An Electrochemical Enzyme Immunoassay for Chicken Luteinizing Hormone: Extension of the Detection Limit by Adequate Control of the Nonspecific Adsorption

Qu, Ying; Berghman, Luc; Vandesande, Frans

doi:10.1006/abio.1998.2648

Cited by 20 publications

(7 citation statements)

References 15 publications

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“…Nonspecific adsorption is a common phenomenon in immunoassays, and numerous blocking agents have been used to suppress the background signals. [37][38][39][40][41] In the present study, significant background ECL signals were observed in the absence of CRP when no blocking agent was used. Several blocking agents were examined, including 0.5% Triton X-100, 10% SuperBlock blocking buffer in PBS, 2.0% BSA, and combinations of these.…”

Section: Synthesis Of Hydrophobic Ru(bpy) 3 2+ Ecl Labels Loading Of ...mentioning

confidence: 56%

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

2004

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Biotinylated anti-C-reactive protein (CRP) species were attached to the surface of streptavidin-coated magnetic beads (MB) and avidin-coated polystyrene microspheres/beads (PSB) entrapping a large number of electrogenerated chemiluminescence (ECL) labels ( approximately 10(9) Ru(bpy)(3)[B(C(6)F(5))(4)](2)/bead) to form anti-CRP<-->MB and Ru(II) subsetPSB/avidin<-->anti-CRP conjugates, respectively. Sandwich-type Ru(II) subsetPSB/avidin<-->anti-CRP CRP anti-CRP<-->MB aggregates were formed when Ru(II) subsetPSB/avidin<-->anti-CRP was mixed with anti-CRP<-->MB conjugates in the presence of analyte CRP. The newly formed aggregates were magnetically separated from the reaction media and dissolved in MeCN containing tri-n-propylamine as an ECL coreactant. ECL was carried out with a potential scan from 0 to 2.8 V vs Ag/Ag(+), and the ECL intensity was found to be proportional to the analyte CRP concentration over the range of 0.010-10 mug/mL. The CRP concentration of an unknown human plasma specimen was measured by the standard addition method based on this technique. Elimination of the nonspecific adsorption of the CRP system with several different blocking agents was also studied, and 2.0% bovine serum albumin was found to be best.

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Section: Synthesis Of Hydrophobic Ru(bpy) 3 2+ Ecl Labels Loading Of ...mentioning

confidence: 56%

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

2004

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“…Generally, the reagent of choice is an inert protein(s), added after coating the microwells with the specific ligand which minimally interacts with the other reagents included in the assay. Beside BSA, gelatin, FCS, lowfat milk, and other proteins used for saturation in ELISA, Tween 20 has proven to be a good blocking reagent too, and its synergistic effect with protein has also been described (6,8).…”

Section: Discussionmentioning

confidence: 98%

Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells

Steinitz

2000

Analytical Biochemistry

127

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“…Dual-label fluorescent IHC for FSH and ␣ subunit was performed as described above utilizing a rabbit polyclonal antiserum to chicken ␣ subunit [25] at a dilution of 1:5000. Cells that contained only ␣ subunit yielded green fluorescence, while cells that contained both FSH and ␣ subunit were yellow/orange.…”

Section: Ihc Characterization Of Mabsmentioning

confidence: 99%

Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary1

Proudman

Vandesande

Berghman

1999

Biology of Reproduction

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As is the case in other tetrapod species, the chicken gonadotropins LH and FSH consist of a common alpha subunit and a hormone-specific beta subunit. Gonadotrophs containing LH were shown earlier to be distributed throughout both the caudal and cephalic lobes of the chicken anterior pituitary, but the cellular distribution of FSH in avian species is still uncertain. The purpose of this study was to determine the cellular distribution of FSH-containing chicken gonadotrophs by use of FSH-specific monoclonal antibodies (mAbs). Three new mAbs toward chicken FSH were proven hormone specific by immunodetection of purified hormones on dot blots and by dual-label immunohistochemistry (IHC) on sagittal sections of chicken pituitaries. A rabbit antibody was used to detect chicken LH. Results showed that LH-containing gonadotrophs were densely distributed throughout the anterior pituitary, whereas gonadotrophs containing FSH were much less numerous; in addition, while also present in both lobes, FSH-positive cells were largely absent from the outer margin of the gland. Dual-label IHC revealed that LH and FSH reside almost exclusively in separate gonadotrophs. The identity of FSH-containing cells was further confirmed through use of an antibody to the chicken alpha subunit, which showed that FSH immunoreactivity was always colocalized with the alpha subunit. Our results suggest the possibility that production and secretion of LH and FSH may be regulated differently in chickens than in most other species studied to date.

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An Electrochemical Enzyme Immunoassay for Chicken Luteinizing Hormone: Extension of the Detection Limit by Adequate Control of the Nonspecific Adsorption

Cited by 20 publications

References 15 publications

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells

Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary1

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An Electrochemical Enzyme Immunoassay for Chicken Luteinizing Hormone: Extension of the Detection Limit by Adequate Control of the Nonspecific Adsorption

Cited by 20 publications

References 15 publications

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)3]2+-Containing Microspheres

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)3]2+-Containing Microspheres

Quantitation of the Blocking Effect of Tween 20 and Bovine Serum Albumin in ELISA Microwells

Immunohistochemical Evidence That Follicle-Stimulating Hormone and Luteinizing Hormone Reside in Separate Cells in the Chicken Pituitary1

Contact Info

Product

Resources

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Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres

Electrogenerated Chemiluminescence. 80. C-Reactive Protein Determination at High Amplification with [Ru(bpy)₃]²⁺-Containing Microspheres