SEARCH · PubMed Health

Results for “ELISA assay variability”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources

PubMed2001

Serum levels of E-selectin, ICAM-1 and VCAM-1 in colorectal cancer patients: correlations with clinicopathological features, patient survival and tumour surgery.

The serum concentrations of the cell adhesion molecules E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were investigated in 63 patients with colorectal cancer and in 51 controls by an enzyme-linked immunosorbent assay (ELISA). Their relationship to clinicopathological variables and patient survival and changes in their levels after surgery were examined. Colorectal cancer patients showed significantly higher serum levels of E-selectin, ICAM-1 and VCAM-1 compared with healthy controls. There was a significant association between the serum levels of these molecules, disease stage and the presence of both lymph node and distant metastases. Both ICAM-1 and VCAM-1 levels correlated with serum E-selectin and carcinoembryonic antigen (CEA) levels. Serum levels of all three molecules decreased significantly after radical resection of the tumour. Elevated pre-operative E-selectin, ICAM-1 and VCAM-1 levels were significant prognostic factors, although not independent of stage, for patient survival. These findings suggest that serum concentrations of E-selectin, ICAM-1 and VCAM-1 may reflect tumour progression and metastasis. Since these markers are linked to CEA levels, it is uncertain whether their measurement will prove cost-effective in colorectal cancer management.

Aged↗

PubMed1984

Maximizing the detection capability of a beet western yellows virus ELISA system.

Conditions for maximizing detection of a California isolate of beet western yellows virus (BWYV) were investigated with the double-sandwich enzyme-linked immunosorbent assay (ELISA) system. Within-plate variability was found to account for less than 1% of the total variation observed on individual microtiter plates. Variability across plates was greater than within plates and accounted for less than 10% of the total variation. Significant reductions in absorbance were observed when either coating or conjugated IgGs were incubated for 2 h at 37 degrees C. Incubation of coating IgGs overnight at 4 or 22 degrees C gave good results. No differences in absorbance were observed when antigen sources were incubated at 37, 22, or 4 degrees C. Absorbance at 1, 3 or 6 h incubation of conjugated IgGs at 4 or 22 degrees C was low to moderate but after 18 or 24 h absorbance was markedly increased without increasing background. Efficient extraction of virus from host and vector tissues increased absorbance. Carborundum added to the extraction buffer worked well when tissue samples were ground in a mortar and pestle but highest absorbance resulted when test samples were prepared with a high-speed tissue homogenizer. Background levels were unacceptably high when 10 or more Myzus persicae were pooled for analysis. Since virus can be detected in 3 or even 1 aphid, pooling for detection is unnecessary. Optimization of the BWYV ELISA system made possible the detection of virus quantities less than 2 ng/ml.

Antibodies, Viral↗

PubMed1999

Endotoxin and tumor necrosis factor-alpha in middle ear effusions in relation to upper airway infection.

OBJECTIVES/HYPOTHESIS: This study was performed to elucidate the role of endotoxin and tumor necrosis factor-alpha (TNF-alpha) in the middle ear effusions (MEEs) of children with otitis media with effusion (OME) in relation to the chronicity of the disease and the presence of upper respiratory tract infection (URTI). STUDY DESIGN: In a retrospective study 140 MEEs were collected from 101 children between 2 and 12 years of age, and evaluated for the cytokine TNF-alpha and the lipopolysaccharide endotoxin. The amounts were quantified and correlated with the type of MEE, OME duration, and the presence of URTI. METHODS: Endotoxin levels were measured using a limulus amebocyte lysate assay and TNF-alpha levels were measured with an enzyme-linked immunosorbent assay (ELISA). Means of the different variables were compared using the one-way ANOVA least significance difference test with P<.05. RESULTS: In MEEs classified as mucopurulent (22.8%) both endotoxin and TNF-alpha levels (11.9+/-3 ng/mg total protein and 61.1+/-21 pg/mg total protein, respectively) were significantly higher compared with serous- (23.6%) or mucoid- (53.6%) type effusions. Fifty-five percent of the children who were classified as having chronic OME also had significantly higher amounts of endotoxin and TNF-alpha. The majority of the children (61%) had no URTI, although children with URTI (36%) did also have significantly higher levels of endotoxin and TNF-alpha in their middle ears. CONCLUSIONS: These results indicate that there is a strong correlation between the endotoxin and the TNF-alpha concentration in the middle ear and the type of MEE, the presence of URTI, and the chronicity of the disease.

