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Results for “ELISA assay variability”

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PubMed1984

Measurement of serum antibody in swine vaccinated with Bordetella bronchiseptica: comparison of agglutination and enzyme-linked immunosorbent assay methods.

The immune responses of 514 pigs from 5 swine breeds (Chester White, Duroc, Hampshire, Landrace, Yorkshire) to vaccination with Bordetella bronchiseptica were measured by agglutination and enzyme-linked immunosorbent assay (ELISA) methods. Both assays showed higher antibody levels in sera of pigs after vaccination than in sera of the pigs before vaccination. The variability of the responses among breeds was greater for the agglutination method than for the ELISA method. The ELISA proved to be more sensitive than the agglutination method and could detect antibody in serum samples diluted at least 100-fold more than those used in the agglutination procedure. The correlation of antibody titers obtained by the 2 methods was small, but statistically (P less than 0.01) significant. The ELISA should be useful for determinations of antibody responses of swine to B bronchiseptica.

Agglutination Tests↗

PubMed1996

Development of enzyme-linked immunoassays for human angiotensin I converting enzyme suitable for large-scale studies.

OBJECTIVE: To develop and validate a simple immunological assay for human angiotensin converting enzyme (ACE) based on monoclonal antibodies. METHODS: Microtitre plates were coated with mouse monoclonal antibody (MoAb) to human ACE (9B9) and incubated with diluted samples of human plasma. In the sandwich enzyme-linked immunosorbent assay (ELISA), the plasma ACE, bound to MoAb 9B9, was revealed using polyclonal anti-ACE antibodies and alkaline phosphatase conjugated to goat anti-rabbit immunoglobulin G. In the plate precipitation assay the ACE activity, quantitatively precipitated from human plasma by MoAb 9B9, was measured by enzymatic fluorimetric assay with p-benzyloxycarboxyl-glycyl-L-histidyl-L-leucine or p-benzyloxycarboxyl-L-phenylalanyl-L-histidyl-L-leucine as substrate, directly in the wells. RESULTS: These assays are specific for the amino-terminal domain of ACE and recognize differences in the conformations of native and recombinant ACE. The sensitivity of the sandwich ELISA was 200 pg/ml assay medium; it quantifies the ACE in 10 microliters human plasma or less. Intra- and inter-assay variability coefficients were 6.2 and 13.6%, respectively. Both variants of the assay determined the plasma ACE concentration in the presence of ACE inhibitors or EDTA. The ACE concentrations were determined by sandwich ELISA in a population of 138 middle-aged healthy Caucasian subjects. They were strongly correlated with the ACE gene insertion/deletion (I/D) polymorphism, which accounted for 20% of the variance of plasma ACE concentration in this population and 16-24% of the variance in plasma ACE activity as measured with three different enzymatic assays. CONCLUSION: The ACE concentration (but not inhibition) can be determined by this ELISA which is suitable for large-scale studies of plasma ACE levels.

Animals↗

PubMed2006

Influenza A virus infection engenders a poor antibody response against the ectodomain of matrix protein 2.

