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 < or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually < or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently > 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.

scholarly journals Hemoglobin-Specific Antibody in a Multiply Transfused Patient With Sickle Cell Disease

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2155-2158 ◽  
Author(s):  
Peter A. Noronha ◽  
Loyda N. Vida ◽  
C. Lucy Park ◽  
George R. Honig
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Cell Disease ◽  
Serum Samples ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Level Of Activity

Abstract Human hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to κ chains, the anti-Hb antibody demonstrated specificity for the region of the Hb β chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.

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 Development of a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay for detecting canine distemper virus

2020 ◽  
Vol 104 (24) ◽  
pp. 10725-10735
Author(s):  
Yuan Zhang ◽  
Gang Xu ◽  
Lu Zhang ◽  
Jiakai Zhao ◽  
Pinpin Ji ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Colloidal Gold ◽  
Canine Distemper Virus ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Test Results ◽  
Canine Distemper ◽  
Serum Samples ◽  
Ideal Method

Abstract Canine distemper virus (CDV) infection causes mass mortality in diverse carnivore species. For effective virus surveillance, rapid and sensitive assays are needed to detect CDV in field samples. In this study, after BABL/c mice were immunized with recombinant CDV-fusion (F) protein, monoclonal antibodies (mAbs) against recombinant CDV-F protein (designated 1A5, 1A6, and 7D5) were produced using traditional hybridoma cell technology. Next, capture antibody (1A6, 800 ng/well) and horseradish peroxidase (HRP)–conjugated detection antibody (HRP-7D5, 1:100, 500 ng/well) were used in a double monoclonal antibody–based sandwich enzyme-linked immunosorbent assay (ELISA) for CDV detection after optimization of both mAb amounts per well using a checkerboard titration test. Based on sandwich ELISA test results for 120 known CDV-negative samples, the cutoff value for a positive result was set to an OD450 nm value ≥ 0.196. As compared with test results obtained from commercial immune colloidal gold test strips, the low limits of detection for the two assays were revealed to be 100 TCID50 per 100 μL. In addition, the sandwich ELISA agreed 100% and 96.4% with commercial immune colloidal gold test strips when testing serum and stool samples. The sandwich ELISA assay provided statistically similar CDV detection. Thus, the sandwich ELISA developed here to detect CDV in fecal and serum samples provided good sensitivity, high specificity, and good reproducibility and should serve as an ideal method for large-scale surveillance of CDV infections in carnivores. Key points • Three CDV mAbs that recognized different epitopes and bound to virion were generated. • The sandwich ELISA based mAbs to detect CDV in fecal and serum samples was developed. • The sandwich ELISA is an ideal method for detecting CDV infections in the field.

Direct chemiluminescence immunoassay for estradiol in serum.

1986 ◽  
Vol 32 (10) ◽  
pp. 1895-1900 ◽  
Author(s):  
J De Boever ◽  
F Kohen ◽  
C Usanachitt ◽  
D Vandekerckhove ◽  
D Leyseele ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Chemiluminescence Immunoassay ◽  
Binding Reaction ◽  
High Affinity ◽  
Microtiter Plates ◽  
Analytical Recovery

Abstract In this simple, reliable, fast solid-phase chemiluminescence immunoassay for directly measuring (i.e., without prior extraction) estradiol-17 beta in serum, a monoclonal antibody is used that binds estradiol with high affinity (Ka = 10(10) L/mol), and does not bind other steroids tested, the highest cross reactivity observed being 0.1% for estradiol-17 alpha. In this system the monoclonal antibody is bound to the wells of microtiter plates via a second antibody directed against the monoclonal antibody. Fifty microliters of serum and estradiol-displacing agents are added, followed by 100 pg of estradiol-isoluminol conjugate, and the label is measured by luminometry after the binding reaction. The sensitivity of the assay is 180 pmol per liter of serum, and the effective working range at less than or equal to 10% CV is 270 to 6700 pmol/L. Analytical recovery of added estradiol averaged 99.7% (SD 6.5%). Within- and between-assay CVs ranged between 5 and 12.7%. Thirty-five unknown serum samples can be assayed within 4 h. Results correlated well with those obtained with a direct RIA: r = 0.94 (n = 149). This assay opens new perspectives for chemiluminescence immunoassays.