Child↗

PubMed1993

Prevalence of a Vibrio cholerae 18-kDa antigen in vibrios.

An 18-kDa protein that occurs in Vibrio cholerae has been described as an in vivo and low-iron regulated outer membrane antigen. Monoclonal antibodies which recognized this antigen were protective as passive vaccines in the infant rabbit model of cholera disease. In this study, those monoclonal antibodies were used in three immunological assays for surveillance of various bacteria for the 18-kDa antigen. ELISA, and Western blot assays gave variable results with bacteria or outer membrane preparations. The biodot assay was the most sensitive test, detecting the 18-kDa antigen in 29 of 29 V. cholerae strains, independent of biotype or serotype. A few other Gram-negative bacteria and V. parahaemolyticus reacted weakly with our antibodies and antiserum.

Animals↗

PubMed2002

Tumor cell-specific gene transfer with retroviral vectors displaying single-chain antibody.

OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid. To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. RESULTS: The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. CONCLUSION: These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.

Gene Transfer Techniques↗

PubMed2005

Development of an ELISA for quantifying lysozyme in hen egg white.

An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.

Animals↗

PubMed2005

ELISA methods to measure cholinergic markers and nerve growth factor receptors in cortex, hippocampus, prefrontal cortex, and basal forebrain from rat brain.

The central cholinergic system has a fundamental role in normal cognitive function, and in diseases that exhibit cognitive dysfunction. The purpose of this study was to design ELISA methods to measure proteins that have essential functions in the central cholinergic system. We were particularly interested in quantifying proteins that respond directly or indirectly to nerve growth factor (NGF). ELISAs offer advantages over Western blot analyses and other methods, such as increased sensitivity, decreased assay variability, increased efficiency, and decreased cost. We developed indirect ELISA methods for: choline acetyltransferase (ChAT); the vesicular acetylcholine transporter (VAChT); the high affinity choline transporter (HACT/CHT); TrkA, the high affinity NGF receptor; the p75 neurotrophin receptor (p75(NTR)). A sandwich ELISA was developed to measure tyrosine-phosphorylated TrkA in brain lysates. We used these ELISAs to compare levels of the above proteins in important memory-related brain regions--basal forebrain, hippocampus, cortex, and prefrontal cortex--from old and young rats. We identified age-related differences in the levels of the aforementioned proteins (e.g., VAChT and HACT/CHT in hippocampus). Thus, these ELISA methods should be particularly useful for comparing the effects of age, disease, drugs, and toxicants on brain levels of key cholinergic and growth factor-related proteins.

Acetylcholine↗

PubMed1981

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of antisperm antibodies.

Several recent comparative investigations using various assays to detect and quantitate levels of antibody to human spermatozoa have produced widely varying results. In an attempt to reduce test variability, an indirect enzyme-linked immunosorbent assay (ELISA) was devised to measure antisperm antibodies. A standardized protocol was adapted employing sperm adsorption to polystyrene microtiter plates, at a density of 10(5) sperm per well, serum and enzyme-conjugate incubation conditions at 37 degrees C for 60 min, and three ten-minute washes between incubations, using phosphate-buffered saline containing Tween-20. Using antihuman sperm antisera generated in rabbits, the ELISA was shown to yield significantly detectable antibody at dilutions of 1/16,384. The ELISA demonstrated approximately 89% reproducibility (ie, 100% minus the coefficient of variation) for an "intraassay" study wherein 300 determinations were performed on the same day on sperm from ten donors. However, when sperm from one donor were used in 30 determinations during ten assays over a six-month period, "interassay" reproducibility was approximately 51%. The ELISA was compared with macroagglutination, microagglutination, and immobilization tests, using rabbit antisperm serum and human sera from vasectomized males. Results of this study indicated that the ELISA was more sensitive, less subjective, and easier to perform than these other commonly used techniques.

Antibodies↗

PubMed1987

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↗

PubMed1989

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↗

PubMed1994

Standardisation of a microplate in situ ELISA (MISE-test) for the susceptibility testing of herpes simplex virus to acyclovir.