BACKGROUND: Matrix protein 2 (M2) is an integral tetrameric membrane protein of influenza A virus (IAV). Its ectodomain (M2e) shows remarkably little diversity amongst human IAV strains. As M2e-specific antibodies (Abs) have been shown to reduce the severity of infection in animals, M2e is being studied for its capability of providing protection against a broad range of IAV strains. Presently, there is little information about the concentration of M2e-specific Abs in humans. Two previous studies made use of ELISA and Western blot against M2e peptides and recombinant M2 protein as immunosorbents, respectively, and reported Ab titers to be low or undetectable. An important caveat is that these assays may not have detected all Abs capable of binding to native tetrameric M2e. Therefore, we developed an assay likely to detect all M2e tetramer-specific Abs. RESULTS: We generated a HeLa cell line that expressed full length tetrameric M2 (HeLa-M2) or empty vector (HeLa-C10) under the control of the tetracycline response element. These cell lines were then used in parallel as immunosorbents in ELISA. The assay was standardized and M2e-specific Ab titers quantified by means of purified murine or chimeric (mouse variable regions, human constant regions) M2e-specific Abs in the analysis of mouse and human sera, respectively. We found that the cell-based ELISA was substantially more effective than immobilized M2e peptide in detecting M2e-specific Abs in sera of mice that had recovered from repetitive IAV infections. Still, titers remained low (< 5 microg/ml) even after two consecutive infections but increased to approximately 50 microg/ml after the third infection. Competition with free M2e peptide indicated that approximately 20% of M2e-specific Abs engendered by infection reacted with M2e peptide. In humans presenting with naturally acquired influenza virus infection, 11 of 24 paired sera showed a > or = 4-fold increase in M2e-specific Ab titer. The Ab response appeared to be of short duration as titers were very low (average 0.2 mug/ml) in all patients at onset of infection and in controls, in spite of evidence for previous exposure to IAV. CONCLUSION: The results provide convincing evidence that M2e-specific Ab-mediated protection is currently lacking or suboptimal in humans.

Animals↗

PubMed2006

Concentrations of isoflavones in plasma and urine of post-menopausal women chronically ingesting high quantities of soy isoflavones.

Soy food or food supplements based on soy containing isoflavones (Isos) are increasingly available in Western countries. However, the variability of Isos levels in urine and plasma in humans during chronic ingestion is poorly documented. Nevertheless, this is the way these compounds will most probably be used in the future, especially if the soy-based supplements market goes on increasing. Here, glycosilated Isos in an enriched extract of Prevastein equal to 100 mg of equivalent Isos aglycone was given daily to 27 post-menopausal women for 30 days and to 12 post-menopausal women for 60 days. Volunteers were given Prevastein in a cereal bar (25 mg Isos) and in a yoghurt (25 mg Isos) both at breakfast and dinner. Plasma samples were collected after overnight fasting. Urine samples were aliquots of a 24 h collection checked on volume and creatinin excretion levels. Genistein, daidzein and equol were measured at day 0 and every 15 days afterwards, using original specific ELISAs. Constant levels were reached from the 15th day. About 59.2% of the volunteers were significant equol producers in the first experiment and 58.3% in the second. A large variability in plasma and urine levels was observed among post-menopausal women consuming 100 mg Isos per day, although remaining relatively stable in each individual subject. This could partly account for the controversial effects of Isos recorded so far in clinical studies. So Isos plasma levels would have to be assayed during chronic exposures, and could help to better understand the large variability of the effects classically observed in clinical studies. ELISA techniques could be easily exported to analytical laboratories to help physicians and nutritionists with their prescriptions.

Aged↗

PubMed1993

Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein.

Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzyme-peptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc.

Alkaline Phosphatase↗

PubMed1997

p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.

Antigen capture enzyme-linked immunosorbent assay (ELISA) for quantitation of the p24 gag protein of human immunodeficiency virus type-1 (HIV-1) is currently the most common method used to demonstrate virus replication both in vivo and in vitro. The present paper describes an ELISA employing readily available inexpensive reagents and gives detailed suggestions for optimizing the variables for specific purposes. The assay is as sensitive as commercial kits (25 pg/ml) and has a linear dose response over a wide range of p24 concentrations (25-1000 pg/ml). For these reasons, as well as its low cost, this assay has proven useful in a variety of research applications. This assay also has been found to be effective in detecting the gag protein of human immunodeficiency virus type-2 and simian immunodeficiency virus.

Bacterial Proteins↗

PubMed2006

Analysis of the antibody response to an immunodominant epitope of the envelope glycoprotein of a lentivirus and its diagnostic potential.

