Enzyme-linked immunosorbent assay (ELISA) with variable antigen for serodiagnosis of T. B. gambiense trypanosomiasis.
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Antigens↗
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Results for “ELISA assay variability”
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Assay-dependent variability in peptide biomarker quantification: experimental evidence from renalase in chronic kidney disease.
BACKGROUND: Renalase is a promising biomarker for kidney disease, but published levels vary widely between studies. We hypothesised that variability in commercial enzyme-linked immunosorbent assays (ELISAs) kits and matrix effects (serum vs plasma) drive these inconsistencies. METHODS: Paired serum and plasma samples from 56 participants (28 chronic kidney disease (CKD) stages 2-5, 28 healthy controls) were tested using three commercial renalase ELISAs (BTLAB, Cloud-Clone, EIAab). We assessed intra-assay precision, inter-assay agreement (Spearman's rank correlation and Bland-Altman analysis on log10-transformed values), matrix effects, and associations with estimated glomerular filtration rate (eGFR). Diagnostic performance was evaluated by Receiver operating characteristic (ROC) analysis. RESULTS: Inter-assay renalase concentrations differed markedly (up to orders of magnitude), with weak inter-assay correlations (r ≤ 0.25). Bland-Altman analyses revealed large, systematic biases between kits. Only the BTLAB assay showed consistent serum/plasma agreement, a significant correlation with eGFR (ρ ≈ 0.32-0.42, p < 0.05), and moderate discriminatory performance for CKD in serum (AUC = 0.70) and plasma (AUC = 0.68). Cloud-Clone and EIAab produced divergent results and strong matrix-dependent biases. CONCLUSIONS: Observed variability among commercial ELISA platforms may compromise comparability between studies. Harmonisation, standardised reference materials, and cross-validation are necessary before renalase assays can be used reliably in clinical practice.
Humans↗
Detection of autoantibodies to Sm antigen in systemic lupus erythematosus by immunodiffusion, ELISA and immunoblotting: variability of incidence related to assays and ethnic origin of patients.
Autoantibodies to small nuclear ribonucleoproteins (snRNP) were studied using the techniques of immunodiffusion, ELISA, and immunoblotting in the sera of 150 patients with systemic lupus erythematosus (SLE), and of 29 patients with mixed connective tissue disease; 900 control patients and 100 normal blood donors were examined simultaneously. The incidence of anti-Sm antibodies in French SLE patients was low compared with the occurrence observed in similar studies in USA (even when highly sensitive assays were used) but was of the same magnitude as European results. Frequency of anti-Sm antibodies in SLE patients varied moderately when detected by immunodiffusion (12%), or by immunoblotting (17%), however, it seems that the ethnic and/or genetic background of patients induces more significant differences. SLE patients from the French West Indies had anti-Sm antibodies in 39% of cases when detected by immunodiffusion and in 50% when immunoblotting was used. In these patients the incidence of the antibodies was five times more frequent than that of mainland French patients. Immunization against snRNP does not seem to be a common feature of all SLE patients.
Antibody Specificity↗
A cross-sectional study of bluetongue virus and Mycoplasma bovis infections in dairy cattle: II. The association between a positive antibody response and reproduction performance.
In a cross-sectional study to determine the possible relationship between a positive antibody test to bluetongue virus (BTV) or Mycoplasma bovis infections and reproductive performance of dairy cows, data were collected on 572 California dairy cows during December 1986 for analysis. Serum samples were tested using an enzyme-linked immunosorbent assay (ELISA). Data on reproduction variables were extracted from the individual cow sheets of the California Dairy Herd Improvement Association records and interfaced with the serological results for analysis. Similar data analyses for both BTV and M. bovis were performed to identify and quantitatively assess the association of the reproduction variables and each agent. These associations were evaluated unconditionally using the chi 2 for categorical variables and Student's t-test for continuous variables. Logistic regression analysis was used to determine if reproduction variables with significant unconditional associations remained significant when adjusted for the effects of possible confounding factors. Both the BTV and M. bovis ELISA antibody titres indicated exposure to the agents. The results of the multiple logistic regression analyses indicated that cows seropositive for BTV were significantly older at first calving (p less than 0.03). For M. bovis, seropositive cows were more likely to have longer intervals from calving to last service and longer intervals from calving to pregnancy diagnosis than seronegative cows (p less than 0.05). The other reproduction variables examined were not significantly associated with ELISA seropositivity.
