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Results for “ELISA assay variability”

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PubMed1994

Human intestinal versus tissue-nonspecific alkaline phosphatase as complementary urinary markers for the proximal tubule.

The availability of early biological markers of renal damage is important for the identification of risk factors and for starting therapeutic intervention in the reversible phase of renal pathology. The usefulness of such markers relies upon their capacity to detect alterations in distinct nephron segments. Using specific monoclonal antibodies against the intestinal isoenzyme of alkaline phosphatase (IAP) and against the tissue-nonspecific isoenzyme (TNAP), we demonstrated that IAP expression in the human kidney is restricted to the straight part of the proximal tubule (the S3 segment), whereas TNAP is expressed mainly in the proximal convoluted tubule (the S1 and S2 segments) but also in the S3 segment. This complementarity opens perspectives for IAP and TNAP as distinct proximal tubular markers, particularly for IAP, since there are no other markers available that are specific for the S3 segment. Based on these monoclonal antibodies, specific and easy to use enzyme-antigen immunoassay (EAIA) procedures were developed to detect IAP and TNAP in human urine samples. The detection limits are below the lowest enzyme activities found in the urine of normal subjects, the intra- and inter-assay variability is low, the analytical recovery approaches 100%, and EAIA enzyme activity values correlate with ELISA immunoreactivity values. Furthermore, easy urine sample preconditioning allows antigen preservation over an extended time period at 4 degrees and -80 degrees C. Using these assays, it could be demonstrated in more than 20 occupationally and environmentally exposed cohorts and clinical patient groups that urinary IAP is indeed a marker of early alterations in the S3 segment, and that it behaves largely independently from urinary TNAP.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1997

Preference for IgG mAb binding insulin in solution or on surfaces is related to immunoglobulin variable region structures.

Studies on insulin autoantibodies often show a lack of correlation between enzyme-linked immunosorbent assays (ELISAs) and fluid phase radioimmunoassays (RIAs). Similarly, a set of IgG anti-insulin monoclonal antibodies (mAb) from BALB/c mice are found to differ in their binding in ELISAs and RIAs. To understand the structural basis for differential insulin binding, soluble and complexed biotinylated insulin is used to confirm binding properties independent of insulin-coated plastic and radioiodination. The binding properties of intact mAb are also present in Fab fragments, indicating ligand preference is not related to valence or to the Fc portion of Ig. Analysis of binding to soluble or bound ligand in relationship to antibody variable (V) region structures indicates that differential binding in the two assays is a property of heavy chain variable region structure. Studies also show that limited amino acid replacements arising during maturation of the immune response may change the binding preference for an individual mAb. These findings indicate that differences in detection of insulin binding in solid phase and fluid phase are not artefactual but reflect intrinsic structural features of immunoglobulin interaction with insulin.

Amino Acid Sequence↗

PubMed2002

Variance components of an enzyme-linked immunosorbent assay for detection of IgG antibodies in milk samples to Mycobacterium avium subspecies paratuberculosis in dairy cattle.

Milk samples from 120 cows were tested up to 10 times in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycobacterium avium ssp. paratuberculosis. The purpose of the study was to estimate variance components of the assay attributable to laboratory factors using mixed model theory. Because of significant interaction between the between-run, between-day and between-plate variables, the ELISA-plate variable was nested in run-number and run-number was nested in day-number. The nested variable accounted for 68% of the laboratory variability (P < 0.001), whereas the intraplate variability accounted for only 0.04% of the laboratory variability (P > 0.9). Therefore, it was concluded that the intraplate variability could be ignored whereas the variability from the combined run-day-plate variable should be considered in any analyses based on the ELISA.

Animals↗

PubMed1986

Clinical advantages from measurement of IgM-rheumatoid factor by enzyme immunoassay.

