Indirect Enzyme Immunoassay Research Articles

SEROEPIDEMIOLOGIC STUDY OF LEISHMANIA INFANTUM INFECTION IN CASTILLA-LEON, SPAIN

Leishmaniasis has increased in importance in recent years because infection with human immunodeficiency virus (HIV) has emerged as a risk factor for this disease. However, the actual prevalence of leishmaniasis in the general population of Spain is unknown. We present a study of the seroprevalence of infection with Leishmania infantum in the general population of Castilla-Leon, Spain. A random sample of individuals presenting to health care clinics (4,825 sera) and of HIV-infected patients in the autonomous community of Castilla-Leon was collected in 1996. The sero-prevalence of antibodies to L. infantum was determined by an indirect enzyme immunoassay and found to be 4.9% in the general population. There was a significant increase in seroprevalence with age (P = 0.001), from 3.96% in those 14-20 years old to 7.2% in those > 70 years old. There were no significant differences between women and men (5.0% versus 4.9%; P = 0.9534). Seroprevalence was significantly higher in people from rural areas than in those from cities (6.0% versus 3.4%; P = 0.001). Patients infected with HIV had a seroprevalence for L. infantum of 64.0%. No differences were observed between women and men, and prevalence did not increase with age.

Development of an indirect competitive ELISA for detection of Campylobacter jejuni subsp.jejuni O:23 in foods.

An indirect enzyme immunoassay for rapid detection of Campylobacter jejuni subsp. jejuni 0:23 has been developed. Optimum concentrations of immobilized cells, polyclonal chicken IgY, and rabbit anti-IgY antibody-horseradish peroxidase conjugate were 3.1 CFU/nL, 10 microg/mL, and 8 microg/mL, respectively. Under such conditions, the detection limit reached 50 CFU/microL, limit of quantification being 480 CFU/microL. By testing 5 chromogens, viz. 1,2-benzenediamine, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3',5,5'-tetramethylbenzidine, bi(4,4'-anisidine) and 3-methyl-2-benzothiazolinone hydrazone, in horseradish peroxidase substrate, 1,2-benzenediamine or 3,3',5,5'-tetramethylbenzidine as H-donors in the enzyme substrate provided the highest ELISA sensitivity. The applied polyclonal antibody was specific for homogeneous antigen. The cross-reactions were observed only with one strain of C. sputorum subsp. sputorum (21.5 %) and with G+ bacterium Micrococcus luteus (6.1 %). Preliminary tests have been performed with a limited number of artificially contaminated food samples. No matrix effects on the ELISA sensitivity were observed. The results (by means of ELISA) were comparable with those given by both a standard cultivation method performed according to CSN ISO 10272 and commercially available Singlepath Campylobacter GLISA-Rapid Test.

Evaluation of serological tests for detection of caprine antibody to Brucella melitensis

A total of 2213 goat sera were tested by the indirect enzyme immunoassay (IELISA), the competitive enzyme immunoassay (CELISA), the fluorescence polarization assay (FPA), complement fixation test (CFT) and the buffered antigen plate agglutination test (BPAT) for their antibody against Brucella melitensis. Of these, 528 blood samples collected in EDTA were tested in the field by the FPA (bFPA). In addition, 249 goat milk samples were tested by IELISA (mELISA). The serum samples were classified based on their reactivity in the BPAT and CFT. A total of 716 sera were positive while 1497 gave negative reactions in both tests. Relative to the BPAT and CFT reactivity, the sensitivity and specificity of the assays were as follows: IELISA: 96.2 and 99.7%, CELISA: 93.6 and 99.4% and FPA: 88.7 and 98.9%, respectively. The bFPA gave sensitivity and specificity values relative to the BPAT and CFT of 92.3 and 61.6% while the mELISA, based on the serum BPAT and CFT of the paired animals gave a relative sensitivity value of 90.9% and a relative specificity value of 92.4%.

Enzyme immunoassay for the diagnosis of brucellosis: chimeric Protein A–Protein G as a common enzyme labeled detection reagent for sera for different animal species

A recombinant protein combining the immunoglobulin binding sites of Proteins A and G, conjugated with horseradish peroxidase was used as a universal detection reagent for the assessment of antibodies against Brucella spp. The reagent was applied in an indirect enzyme immunoassay for detection of antibodies to smooth lipopolysaccharide antigen in sera from Brucella spp. exposed and non-exposed cattle, sheep, goats and pigs and to antibodies to rough lipopolysaccharide in sheep, dogs and cattle. The results were similar to those obtained when murine monoclonal antibody–enzyme conjugates were used. An added advantage was that a universal cut-off for all tests using the proteins A and G detection reagent could be established, simplifying diagnostic interpretation of the data.