Age Factors↗
Direct detection of human cytomegalovirus in urine specimens from renal transplant patients following polymerase chain reaction amplification.
A polymerase chain reaction (PCR) assay was used to amplify human cytomegalovirus (HCMV) directly from urine specimens taken from renal transplant patients. In serial urine samples from patients who had at least one specimen positive for HCMV; the PCR assay consistently detected the presence of HCMV DNA sequences, whereas virus detection by other tests such as enzyme-linked immunosorbent assay (ELISA), nonradioactive DNA hybridization assay, and virus isolation were variable. Of 37 specimens positive by PCR, 36 were positive by either ELISA, hybridization assay, or virus isolation. Infectious virus was detected in 13 of the 37 PCR-positive urines. HCMV DNA was detected by PCR in all samples that were positive for HCMV by either hybridization assay or virus isolation. The viral genome copy number was determined by PCR assay for several urine samples that were positive by virus isolation but negative for HCMV by ELISA or hybridization assay. Viral genome copy number estimates indicated the presence of HCMV at very low levels in these urines verifying the fidelity of the virus isolation procedures. The consistency of the PCR assay makes it an ideal method for detection of infection and monitoring antiviral drug therapy in patients infected with HCMV.
Antigens, Viral↗
The von Willebrand factor.
Von Willebrand factor (vWf) is a multimeric and multivalent adhesive protein which is essential for platelet adhesion to subendothelium and for stabilization of factor VIII procoagulant activity in circulation. The quantitative measurement of vWf involves essentially two different approaches. The first is based on the interaction between vWf and Gp Ib of the platelet membrane in presence of ristocetin (ristocetin cofactor activity, RiCof) and depends not only on the amount of the factor but also on its ability to bring about this interaction, large multimers being more active. The second approach involves the immunological quantitation of vWf (vWf:Ag) by its interaction with specific polyclonal or monoclonal antibodies as measured by several methods, i.e., electroimmunoassay, immunoradiometric assay and immunoenzymatic assay. Although in the majority of type II von Willebrand disease (vWd) with dysfunctional vWf there is a discrepancy between RiCof and vWf:Ag, it should be emphasized that RiCof activity does not entirely reflect the 'true' activity of vWf since it does not explore all the functions of this factor; furthermore, the relationship between degree of multimerization and RiCof level is not always tenable, as for example in vWd 'Vicenza'. For the diagnosis of congenital and acquired vWd RiCof assay together with family investigation is the eligible test, with an estimated ability to detect at least 50% of the carriers of the abnormal gene, including mildly affected patients; vWf:Ag appears less sensitive and, on the basis of studies carried out in our laboratory, a relative sensitivity of 64% is proposed. Both assays require the definition of separate normal ranges for children and adults and for 0 and non-0 blood group subjects; a nonparametric approach in a large sample of normal subjects is advisable. With RiCof assay performed by an aggregometric method using formalin-fixed platelets an interassay variability of 6% and 8.5% respectively for high- and low-control plasma was found in our laboratory. With vWf:Ag assayed by an ELISA method a variability of 7% for low- and 6% for high-control plasma was found. Thus, both methods appear sufficiently precise for clinical use. The use of an internal pool calibrated against an international standard allows to perform comparable interlaboratory measurements. To further improve standardization of these assays, collaborative studies seem urgently required.
Adult↗
Clinical associations of IgG antibodies to the ribonucleoprotein p67 polypeptide in patients with systemic lupus erythematosus.
The ribonucleoprotein (RNP) p67 antigen was purified from rabbit thymus and used in an enzyme linked immunosorbent assay (ELISA) with low interassay variability to detect IgG antibodies to p67 in patients with autoimmune connective tissue diseases. These antibodies were found in eight (80%) patients with a clinical diagnosis of mixed connective tissue disease (MCTD) but also in 27 (40%) patients with systemic lupus erythematosus (SLE). Sixty six per cent of the 12 patients with SLE with high levels of antibodies to p67 (> 50 U) had three or more features of MCTD, including myositis, fibrosing alveolitis, Raynaud's phenomenon, and sclerodactyly. Antibodies to the p67 RNP were not associated with the presence or absence of renal disease in the patients with SLE. This study suggests that antibodies against the p67 RNP are markers for clinical features of MCTD even in the context of SLE.