Two enzyme immunoassays for rheumatoid factor (RF) were compared with the traditional latex agglutination test. Preference is expressed for an ELISA specific for IgM-RF because it yields specific, quantitative results. Variability of this assay was least in sera containing moderate levels of RF and it was less precise at low concentrations. Survey of a random selection from the local population showed a similar prevalence of IgM-RF positivity as revealed by previous surveys using agglutination techniques. We conclude that measurement by ELISA yields no great increase in the discriminative ability of RF testing.

Adult↗

PubMed1997

Clinical relevance of anti-HLA antibodies pre and post transplant.

Pretransplant histocompatibility testing seeks to select compatible donor-recipient pairs for transplantation. Sera from prospective renal transplant recipients are screened for the presence of human leukocyte antigen (HLA) antibodies to determine humoral alloimmunization. Present techniques screen patient sera using a complement-dependent cytotoxicity assay and express the results as percent of panel reactive antibody (PRA). However, the standard assay suffers because it needs viable target cells, a variable sensitivity of cells for complement, subjective evaluation, a lack of standardized methodology, and a variable correlation with clinical outcomes. Alternatively, an enzyme-linked immunosorbent assay (ELISA) methodology can detect IgG anti-HLA reactivity based on the binding of immunoglobulin to soluble HLA class I antigens. This method provides increased objectivity and reproducibility, does not require use of viable target cells, and most importantly, detects immunoglobulin that is reactive to HLA class I antigens. Data discussed herein suggest that identifying reactive recipient sera using the enzyme-linked immunosorbent assay (ELISA) (PRA-STAT, Sang Stat Med, Menlo Park, CA) methodology may be more informative clinically than current standard percent of panel reactive antibody (PRA) assays.

Cytotoxicity Tests, Immunologic↗

PubMed1995

Urinary immunoglobulins in patients with continent urinary reservoirs and ileal conduits.

BACKGROUND: Although bowel segments are commonly used for reconstructing the urinary tract, a high incidence of bacteriuria is observed in patients with urinary intestinal diversion. The normal gastrointestinal tract possesses a potent mucosal immune system characterized by secretory immunoglobulin A (sIgA) as the major humoral defense factor. However, the significance of urinary IgA secretion as the mucosal defense mechanism in patients with urinary intestinal diversion has remained obscure. In this study, urinary levels of sIgA as well as serum-type IgA were measured in patients with continent urinary reservoirs (Kock and Indiana pouches) and ileal conduits. METHODS: Twenty-four-hour urine samples were collected in a total of 80 patients with urinary intestinal diversion (22 Kock pouch patients, 21 Indiana pouch patients and 37 ileal conduit patients). The amount of sIgA and serum-type IgA were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Urinary IgA levels showed great inter- and intra patient variability in all three groups. Indiana reservoir urine contained significantly greater amounts of sIgA (mean 32.0 mg/24 hrs) than Kock reservoir urine (11.9 mg) and conduit urine (4.9 mg), whereas Kock reservoir urine contained significantly more sIgA than conduit urine. However, the corresponding difference was not observed in regard to serum-type IgA. In none of the three modes of urinary diversion did 24-hour sIgA excretion show any correlation with the length of time after surgery. CONCLUSIONS: Since the amounts of sIgA in these patients were much greater than reported in urinary tract infection as well as in normal subjects, the major portion of urinary sIgA seemed to be secreted by the intestinal segments. Long-term sIgA secretion in urinary intestinal diversion, especially continent urinary reservoirs, may be an important host defense system.

Humans↗

PubMed1992

Enzyme immunoassay for Q fever: comparison with complement fixation and immunofluorescence tests and dot immunoblotting.