Viral susceptibility testing has been traditionally performed by plaque reduction assay (PRA) which is labour intensive, time consuming and requires subjective input by the reader. An in situ enzyme-linked immunosorbent assay (ELISA) method has been developed with the potential to overcome many of the limitations of PRA, and has been applied to a variety of viruses. Previous reports of ELISA susceptibility assays have shown little standardisation between these methods, or any significant analysis of the variable factors which may influence the outcome of the assay. This study optimised the sensitivity of a microplate in situ ELISA (MISE-test) for the detection of viral growth, manipulated the interaction between cells, virus and acyclovir to determine the effect of their relationship on susceptibility results, and established standard assay conditions based on quality controlled parameters such as assay variability and linear ranges. 33 isolates of HSV-2 were tested for susceptibility to acyclovir by PRA, and the standardised MISE. Factors which were critical to the performance of the MISE included inoculum size, inoculation method, duration of incubation, fixative type, immunoglobulin working strengths and choice of chromogenic substrate. Using the ELISA it was possible to separate sensitive HSV-2 isolates from resistant isolates applying a cutoff ID50 value of 2.0 mg/l. The correlation coefficient between PRA and MISE was 0.65. The standardised microplate in situ ELISA was found to be an acceptable alternative to the plaque reduction assay.

Acyclovir↗

PubMed2000

Antibody responses in patients with staphylococcal septicemia against two Staphylococcus aureus fibrinogen binding proteins: clumping factor and an extracellular fibrinogen binding protein.

We analyzed the serum antibody responses against two Staphylococcus aureus fibrinogen binding proteins, the cell-bound clumping factor (Clf) and an extracellular fibrinogen binding protein (Efb). The material consisted of 105 consecutive serum samples from 41 patients suffering from S. aureus septicemia and 72 serum samples from healthy individuals. An enzyme-linked immunosorbent assay (ELISA) was developed. Healthy individuals showed variable levels of antibodies against the studied antigens, and cutoff levels (upper 95th percentile) against these antigens were determined. No correlation was seen between serum antibody levels against Clf and Efb. In acute-phase samples 27% of patients showed positive antibody levels against Clf and 10% showed positive levels against Efb, while in convalescent-phase samples 63% (26 of 41) showed a positive serology against Clf and 49% (20 of 41) showed a positive serology against Efb. Antibody levels against Efb were significantly lower in the acute-phase sera than in sera from healthy individuals (P = 0. 002). An antibody response against Clf was most frequent in patients suffering from osteitis plus septic arthritis and from endocarditis (80% positive). The antibody response against Efb appeared to develop later in the course of disease. A possible biological effect of measured antibodies was demonstrated with the help of an inhibition ELISA, in which both high-titer and low-titer sera inhibited the binding of bacteria to fibrinogen. In conclusion, we have demonstrated in vivo production of S. aureus fibrinogen binding proteins during deep S. aureus infections and a possible diagnostic and prophylactic role of the corresponding serum antibodies in such infections.

Adolescent↗

PubMed1999

Taguchi optimisation of ELISA procedures.

We propose a new method in the field of ELISA optimization using an experimental design called the Taguchi method. This can be used to compare the net effects of different conditions which can be both qualitative and quantitative in nature. The method reduces the effects of the interactions of the optimized variables making it possible to access the optimum conditions even in cases where there are large interactions between the variables of the assay. Furthermore, the proposed special assignment of factors makes it possible to calculate the biochemical parameters of the ELISA procedure carried out under optimum conditions. Thus, the calibration curve, the sensitivity of the optimum assay, the intra-assay and inter-assay variability can be estimated. The method is fast, accessing the results in one step, compared to the traditional, time-consuming 'one-step-at-a-time' method. We exemplify the procedure with a method to optimize the detection of ScFv (single chain fragment of variable) phages by ELISA. All the necessary calculations can be carried out by a spreadsheet program without any special statistical knowledge.

Analysis of Variance↗

PubMed2000

Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein.