Immunodiagnosis of paramphistomosis using monoclonal antibody-based sandwich ELISA for detection ofParamphistomum gracilecirculating 16 kDa antigen

Parasitology ◽  
2017 ◽  
Vol 144 (7) ◽  
pp. 899-903 ◽  
Author(s):  
PANAT ANURACPREEDA ◽  
KULLANID TEPSUPORNKUL ◽  
RUNGLAWAN CHAWENGKIRTTIKUL
Keyword(s):  
Monoclonal Antibody ◽  
Detection Method ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Microtitre Plate ◽  
Serum Samples ◽  
Fecal Samples ◽  
Lower Detection Limit ◽  
Lower Detection ◽  
Sensitivity Specificity

SUMMARYIn this study, we have produced a monoclonal antibody (MoAb) against 16 kDa antigen ofParamphistomum gracile(16 kDaAgPg), and developed an accurate sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of circulating 16 kDaAg in the serum and fecal samples from cattle naturally infected withP. gracile. MoAb 1D10 was immobilized on a microtitre plate, and the antigen in the samples was captured and detected with biotinylated rabbit anti-16 kDaAgPg antibody. The lower detection limit of sandwich ELISA was 3·5 pg mL−1, and no cross-reaction with other parasite antigens was evaluated. The reliability of the assay was examined using the serum and fecal samples from cattle naturally infected withP. gracile, Fasciola gigantica, Moniezia benedeni, Trichurissp.,Strongyloidessp., strongylids and non-infected animals. The sandwich ELISA showed the sensitivity, specificity and accuracy at 98·33, 100 and 99·55% (serum samples), and 96·67, 100 and 99·09% (fecal samples). Therefore, this detection method is a rapid and excellent potential assay for the accurate diagnosis of paramphistomosis.

Solid-phase enzymoimmunoassay for osteocalcin in human serum or plasma, with use of a monoclonal antibody.

1989 ◽  
Vol 35 (10) ◽  
pp. 2087-2092 ◽  
Author(s):  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Detection Limit ◽  
Horseradish Peroxidase ◽  
Human Serum ◽  
Solid Phase ◽  
Sample Volume ◽  
Microtiter Plates ◽  
Analytical Recovery ◽  
Peroxidase Conjugate

Abstract In this solid-phase enzymoimmunoassay on microtiter plates for osteocalcin in serum or plasma, we use an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin. We thoroughly standardized the assay for measurement of osteocalcin in both serum and plasma, demonstrating independence of sample volume, and determining the analytical recovery and within-and between-assay CVs. The detection limit was between 0.6 and 1.1 micrograms/L and the ED50 was 16 micrograms/L for a 5-microL sample volume. The intra-assay CV over the range 3 to 74 micrograms/L was less than or equal to 15%. The interassay CV over the range 3.6 to 46 micrograms/L was less than or equal to 16%. Results by this assay and by an in-house radioimmunoassay in which the same monoclonal antibody was used correlated well (r2 = 0.948). Osteocalcin concentrations in serum and plasma as measured with the present assay agreed well with published values.

An enzyme-linked immunosorbent assay for lactose synthase (galactosyltransferase) in serum and its application as a tumor marker in ovarian carcinoma.

1983 ◽  
Vol 29 (11) ◽  
pp. 1928-1933 ◽  
Author(s):  
B Verdon ◽  
E G Berger ◽  
S Salchli ◽  
A Goldhirsch ◽  
A Gerber
Keyword(s):  
Ovarian Carcinoma ◽  
Patient Monitoring ◽  
Blood Donors ◽  
Reference Interval ◽  
Tumor Burden ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fixed Amount ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Lactose Synthase

Abstract This assay for lactose synthase (galactosyltransferase, EC 2.4.1.22) in serum involves two sequential incubations: serially diluted standard or sample antigen is reacted with a fixed amount of antibody; unbound antibody is then adsorbed to wells of antigen-coated microtiter plates and determined by a second antibody directed against the first antibody and coupled to phosphatase. The standard curve is linear for galactosyltransferase concentrations of 10 to 600 micrograms/L. The within-assay CV of a serum sample was 9.3% (SD 4.1%), the between-assay was 3.8% (SD 2.4%). Serum galactosyltransferase concentrations computed from three different dilutions yielded CVs of 6.5% (SD 5.7%, n = 14). We evaluated the method's accuracy by recovery analysis and by comparing enzyme activity in serum with that of purified galactosyltransferase from human milk. The normal reference interval, as estimated from data on 27 healthy blood donors, was 60-436 micrograms/L (mean 224, SD 101 micrograms/L). We applied the assay to samples of serum from ovarian carcinoma patients grouped according to tumor burden. We also determined galactosyltransferase in ascites fluid and found these values useful for diagnosis, whereas determinations in serum may serve mainly for patient monitoring.