Autoantibodies↗
The dissociation of GM-CSF efficacy from toxicity according to route of administration: a pharmacodynamic study.
The pharmacokinetics and pharmacodynamics of bacterially synthesized granulocyte-macrophage colony stimulating factor (GM-CSF) were studied in 33 patients. GM-CSF (0.3-30 micrograms/kg/d) was administered subcutaneously (s.c.) or intravenously (i.v.) by bolus or 2 h infusion for 10 d to three patients at each dose level, and serum levels measured by enzyme-linked immunosorbent assay (ELISA). Relationships between pharmacokinetic variables and GM-CSF efficacy and toxicity were investigated. GM-CSF appeared more effective s.c. than i.v. Correlations with response improved when patients with neutropenia or marrow infiltration by tumour were excluded. For the remaining patients, the correlation coefficients between the day 10 leucocyte count was highest with dose (R = 0.629, P less than 0.01), the duration GM-CSF concentrations exceeded 1 ng/ml (r = 0.524, P less than 0.05) and area under the concentration-time curve (R = 0.508, P less than 0.05). There was no correlation with peak serum GM-CSF (Cmax) (R = 0.188, P = NS). In contrast, the first dose reaction (characterized by hypoxaemia and hypotension) was associated with high Cmax (P less than 0.01) and i.v. administration. Fever, liver enzyme elevation and pericarditis occurred at 10-30 micrograms/kg and were not influenced by route. Since the variables correlating with response differed from those correlating with these toxicities, it appears that the therapeutic index for GM-CSF can be increased by modifying the method of administration.
Dose-Response Relationship, Drug↗
Development and Validation of a Novel LC-MS/MS Based Proteomics Method for Quantitation of Retinol Binding Protein 4 (RBP4) and Transthyretin (TTR).
Retinol binding protein 4 (RBP4), the circulating carrier of retinol, complexes with transthyretin (TTR) and is a potential biomarker of cardiometabolic disease. However, RBP4 quantitation relies on immunoassays and western blots without retinol and TTR measurement. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous absolute quantitation of circulating RBP4 and TTR is critical to establishing their biomarker potential. Surrogate peptides with reproducible, linear LC-MS/MS response were selected. Purified proteins were used as quantitation standards and heavy-labelled peptides as internal standards. Matrix effects were evaluated. The validated method was applied to measure inter- and intra-individual variability in RBP4 and TTR concentrations in healthy individuals and patients with diabetic kidney disease. Quantitation was linear for the clinically relevant concentration ranges of RBP4 (0.5-6 μM) and TTR (5.8-69 μM). Assay inter-day variability was <12% and precision within 5%. The inter-individual variability for RBP4 and TTR concentrations was 18-26%, while intra-individual variability was similar to assay variability. RBP4 and TTR quantitation correlated with commercially available ELISA assays. The developed LC-MS/MS method enables simultaneous absolute quantitation of RBP4 and TTR in serum and plasma that can be applied to clinical biomarker studies and stoichiometric measurements of circulating RBP4, TTR, and retinol.
Retinol binding protein 4 (RBP4)↗
ELISA assays for IgM and IgG rheumatoid factors: their clinical correlations during therapy with slow-acting anti-rheumatic drugs.
A longitudinal study of changes in rheumatoid factor levels and their correlations with other clinical and laboratory assessment of disease activity followed 45 rheumatoid arthritis patients during 12 months continuous therapy with gold and penicillamine. Rheumatoid factors were measured by the titred latex assay and by ELISA assays for IgM and IgG rheumatoid factor. The IgM ELISA assay correlated strongly with the titred latex assay and weakly with the IgG rheumatoid factor assay at 0, 6 and 12 months. All showed significant falls with treatment, and ELISA assays were not more sensitive indicators than the latex assay. Correlations of initial rheumatoid factor levels by all 3 assays to 34 other clinical and laboratory assessments showed that only a small number of weak correlations existed; there were more at 6 and 12 months, but still not many. Changes in rheumatoid factor levels over 0-6 months showed no significant correlation to changes in other variables. There were no more correlations with ELISA assays than with titred latex rheumatoid factor. We conclude that ELISA rheumatoid factor assays have no clinically relevant advantage over the titred latex assay when following rheumatoid patients treated with gold or penicillamine.
Arthritis, Rheumatoid↗
Detection of murine typhus infected fleas with an enzyme-linked immunosorbent assay.
An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.