Enzyme-linked immunosorbent assays (ELISA) for the detection of specific immunoglobulin G (IgG) and IgM antibodies were developed by using purified Coxiella burnetii cells. Variables, including type of microtiter plate, blocking agent, incubation conditions, antigen stability, and substrate type, were examined to achieve optimal ELISA performance. The reliabilities of the assay systems were compared with those of complement fixation (CF) and enhanced immunofluorescence (EIF) tests with 600 human serum samples from defined clinical cases of Q fever, routine samples, and serum specimens from farmers. ELISA and EIF test results agreed in all cases. Dot immunoblotting was also used to test some of these sera and gave a rapid, qualitative result, which agreed with ELISA and EIF test results in all cases. No instances were found in which both ELISA and EIF test results were negative and the CF test results was positive. However approximately 5% of the sera were positive by ELISA and the EIF test while the CF test result was either negative or unreadable because of serum anticomplementary activity. We conclude that dot immunoblotting is a useful screening test, whereas ELISA and the EIF test are both rapid and sensitive tests when used for the serodiagnosis of Q fever and should be considered to be replacements for the CF test.

Antibodies, Bacterial↗

PubMed1990

Indirect enzyme-linked immunosorbent assay for detection of antibody to a 110,000-molecular-weight hemolysin of Actinobacillus pleuropneumoniae.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect swine antibody to a 110,000-molecular-weight hemolysin (110K hemolysin) of Actinobacillus pleuropneumoniae. Affinity-purified rabbit polyclonal or mouse monoclonal immunoglobulin G to the hemolysin of A. pleuropneumoniae serotype 5 strain J45, followed by hemolysin-rich concentrated culture supernatant, was used to bind swine antibody to hemolysin to microdilution plates. Sixty-nine serum samples from swine that were clinically normal, presented with clinical evidence of pleuropneumonia, were experimentally immunized or challenged, or were free of pleuropneumonia were tested, and their ELISA titers were compared with complement fixation (CF) titers. On the basis of serum samples from swine that were clinically normal and negative by CF, an ELISA titer of 1:320 or greater was considered positive. In comparison with CF, the sensitivity of the ELISA was 98.1% and the specificity was 90%. The two samples negative by CF and positive by indirect ELISA were, however, also positive for antibody to serotype 5 capsule by ELISA. Immunization of normal pigs with whole cells or purified hemolysin boosted titers 4- to 128-fold within 4 weeks. Immunoblotting demonstrated that the affinity-purified immunoglobulin G to hemolysin used for capture in the assay recognized only a 110K protein of A. pleuropneumoniae serotypes 1 to 7, although the reactivity was quantitatively variable between serotypes. Therefore, the indirect ELISA is capable of identifying animals infected with or exposed to most, if not all, serotypes of A. pleuropneumoniae. If an indirect ELISA titer of 1:320 or greater is considered positive, the assay can be a valuable diagnostic tool in both clinical and research laboratories.

Actinobacillus↗

PubMed1990

Parasite-specific antibody responses of ruminants infected with Trypanosoma vivax.

Sera from goats and cattle that were infected with two Trypanosoma vivax clones (ILDat 1.2 and ILDat 2.1) derived from different stocks were analysed for antibody activity against the variable surface glycoproteins (VSGs) of the infecting clones by enzyme-linked immune assays (ELISA) and immune lysis. To obtain purified VSG, lysed trypanosomes were separated on dodecyl sulphate-polyacrylamide gels. The gels were copper stained and the VSG protein band was excised from the gel. After destaining, the proteins were electroeluted from the gel slices and used as antigens in ELISA. High titres of IgM and IgG1 antibodies and lytic antibodies against the VSG of the infecting clone were detected. The IgG1 response appeared about 4 days later than the IgM response. IgG2 antibodies were only detected in goats and cattle that were infected with ILDat 1.2. Two goats and two calves that were infected with ILDat 1.2 showed recurrent peaks in lytic activity and of IgM and IgG1 antibody activity to the VSG of the infecting variable antigenic type (VAT). Two goats that were infected with ILDat 2.1 showed a similar pattern, but in two other goats there was a recurrent peak only in the IgM class. Recurrent peaks of antibody activity to the VSG of ILDat 1.2 and ILDat 2.1 were not detected in the sera of goats that had been inoculated with irradiated trypanosomes or that had been infected with an unrelated T. vivax clone. The recurrence of antibody peaks against the VSG of infecting VATs suggests that trypanosomes with completely or partially identical surface determinants reappear during T. vivax infection of ruminants.