Structural and immunochemical relationship between the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 396/C-1 and Escherichia coli O126

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 396/C-1 has been determined. Sugar and methylation analyses together with 1H and 13C NMR spectroscopy were the main methods used. Inter-residue correlations were determined by 1H, 1H-NOESY, 1H, 13C-heteronuclear multiple-bond correlation and dipole–dipole cross-correlated relaxation experiments. The PS is composed of pentasaccharide repeating units with the following structure: Analysis of NMR data reveals that on average the PS consists of ∼13 repeating units and indicates that the biological repeating unit contains an N-acetylglucosamine residue at its reducing end. This structure is different to that reported for the O-antigen polysaccharide from E. coli O126. Monospecific anti- E. coli O126 rabbit serum from The International Escherichia and Klebsiella Centre did not distinguish between the E. coli strain 396/C-1 and the E. coli O126 reference strain, neither in slide agglutination nor in an indirect enzyme immunoassay. Subsequent successful serotyping of the E. coli strain 396/C-1 showed it to be E. coli O126:K+:H27.

Brucellosis in wild boar (Sus scrofa) in the Republic of Croati

During the years 2001 and 2002 on seven localities in Croatiaa survey on the prevalence of brucellosis in wild boar was carried out. The survey included 271 (52.7%) female and 243 (47.3%) male animals between 7 months and 4 years of age and weighing from 14 to 135 kg. On that occasion 514 blood samples of wild boar were serologically analysed. For serological analysis indirect enzyme immunoassay (iELISA), Rose Bengal test (RBT), complement fixation test (CFT) and slow agglutination test (SAT) were used. In all of the wild boar from all of the localities investigated positive reactions to brucellosis were established. Most of the positive reactions were established by iELISA (13.6%), then by RBT (11.5%), CFT (10.5%) and SAT (8.9%). Tissue samples of 106 animals: testes samples from 67 animals, uterus tissue from 38 animals and 5 fetuses of piglets from 1 mother were analysed bacteriologically. Brucella suis biovar 2 was isolated from 18 (17.0%) animals that originated from all of the localities investigated. Isolates were identified by PCR using BRU-UP and BRU-LOW primers specific for genus Brucella and primers specific for IS711. Based on our results it could be concluded that in Croatia wild boar are natural vector and/or reservoirs of B. suis biovar 2. This permanent risk factor is hazardious for domestic and wild animals in theRepublic ofCroatia.

Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7

Sera from cattle naturally infected with Brucella abortus ( n=160), vaccinated with B. abortus S19 ( n=88) or immunized with Yersinia enterocolitica O:9 ( n=25) or Escherichia coli O157:H7 ( n=80) were collected. The sera were compared for antibody content to the same bacteria by indirect enzyme immunoassay (IELISA), fluorescence polarization assay (FPA) and competitive enzyme immunoassay (CELISA). Cattle sera ( n=523) collected randomly from across Canada were tested in the same tests. Sera from the B. abortus infected group reacted positively in the brucellosis IELISA (IELISA Br), CELISA and FPA (FPA Br) and the Y. enterocolitica IELISA (IELISA Ye) while the Y. enterocolitica FPA (FPA Ye) detected antibody in 93.8% and the E. coli IELISA (IELISA Ec) 86.9% and the E. coli FPA (FPA Ec) 48.1%. About 70% of the sera from B. abortus S19 vaccinated animals reacted in the three IELISAs, 45% in the CELISA, and 37.7% in the FPA Ec, 21.6% in the FPA Br and 5.7% in the FPA Ye. Sera from E. coli O:157 exposed cattle reacted mainly in the IELISA Ec and FPA Ec although surprisingly 87.5% reacted in the IELISA Ye and only 3.8% in the IELISA Br. No reactions were observed with these sera in the FPA Br and FPA Ye but one serum gave a low positive reaction in the CELISA. All sera from Y. enterocolitica O:9 exposed cattle reacted in the IELISA Br and IELISA Ye and 80% in the IELISA Ec. In the CELISA, 44% gave a positive reaction and 64% were positive in the FPA Br, 28% in the FPA Ye and 12% in the FPA Ec. Of the 523 Canadian sera, about 50% reacted in the E. coli tests with only minor reactions in the Y. enterocolitica O:9 and B. abortus assays. From the data, the cross reaction between E. coli O157:H7, Y. enterocilitica O:9 and B. abortus is dependent on the test used. Thus, extensive cross reaction was observed with the IELISA with much less reactivity in the FPA and the CELISA.