Animals↗
A sensitive antiviral neutralization bioassay for measuring antibodies to interferons.
An improved bioassay for measuring neutralizing antibodies to interferons (IFN) is described. The assay is based upon an objective and precise quantification of the viral cytopathic effect. This effect is measured via the dehydrogenase-system in cells, and quantified spectrophotometrically. Virus-infected cells, in contrast to non-infected cells, possess low enzyme activity resulting in low OD signals. This fall in OD can be prevented by the addition of a small, but fixed amount of IFN before the addition of virus. Anti-IFN sera will neutralize the protective effect of IFN. This effect can be quantified by measurement of the reduction in the OD signals. Antibodies to recombinant IFN were found to cross-react with human leukocyte IFN although to a ten-fold lower degree. The assay requires no expensive reagents, it is performed in 96-well microtrays and the results can be measured in an ordinary ELISA scanner. The assay is highly reproducible, yielding inter- and intra-assay variability of less than 10%. The sensitivity is much higher than that reported previously for the CPE technique and that of ELISA techniques.
Animals↗
Elementary school students' performance with two ELISA test systems.
OBJECTIVE: To examine analytic performance by previously untrained and inexperienced subjects using enzyme-linked immunosorbent assay (ELISA) tests developed for decentralized laboratories. Performance variability between tests assigned to the "simple" and "moderately complex" Health Care Financing Administration laboratory levels was evaluated. DESIGN: A nonrandomized trial of the Surecell Strep-A chorionic gonadotropin ELISA tests. Each subject processed nine unknown specimens (three negative, three weakly positive, and three strongly positive) for each ELISA test. Subjects were blinded to expected test results. SETTING: An elementary school. SUBJECTS: A convenience sample of 52 students enrolled in the sixth and seventh grades. This age group was chosen because of their ability to generally comprehend instructions and remain attentive to the testing task. INTERVENTIONS: Subjects were either self-trained by reading package insert directions or trained by a manufacturer's sales representative. MAIN OUTCOME MEASURES: Performance was measured as the percentage of correct test results for the unknown specimens. The sensitivity and specificity for each test by operator group were calculated. RESULTS: Subjects demonstrated an overall sensitivity of 97.1% and specificity of 94.7% for human chorionic gonadotropin unknown specimens and a 95.9% sensitivity and 96.8% specificity for group A streptococcus unknown specimens. No significant differences between the self-trained group and the representative-trained group were observed for either group A streptococcus or human chorionic gonadotropin tests. Performance was so high with the first specimen that improvement over time (ie, a "learning curve") could not be demonstrated. CONCLUSION: These ELISA test systems are able to achieve high levels of performance by subjects with no formal laboratory background, no previous method specific experience, and limited self-training.
Adolescent↗
The repertoire diversity and magnitude of antibody responses to bacterial antigens in aged mice: I. Age-associated changes in antibody responses differ according to the mouse strain.
Aging influences the host immune responses in various ways. In aging mice we have studied the antibody responses to two unrelated bacterial antigens. Streptococcus pneumoniae R36a vaccine (Pn) and TNP coupled to Brucella abortus (TNP-BA). Aged animals (20-24 months old) of the C57BL/6 strain had markedly reduced numbers of IgM antibody plaque-forming cells (PFC) to Pn as compared to young/adult mice (2-3 months old). In contrast, the anti-Pn IgM PFC responses of aged BALB/c mice were consistently higher than they were in the young/adult mice. The increased anti-Pn responses were not due to a nonspecific immunostimulation, because the responses of aged BALB/c mice to TNP-BA were lower as compared to the adults. However, the aged BALB/c mice responded relatively poorly to Pn challenge, and their IgG responses (as determined by ELISA plaque assay) demonstrated a very high individual variability. The clonotypic diversity of anti-Pn response in young BALB/c and C57BL/6 is limited, such that the majority of PFC produce antibody that express all idiotopes (Id) of the T15 immunoglobulin encoded in the VH-S107/Vk22 genes. In contrast, the PFC from aged mice are diverse, expressing incomplete T15 Id or none at all, suggesting that the antibodies are encoded by altered T15 genes and by different, non-T15 genes. Our data demonstrate that the age-related changes in the magnitude of antibody response to certain antigens are influenced by the host genetic make-up, and that the changes in magnitude and diversity of antibody response may be unrelated to each other.
Aging↗
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↗
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↗
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↗
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↗