Animals↗

PubMed2006

CSF serotonin, 5-hydroxyindolacetic acid and neuropeptide Y levels in severe major depressive disorder.

Serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) have been the putative markers of MDD. Neuropeptide Y (NPY) may have an important role in the pathophysiology of major depressive disorder (MDD). However, direct measures of cerebrospinal fluid (CSF) 5-HT and NPY in severe MDD have been lacking. In the present study, we examined CSF 5-HT, 5-HIAA and NPY levels and correlate them with gender and suicidal behavior of severe major depressive disorder. Forty drug-free subjects with a severe major depressive disorder and forty control subjects underwent lumber puncture and psychiatric evaluation. Cerebrospinal fluid levels of 5-HT, 5-HIAA and NPY were assayed by enzyme-linked immunosorbent assay (ELISA) test. The relationships among 5-HT, 5-HIAA, NPY and clinical variables were statistically evaluated. There were no differences between severe major depressive disorder and controls in all parameters measured. In severe MDD group, significantly lower CSF 5-HT and higher 5-HT turnover (5-HIAA/5-HT) were found in female patients compared with male patients. The patients with intense suicidal intents and suicidal attempts had significantly lower CSF 5-HT compared to patients with nonsuicidal intents. Additionally, significantly lower CSF NPY was found in first episode depressed patients compared with recurrent depressed patients. Gender-related difference in CSF 5-HT implied a female preponderance in major depression to some extent. Cerebrospinal fluid 5-HT levels and 5-HT turnover (5-HIAA/5-HT) could be valuable tools for prediction of suicidality and potential markers for evaluating major depressive disorder. NPY was perhaps a marker for first episode depression.

Adult↗

PubMed2001

Soluble CD154 in rheumatoid arthritis: elevated plasma levels in cases with vasculitis.

OBJECTIVE: To determine the levels of soluble CD154 (sCD154) in the plasma of patients with rheumatoid arthritis (RA) and rheumatoid vasculitis (RV). and to examine the relationship between the levels of sCD154 in plasma and the clinical variables. METHODS: Levels of sCD154 were quantified in 39 plasma samples from patients with RA, including 9 patients who were also diagnosed with RV, and compared with those of 20 healthy subjects. An ELISA was established and specificity of the ELISA was tested by control ELISA using isotype-matched IgG and preabsorption assay. The titers of IgM and IgG rheumatoid factor (IgM-RF, IgG-RF) for each patient were determined simultaneously, and values of other laboratory variables were also determined. RESULTS: Levels of sCD 154 in plasma were higher in patients with RA than in the healthy subjects (p < 0.02). Compared with RA patients without vasculitis, patients with RV had significantly higher levels of sCD154 in their plasma (p < 0.001). Control ELISA and absorption assay of sCD154 indicated that our ELISA system was capable of measuring plasma sCD154 in RA patients. Levels of sCD154 in RA plasma correlated significantly with both IgM-RF and IgG-RF titers (r = 0.64 and 0.61, respectively, both p < 0.001). The levels of sCD154 decreased after commencement of treatment for vasculitis in cases with RV. CONCLUSION: We identified the presence of sCD154 in RA plasma, with especially high levels in cases with vasculitis. Correlation between sCD154 and RF titers indicates the CD154-CD40 pathway is likely related to pathogenic RF production.

Adult↗

PubMed1984

The identification of monoclonal class switch variants by sib selection and an ELISA assay.

Monoclonal antibodies can now be generated against a wide variety of antigens. However, a potentially useful monoclonal antibody may be of the wrong class or subclass for a particular task. Antibodies of the desired class can be obtained by identifying rare subclones in which the variable region has been rearranged to a new constant region. We describe here the use of sib selection and an enzyme-linked immunoassay (ELISA) for isolating such class and subclass switch variants from 2 IgM and 1 IgG3 producing hybridoma. The relative simplicity of ELISA assays makes it feasible to apply this approach to many different hybridomas. Since commercial affinity purified class and subclass specific antibodies are now available, the method can be used by any laboratory. Furthermore, the technique does not require that the switch variants express surface immunoglobulin and enriches for hybridomas secreting higher amounts of antibody.

