Indirect Enzyme Immunoassay Research Articles

Characterisation of antibodies for the immunochemical detection of Enterobacter sakazakii

An indirect competitive enzyme immunoassay of Enterobacter sakazakii has been developed. The rabbit polyclonal antibodies to heat-labile or heat-stable antigen of the type strain E. sakazakii CNCTC 5739<sup>T</sup> were prepared for these purposes. The detection limits of enzyme immunoassays were within the range 0.6–14.4 × 10<sup>5</sup> cells/ml. Antibodies raised to heat-labile antigen were serotype-specific. Although they contain non-specific IgG fractions binding periplasmatic and cytosol proteins, the interactions of these immunoglobulins are not manifested under conditions of ELISA developed.

Comparative Study of Antinuclear Antibody Detection by Indirect Immunofluorescence and Enzyme Immunoassay in Lupus Patients

To evaluate the value of immunofluorescent and ELISA techniques in early diagnosis of systemic lupus erythematosus (SLE) and to find out whether there is a correlation between Antinuclear antibodies (ANA) pattern and prognosis by observing clinical score changes using British Isles Lupus Assessment Group score. The study included 75 SLE patients, 11 disease control group, and 18 healthy control group. ANA and ds-DNA antibodies detection were done by ELISA and Immunofluorescence for all groups. Immunofluorescence technique is more sensitive for ANA and ds-DNA detection than ELISA technique (100% versus 90.7%,and 93% versus 89.3% respectively); ELISA showed 89.7 % specificity for ANA detection compared to 86.2% for Immunofluorescence, and both have 100% specificity for ds-DNA detection ;homogenous ANA pattern, showed statistically significant higher BILAG score compared to speckled pattern either at the start of the study or after the follow up period (p = 0.000); and ds-DNA titer showed statistically significant decrease in titer after therapy (p = 0.01). ANA detection by Immunofluorescence is more sensitive and effective screening assay in patients with clinical features of SLE and both ds-DNA titer by ELISA and BILAG score for severity index are considered the best markers for follow-up.

Production and characterization of antibodies against fumigaclavine A

Polyclonal antibodies against fumigaclavine A (FuA) were obtained from rabbits after immunization with a FuA-keyhole limpet hemocyanine conjugate prepared by formaldehyde condensation. Using these antibodies and a FuA-bovine serum albumine conjugate, a competitive indirect enzyme immunoassay (EIA) was established. The antiserum obtained from one rabbit enabled highly sensitive detection of FuA, with an IC50 level and detection limit of the standard curve of 3.3ng/ml and approx. 1ng/ml, respectively. The EIA was very specific for FuA, with 1.3% cross-reactivity with FuB. Several other lysergic acid derivatives (ergonovine, ergotamine, alpha-ergocryptine) were tested but did not cross-react in the FuA EIA. This is the first description of antibodies against FuA and the first development of an EIA for FuA.

Development of an Indirect Enzyme Immunoassay using Monoclonal Antibodies for the Measurement of 17α-Hydroxyprogesterone in Human Serum

An indirect enzyme immunoassay for the measurement of total 17α-hydroxyprogesterone (17OHP) in serum using monoclonal antibodies generated in our laboratory was developed. Here, (a) instead of extraction with solvents, serum was heated to free protein-bound 17OHP and assay was performed at pH 9.6, (b) to ensure uniform assay conditions for both standards and samples, buffer for standards contained charcoal-stripped pre-heated pooled cord serum. Assays were done in 96-well EIA microplates pre-coated with 17α-hydroxyprogesterone-3-(o-carboxymethyl)oxime: bovine serum albumin. Secondary antibody was horseradish peroxidase-linked sheep anti-mouse IgG polyclonal antibody. The method was accurate and suitable for screening for congenital adrenal hyperplasia.

Synthesis and Characterization of Mercapturic Acid (N-acetyl-L-cysteine)-Aflatoxin B1 Adduct and Its Quantitation in Rat Urine by an Enzyme Immunoassay

