scholarly journals Excess EDTA interferes with cortisol measurement using a solid-phase, chemiluminescent enzyme immunoassay

2018 ◽  
Vol 30 (3) ◽  
pp. 438-441 ◽  
Author(s):  
Robert J. Kemppainen ◽  
Ellen N. Behrend ◽  
Stephanie F. Carter ◽  
Janeva E. Cole
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Concentration ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Serum Samples ◽  
Edta Plasma ◽  
Edta Concentration ◽  
Canine Serum ◽  
Chemiluminescent Enzyme Immunoassay

Hormone assays that use a solid-phase, automated, chemiluminescent enzyme immunoassay (CEIA) with an alkaline phosphatase–tagged hormone or antibody as a reporter are performed on serum or EDTA plasma in our laboratory. CEIA cortisol results appeared to increase in the presence of excess EDTA. We investigated the effect of the addition of different amounts of EDTA on cortisol concentrations in pooled canine serum samples. The recommended EDTA plasma concentration of 4.1 mmol/L (1.8 mg/mL) did not alter cortisol concentrations when added to serum pools; however, the addition of ≥5.1 mmol/L (2.25 mg/mL) of EDTA increased apparent concentrations of cortisol. Supplementation of serum samples with MgCl2 to 5 mmol/L reversed the effect of EDTA up to a concentration of ~8.1 mmol/L (3.6 mg/mL). Our findings show that CEIA cortisol results on EDTA plasma can be artificially increased if the EDTA concentration exceeds 5.1 mmol/L.

Rapid and sensitive chemiluminescent enzyme immunoassay for measuring tumor markers

1991 ◽  
Vol 37 (9) ◽  
pp. 1639-1644 ◽  
Author(s):  
I Nishizono ◽  
S Iida ◽  
N Suzuki ◽  
H Kawada ◽  
H Murakami ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Carcinoembryonic Antigen ◽  
Enzyme Immunoassay ◽  
Tumor Markers ◽  
Lower Limit ◽  
Limit Of Detection ◽  
Solid Phase ◽  
Serum Samples ◽  
Coated Particles ◽  
Chemiluminescent Enzyme Immunoassay

Abstract We developed a chemiluminescent enzyme immunoassay (CLEIA) to quantify such tumor markers as carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), CA19-9, and CA125. We used a novel chemiluminescent substrate, a derivative of 1,2-dioxetane phosphate (AMPPD), to measure alkaline phosphatase as a labeling enzyme to Fab' fragments of antibody. Regardless of the solid phase, i.e., polystyrene beads (6 mm diameter) or ferrite-coated particles (0.3 microns diameter), the standard curves within the dynamic ranges of the conventional RIA or enzyme immunoassay (EIA) were linear in all cases except for those with AFP. Use of the ferrite particles further shortens the immunoreaction, so the assay can be performed in 30 min. In addition, the relationships between concentrations of the marker and chemiluminescent signals for CA19-9, CA125, and CEA were linear up to concentrations about 10-fold greater than the ordinary dynamic ranges. Intra- and interassay CVs (averages for individual analyte) were 2.2%-4.9% and 2.0%-5.8%, respectively. In an analysis of serum samples, results of the CLEIA correlated reasonably well with those of RIA or EIA. The lower limit of detection by CLEIA with ferrite particles was 390 arb. units/L for CA19-9, 990 arb. units/L for CA125, 0.06 micrograms/L for CEA, and 0.03 micrograms/L for AFP. Thus, the sensitivity increased to between two- and 10-fold that of RIA or colorimetric EIA, depending on the analytes.

scholarly journals Evaluation of Solid-Phase Chemiluminescent Enzyme Immunoassay, Enzyme-Linked Immunosorbent Assay, and Latex Agglutination Tests for Screening Toxoplasma IgG in Samples Obtained from Cats and Pigs

2000 ◽  
Vol 12 (2) ◽  
pp. 136-141 ◽  
Author(s):  
Ashok K. Singh
Keyword(s):  
Enzyme Immunoassay ◽  
Immunosorbent Assay ◽  
Solid Phase ◽  
Latex Agglutination ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Method ◽  
Serum Samples ◽  
Positive Results ◽  
Chemiluminescent Enzyme Immunoassay