The envelope glycoprotein of small ruminant lentiviruses (SRLV) is a major target of the humoral immune response and contains several linear B-cell epitopes. We amplified and sequenced the genomic segment encoding the SU5 antigenic site of the envelope glycoprotein of several SRLV field isolates. With synthetic peptides based on the deduced amino acid sequences of SU5 in an enzyme-linked immunosorbent assay (ELISA), we have (i) proved the immunodominance of this region regardless of its high variability, (ii) defined the epitopes encompassed by SU5, (iii) illustrated the rapid and peculiar kinetics of seroconversion to this antigenic site, and (iv) shown the rapid and strong maturation of the avidity of the anti-SU5 antibody. Finally, we demonstrated the modular diagnostic potential of SU5 peptides. Under Swiss field conditions, the SU5 ELISA was shown to detect the majority of infected animals and, when applied in a molecular epidemiological context, to permit rapid phylogenetic classification of the infecting virus.

Amino Acid Sequence↗

PubMed2005

[Detection of SARS-associated coronavirus N protein by time-resolved fluoroimmunoassay].

OBJECTIVE: To develop a method for quantitative detection of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) N protein by timed-resolved fluoroimmunoassay (TRFIA). METHODS: Using a monoclonal antibody (mAb) against SARS-CoV N protein, screened by SARS-CoV N protein and matching experiment, a method for quantitative detection of SARS-CoV N protein by TRFIA was established on the basis of double sandwich enzyme-linked immunosorbent assay (ELISA) and evaluated against the ELISA kit. RESULT: The measurement range of the assay was 0.02-150 ng/ml with a sensitivity of 0.02 ng/ml, the coefficient of variability within runs of 3.3%;-6.2%;, and coefficient of variability between days of 5.3%;-9.6%;. The results of detection were consistent between ELISA and TRFIA. CONCLUSION: TRFIA is a new, sensitive and specific immunoassay for detecting SARS N protein with potential value in clinical applications.

Antibodies, Monoclonal↗

PubMed1988

The role of HTLV-I in tropical spastic paraparesis in Jamaica.

We report clinical and laboratory investigations of 47 native-born Jamaican patients with endemic tropical spastic paraparesis and of 1 patient with tropical ataxic neuropathy. Mean age at onset was 40 years, with a female-male preponderance (2.7:1). Neurological features of endemic tropical spastic paraparesis are predominantly those of a spastic paraparesis with variable degrees of proprioceptive and/or superficial sensory impairment. Using enzyme-linked immunoabsorbent assay (ELISA), IgG antibodies to human T-lymphotropic virus type I (HTLV-I) were present in 82% of sera and 77% of cerebrospinal fluids. On Western blot analysis, IgG antibodies detected the p19 and p24 gag-encoded core proteins in both serum and cerebrospinal fluid. Titers were tenfold higher by ELISA in serum than in cerebrospinal fluid, and some oligoclonal bands present in fluid were not seen in serum. Serum-cerebrospinal fluid albumin ratios were normal, and IgG indexes indicated intrathecal IgG synthesis. Histopathological changes showed a chronic inflammatory reaction with mononuclear cell infiltration, perivascular cuffing, and demyelination that was predominant in the lateral columns. In 1 patient, a retrovirus morphologically similar to HTLV-I on electron microscopy was isolated from spinal fluid. Our investigations show that endemic tropical spastic paraparesis in Jamaica is a retrovirus-associated myelopathy and that HTLV-I or an antigenically similar retrovirus is the causal agent.

Adolescent↗

PubMed1996

High incidence of anti-FVIII antibodies against non-coagulant epitopes in haemophilia A patients: a possible role for the half-life of transfused FVIII.