A simple and sensitive indirect noncompetitive enzyme immunoassay to quantitate mercapturic acid-aflatoxin B1 (AFB1) adduct in rat urine is reported. A novel procedure was developed for in vitro synthesis of an immunogen, bovine serum albumen-glutathione-aflatoxin B1 (BSA-GSH-AFB1) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. Sulphydryl group's analysis confirmed the conjugation of-SH groups to AFB1. Thin-layer chromatography and spectral analysis (absorption, fluorescence, and Fourier transform infrared) of the conjugates further confirmed the formation of the adducts. Polyclonal antibodies specific to mercapturic acid-AFB1 adduct were produced against BSA-GSH-AFB1. The assay was found to be linear in the range of 100 pg-100 ng of the analyte (y = a + bx). A 50% displacement of BSA-GSH-AFB1 antibodies was achieved at an inhibitory concentration (IC50) of 11.9 ng GSH-AFB1 (r2 = 0.98) and 1.22 ng N-acetyl-L-cysteine (NAC)-AFB1 (r2 = 0.98). Spiking 5 microg/mL of reference standard to the control rat urine showed a recovery of 98 +/- 2%. The immunoassay was validated in a rodent model exposed to a single oral dose of 1 mg/kg body mass of pure AFB1. The excretion of NAC-AFB1 adduct was quantitated at the end of 24 h. The concentration of the NAC-AFB1 adduct excreted in urine as determined by the immunoassay was found to be in the range of 3.22-5.97 microg/mg creatinine. The present method may find wide application as a biochemical tool in molecular epidemiological and intervention studies with respect to human exposure to dietary aflatoxins.

Systemic Infection with Enteric Adenovirus in Immunocompetent Child withHaemophilus influenzaeDisease

Systemic Infection with Enteric Adenovirus in Immunocompetent Child with<i>Haemophilus influenzae</i>Disease

Survey of antibodies to Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) in dromedaries ( Camelus dromedarius) in close contact with an infected goat population

Given recent evidence of a possible role of camelids in the epidemiology of diseases caused by mycoplasma species in other ruminants, we examined the prevalence of the mycoplasmas most commonly responsible for contagious agalactia (CA) in small ruminants among the dromedaries ( Camelus dromedarius) of Lanzarote. On this island, one of the most arid regions of Spain in which CA is considered endemic, dromedaries and infected small ruminants such as goats and sheep, live in close contact. Specific IgG antibodies against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony biotype) were determined using two indirect enzyme immunoassays in 28% of the island's dromedary population. None of the serum samples tested were found to have antibodies against the two mycoplasma species, suggesting that the camels are not potential carriers of the main causative agents of CA.

Confirmation of Rubella within 4 Days of Rash Onset: Comparison of Rubella Virus RNA Detection in Oral Fluid with Immunoglobulin M Detection in Serum or Oral Fluid

Rubella virus infection is typically diagnosed by the identification of rubella virus-specific immunoglobulin M (IgM) antibodies in serum, but approximately 50% of serum samples from rubella cases collected on the day of rash onset are negative for rubella virus-specific IgM. The ability to detect IgM in sera and oral fluids was compared with the ability to detect rubella virus RNA in oral fluids by reverse transcription-PCR (RT-PCR) by using paired samples taken within the first 4 days after rash onset from suspected rubella cases during an outbreak in Perú. Sera were tested for IgM by both indirect and capture enzyme immunoassays (EIAs), and oral fluids were tested for IgM by a capture EIA. Tests for IgM in serum were more sensitive for the confirmation of rubella than the test for IgM in oral fluid during the 4 days after rash onset. RT-PCR confirmed more suspected cases than serum IgM tests on days 1 and 2 after rash onset. The methods confirmed approximately the same number of cases on days 3 and 4 after rash onset. However, a few cases were detected by serum IgM tests but not by RT-PCR even on the day of rash onset. Nine RT-PCR-positive oral fluid specimens were shown to contain rubella virus sequences of genotype 1C. In summary, RT-PCR testing of oral fluid confirmed more rubella cases than IgM testing of either serum or oral fluid samples collected in the first 2 days after rash onset; the maximum number of confirmations of rubella cases was obtained by combining RT-PCR and serology testing.

Efficiency of a recombinant MSA-2c-based ELISA to establish the persistence of antibodies in cattle vaccinated with Babesia bovis