Serum samples from cats and pigs were analyzed by the solid-phase chemiluminescent enzyme immunoassay (SPCEI), enzyme-linked immunosorbent assay (ELISA), and indirect latex agglutination (ILA) methods. The SPCEI and ILA methods accurately analyzed Toxoplasma IgG (T-IgG) in both clinical and spiked samples from pigs and cats. The ELISA method accurately analyzed T-IgG in spiked samples from cats and pigs or clinical samples from pigs, but it did not accurately analyze T-IgG in clinical samples from cats. The antibody used in the ELISA kit did not cross-react with cat T-IgG. The SPCEI method that uses a stand-alone automated analyzer provided quantitative analysis, whereas the ELISA and ILA methods provided qualitative or, at best, semiquantitative analysis of T-IgG. The SPCEI and ELISA methods were rapid (60–90 minutes for 30 samples), whereas the ILA method required 13–15 hours for 30 samples. Although the three methods accurately distinguished positive from negative samples, the ILA method yielded many weakly positive results that were not confirmed by either the ELISA or SPCEI method. Thus, the indirect agglutination tests may give nonspecific responses at lower T-IgG concentrations.

scholarly journals Effect of EDTA on measurement of cortisol and thyroxine by chemiluminescent enzyme immunoassay in dogs

2020 ◽  
Vol 32 (3) ◽  
pp. 363-368 ◽  
Author(s):  
Dana A. Schechter ◽  
Hollie P. Lee ◽  
Robert J. Kemppainen ◽  
Ellen N. Behrend
Keyword(s):  
Acetic Acid ◽  
Enzyme Immunoassay ◽  
Clinical Setting ◽  
Plasma Concentrations ◽  
Plasma Samples ◽  
Serum Samples ◽  
Measured Concentration ◽  
Edta Plasma ◽  
Clinical Conditions ◽  
Chemiluminescent Enzyme Immunoassay

The addition of ethylenediamine tetra-acetic acid (EDTA) to serum can affect the measurement of cortisol by chemiluminescent enzyme immunoassay (CEIA); addition of magnesium chloride (MgCl2) may reverse the effects. However, similar characteristics for thyroxine (T4) measurement are unknown. We measured cortisol and T4 in paired EDTA-anticoagulated plasma and serum samples from 50 dogs. Additionally, both hormones were measured in 15 samples of each type after the addition of MgCl2. Samples were collected under routine clinical conditions; therefore, specific EDTA concentrations in plasma samples were unknown. Cortisol and T4 values were significantly different comparing plasma and serum samples in the absence of MgCl2. For cortisol and T4, EDTA-plasma concentrations were 51.2% and 43.7% higher than serum, respectively ( p < 0.001 for both). The addition of MgCl2 to plasma significantly decreased the measured cortisol concentrations ( p < 0.001) but not T4 ( p = 0.44). After addition of MgCl2, cortisol concentrations in EDTA-plasma were no longer significantly different from serum, whereas T4 concentrations in EDTA-plasma remained significantly different from serum. In the clinical setting in which tubes may be underfilled, use of EDTA-plasma significantly increases the measured concentration of cortisol and T4 obtained by CEIA. Addition of MgCl2 to EDTA-plasma can overcome the effects of EDTA when measuring cortisol, but not T4. Thus, T4 should not be measured in EDTA-plasma.

Column enzyme immunoassay for secretory immunoglobulin A in serum.

1983 ◽  
Vol 29 (1) ◽  
pp. 151-153 ◽  
Author(s):  
R Yamamoto ◽  
S Kimura ◽  
S Hattori ◽  
Y Ishiguro ◽  
K Kato
Keyword(s):  
Enzyme Immunoassay ◽  
Sodium Carbonate Solution ◽  
Solid Phase ◽  
Immunoglobulin A ◽  
Enzyme Reaction ◽  
Secretory Component ◽  
Secretory Immunoglobulin A ◽  
Serum Samples ◽  
Sepharose 4B ◽  
Secretory Immunoglobulin

Abstract This enzyme immunoassay for specific measurement of secretory immunoglobulin A concentrations in human serum involves use of a small chromatographic column as a solid-phase. Serum samples are incubated for 2 h with beta-D-galactosidase-labeled antibody to secretory component, then passed through a 0.1-mL Sepharose 4B column containing antibodies to human immunoglobulin A. After the column is washed to remove the unbound label, the buffer in the column is replaced by a solution of o-nitrophenyl-beta-D-galactoside (a beta-D-galactosidase substrate) and incubated at 25 degrees C overnight. The enzyme reaction is stopped by washing the column with sodium carbonate solution, and the absorbance of the eluate is measured at 420 nm. The concentration of secretory immunoglobulin A can be determined with a minimum detectable sensitivity of 3 mg/L, without interference from free immunoglobulin A and secretory component in the same samples.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was &lt; or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually &lt; or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently &gt; 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland

1993 ◽  
Vol 39 (11) ◽  
pp. 2322-2325 ◽  
Author(s):  
S M Yie ◽  
E Johansson ◽  
G M Brown
Keyword(s):  
Pineal Gland ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Linear Regression Analysis ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Rat Serum ◽  
Practical Applications ◽  
Pineal Function ◽  
Basic Studies

Abstract We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.