Evaluations of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation.

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.

Rough Lipopolysaccharide of Brucella abortus RB51 as a Common Antigen for Serological Detection of B. ovis, B. canis, and B. abortus RB51 Exposure Using Indirect Enzyme Immunoassay and Fluorescence Polarization Assay

Rough lipopolysaccharide (RLPS) antigens were prepared from cultures of Brucella abortus RB51, B. ovis, and B. canis. The preparations were standardized by weight and tested with sera from cattle immunized with B. abortus RB51, sheep infected with B. ovis, and dogs infected with B. canis. Populations of unexposed animals of each species were also tested. The tests used were the indirect enzyme immunoassay (IELISA) using RLPS and the fluorescence polarization assay (FPA) using RLPS core fractions, labeled with fluorescein isothiocyanate. The IELISA using B. abortus RB51 RLPS antigen resulted in sensitivity and specificity values of 94.8% and 97.3%, respectively, when testing bovine sera, 98.5% and 97.8% when testing ovine sera, and 95.8% and 100% when testing dog sera. The IELISA using B. ovis RLPS antigen gave sensitivity and specificity values of 80.5% and 91.7%, respectively with bovine sera, 98.9% and 93.8% with sheep sera, and 70.8% and 79.8% with dog sera. The IELISA using B. canis RLPS antigen resulted in sensitivity and specificity values of 97.0% and 97.4%, respectively, with bovine sera, 96.2% and 96.3% with sheep sera, and 95.8% and 98.8% with dog sera. Labeling RLPS core from B. ovis and B. canis with fluorescein was not successful. B. abortus RB51 core labeled with fluorescein resulted in sensitivity and specificity values of 93.5% and 99.8%, respectively, with bovine sera and 78.1% and 99.0% with sheep sera. It was not possible to test the dog sera in the FPA.

Electrochemical studies of 1,5-dihydroxynaphthalene as substrate for voltammetric enzyme immunoassay

A sensitive voltammetric enzyme immunoanalytical method is described for assay of horseradish peroxidase (HRP), labelled-HRP and antigen:antibody:labelled-HRP conjugate (Ag︰AB︰IgG-HRP) using 1,5-dihydroxynaphthalene (1,5-DHN) as substrate. 1,5-DHN is an electrochemical inactive substance, but it may be oxidized by O 2 in air to 5-hydroxy-1,4-naphthoquinone in basic solution, which produces a well-defined voltammetric reduction peak. After adding H 2 O 2 and HRP, HRP catalyzes H 2 O 2 oxidizing 5-hydroxy-1,4-naphthoquinone to 3-hydroxyphthalic acid, leading to the decrease of the voltammetric reduction peak, with which the activity of HRP and the labelled-HRP, further the antigen and antibody may be determined. The mechanism of this voltammetric enzyme-linked immunoassay system is different from the previous reports, in which there are no voltammetric reduction peaks before the addition of H 2 O 2 and HRP, and the reduction peaks appear after the addition of H 2 O 2 and HRP. The height of the voltammetric peak is directly proportional to the concentration of HRP in the previous systems, while the decrease of the voltammetric peak is linear with the concentration of HRP in this system. This new electrochemical method is combined with an indirect enzyme immunoassay using direct antigen-coating format for the detection of the purified tobacco mosaic virus (TMV) with a linear range from 25.0 to 340.0 ng/mL, and a detection limit 25.0 ng/mL.

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Diagnosis of Norwalk Virus Infection by Indirect Enzyme Immunoassay Detection of Salivary Antibodies to Recombinant Norwalk Virus Antigen

Detection of O-acetylated Vi polysaccharide of Salmonella enterica subspecies typhi by Enzyme Immunoassay

Immunisation with capsular Vi polysaccharide (Vi PS) of Salmonella enterica serovar Typhi ( S. typhi) protects against typhoid. This protection depends on the presence of O-acetyl groups on the Vi PS, which form an immunodominant epitope. An antiserum raised against conjugated Vi PS was used as the basis for an indirect Enzyme Immunoassay (EIA). The antiserum did not react with lipopolysaccharide of five gram negative bacteria including S. typhi. Vi PS from three different sources was tested, and all but one of 18 native Vi PS preparations had EIA values comparable to a standard Vi PS preparation. The sensitivity of the EIA for the detection of O-acetyl groups on Vi PS was compared to an NMR spectroscopy assay (Biologicals 28 (2000) 17–24). The EIA distinguished between O-acetylated and de- O-acetylated Vi PS preparations. However, significantly lower EIA reactivity was observed only for samples which had O-acetylation levels of 25% or less. This assay should facilitate batch control of Vi vaccines.