scholarly journals PRRSV Vaccine Strain-Induced Secretion of Extracellular ISG15 Stimulates Porcine Alveolar Macrophage Antiviral Response against PRRSV

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1009
Author(s):  
Hongbin Liu ◽  
Bingjun Shi ◽  
Zhigang Zhang ◽  
Bao Zhao ◽  
Guangming Zhao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Vaccine Strain ◽  
Sandwich Elisa ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Porcine Alveolar Macrophage ◽  
Type I ◽  
Swine Industry ◽  
Host Interactions ◽  
Isg15 Expression

Porcine reproductive and respiratory syndrome virus (PRRSV) has disrupted the global swine industry since the 1980s. PRRSV-host interactions are largely still unknown but may involve host ISG15 protein. In this study, we developed a monoclonal antibody (Mab-3D5E6) specific for swine ISG15 (sISG15) by immunizing mice with recombinant sISG15. A sandwich enzyme-linked immunosorbent assay (ELISA) incorporating this sISG15-specific Mab was developed to detect sISG15 and provided a lower limit of sISG15 detection of 200 pg/mL. ELISA results demonstrated that infection of porcine alveolar macrophages (PAMs) with low-virulence or attenuated PRRSV vaccine strains induced intracellular ISG15 expression that was independent of type I IFN production, while PAMs infection with a PRRSV vaccine strain promoted extracellular ISG15 secretion from infected PAMs. Conversely, the addition of recombinant sISG15 to PAMs mimicked natural extracellular ISG15 effects whereby sISG15 functioned as a cytokine by activating PAMs. Once activated, PAMs could inhibit PRRSV replication and resist infection with PRRSV vaccine strain TJM. In summary, a sandwich ELISA incorporating homemade anti-ISG15 Mab detected ISG15 secretion induced by PAMs infection with a PRRSV vaccine strain. Recombinant ISG15 added to cells exhibited cytokine-like activity that stimulated PAMs to assume an anti-viral state that enabled them to inhibit PRRSV replication and resist viral infection.

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.

Monoclonal antibodies specific for prothrombin fragment 1.2 and their use in a quantitative enzyme-linked immunosorbent assay

1993 ◽  
Vol 39 (4) ◽  
pp. 583-591 ◽  
Author(s):  
M J Hursting ◽  
B T Butman ◽  
J P Steiner ◽  
B M Moore ◽  
M C Plank ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Sandwich Elisa ◽  
Peptide Sequence ◽  
Carboxyl Terminus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombotic Risk ◽  
Prothrombin Fragment ◽  
Amino Terminal

Abstract Prothrombin fragment 1.2 (F1.2) is an activation peptide generated during a critical event of blood coagulation, the conversion of prothrombin to thrombin. As a marker of thrombin generation, F1.2 has clinical potential in assessing thrombotic risk and monitoring anticoagulant therapy. In developing a highly specific, monoclonal antibody-based immunoassay of human plasma F1.2, we generated six murine anti-F1.2 monoclonal antibodies, using as immunogen a synthetic peptide (sequence: CGSD-RAIEGR) similar to the unique carboxyl terminus of F1.2. Each antibody bound F1.2 but not prothrombin. Epitope mapping studies with one antibody (5-3B) showed that optimum binding required six to eight amino acids plus a terminal arginine to emulate the F1.2 carboxyl terminus. A quantitative sandwich ELISA for human plasma F1.2 was configured with monoclonal antibody 5-3B as the capture antibody and peroxidase-labeled polyclonal antibodies to the F1.2 amino-terminal region as detector antibodies. Calibrators were prepared by adding purified F1.2, 0-10 nmol/L, to F1.2-depleted plasma. Assay characteristics included the following: mean (+/- SD) analytical recovery of 98% +/- 13%; no interference from lipemia, hemolysis, icterus, or thrombolytic agents; 0.08 nmol/L sensitivity; and mean intra- and interassay imprecision (three lots) < 12% at both low and high concentrations of F1.2.

Sign in / Sign up

Export Citation Format

Share Document