Specific enzyme immunoassay for human chorionic gonadotrophin

1981 ◽  
Vol 97 (4) ◽  
pp. 562-568 ◽  
Author(s):  
Tatsuhiro Sekiya ◽  
Yoshihito Furuhashi ◽  
Setsuko Goto ◽  
Shigeaki Kaseki ◽  
Yutaka Tomoda ◽  
...  
Keyword(s):  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Serum Samples ◽  
Coefficients Of Variation ◽  
Highly Sensitive ◽  
Assay Tube ◽  
Sandwich Type ◽  
Serial Samples

Abstract. A sandwich-type enzyme immunoassay system specific for human chorionic gonadotrophin (hCG) was prepared with the antibody Fab'-β-D-galactosidase complex and the antibody F(ab')2-immobilized silicone rubber solid phase by using a purified antibody to β subunit of hCG (hCGβ). The assay system cross-reacted less than 4% with human luteinizing hormone (hLH) and human follicular stimulating hormone (hFSH), and proved to be highly sensitive with hCG measurable at levels as low as 0.3 mIU per assay tube. Using 50 μl of serum sample, 6–600 mIU/ml of hCG in serum could be determined specifically with the same degree of precision as in radioimmunoassay but without sample interference with the assay. The coefficients of variation within-run and between-run were 8.6–8.9%, and 4.9– 10.7%, respectively. Values obtained with the enzyme immunoassay correlated well with those of radioimmunoassay ([unk] = 0.98, slope = 0.94, y-intercept = 10.2 mIU/ml for 75 serum samples). Results of the immunoassay of hCG levels in serial samples of serum from healthy women and patients with choriocarcinoma show that this method is useful in the clinical diagnosis of trophoblastic disease.

scholarly journals Solid-phase enzyme immunoassay for immunoglobulin M antibodies to cytomegalovirus

1977 ◽  
Vol 5 (6) ◽  
pp. 629-634
Author(s):  
H Schmitz ◽  
H W Doerr ◽  
D Kampa ◽  
A Vogt
Keyword(s):  
Alkaline Phosphatase ◽  
Horseradish Peroxidase ◽  
Enzyme Immunoassay ◽  
Indirect Immunofluorescence ◽  
Solid Phase ◽  
Immunoglobulin M ◽  
Nuclear Antigen ◽  
Immunofluorescence Method ◽  
Infected Cells

A sensitive, solid-phase enzyme immunoassay for the detection of immunoglobulin M antibodies to cytomegalovirus is described. The results of the enzyme immunoassay correlated well with those obtained by an indirect immunofluorescence method. Horseradish peroxidase proved to be a more sensitive label than alkaline phosphatase. Nonspecific reactions, occurring with commercially available cytomegalovirus antigens, could be avoided by using a nuclear antigen prepared from sonically disrupted nuclei of cytomegalovirus-infected cells.

Enzyme immunoassay of antibodies to Sjögren's syndrome B antigen.

1981 ◽  
Vol 27 (8) ◽  
pp. 1341-1345 ◽  
Author(s):  
A M Teppo
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Immunoassay ◽  
Sjögren’S Syndrome ◽  
Sjögren's Syndrome ◽  
Solid Phase ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Nitrophenyl Phosphate ◽  
Purified Antigen ◽  
Sjögren‘S Syndrome

Abstract I describe a solid-phase enzyme immunoassay of IgG-IgA-, and IgM-type antibodies to Sjögren's syndrome B antigen. Polystyrene tubes are coated with the purified antigen. The antibodies are allowed to bind with their antigens, and then detected with alkaline-phosphatase-conjugated anti-human IgG, IgA, or IgM sera. The amount of alkaline phosphatase fixed to the tubes is determined in pH 10.0 diethanolamine buffer at 37 degrees C with p-nitrophenyl phosphate as substrate. The absorbance of the p-nitrophenolate ion liberated in 1 h at 37 degrees C is measured at 406 nm. When rheumatoid factor is present, the values obtained for IgG antibodies are too low, and those for IgM antibodies are too high.

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