Factors affecting enzyme-linked immunosorbent assay (ELISA) for insulin antibodies in serum

We have evaluated the factors affecting an enzyme-linked immunosorbent assay (ELISA) for circulating insulin antibodies. Dilutions of patients' sera were incubated in polystyrene tubes coated with procine insulin. The second incubation was with alkaline phosphatase-conjugated anti-human Fab. Each of these reactions was complete after 4 h. Specificity of the reaction for insulin antibodies was demonstrated by removal of anti-insulin activity after preincubation with insulin, but not with glucagon at similar concentrations. Sensitivity of the ELISA system, assessed by performing the reaction with affinity-column purified insulin antibodies, was 10 microgram of specific antibody per liter. Using this system, we examined sera from 22 patients who had been determined by radioimmunoassay to have insulin antibodies, and sera from 23 normal individuals. The ELISA results correlated reasonably well with those of RIA (r = +0.84). Besides detecting insulin antibodies in diabetic patients who are being treated with insulin, we use this ELISA test as a screening procedure to be certain insulin antibodies are not present when we use indirect immunofluorescence methods of fixed pancreatic substrate to detect islet-cell antibodies.     

One-step enzyme-linked immunosorbent assay (ELISA) for measurement of serum free leptin

In man, circulating leptin levels are increased with obesity and are regulated by a complex of hormonal, feeding and body-weight changes. Accurate and precise methods to quantitate circulating serum free leptin (f-leptin) concentrations are needed for physiological and clinical studies. We developed a one-step enzyme immunoassay to measure human f-leptin in serum. The detection limit was 0.40 ng/ml. The recovery of leptin added to serum was 90.8-102.8%. The within-run and between-day coefficients of variation (C.V.) ranged from 2.8 to 7.7 and 5.7 to 9.7%, respectively, and the immunoassay had an overall recovery rate for serial dilution in the range of 94. 0-109.9%. Measured serum f-leptin concentrations in 201 adults correlated (r=0.449, P<0.001) directly with body mass index (BMI kg/m(2)), particularly when results were separated by gender (r=0. 709 for male, P<0.001; r=0.643 for female, P<0.001). We conclude that this one-step enzyme immunoassay is accurate for measuring f-leptin in human serum.     

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA)

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.     

Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)

This brief note addresses the historical background of the invention of the enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA). These assays were developed independently and simultaneously by the research group of Peter Perlmann and Eva Engvall at Stockholm University in Sweden and by the research group of Anton Schuurs and Bauke van Weemen in The Netherlands. Today, fully automated instruments in medical laboratories around the world use the immunoassay principle with an enzyme as the reporter label for routine measurements of innumerable analytes in patient samples. The impact of EIA/ELISA is reflected in the overwhelmingly large number of times it has appeared as a keyword in the literature since the 1970s. Clinicians and their patients, medical laboratories, in vitro diagnostics manufacturers, and worldwide healthcare systems owe much to these four inventors.     

An enzyme-linked immunosorbent assay (ELISA) for the detection of serum IgA against Candida albicans mannan

An enzyme-linked immunosorbent assay (ELISA) is described for measuring serum IgA against highly purified preparations of the Candida albicans immunodominant surface carbohydrate, mannan. Optimal assay conditions are presented for the test to be run in less than four hours. The assay is reliable and easy to use in the diagnostic laboratory with an intraassay variation of less than 10%. Blood donors showed a wide variation of serum levels. Two groups of patients awaiting organ transplants, with a high degree of mucosal colonization by Candida species, showed significantly higher levels of serum IgA against the mannan as compared to the blood donors. This test is proposed to be a useful tool in the surveillance of Candida colonization as one of the factors predisposing for infection in both immunocompetent and immunocompromised patients.     

