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Results for “ELISA assay variability”

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PubMed1998

Anticardiolipin antibodies in classic pediatric hemolytic-uremic syndrome: a possible pathogenic role.

Anticardiolipin (aCL) antibodies have been associated with thrombocytopenia, hemolytic anemia and an increased risk of thrombosis in different vascular locations, even in the absence of lupus. The classic hemolytic-uremic syndrome is a postinfectious acute renal failure characterized by hemolytic anemia, thrombocytopenia and the presence of widespread glomerular thrombosis in the kidney, with pathogenic mechanisms that remain to be identified. In order to establish the frequency of aCL antibodies in this syndrome and to identify a possible role in the pathogenesis and clinical manifestations, 17 patients were studied during the reactant phase of the disease looking for an association between the presence of aCL antibodies (isotypes IgG, IgA and IgM) and the main clinical variables of the syndrome. In 8 patients IgG aCL was present, 2 patients had IgM aCL, and 1 had IgA antibodies on the solid-phase ELISA aCL assays, but no association could be demonstrated with the clinical variables studied. Although it might correspond to an epiphenomenon related to the triggering intestinal infection, a pathogenic role cannot be discarded and additional studies should be performed.

Antibodies, Anticardiolipin↗

PubMed2006

Serum heat shock protein 70 and its correlation with clinical characteristics in patients with sudden sensorineural hearing loss.

OBJECTIVES: Previous studies have reported that in cochlear cells, heat shock protein (HSP)70 is expressed after ototoxic stimuli. We performed this study to investigate the possibility of HSP70 being found in circulation and to observe its possible clinical role in patients with sudden sensorineural hearing loss (SSNHL). METHODS: A total of 67 patients with SSNHL and age-sex matched normal controls were included in this study. Their serum HSP70 levels were measured with highly sensitive enzyme-linked immunoadsorbent assay (ELISA) test and confirmed with Western blot immunoassay. Clinical variables including initial and final audiometric findings and accompanying symptoms were analyzed to find the possible clinical value of serum HSP70 level in the patients with SSNHL. RESULTS: Serum HSP70 levels were significantly higher in the patients with SSNHL than those of normal controls by ELISA test (P < .01), and their presence were confirmed as strong bands in the Western blot assay. There was significant correlation between serum HSP70 levels and the recovery of HL in the patients with SSNHL (P < .01). CONCLUSION: This is the first report showing elevated serum HSP70 levels in the patients with SSNHL compared with normal controls. We suggest that serum HSP70 levels might have a clinical role for predicting prognosis of HL in the patients with SSNHL.

Adolescent↗

PubMedDate not supplied

Nonspecific reactions in an enzyme-linked immunosorbent assay caused by binding of immunoglobulins in situ to egg-propagated infectious bronchitis virus.

High levels of nonspecific background absorbance and increased variability were found in a previously optimized enzyme-linked immunosorbent assay (ELISA) for infectious bronchitis virus (IBV) antibody after changing to commercially available non-pathogen-free eggs for viral antigen production. An increase in bound viral antigen in the assay caused a proportionate increase in the nonspecific binding of the conjugate, independent of other variables, in the absence of serum. Virus was propagated in non-pathogen-free eggs, and individual viral proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Localization of chicken IgG-virus complexes were identified by immunoprecipitation with peroxidase-conjugated anti-chicken IgG. Specific staining at molecular weights corresponding to major proteins of IBV was demonstrated in these viral preparations. Virus grown in specific-pathogen-free eggs and treated in the same manner showed only slight amounts of staining. This evidence suggests that viral antigens grown in eggs from a non-pathogen-free flock bind with maternal chicken immunoglobulins present in the allantoic cavity of eggs. This IgG caused nonspecific reactions in our chicken ELISA system and gives cause for concern in any diagnostic system requiring the propagation of agents in fertile eggs.

Animals↗

PubMed1999

Soluble TNF-alpha receptor and IL-1 receptor antagonist elevation in BAL in active pulmonary TB.