The occurrence of antibodies (Abs) capable of inhibiting factor VIII (FVIII) coagulant activity is a severe complication in haemophilia A, leading to the inhibition of transfused FVIII activity. It is not known whether, or to what extent, post-transfusion antibodies may also arise against non-coagulant epitopes. Therefore we set up a system capable, in theory, to detect all the FVIII-induced antibodies by use of an enzyme-linked immunoassorbent assay (ELISA) based on coating human recombinant FVIII onto polystyrene microtitre plates. Serum samples from 23 patients affected by haemophilia A of different gravity (22 referred to our Centre and one to the Bari Centre) were analysed. Although only one patient was positive at Bethesda assay, the presence of antibodies in ELISA was detected in 39% of patients in variable degrees; transfusion with FVIII was found to induce a raise in antibody titre, arguing in favour of the specificity of the phenomenon. The clinical relevance of these non-inhibitory antibodies was evaluated in three patients; although half-life did not show any change in the patients without or with low amount of antibodies, FVIII clearance was found enhanced in the patient displaying high titre antibodies. We propose detection of anti-FVIII antibodies by ELISA when routinely assessing haemophilia A patients.

Adolescent↗

PubMed1996

Collaborative study for the evaluation of enzyme-linked immunosorbent assays used to measure human antibodies to Bordetella pertussis antigens.

Acellular pertussis vaccines are being evaluated in multiple clinical studies, and human immunogenicity data will likely be pivotal in the appraisal of vaccine responses between populations and the responses to different vaccine combinations. Antibody response to pertussis antigens is also used in the diagnosis of pertussis. An international study was designed to assess the comparability of data generated in different laboratories by enzyme-linked immunosorbent assays (ELISAs). Thirty-three participating laboratories were asked to quantitate specific antibody to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), or fimbrial proteins (FIM) in 21 samples. Samples were to be assayed in triplicate in five independent assays by each ELISA routinely performed in the laboratory to assess intra-assay, interassay, and population variability. The mean sample values were used to compare quantitative results among the laboratories. Thirteen of the 32 laboratories which submitted evaluable data for an assay to measure antibodies to PT, 12 of 30 laboratories with assays for FHA, 10 of 17 laboratories with assays for PRN, and 6 of 13 laboratories with assays for FIM maintained a coefficient of variation below 20% for 75% of the samples tested. Assays that measure antibodies to FIM appear to be less precise than the other assays. Precision varied among laboratories that used similar methods. The relative values of intra- and interassay variabilities were not consistent for a given assay within a laboratory, indicating that the sources of these variability components may be unrelated. Precision and agreement appeared less reliable for samples with low antibody levels. Ranking and regression analyses suggest that some laboratories generated comparable quantitative results, although direct comparison or combination of results from different laboratories remains difficult to support. Calibration to the U.S. Reference Pertussis Antisera appears to have been successful at standardizing the results in some laboratories. Statistical analyses are affected by assay precision and are not necessarily reliable sole predictors of biologically relevant differences in quantitative results. If results from different laboratories must be compared, appropriate studies of precision and quantitative agreement should be conducted to support the specific comparisons.

Antibodies, Bacterial↗

PubMed1991

Correlation of serological, quantitative and cell-mediated functional assays of maternal alloantibodies with the severity of haemolytic disease of the newborn.

Serum samples containing IgG red blood cell (RBC) antibodies were collected without reference to clinical information from 131 pregnant alloimmunized women. Anti-D and anti-K were present in sera from 75 and 20 patients respectively. Antibody titres were determined by indirect antiglobulin test (IAGT), anti-D levels were measured by AutoAnalyzer, RBC-binding IgG was quantified using an enzyme-linked immunosorbent assay (SOL-ELISA), and functional activities were measured using the monocyte chemiluminescence (CL) test, antibody-dependent monocyte-mediated and K cell-mediated cytotoxicity (ADCC) assays, and rosette formation with U937 cells. Details of clinical outcomes were obtained retrospectively from 104 pregnancies. Forty-one babies were 'antigen-negative', and of the remainder, four required top-up transfusions, 12 required exchange transfusions, three received intrauterine transfusions, and two died in utero. A comparison of test results with severity of haemolytic disease of the newborn (HDN) showed that, provided sera tested were collected within 8 weeks of the expected delivery date, the CL test and the monocyte-mediated ADCC assay differentiated those D-positive babies which required exchange transfusions from those unaffected or only mildly affected. The usefulness of results from the AutoAnalyzer and IAGT in predicting disease severity was compromised by the wide range of results from mothers of unaffected babies. This variability was less apparent in the SOL-ELISA which predicted severe HDN with greater precision. Results from the U937 rosette assay and the K cell-mediated ADCC assay failed to correlate with disease severity.