Antibodies to Aspergillus fumigatus in farmers' lung patients measured by enzyme-linked immunosorbent assay (ELISA)

Enzyme-linked immunosorbent assay (ELISA) was used to measure antibodies against Aspergillus fumigatus in farmers' lung patients, various other pulmonary diseases and healthy controls. Both the prevalence and the mean titre of antibodies were significantly higher in farmers' lung patients than in the other groups. There was no difference between controls and patients with bronchial asthma or non-allergic pulmonary diseases. On the other hand, in the group of patients with respiratory diseases of undefined aetiology the mean antibody titre was significantly higher than that of the controls. Comparison of ELISA and the precipitin test showed the higher sensitivity of ELISA, but otherwise the two tests were in a close agreement.     

An optical pickup enzyme-linked immunosorbent assay (ELISA) with a microfluidic disk

We evaluated optical pickup ELISA with an original microfluidic disk that contains eight radially arranged channels, which enable semi-automatic sample loading and washing. This disk-shaped chip composed of acrylic plates was fabricated by CO₂ laser machining and capture antibodies were immobilized in the channels. After the immunoreaction with antigens and enzyme-linked secondary antibodies, an enzyme-catalyzed nanoaggregation of o-phenylenediamine was detected by measuring the reflectivity change of a laser beam focused in the channel. The assay of C-reactive protein (CRP) was successfully performed in a short amount of time (approximately 20 min from CRP loading). The limit of detection was determined to be 2 ng mL⁻¹, which is more sensitive as compared with conventional ELISA using microplates.

Optical pickup ELISA with an original microfluidic disk, which enable semi-automatic sample loading and washing, was developed. The rapid and sensitive assay of C-reactive protein (CRP) was successfully performed.     

Detection of antibodies in Schistosoma japonicum infections by a micro-technique of enzyme-linked immunosorbent assay (ELISA)

Antibodies in Schistosoma japonicum infections were successfully detected by micro-technique (0.3ml/well) of ELISA on polystyrene microtiter plates using peroxidase labelled anti-human IgG and 5-amino salicylic acid as conjugate and substrate, respectively. Serum samples collected from 22 proven patients in Leyte, Philippines showed titers of higher than 1:960 in 18 cases, 1:240 in 2 cases and 1:120 in 2 cases whereas titers of 22 proven negative sera collected in Tokyo were less than 1:15. Though the reaction was read by spectrophotometry in the present study, the difference of reactions between positive and negative sera was so clear as to be recognizable readily by visual readings.     

Measurement of beta 2-microglobulin, retinol-binding protein, alpha 1-microglobulin and urine protein 1 in healthy children using enzyme-linked immunosorbent assay.

Enzyme-linked immunosorbent assays (ELISA) have been developed for the measurement of beta 2-microglobulin (B2M), retinol-binding protein (RBP), alpha 1-microglobulin (A1M) and urine protein 1 (UP1) in children. Results from random urine samples in 43 children (31 for B2M) are, when corrected for urine creatinine (geometric mean (range)): B2M 9.8 (6.0-40.7) micrograms/mmol, RBP 8.1 (less than 1-24.5) micrograms/mmol, A1M 0.4 (0.1-2.2) mg/mmol and UP1 17.8 (less than 2-309.4) micrograms/mmol. Fractional excretions (FE) in 23 children (14 for B2M) are (geometric mean (range)): FEB2M 0.04% (0.02-0.10%) and FEUP1 0.10% (0.01-1.21%). Results in overnight urine collections are also presented. Our results extend existing data for normal ranges in adults to include children and provide data on UP1 concentrations.     

血筛实验室HBsAg酶联免疫吸附试验灰区设置分析

目的 评价本实验室乙肝表面抗原(HBsAg)酶免试剂灰区设置0.8倍临界值(CO值)的必要性及合理性.方法 1)采用2种HBsAg的ELISA试剂分别检测卫生部临检中心"化学发光多中心合作项目"血清盘标本792份,计算2种试剂真阳性检出率、灰区标本确证阳性率;绘制ROC曲线确定2种试剂最佳CO值,比较不同CO值下(最佳...     