Accumulating evidence suggests that patients with active pulmonary tuberculosis (TB) have an alveolar inflammation resulting in the release of tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta in bronchoalveolar epithelial fluid. It was proposed that the levels of these cytokines would correlate with clinical status parameters (extent of pulmonary involvement, fever, and body weight loss) and that their naturally occurring inhibitors would be concomitantly released in the local inflammatory sites. To test this hypothesis lung epithelial lining fluid (ELF) obtained by bronchoalveolar lavage and serum were collected from 29 patients with active pulmonary TB and 15 healthy subjects to determine the levels of these variables using a sandwich enzyme-linked immunosorbent assay (ELISA). ELF levels of TNF-alpha, soluble (s)TNF receptor I (RI), sTNF-receptor II (RII) and interleukin-1 receptor antagonist (IL-1RA) but not IL-1beta, and their serum levels except for sTNF-RII and IL-1beta were significantly higher in TB patients. Nevertheless, only ELF levels of TNF-alpha and IL-1beta were significantly correlated with disease status. No correlation was found between TNF-alpha levels and those of sTNF-RI and sTNF-RII, nor between IL-1beta and IL-1RA in ELF and serum of TB patients, although there was a significant correlation between sTNF-RI and sTNF-RII levels both in ELF and serum. These findings suggest local release of tumour necrosis factor-alpha and interleukin-1beta and a correlation with disease status. Soluble tumour necrosis factor-alpha receptors and interleukin-1beta receptor antagonist, although increased in lung epithelial lining fluid and serum in tuberculosis patients, were not correlated with tumour necrosis factor-alpha and interleukin-1beta or with disease status.

Adult↗

PubMed1993

Detection of antibodies to Cowdria ruminantium in the serum of domestic ruminants by indirect ELISA.

A solid phase enzyme immunoassay for the detection of antibodies to Cowdria ruminantium in the serum of domestic ruminants was developed by using microorganisms cultivated on bovine umbilical endothelial cells as antigen. When the culture showed 90% lysis, the supernatant was centrifuged, sonicated and coated on polystyrene microtiter plates. Antibodies were detected as early as 9 days after experimental immunization of goats. The sensitivity of the ELISA calculated with 73 ruminant sera ranged between 97.3% and 98.6%. The overall specificity of the test was 97% (N = 2925). However, the specificity was far lower for sheep (94.4%, N = 881) than for goats (98.6%, N = 651) and cattle (97.8%, N = 1393). Crossreactivity which could explain some of the false positive reactions, was found between Cowdria antigen and sera raised against Ehrlichia bovis (1 bovine positive out of 2 inoculated) or E. ovina (2 sheep out of 2 inoculated became positive) but not with E. phagocytophila. The intra-assay and inter-assay variability were 7.5% and 7.8% respectively, indicating a good reproducibility of the ELISA.

Animals↗

PubMed1999

A tandem repeat of MUC1 core protein induces a weak in vitro immune response in human B cells.

We have recently described an efficient method to study the human humoral immune response in vitro and to generate isotype-switched, antigen-specific human B cells, which has allowed us to produce high-affinity IgG antibodies against different peptides. In an attempt to study the in vitro immune response against self-antigens, such as tumour-associated antigens, this protocol was used to immunise resting human peripheral blood B cells with a peptide epitope from the human-adenocarcinoma-associated antigen, MUC1. After the two-step in vitro immunisation, the secondary immunised cultures were tested for MUC-1-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Phage molecular libraries were subsequently constructed, using the variable parts of Ig genes derived from cells taken from ELISA-positive wells. The libraries were selected on the MUC1 core peptide. Antigen-specific Fab fragments, specific for the self antigen MUC1, were found in the library of secondary immunised IgG+ B cells and these antibodies were evaluated by BIAcore analysis. The specific Fab fragments exhibited an unusually rapid dissociation rate constant and the overall response frequency was lower, as compared to other antibodies generated by this protocol, which might be explained by the repetitive nature of the core peptide used for immunisation.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of cDNAs encoding VH and VL of a monoclonal anti-CEA antibody (CEA 79) cross-reactive with NCA-95 and generation of a single-chain Fv molecule (scFv).

We cloned complementary DNA (cDNA) encoding the variable regions of heavy chain (VH) and of light chain (VL) of a monoclonal anti-carcinoembryonic antigen (CEA) antibody cross-reactive with nonspecific cross-reacting antigen-95 (NCA-95), which had been previously prepared and designated as CEA 79 (gamma 2a, kappa). From these cDNAs, a phagemid expression vector for the CEA79 single chain variable fragment (scFv) was generated. Enzyme-linked immunosorbent assay (ELISA), competitive ELISAs, and Western blotting confirmed that the scFv displayed on the surface of the bacteriophage had retained affinity for CEA and NCA-95. We then determined the nucleotide sequences of the cloned cDNAs for VH and VL. The sequence analysis revealed that VH and VL of the CEA 79 antibody represent new members of the mouse heavy chain subgroup "miscellaneous" and the kappa light chain subgroup "V", respectively.