Antibody-Dependent Cell Cytotoxicity↗

PubMed2000

Tissue transglutaminase antibodies in celiac disease: assessment of a commercial kit.

OBJECTIVE: Tissue transglutaminase was identified as the autoantigen eliciting endomysial antibody. A homemade enzyme-linked immunosorbent assay (ELISA)-based test was recently developed to determine quantitative titers of IgA antitissue transglutaminase antibody. Our objective in this study was to assess the suitability of a newly developed commercial kit for quantitative determination of antibody in patients with untreated celiac disease. MATERIALS: We tested serum samples from 79 untreated celiac patients, 42 healthy blood donors, and 18 patients with nonceliac intestinal disorders evaluated in two different centers. Samples were tested for antitissue transglutaminase, and antiendomysial and antigliadin antibodies in the center where diagnosis was performed. To assess interlaboratory variability of methods, 24 samples randomly selected were blindly tested in both centers. Antitissue transglutaminase antibodies were determined using a commercial kit (INOVA Diagnostics, Inc., San Diego, CA). RESULTS: Untreated celiac patients had significantly higher titers of antitissue transglutaminase than healthy and disease controls (p < 0.00001). According to the cut-off provided by the manufacturers (20 AU/mL), overall sensitivity was 92% (85% for one center and 100% for the other) and specificity was 98% (100% and 95%, respectively). Antiendomysial antibody was 86% sensitive and 100% specific. Discordance between antitissue transglutaminase and antiendomysial antibodies was detected in 13% of patients. Although two antitissue transglutaminase-negative cases had a positive antiendomysial antibody, the inverse situation was found in eight cases. A blind determination of antitissue transglutaminase on the same samples evidenced a good agreement (kappa statistic: 0.66) between both centers when assessment was qualitative (based on the decision of positive or negative). Although correlation of titers for both determinations was highly significant (r: 0.902, p < 0.00001), a very wide interlaboratory variability (median: 50%) was detected when absolute values were considered. CONCLUSIONS: The quantitative determination of antitissue transglutaminase using a commercial kit was highly sensitive and specific for detection of celiac disease. We observed an incomplete overlapping with antiendomysial antibody. The very high variability of values between laboratories still remains to be solved so as to propose the commercial ELISA assay for the screening of celiac disease.

Adolescent↗

PubMedDate not supplied

Western blot technique in the serological evaluation of three LAV/HTLV III-infected Italian families.

In order to confirm suspected LAV/HTLV III infection, serological evaluation of patients is of utmost importance. ELISA is currently being employed on a large scale for screening, but like the immunofluorescence assay, it has a variable rate of possible non-specific positivity. On the other hand, the Western Blot (WB) technique can detect antibodies to different viral proteins. In this paper we are reporting the serological patterns of three LAV/HTLV III-infected families. In particular, their viral protein-specific antibody patterns are described. With the exception of one child, all the patients tested showed seropositivity in both ELISA and WB. In the one child mentioned above, ELISA and immunofluorescence positivity were due to non-specific binding. Two out of three children tested showed a close correlation between a severe clinical course and the absence of p25-specific IgM. In contrast, one child showing a switch from IgM to p25-specific IgG antibodies had a favorable clinical course. We observed a family in which vertical transmission of LAV/HTLV III from the mother to her neonate seems not to have happened; the child was seronegative and healthy at the age of one. At birth, this neonate had LAV/HTLV III-specific IgG corresponding to the mother's pattern, but it lacked viral-specific IgM. Its mother had transmitted the viral infection to her first child, who died of AIDS. Preliminary suggestions are made about the detection of different specific antibodies and clinical features; the utility of WB is emphasized.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Lack of specificity for antibodies to double stranded DNA found in four commercial kits.