Bovine babesiosis is caused by Babesia bovis and B. bigemina in Argentina. These protozoans are prevalent north of parallel 30°S, where their natural vector Rhipicephalus ( Boophilus) microplus is widespread. To prevent babesiosis outbreaks in endemic areas, an increasing population of 4–10-month-old calves are vaccinated with low virulence B. bovis R1A (BboR1A) and B. bigemina S1A (BbiS1A) strains. In non-endemic areas, an additional calf population is also vaccinated and boostered as adults, before they are relocated to R. microplus-endemic areas of the country. Serological tests are currently utilized not only to determine the status of natural Babesia spp. infections, but also to confirm the infection caused by vaccine strains. For this purpose, an indirect enzyme immunoassay (ELISA) based on the recombinant major surface antigen-2c (rMSA-2c) of B. bovis expressed in Escherichia coli, was standardized using sera from Babesia spp. experimentally infected cattle. ELISA rMSA-2c was validated using sera obtained weekly during 336 days from steers primed and boostered with BboR1A and/or BbiS1A on days 0 and 154, then compared with the immunofluorescent-antibody test (IFAT). Western blot (WB) protein analysis was used to confirm the specificity of the immune response to rMSA-2c. The sensitivity and specificity for ELISA rMSA-2c were 92 and 96% after the Babesia spp. priming and 88 and 73% after the boostering immunization, respectively. The sensitivity and specificity for IFAT were 99 and 90% after priming and 92 and 98% after boostering, respectively. Unlike IFAT, ELISA rMSA-2c detected a remarkable delayed booster response and a significant drop in specificity between 35 and 84 days after the booster immunization. Simultaneously, 87.5% of cattle boostered with B. bigemina showed cross-reactions in the ELISA rMSA-2c, particularly between 63 and 77 days after the inoculation. A reaction against E. coli was observed, since bands of approximately 40 and/or 42 kDa were detected using sera from cattle before and after Babesia spp. inoculations. ELISA rMSA-2c showed to be useful between 42 and 98 days after priming with Babesia spp. live vaccine to evaluate the success of infecting cattle. However, after boostering the test showed low specificity.

Development of a high‐throughput, indirect antibody immobilization format chemiluminescence enzyme immunoassay (CLEIA) for the determination of progesterone in human serum

A high-throughput and simple chemiluminescence (CL) enzyme immunoassay (CLEIA) for the determination of progesterone (P) in human serum was developed, with the highly sensitive 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2'-adamantane) (AMPPD)-alkaline phosphatase (ALP) system as the CL detection system. The results showed that the indirect immobilization of rabbit anti-progesterone polyclonal antibody (RAPA) through secondary antibody exhibited apparent advantages over direct coating in terms of antibody saving and improvement of the coating stability and uniformity. The direct analysis of P in human serum without extraction was realized by using 8-anilino-1-naphthalenesulphonic acid (ANS) to displace P from its binding proteins. The effect of several relevant parameters of the immunoreaction were examined and optimized. Compared with some commercial progesterone kits, the presented CLEIA has higher sensitivity with detection limitation as low as 0.06 ng/mL. The recoveries were 95.9-101%. The coefficient of variation was <8.4% and 9.9% for intra- and inter-assay precision, respectively. This method has been successfully applied to the evaluation of P in human serum.

Investigação sorológica de casos subclínicos de leishmaniose tegumentar após um surto em uma localidade endêmica

Subclinical cases of cutaneous leishmaniasis were investigated in a district of Maringá, State of Paraná, by means of the indirect immunofluorescence reaction and enzyme immunoassay. Among the 130 samples analyzed, one presented titer 40 and three presented titer 20 in the indirect immunofluorescence reaction, while 11 samples were positive in the enzyme immunoassay reaction. These data suggest that subclinical cases exist at this locality.

Antigen‐antibody combination assays for blood donor screening: weighing the advantages and costs

Antigen‐antibody combination assays for blood donor screening: weighing the advantages and costs

Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals

A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples.

Detection of anti-endomysial and anti-tissue transglutaminase autoantibodies in media following culture of oral biopsies from patients with untreated coeliac disease

Background and aims Coeliac disease is an autoimmune disorder characterised by high levels of anti-endomysial and anti-tissue transglutaminase autoantibodies in sera and media of cultured intestinal mucosa biopsies from affected patients. In this study, we wished to investigate whether anti-endomysial and anti-tissue transglutaminase antibodies can also be detected in culture media of oral mucosa specimens, and whether the mouth can be used as an area of immunological testing for coeliac disease. Methods Small intestine and cheek biopsy samples taken from 16 patients with active coeliac disease and from 11 controls were cultured in vitro for 48 h at 37 °C in presence of medium alone. Anti-endomysial and anti-tissue transglutaminase were detected in sera and in supernatants of these cultured biopsy samples by indirect immunofluorescence and enzyme immunoassay (EIA), respectively. Results Anti-endomysial and anti-tissue transglutaminase were positive in sera of 15/16 coeliac disease patients. Culture media of intestinal mucosa samples from 14/16 coeliac disease patients were anti-endomysial positive, while the same antibodies were positive in supernatants of cultured oral mucosa samples from 15/16 coeliac disease patients. Anti-tissue transglutaminase were positive in both intestinal and oral culture media of 15/16 coeliac disease patients. Neither anti-endomysial nor anti-tissue transglutaminase were found in sera or in culture supernatants of both intestinal and oral biopsy samples from 11 controls. Conclusions Our study suggests a new immunological site to detect the pathognomonic autoantibodies of coeliac disease and confirms that the mouth is involved in this illness.