Animals↗

PubMed1998

Immunogenicity of DNA vaccines expressing human immunodeficiency virus type 1 envelope glycoprotein with and without deletions in the V1/2 and V3 regions.

DNA vaccines that express the human immunodeficiency virus type 1 HXB-2 envelope glycoprotein (Env) with or without deletions of the major variable regions V1/V2 and V3 were tested for the ability to raise enzyme-linked immunosorbent assay (ELISA) and neutralizing antibody in New Zealand White (NZW) rabbits. Three forms of the Envs were examined: gp120, the surface (SU) receptor-binding domain; gp140, the entire extracellular domain of Env; and gp160, the complete form of Env. For the forms of Env containing the variable regions, the gp120-expressing DNA plasmid was more immunogenic than the gp140- or gp160-expressing DNA plasmids. Removing the V1/2 and V3 variable regions increased the immunogenicity of the gp140- and gp160-expressing DNAs. Deletion of the variable regions also resulted in antibody responses against determinants that were not presented by the forms of Env containing the variable regions. Despite the improved immunogenicity, removing the V1/V2 and V3 domains did not improve the ability of Env to raise neutralizing antibodies. These results suggest that increasing the exposure of internal structures of Env that include the CD4-binding site does not necessarily result in the generation of better neutralizing antibody.

AIDS Vaccines↗

PubMed2003

Molecular and immunochemical characteristics of monoclonal and recombinant antibodies specific to bisphenol A.

Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA). Among these mabs, BBA-2187 was the most reactive towards bisphenol A. The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported. Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells. The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs. The most sensitive assay was achieved with BBA-2187scFv. Its cross-reactivity to the related compounds was similar to that of the parent mab. Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH.

Amino Acid Sequence↗

PubMed2006

Fecal pancreatic elastase: a reproducible marker for severe exocrine pancreatic insufficiency.

BACKGROUND: In order to apply fecal pancreatic elastase for follow-up of exocrine pancreatic function in chronic pancreatitis and cystic fibrosis, we examined the sensitivity, specificity, and long-term variability of a new polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). METHODS: Patients with definite chronic pancreatitis (n = 23), probable or possible chronic pancreatitis (n = 14), autoimmune pancreatitis (n = 7), or acute pancreatitis (n = 11), and 51 healthy subjects and 11 healthy infants participated in this study. Pancreatic function was graded as normal (n = 3), mild (n = 18), moderate (n = 9), or severe (n = 18) exocrine insufficiency on the basis of secretin tests. Fecal pancreatic elastase was measured by a new ELISA. RESULTS: Fecal pancreatic elastase concentration in control subjects varied widely, with a median of 478 microg/g. The specificity of this test was 90.2% with a cutoff value of >200 microg/g. The sensitivities were 60.9% for detecting definite chronic pancreatitis, 76.5% for calcifying pancreatitis, 71.4% for autoimmune pancreatitis, and 7.1% for probable or possible chronic pancreatitis. The sensitivities were 16.7% for mild, 12.5% for moderate, and 72.2% for severe exocrine pancreatic insufficiency. Forty patients were reexamined after a median interval of 347 days. The fecal pancreatic elastase levels between the first and second tests were not significantly different. Two infants, 4.5 and 5 months old, had abnormally low values, but after a median of 304 days all infants showed normal levels (median, 444 microg/g). CONCLUSIONS: Fecal pancreatic elastase is a reproducible marker for severe exocrine pancreatic insufficiency. This test is valuable for longitudinal follow-up of exocrine pancreatic function.

