantly raised in sera from patients with rheumatoid arthritis compared to normal controls. This was not correlated with other indices of disease activity such as erythrocyte sedimentation rate or C reactive protein and was unrelated to glucocorticoid treatment. CONCLUSIONS: Extracellular annexin V provides an antigenic stimulus for autoantibody production and its in vivo expression is independent of glucocorticoid control. Such autoantibodies may have a detrimental role in the arthritic condition by interfering with putative functions of annexin V, including collagen type II binding, inhibition of phospholipase A2 activity, and Fc receptor activity.     

Detection of anti-topoisomerase I antibodies using a full length human topoisomerase I recombinant protein purified from a baculovirus expression system

Topoisomerase I (topo I) is a major systemic sclerosis (SSc)-associated autoantigen. A cDNA construct encoding full length human topo I in a recombinant baculovirus transfer vector was used to infect insect cells in culture from which recombinant protein was purified. An ELISA using recombinant protein was evaluated in 340 sera including sera from 134 patients with SSc, of whom 33 had anti-topo I antibodies detected by immunodiffusion. A high yield of pure topo I of expected molecular mass and catalytic activity was obtained. The recombinant topo I ELISA was 92% sensitive and 98% specific in detecting anti-topo I antibodies which were present almost exclusively in patients with SSc. Therefore, the potential advantages of expressing human autoantigens in eukaryotic systems for diagnostic purposes were confirmed.     

Assessment using ELISA of the herd immunity levels induced in cattle by foot-and-mouth disease oil vaccines

The development of a liquid-phase blocking sandwich ELISA (LPBE) to measure antibodies (Ab) produced in cattle with the O, A and C foot-and-mouth disease virus (FMDV) types of commercial vaccines used in Argentina is described. The test was specific: 99% of na?ve cattle sera (n = 130) gave titres below log10 = 1.2, and none had a titre above log10 = 1.5. Comparative studies with serum neutralization test (SNT) using sera from cattle which received one or more vaccine doses is reported. The overall rank correlation coefficient (Spearman's rho, rs) between SNT and LPBE were highly significant (rs > 0.67, P < 0.0001) for all vaccine strains. LBPE Ab titres on sera collected 90 days post vaccination were compared with results of cattle protection tests by applying a logistic regression. The minimum Ab titres at which 85% and 75% of the cattle were protected for each FMDV type were determined in order to interpret field Ab data in terms of protection. Application of this method allows large scale serological examinations to monitor antibody levels in vaccinated animals as an indirect indicator of the FMD control program status in the field. Its use in the evaluation of commercial batches of FMD vaccine is discussed.