Antibodies against astrocyte M1 and M2 muscarinic cholinoceptor from schizophrenic patients' sera

We demonstrated the presence of circulating antibodies from schizophrenic patients able to interact with cultured astrocytes activating muscarinic acetylcholine receptors (mAChRs). Sera and purified IgG from 15 paranoid schizophrenic and 15 age-matched normal subjects were studied by indirect immunofluorescence (IFI), flow cytometry, dot blot, enzyme immunoassay (ELISA), and radioligand competition assays. Astrocyte membranes and/or a synthetic peptide, with identical amino acid sequence of human M(1) and M(2) mAChR, were used as antigens. By IFI and flow cytometry procedures, we proved that serum purified IgG fraction from schizophrenic patients, reacted to astrocyte cell surface. The same antibodies were able to inhibit the binding of the specific mAChR radioligand (3)H-QNB. Using synthetic peptide for dot blot and ELISA, we demonstrated that these antibodies reacted against the second extracellular loop of human cerebral M(1) and M(2) mAChR. Also, the corresponding affinity-purified antipeptide antibody displayed an agonistic-like activity associated to specific M(1) and M(2) mAChR activation, increasing inositol phosphates accumulation and decreasing cyclic AMP production, respectively. This article gives support to the participation of an autoimmune process in schizophrenia disease.

Immunochemical rapid test for multiresidue analysis of antimicrobial drugs in milk using monoclonal antibodies and hapten–glucose oxidase conjugates

Based on monoclonal antibodies (Mabs) against streptomycin, sulfadiazine, sulfamethazine and cloxacillin, a semiquantitative immunochemical rapid test for multiresidue analysis has been developed. The test format is that of an indirect competitive enzyme immunoassay. Glucose oxidase conjugates of the individual compounds were found to be suitable coating antigens. Individual immunoassays exhibited sensitivities far below the respective maximum residue limits (MRLs) in milk, i.e. 3.2 ng ml−1 for streptomycin, 9.0 ng ml−1 for sulfadiazine, 7.0 ng ml−1 for sulfamethazine and 0.8 ng ml−1 for cloxacillin. By optimising the assay conditions, total assay time was reduced to 15 min. The individual tests were then integrated onto a single microtitre plate array, in which the milk samples were simultaneously incubated with a mixture of the specific Mabs. The detection limits obtained with the multitest system again were below the respective MRLs, i.e. 14, 20, 13 and 1.7 ng ml−1, respectively, for streptomycin, sulfadiazine, sulfamethazine and cloxacillin. The coefficients of variation were less than 10%. Cross-reactivities with structurally related substances have been determined for both the individual assays and the multitest system.

Immunochemical rapid test for multiresidue analysis of antimicrobial drugs in milk using monoclonal antibodies and hapten–glucose oxidase conjugates

Based on monoclonal antibodies (Mabs) against streptomycin, sulfadiazine, sulfamethazine and cloxacillin, a semiquantitative immunochemical rapid test for multiresidue analysis has been developed. The test format is that of an indirect competitive enzyme immunoassay. Glucose oxidase conjugates of the individual compounds were found to be suitable coating antigens. Individual immunoassays exhibited sensitivities far below the respective maximum residue limits (MRLs) in milk, i.e. 3.2 ng ml −1 for streptomycin, 9.0 ng ml −1 for sulfadiazine, 7.0 ng ml −1 for sulfamethazine and 0.8 ng ml −1 for cloxacillin. By optimising the assay conditions, total assay time was reduced to 15 min. The individual tests were then integrated onto a single microtitre plate array, in which the milk samples were simultaneously incubated with a mixture of the specific Mabs. The detection limits obtained with the multitest system again were below the respective MRLs, i.e. 14, 20, 13 and 1.7 ng ml −1, respectively, for streptomycin, sulfadiazine, sulfamethazine and cloxacillin. The coefficients of variation were less than 10%. Cross-reactivities with structurally related substances have been determined for both the individual assays and the multitest system.