Adult↗

PubMed2000

Sialochemical markers of salivary gland involvement with Sjögren's syndrome secondary to rheumatoid arthritis and primary biliary cirrhosis.

Sjögren's syndrome is an autoimmune condition affecting the lacrimal and salivary glands and can be associated with rheumatoid arthritis and primary biliary cirrhosis. Parotid salivas collected from patients and normal controls were analysed for lactoferrin, IgA and beta2-microglobulin (measured by ELISA), and cystatin (measured by a enzyme inhibition assay). Output data provided less variable means, whilst expressing results as a proportion of the total protein provided greater specificity as markers for Sjögren's syndrome. Levels of specificity for IgA, lactoferrin and beta2-microglobulin were all high (100, 95 and 100%, respectively). Sensitivity levels of these markers (but not cystatin) tended to be similar for Sjögren's syndrome secondary to primary biliary cirrhosis (IgA, 25%; lactoferrin, 63%; and beta2-microglobulin, 50%), compared to Sjögren's syndrome secondary to connective tissue diseases such as rheumatoid arthritis (IgA, 50%; lactoferrin, 86%; and beta2-microglobulin; 38%).

Arthritis, Rheumatoid↗

PubMed1980

A quantitative enzyme immunoassay for IgM rheumatoid factor using human immunoglobulin G as substrate.

A quantitative "sandwich" enzyme-immunoassay for IgM rheumatoid factor (RF) using human immunoglobulin G as substrate covalently bound to small plastic discs is described. The findings indicate that it is a simple, sensitive, reproducible, and specific method for measuring RF activity. Test results are reported in IU/ml on the basis of the World Health Organization reference RF serum, permitting standardized reporting of this determination between laboratories using this method. Interassay and interlaboratory variability of results are less than 7%. The enzyme-linked immunosorbent assay (ELISA) results correlated well with those found by latex and bentonite flocculation titrations. A number of ELISA RF-positive specimens that were latex-negative and/or Waaler-Rose-negative were confirmed positive by inhibition of the assay with soluble aggregated human IgG, but not with nonaggregated human IgG. Comparison was made between ELISA RF results when using goat or human IgG as substrate. Human IgG was usually more reactive with RFs than was goat IgG.

Agglutination Tests↗

PubMed2005

Urinary kidney injury molecule-1: a sensitive quantitative biomarker for early detection of kidney tubular injury.

Sensitive and specific biomarkers are needed to detect early kidney injury. The objective of the present work was to develop a sensitive quantitative urinary test to identify renal injury in the rodent to facilitate early assessment of pathophysiological influences and drug toxicity. Two mouse monoclonal antibodies were made against the purified ectodomain of kidney injury molecule-1 (Kim-1), and these were used to construct a sandwich Kim-1 ELISA. The assay range of this ELISA was 50 pg/ml to 5 ng/ml, with inter- and intra-assay variability of <10%. Urine samples were collected from rats treated with one of three doses of cisplatin (2.5, 5, or 7.5 mg/kg). At one day after each of the doses, there was an approximately three- to fivefold increase in the urine Kim-1 ectodomain, whereas other routinely used biomarkers measured in this study [plasma creatinine, blood urea nitrogen (BUN), urinary N-acetyl-beta-glucosaminidase (NAG), glycosuria, proteinuria] lacked the sensitivity to show any sign of renal damage at this time point. When rats were subjected to increasing periods (10, 20, 30, or 45 min) of bilateral ischemia, there was an increasing amount of urinary Kim-1 detected. After only 10 min of bilateral ischemia, Kim-1 levels on day 1 were 10-fold higher (5 ng/ml) than control levels, whereas plasma creatinine and BUN were not increased and there was no glycosuria, increased proteinuria, or increased urinary NAG levels. Thus urinary Kim-1 levels serve as a noninvasive, rapid, sensitive, reproducible, and potentially high-throughput method to detect early kidney injury in pathophysiological studies and in preclinical drug development studies for risk-benefit profiling of pharmaceutical agents.

Animals↗