Prevalence of Anti-Hantavirus Antibodies in Patients with Hypertransaminemia in Madrid (Spain)

The hantaviruses are involved in a number of clinical syndromes of different severity and prognosis. Hantaviruses are widely distributed around the world, but the spectrum of illnesses they cause outside recognized endemic areas is unclear. A retrospective analysis was performed to detect anti-hantavirus antibodies in the serum of patients with hypertransaminemia of unknown etiology and in that of healthy members of the general population of Madrid (Spain). Antibodies were detected by indirect immunofluorescence and enzyme immunoassay; positive results were confirmed by Western blotting. Of the 182 patients with hypertransaminemia, 11 (6%) were positive for anti-hantavirus IgG antibodies; Western blotting using recombinant Puumala virus N antigen showed one of these patients to have hantavirus-specific IgM antibodies. Among the 146 healthy subjects from the general population, 3 (2%) were positive for anti-hantavirus IgG antibodies. These results show that anti-hantavirus antibodies are more commonly detected in patients with hypertransaminemia than in healthy people.

Detection of Ovine Antibody to Brucella ovis by Indirect Enzyme Immunoassay

Because some batch‐to‐batch variation in the preparation of rough lipopolysaccharide (RLPS) from Brucella ovis has been experienced, several protocols were tested to establish the most reliable method for detection of antibody in indirect enzyme immunoassay. An early version of the assay gave a performance index (PI=sum of optimum percent sensitivity and percent specificity, determined by receiver operator characteristic analysis) of 198.6. This assay used RLPS from B. ovis as the antigen and a monoclonal antibody specific for bovine IgG1 heavy chain‐enzyme conjugate for detection. This was not repeatable using other batches of antigen. Newer versions of the assay generally had decreased sensitivity values, giving PIs of 193. Use of a recombinant protein A/G‐enzyme conjugate did not improve the PI (PI=190), giving reduced specificity and higher sensitivity. The final version used B. abortus RB51 RLPS as the antigen and protein A/G‐enzyme conjugate for detection, giving a PI of 197. Because of the batch uniformity of the B. abortus RB51 RLPS and the versatility of the protein A/G‐enzyme conjugate, the latter version appears to be the most useful for diagnostic serology.

Detection of Eschericia coli O157:H7 by Fluorescence Polarization Assay and Polymerase Chain Reaction

It is recognized that cattle and other domestic animals can be a reservoir of pathogenic Escherichia coli, including serotype O157:H7. To contain this potential health hazard, the first step is the identification of the carrier animals. For these purposes, a rapid serological screening test, a fluorescence polarization assay (FPA) was developed and results obtained from a randomly selected cattle population as well as cattle immunized with E. coli O157:H7 were compared to those obtained with an indirect enzyme immunoassay (IELISA). To identify pathogenic strains in carrier animals, polymerase chain reactions (PCR) for Shiga‐like toxins I and II were implemented using agarose electrophoresis. The sensitivity of the fecal extracted E. coli for Shiga‐like toxin I and II was approximately 200 CFU per reaction using multiplex hot‐start nested PCR. The sensitivity of the fecal extracted E. coli varied from approximately 5×102 to 2.5×103 CFU per reaction depending on the commercial kits used. The combination of the serological screening FPA and hot‐start nested PCR confirmatory assays provided rapid identification of the pathogen.

Salmonella enterica Serotype Urbana Interference with Brucellosis Serology

Sheep were immunized with killed Salmonella enterica serotype Urbana cells and their sera were tested in various serological tests for antibody to Brucella sp., Yersinia enterocolitica O:9 and Escherichia coli O:157 H:7. Of the eight sheep, all gave a positive agglutination reaction in the brucellosis buffered antigen plate agglutination test (BPAT), seven gave positive brucellosis standard tube agglutination test (TAT) and complement fixation test (CFT) results and four gave slightly positive reactions in a competitive enzyme immunoassay (CELISA). Seven sera were negative in an indirect enzyme immunoassay (IELISA‐SLPS) using B. abortus smooth lipopolysaccharide (SLPS) antigen and all were negative in a fluorescence polarization assay (FPA‐OPS) using B. abortus O‐polysaccharide antigen. Two sheep gave a slight positive reaction in an IELISA using Brucella rough lipopolysaccharide antigen (IELISA‐RLPS) and four sheep were slightly positive in an FPA using Brucella LPS core antigen (FPA‐CORE). All sheep had high antibody responses to S. enterica serotype Urbana, Y. and E. coli O:157 and 7 were positive for antibody to Y. enterocolitica O:9 when tested by IELISA. The sheep were negative when tested in the FPA using OPS from Y. enterocolitica O:9 but all were strongly positive in the FPA using OPS from E. coli O:157 while seven sheep had titers to S. enterica serotype Urbana. The impact on diagnostic serology for brucellosis is discussed.