Amino Acid Sequence↗

PubMed1986

The oral immune system: dynamics of salivary immunoglobulin production in the inbred mouse.

In the oral cavity, salivary immunoglobulins (Igs) are the principal mediators of specific immunity. Using carbachol to stimulate saliva flow, we investigated, in a kinetic study, individual variations in salivary Ig concentrations in 23 adult BALB/c mice using an enzyme-linked immunosorbant assay (ELISA). It appeared that salivary Ig concentrations are highly variable in individual mice (IgA: 3-81 micrograms/mL; IgG: 0-2.9 micrograms/mL; IgM: 0.002-0.14 microgram/mL). In individual mice stimulated at different times over a 3 week period there are considerable variations both in salivary Ig concentrations and in their respective ratio. Broad variations were also found in the levels of specific IgA and IgG antibodies to three indigenous oral murine bacteria. Present data thus indicate that among genetically identical mice of the same age and sex, sharing identical diet, there is considerable heterogeneity in salivary Igs. As this heterogeneity was mimicked at the cellular level in major and minor salivary gland-associated B-cells, it appears that antibody dynamics in the oral cavity could reflect the adaptive capacity of the oral immune system to local antigenic challenge.

Animals↗

PubMed1997

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.

Adult↗

PubMed1992

Pitfalls in determining IgG and IgG subclass antibodies to food antigens.

Several variables were found to influence enzyme-linked immunosorbent assay (ELISA) measurements of IgG and IgG subclass antibodies to food antigens. Two polyclonal rabbit antibody reagents to human IgG, and two sets of murine monoclonal antibodies to human IgG subclasses, were compared as secondary reagents. The choice of both polyclonal and monoclonal reagents affected significantly the results. High levels of IgA to an antigen depressed the measurements of comparable total IgG antibodies but did not influence the percentages of the four IgG subclass activities. Different ways of expressing the IgG subclass results were compared and the validity of the reference measurements used to obtain them was examined. Information from previous studies, together with the present data, suggests that absolute values cannot be reliably determined. We have therefore chosen to express the results for IgG subclass activities on a relative basis with reference to units of total IgG activity against the same antigen in each subject. This approach is practical and appears scientifically acceptable but limits the use of such determinations to comparisons between groups of subjects studied in the same laboratory.

Adolescent↗

PubMed2001

The effect of tourniquet application, tranexamic acid, and desmopressin on the procoagulant and fibrinolytic systems during total knee replacement.

STUDY OBJECTIVE: To assess the influence of tourniquet inflation-deflation as well as desmopressin and tranexamic acid (TA) administration on prothrombin fragment 1.2, fibrinogen, plasmin antiplasmin complex, and D-dimer concentrations during total knee replacement. DESIGN: Randomized, placebo-controlled study. SETTING: Large referral hospital. PATIENTS: 30 ASA physical status I, II, and III patients undergoing total knee replacement. INTERVENTIONS: Patients were randomized to one of three treatment groups. Patients received either tranexamic acid, desmopressin, or an equal volume of saline, intravenously. MEASUREMENTS AND MAIN RESULTS: Cubital blood was drawn immediately before induction of anesthesia, 1 hour after tourniquet application, and 2 and 15 minutes after tourniquet deflation. Fibrinogen and D-dimer levels were measured using the Clauss Method and latex agglutination, respectively. Plasmin antiplasmin complex and prothrombin fragment 1.2 levels were measured by enzyme-linked immunosorbent assay (ELISA). All assays were performed in duplicate, and intra-assay variability was documented. No statistically significant difference in fibrinogen, D-dimer, plasmin antiplasmin complex, or prothrombin fragment 1.2 levels was demonstrated among the groups. Similarly, within each group there were no statistically significant differences in the variables studied. However, despite the lack of statistical significance, when compared with their levels during tourniquet application, an increase in D-dimer and plasmin antiplasmin complex levels was observed in all three groups at 2 and 15 minutes after tourniquet release. In contrast, no increase in prothrombin fragment 1.2 generation was noted. Significantly more allogeneic blood was transfused in the Control and Desmopressin Groups when compared with the tranexamic acid group (p< 0.02). CONCLUSIONS: No evidence of tourniquet-induced fibrinolysis or thrombin generation was demonstrated in the systemic circulation. Desmopressin and tranexamic acid had no significant effect on the variables measured.