The hepatis B virus X protein (HBx) has been thought to be implicated in the development of hepatocellular carcinoma. Although many functions of HBx have been reported, it is not clear which of HBx functions is important in hepatocellular carcinogenesis. To study HBx function, we produced a monoclonal anti-HBx Ab secreted by hybridoma cell clone H7 and mapped its epitope to a region of HBx between amino acids 29 and 48 by Western blot with truncated forms of HBx and by enzyme-linked immunoadsorbent assay (ELISA) with synthetic HBx peptides. The variable regions of H7 anti-HBx Ab were cloned by polymerase chain reaction using the degenerate-primers and by the 5' rapid amplification-cDNA end method. The sequence analyses revealed that the variable gene segments of the heavy and light chains are the members of mouse heavy chain variable gene 1 family and kappa light chain variable gene 2 family, respectively. In addition, J(H)2 or Jkappa4 gene segment at the end of the heavy-chain or light-chain variable region and DSP2.x gene segment in the CDR 3 of heavy chain were identified.

Amino Acid Sequence↗

PubMed1994

Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay.

A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre- and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre- and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the between-laboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 micrograms of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 micrograms of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.

Adult↗

PubMed2000

Angiogenic cytokines in serum and plasma of patients with head and neck squamous cell carcimona

INTRODUCTION: Angiogenesis is critical for tumour growth and dissenmination. Some angiogenic cytokines can be detected in the serum/plasma, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiogenin (ANG) and endostatin. VEGF, bFGF and ANG are pro-angiogenic cytokines implicated in tumour angiogenesis. Endostatin is antiangiogenic and arises from tumour-induced porteolysis. The purpose of this study was to ascertain whether there are differences between healthy controls and patients head and neck squamous cell carcinoma (HNSCC) for each cytokine; and whether there is any association with clinico-pathological variables or prognosis. METHODS: Serum/plasma was assayed by ELISA in the following numbers of patients and controls: serum VEGF (80 : 80); plasma VEGF (32 : 15); serum bFGF and ANG (25 : 15); and plasma endostatin (26:15). RESULTS: Serum VEGF is raised in patients with HNSCC (P < 0.001) but there were no associations with clinico-pathological variables or outcome. Serum VDGF correlated with platelet count (P = 0.05) and plasma VDGF (P = 0.02) although the latter is substantially lower (P < 0.001). bFGF and ANG were not raised in patients with HNSCC. Plasma endostatin was lower in patients with HNSCC than controls (P = - 0.02). However, with the HNSCC group endostatin was positively associated with disease recurrence (P = 0.008). CONCLUSIONS: These data suggest that plasma endostatin may be a clinically useful prognostic marker in patients with HNSCC. Serum VDGF arises from platelet aggregation and does not reflect tumour load, dissemination or prognosis. bFGF and ANG are not raised in patients with HNSCC.

Journal Article↗

PubMed1990

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↗

PubMed1995

Serologic responses of dogs naturally exposed to or vaccinated against Borrelia burgdorferi infection.

OBJECTIVE: To characterize the serologic responses of dogs naturally exposed to or vaccinated against Borrelia burgdorferi and to assess responses at intervals after antibiotic treatment. DESIGN: Prospective, controlled clinical trial. ANIMALS: 19 dogs of various breeds and ages with narrowly defined clinical criteria of limb/joint borreliosis and 10 control dogs of equivalent age were used to determine serologic responses following natural exposure to the organism. Eight seronegative dogs were used to determine serologic responses following vaccination. PROCEDURE: Serologic responses to B burgdorferi and recombinant outer surface protein (Osp)A, flagellin, and P39 were assessed by means of ELISA and western immunoblot. Passive protective activity was assessed by use of a mouse protection assay. RESULTS: Naturally exposed dogs were seropositive, but had variable ELISA titers and immunoblot profiles. Immunoblot analysis did reveal consistent reactions to flagellin, P39, and a 22 kd protein, but not to OspA. Antibody responses did not change appreciably up to 13 weeks after antibiotic treatment. Vaccinated dogs had strong reactions to OspA and OspB, but not to P39. CLINICAL IMPLICATIONS: Dogs with clinical borreliosis are seropositive and remain seropositive after antibiotic treatment, emphasizing that serologic testing is not a useful means of measuring clinical response. Serologic responses of infected dogs can be discriminated from those of vaccinated dogs by means of immunoblot analysis, and recombinant P39 is a potentially useful antigen for that purpose.

Animals↗