Four commercially available kits (three enzyme linked immunosorbent assays and one modified Farr radioimmunoassay) were compared for their ability to detect specifically autoantibodies to double-stranded DNA (dsDNA) using 66 patient sera. This was assessed by comparing the results of the kits with those from an ELISA specifically measuring antibodies against highly purified dsDNA, single-stranded DNA (ssDNA), native DNA and histones. The RIA and two of the ELISAs seemed equally efficient at detecting antibodies to dsDNA, but all three also detected anti-ssDNA (the RIA being particularly bad for this). The need for highly purified dsDNA was clearly shown. The results obtained with one ELISA did not correlate with any variable investigated in this study. A total of 220 sera were assayed with the IDS RIA, of which 130 were recorded as positive. Of these, 50 sera seemed to contain no identifiable autoantibodies. This very high false positive rate may be due, at least in part, to precipitation of nonspecifically bound labelled DNA.

Antibodies, Antinuclear↗

PubMed1995

[Seroepidemiology of human parvovirus B19 using recombinant VP1 + VP2 particle antigen].

Antibody prevalence to B19 virus in Japan was previously reported with 1973 and 1984 serum collections. Since then we have had two big epidemics of erythema infectiosum in Japan: 1986-87 and 1991-92. In an attempt to estimate how much those epidemics have affected seroprevalence to B19 virus infection, we studied seroepidemiology in three separate areas using a newly developed ELISA system consisting of recombinant VP1 + VP2 particle antigen. Total of 900 sera obtained in 1993 from healthy individuals living in northern, central and southwest parts of Japan were assayed for the presence of anti B19 IgG antibody. In the first, the new assay system was compared with our conventional assay system in which native B19 virus particles were used as antigen. Of 220 serum samples tested, 212 (96.4%) gave same results in both assays. Remaining 8 samples which gave variable results were those of near cut off values. We concluded that this new ELISA system can be recommended for seroepidemiological use. Antibody prevalence rates were 10% (0-4 y), 54% (5-9 y), 59% (10-14 y), 46% (15-19 Y), 38% (20-29 y), 48% (30-39 y), 64% (40-49y) and 76% (50 y-). No gender difference was observed. Comparison of antibody prevalences in 1973, 1984 and 1993 suggested that approximately 10% (about 8 million people) of the total population under 40 years of age acquired immunity to B19 virus during one epidemic period.

Adolescent↗

PubMed1991

A unified serotyping scheme for Moraxella bovis.

Fifty-three Australian, seven British, two American and two New Zealand isolates of Moraxella bovis were classified into seven serogroups on the basis of their variable fimbrial (pilus) antigens using whole cell slide agglutination (SA), enzyme-linked immunosorbent assays (ELISA) and tandem-crossed immunoelectrophoresis (TCIE). Although results of serogroup classification by SA and ELISA were identical in 68.7% of isolates, it was found necessary to resolve the discrepancies between the two systems using TCIE. Results suggest that world-wide variation in the potentially host-protective fimbrial antigens of M. bovis may be relatively limited. It is proposed that the previous numerical classifications of British and Australian serogroups are appropriately amalgamated as a result of this latest study and are designated as serogroups A to G inclusive. A protocol for the further serotyping of fresh, fimbriate isolates of M. bovis is suggested.

Agglutination Tests↗

PubMed2004

Seroprevalence of simian immunodeficiency virus in wild and captive born Sykes' monkeys (Cercopithecus mitis) in Kenya.

BACKGROUND: The Sykes' monkey and related forms (Cercopithecus mitis) make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk). We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex. RESULTS: The SIVsyk antigen-specific Western blot assay detected more serum samples as positive than either of the cross-reactive ELISA assays. Using this assay, we found that seroprevalence is higher than previously reported, but extremely variable in wild populations (from 0.0 to 90.9%). Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified. CONCLUSION: We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission.

Animals↗