Aged↗

PubMedDate not supplied

Development, characterization and future prospects of monoclonal antibodies against spores of Glugea atherinae (protozoa-microsporidia-fish parasites).

Monoclonal antibodies against spores of Glugea atherinae were obtained after lymphocytic hybridization made from immunized mouse splenocytes. Screening using an indirect enzyme linked immunosorbent assay (ELISA), revealed seven monoclonal antibodies with an intense but variable reaction with the spores of fish microsporidia, and a moderate reaction with those of an insect microsporidium (Nosema sp.). The reaction was weaker with spores of Encephalitozoon intestinalis found in HIV+ patients. FITC and Dot Blot confirmed the majority of these results. After biotinylation of the seven antibodies, inhibition tests allowed the localization of two different recognition domains on the spores of Glugea atherinae. The multiple antigenic determinants and their probable polysaccharide nature seem to be in accord with the class IgM of the antibodies produced. This work confirms the potential of these antibodies for microsporidian taxonomy and diagnosis, especially the use of Mabs 12F9 and 12H5 for detection of spores in stools of HIV+ patients.

Animals↗

PubMed2003

Preferential induction of prodestructive matrix metalloproteinase-1 and proinflammatory interleukin 6 and prostaglandin E2 in rheumatoid arthritis synovial fibroblasts via tumor necrosis factor receptor-55.

OBJECTIVE: To assess expression and individual functional relevance of tumor necrosis factor receptor 55 (TNF-R55) and TNF-R75 in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFB). METHODS: Seventh to 9th passage RA SFB and OA SFB were analyzed for TNF-R expression by FACS. The SFB were then stimulated with TNF-a (1-10 ng/ml) or agonistic anti-TNF-R55 (HTR-9) and anti-TNF-R75 (UTR-1) monoclonal antibodies (1-25 micro g/ml each). Matrix metalloproteinase-1 (MMP-1), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), interleukin 6 (IL-6), and prostaglandin E(2) (PGE(2)) in culture supernatants were quantified by ELISA, and DNA fragmentation by TUNEL assay. RESULTS: RA SFB variably expressed TNF-R55 (7.2 +/- 2.2% positive cells, mean +/- SEM) and TNF-R75 (0.6 +/- 0.3%), similarly to OA SFB (6.8 +/- 2.1% and 1.6 +/- 0.8%, respectively). RA SFB constitutively secreted large amounts of TIMP-1 (1700 ng/ml), but only small amounts of MMP-1 (23.7 ng/ml), IL-6 (4.4 ng/ml), and PGE(2) (0.34 ng/ml). OA SFB secreted comparable amounts of TIMP-1 (2470 ng/ml), MMP-1 (37 ng/ml), and IL-6 (5.0 ng/ml), but significantly higher amounts of PGE(2) (0.58 ng/ml; p </= 0.05). TNF-a stimulation induced IL-6 secretion by RA SFB (3-fold) and OA SFB (4-fold), as well as MMP-1 secretion (RA, 85-fold; OA, 29-fold), with significant differences between RA and OA. This was exclusively mediated by separate stimulation with agonistic anti-TNF-R55 Mab. Strikingly, RA SFB were completely unresponsive to TIMP-1 mRNA and protein induction by TNF-a, whereas TIMP-1 mRNA and/or protein in OA SFB was significantly upregulated by TNF-a (2-fold; p </= 0.05, OA > RA) and by separate stimulation of both TNF receptors. TNF-a-induced PGE(2) release by RA SFB (92-fold) and OA SFB (56-fold) was mediated by both TNF receptors; however, predominantly by TNF-R55. DNA fragmentation was induced exclusively by high concentrations of anti-TNF-R55 Mab and only in RA SFB. CONCLUSION: These results indicate preferential induction of prodestructive and proinflammatory mediators in RA SFB by the TNF-R55, with potential implications for understanding the pathogenesis of RA and the development of more specific therapeutic strategies.

Aged↗

PubMed2000

Qualifying ELISA data: combining information.