One point dilution enzyme-linked immunosorbent assay (ELISA) for Toxoplasma gondii seroepidemiological surveys

A one point dilution enzyme-linked immunosorbent assay (ELISA) procedure suitable for determining immunoglobulin G (IgG) antibody levels to Toxoplasma gondii (T. gondii) in community seroepidemiological surveys is described. A two-fold serial dilution ELISA procedure was first used to determine the IgG titers in 56 and 83 sera earlier screened by the Sabin-Feldman dye test (DT) and the indirect hemagglutination test (IHA), respectively. The regression rate of the results by the DT and ELISA was 0.92. Comparison of the results by the IHA and the two-fold serial dilution ELISA gave regression coefficient of 0.92. Using the absorbance values for the test sera at dilutions of 1:20, standard curves made by plotting the optical density versus the corresponding dilution factor of a control sera were used to estimate the antibody levels. The regression coefficient of the results by the two-fold serial dilution method and those by the curves for sera with titers of up to 1:320 was 0.97. The curves could not, however, estimate accurately the antibody level in sera with titers above 1:320. The one point dilution ELISA described is a useful epidemiological tool for the screening of IgG antibody to Toxoplasma gondii in the community. However, larger series are required to confirm our observations.     

New enzyme-linked immunosorbent assay for glycocalicin in plasma

A new sandwich-type enzyme-linked immunosorbent assay for quantifying glycocalicin, a proteolytic fragment of platelet membrane glycoprotein Ib, is described. The assay is based on the use of two monoclonal antibodies raised against glycoprotein Ib and involves the avidin-biotin technique. The detection limit is 7 micrograms/L and the range of glycocalicin determined in plasma is 0.01 to 1 mg/L. Assay time is 2 h. The intra-assay CV ranged from 3.6% to 5.2%, the interassay CV from 5.4% to 8.0%. Analytical recovery of purified glycocalicin added to a plasma pool averaged 96%. In 36 healthy subjects, the mean glycocalicin concentration in plasma was 0.36 (SD 0.07) mg/L (2.7 nmol/L). We conclude that this assay is suitable for measuring glycocalicin in plasma and is also more sensitive and precise than the previously published immunoassays based on competitive binding assay.     

Simultaneous detection of multiple proteins with an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL).

Protein arrays hold a promise in basic and clinical applications. As the first step to develop such array system, I used an array-based enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescence (ECL) to demonstrate the feasibility of simultaneous detection of multiple proteins. In the direct ELISA system, different known immunoglobulin Gs (IgGs) were immobilized onto polyvinylidine difloride (PDVF) membrane through 96-well format Bio-Dot unit. The antigens were then individually and collectively detected by incubation of membranes with different antibodies coupled with ECL. In the sandwich ELISA system, the cytokine capture antibodies were immobilized onto PDVF membranes. The membranes were then incubated with single cytokine or a combination of different cytokines. The captured cytokines were detected by biotin-conjugated antibodies coupled with ECL system. Experiments demonstrated that multiple IgGs and cytokines could be simultaneously detected using this approach with high specificity and sensitivity. More importantly, cytokines from biological samples were detected using this approach, which can be used in any general laboratory setting without any sophisticated equipment. This concept could be extended to develop a protein-based array system.     

Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies

This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.     

Development of an enzyme-linked immunosorbent assay of apolipoprotein E-AII complex in plasma

Measurement of apolipoprotein (apo) E-AII complex in human plasma is important in determining the role of apoE in lipoprotein metabolism. In this paper, we demonstrate a new and simple method to determine apoE-All complex by using an enzyme-linked immunosorbent assay. Anti-apoE IgG (goat) was used as a capture antibody, and captured apoE-All complexes were detected by an anti-apoAll (rabbit) horseradish peroxidase-conjugated anti-rabbit IgG (goat) system. With this method, apoE-All complex was specifically determined without the interference of apoAll and was not affected by apoE monomer less than 250 mg/L. The content of the complex in reference serum, a normolipidemic serum pooled from five subjects with phenotype E3/E3, was arbitrarily defined as 100%. The coefficients of variation were 3.5%-6.3% within assay and 8.8%-11.6% between assays.