Determination of DDT in model samples by using solid-phase extraction and indirect competitive enzyme immunoassay

Two-step indirect competitive enzyme immunoassay of DDT has been developed. The limit of detection and limit of quantification were 0.3 nmol l−1 and 4.2 nmol l−1, respectively. To shorten the analysis time, the one-step enzyme immunoassay has been developed by replacing anti-DDT antibody with the complex anti-DDT antibody-HRP. The anti-DDT antibody-HRP conjugate was prepared by periodate method. A concentration of the preparation was 1.9 mg·ml−1 and molar ratio Px/IgG was 1.87. The detection limit of the one-step ELISA reached 0.3 nmol l−1, but the sensitivity of assay was very poor. The two-step enzyme immunoassay of DDT has been adapted for the chemiluminescent detection of the complex antigen-antibody. The detectability of chemiluminescent ELISA was comparable with that of chromogenic ELISA. A limited number of the artificially contamined samples have been tested. All samples were prepared by solid-phase extraction by using the commercial Agilent Zorbax SPE C18 columns. A background was observed in most tested samples. The recoveries obtained from the analysis of DDT spikes reached values between 89.0–161.0%, and 81.5–111.6% at standard addition 50 μg·kg−1, and 200 μg·kg−1, respectively. The overestimated amounts of DDT were determined in spinach and nectarines at standard addition 50 μg·kg−1.

Severe Acute Respiratory Syndrome: The Evolution of a New Epidemic

Severe Acute Respiratory Syndrome: The Evolution of a New Epidemic

Production of fumonisins by Fusarium moniliforme strains isolated from corn grain

Fusarium moniliforme is the predominant fusarium species in the grain mycoflora of corn grown in Northern Caucasus, accounting for 95% of fusarium isolates. Eighty-five Fusarium moniliforme strains were grown on grain substrate and checked for the presence of fumonisins (B1 + B2 + B3) by indirect solid-phase enzyme immunoassay (EIA). All strains were capable of producing fumonisins (0.95 to 32,000 mg/kg). Strains sampled in the Krasnodar krai produced the highest fumonisin levels (averaging 5490 mg/kg).

Development of an Indirect Competitive ELISA of DDT

Indirect competitive enzyme immunoassays of DDT based on polyclonal antibodies have been developed. The limit of detection and limit of quantification were 0.3 nmol l m 1 and 4.2 nmol l m 1 , respectively. Non-specific interaction was eliminated by the saturation of the solid phase with 2% human serum albumin. Different polystyrene microplates were tested. The cross-reactivity of the polyclonal rabbit antibody to DDT with 2,4'-DDE, 4,4'-DDD, 2,4'-DDT, 2,4'-DDD, Methoxychlor and 4,4'-DDE was 1, 9, 12, 13, 36 and 186%, respectively. No cross-reactivity of the tested antibody with 4,4'-DDA, 4,4'-DDOH, 4,4'-DBP, 4,4'-DDM, 4,4'-PCB, 2,3'-PCB, 4-PCB and 3-PCB was observed. With respect to a limited solubility of the analyte in water, the influence of different concentrations of methanol, ethanol, acetone and N,N'-dimethylformamide on the assay sensitivity were tested. A recommended concentration of the organic solvents is 5-10% (v/v) in the reaction mixture. Preliminary tests were done with a limited number of artificially contamined food samples. A background was observed in all tested food samples especially in strawberries, nectarines, and different species of pepper. False negative results were obtained when the DDT concentration was measured in beer, red wine and Pepsi-Cola.

Fluorescence polarization assay for detection of Brucella abortus antibodies in bulk tank bovine milk samples.

A simple, rapid, inexpensive fluorescence polarization assay for the detection of antibodies to Brucella abortus in bulk tank milk samples at the farm level or at dairies with a sensitivity and specificity of 100 and 95.9%, respectively, is described. The assay detects antibodies to B. abortus in 15 min by testing undiluted whey produced by chemical and physical manipulation of milk from bulk tanks. This sampling is noninvasive and therefore costs less and is less stressful than blood-based tests. The assay is specific and can detect antibodies at levels below that of the indirect enzyme immunoassay for milk and the fluorescence polarization assay for individual milk samples. Use of this test would make programs for surveillance of dairy animals and eradication of B. abortus more cost-effective.