Hantavirus infection in Brazil: development and evaluation of an enzyme immunoassay and immunoblotting based on N recombinant protein

The symptoms of hantavirus pulmonary syndrome may resemble those of other febrile illnesses. The development of an accurate diagnostic test should therefore improve clinical prognosis and be useful in epidemiologic studies. We evaluated the use of a recombinant antigen (rN Δ 85 ) based on the S-segment sequences of a Brazilian hantavirus for detecting immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against hantavirus in an indirect enzyme immunoassay (EIA). We assayed 613 serum samples (570 from humans and 43 from rodents). IgM EIA had a sensitivity of 94.1% and a specificity of 99.1%. IgG EIA had a sensitivity of 95.2% and a specificity of 98.4%. This evaluation confirms that rN Δ 85 IgM and IgG EIA tests are potentially useful rapid, sensitive, and cost-effective tools for detecting antibodies against hantaviruses indigenous to Brazil and other South American countries, in patients with acute or convalescent hantavirus infection, and in rodent reservoirs.

Prevalence of Antibodies against Rubella Virus in Spain

The importance acquired by the strategy of anticipating rubella revacunation needs no emphasis1. Recommendations by experts committees and actualization of the prevailing childhood immunization schedules are unanimous when it comes to including the above-mentioned strategy2. In this context, seroepidemiologic survey studies allow the assesment of humoral immunologic response against viral structural antigens3,4 despite the assumed potential bias attached to their design. Being aware of the importance of describing rubella seropositivity real status in our setting we decided to document such situation in children under theoretically proper immunization schedules. The present contribution relies on data from a cross-sectional study carried out between 1999 and 2000 in children from the autonomous region of Spain, Castilla y Leon, (the largest region in the European Economic Community). The chosen framework was restricted to serum samples received in a Microbiology Laboratory of a University Hospital with the request of analyzing infectious markers other than rubella antibodies. According to demographic features a double stratification was made and we evaluated 323 children whose age was between 1 and 5 years and 1166 from 6 to 14 years of age. All samples were alicuoted and frozen (-20oC) until the moment of processing. Antibodies to proteic antigens of rubella viral envelope were determined by means of an indirect enzymeimmunoassay (EIA) (Bio-Whittaker, USA). Results were validated according to manufacturer's instructions and samples that showed a neat absorbance greater the cut-off value plus 15% were considered positive. Our findings reveal that 309 of the investigated samples belonging to children from 1 to 5 years of age (95.7%) had antibodies to rubella virus (95% CI, 93.2% to 98.2%), being the rest of them seronegative at the moment of our study. In the age group ranging from 6 to 14 years the prevalence of antibodies was 90.5% (1055/1166) (95% CI, 88.6% to 92.5%) which resulted lower to that found in the previous group and the difference was statistically significant (p=0.003). This fact reflects a decrease of 5.2% in the seropositivity percentage of the second age group. An additional finding was that when we investigated the 6 to 14 years old group in terms of gender differences, girls (568) reached significantly higher percentages of seroprotection than boys (521) did (94.4 vs 87.1%, p=0.000). Conscious of the caution required in this kind of studies and assuming the internal validity for the evaluated population, our results point out to a age dependent loss of seroprotection. Among the potential causes that maintain this fact we may denote on the one hand the differences in vaccine coverage reached by both groups of children, just as Davidkina et al.5 have recently reported, and on the other hand limitations inherent in the vaccination itself6,7. Moreover, it is of course true that in our country, as well as in all developed countries, systematic vaccination of girls before puberty yields high efficiency, in order to prevent consequences derived from a potential primary infection during pregnancy8,9, all the same it is also certain that we still attend to the presence of small proportions of unprotected persons. The efficiency of new strategies of anticipating combined vaccines needs to be evaluated, and seroepidemiological studies seem to be a good tool for such