With immunoassay or bioassay data, the assay standards often exhibit considerable inter-assay variability. However, the assay controls, which are used to monitor the assay performance and set acceptance criteria, should have no or less interassay variability. In this paper, we develop a mixed-effect calibration model for the assay controls to set new acceptance criteria and qualify the enzyme-linked immunosorbent assay (ELISA) data, which incorporates the interassay variation of assay standards and the nature of the assay controls, and overcomes the problems caused by traditional fixed-effect calibration model.

Calibration↗

PubMed1988

Diagnosis of Indian kala-azar by dot enzyme-linked immunosorbent assay (dot-ELISA).

Serodiagnosis of kala-azar was determined by dot-ELISA under variable conditions such as may be encountered in the field, for example, in the nature of the antigen, the preservation of antigen at different temperatures, variation in incubation temperature and so on. The test was found to be useful under field conditions, and the correlation between ELISA and dot-ELISA was significant. Dot-ELISA has definite advantages over the ELISA test, as it is easy to perform, is stable and does not require expensive equipment. The test can be used for large-scale screening of sera in sero-epidemiological studies.

Animals↗

PubMed2002

Revisiting the anticardiolipin test and its standardization.

Although the importance of the anticardiolipin test in diagnosis of antiphospholipid syndrome (APS) is widely accepted, there remains much misunderstanding about the strengths and weaknesses of this assay. Several disorders result in formation of low levels of the antibody, hence the anticardiolipin test is not specific when results are low positive. In general, the higher the anticardiolipin level the greater the likelihood of a diagnosis of APS. Hence there have been numerous efforts to enable reproducible measurement of anticardiolipin levels. Standard calibrators were introduced to construct calibration curves from which levels of unknown samples can be derived. Those standard calibrators were made by mixing varying quantities of high positive with normal sera. More recently, calibrators derived from monoclonal anticardiolipin antibodies have been introduced. There are advantages and disadvantages with both types of calibrators. Determination of a precise and reproducible anticardiolipin level is difficult, whatever the calibrators used, because the assay is dependent on several variable components, any of which may fail on any given day. Utilization of a semi-quantitative measure (low, medium, high) may suffice in most clinical settings and would be less subject to error. Validated ELISA kits may offer greater reproducibility, since there is less variability than bench assays set up in very different laboratories. Whether using a kit or a bench assay, meticulous attention to detail offers the best opportunity for precision and reproducibility.

Antibodies, Anticardiolipin↗

PubMed2000

Detection of anti-GM1 ganglioside antibodies in patients with neuropathy by a novel latex agglutination assay.

Highly elevated titers of serum anti-GM1 ganglioside antibodies are closely associated with multifocal motor neuropathy, but low titers are commonly present in normal individuals or other diseases. Current systems for measuring anti-GM1 antibodies utilize the enzyme-linked immunosorbent assay (ELISA), in which serum dilutions are tested for binding to excess antigen immobilized on the surface of microwells. The ELISA system, however, is relatively time consuming, labor intensive, and costly, in addition to being prone to methodological variability. We have developed a novel agglutination assay for the detection of anti-GM1 antibodies, utilizing GM1 ganglioside-coated latex beads. In contrast to the ELISA system, antibody titers may be quantified by testing for agglutination using latex beads coated with decreasing amounts of antigen. The agglutination assay compares favorably to the ELISA system in sensitivity and specificity, but is considerably less costly and takes only a few minutes to perform.

Amyotrophic Lateral Sclerosis↗

PubMed1991

The use of recombinant antigens in ELISA procedures for the quantification of intrathecally produced HIV-1-specific antibodies.

An ELISA procedure is described for the quantification of intrathecally synthesized immunoglobulin G antibodies to human immunodeficiency virus (HIV) antigens. Recombinant p17, p24, endonuclease (END), reverse transcriptase (RT), a peptide from the transmembrane region of gp41 (ENV80) and a fusion protein containing HIV-1 and HIV-2 epitopes were compared with a commercially available ELISA. Using a reference serum, antibodies in serum and cerebrospinal fluid (CSF) to all of the antigens could be measured quantitatively in a reliable and reproducible fashion. Despite the fact that the titer varied up to 10(5)-fold between CSF and serum, interassay variability ranged from 3.87% for p17 to 8.41% for RT and intra-assay variability varied from 3.9% +/- 1.2% for p17 to 14.3% +/- 3.9% for the commercial ELISA. Antibody specificity indices (ASI) obtained by relating CSF/serum titers with reference to the corresponding IgG concentrations can be used to detect intrathecal synthesis of virus specific antibodies.

Enzyme-Linked Immunosorbent Assay↗