Document Detail


Quantitation of the blocking effect of tween 20 and bovine serum albumin in ELISA microwells.
MedLine Citation:
PMID:  10873278     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
ELISA provides a highly sensitive procedure for quantitating antigens and antibodies. In that assay, microwells are coated initially with a specific ligand and then saturated with inert molecules to minimize nonspecific background. Coating can be improved by pretreating the microwells with poly-l-lysine (PLL). Proteins and Tween 20 are most often used to block vacant binding sites in enzyme-linked immunosorbent assay (ELISA). In the present study the blocking effects of Tween 20 and bovine serum albumin (BSA) were estimated using an original novel approach. In the assay the magnitude of saturation of the microwells was quantitated by measuring the enzymatic activity of alkaline phosphatase adsorbed to residual vacant sites in the microwell. Tween 20 completely saturated ELISA microwells at concentrations higher than 2 microg/ml. If the microwells were pretreated with PLL, even high concentrations of the detergent did not completely saturate the wells. In contrast, BSA completely saturated both PLL-treated and nontreated microwells at 5 microg/ml. Complementation of Tween 20-induced saturation of PLL-treated microwells was achieved only by addition of BSA at concentration required for BSA alone to reach complete saturation. This approach is applicable for assessing binding to ELISA microwells of any reagent of choice either as a ligand or as a blocking reagent.
Authors:
M Steinitz
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  282     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2000 Jul 
Date Detail:
Created Date:  2000-08-21     Completed Date:  2000-08-21     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  232-8     Citation Subset:  IM    
Copyright Information:
Copyright 2000 Academic Press.
Affiliation:
Experimental Pathology, The Hebrew University-Hadassah Medical School, Jerusalem, 91120, Israel.
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MeSH Terms
Descriptor/Qualifier:
Alkaline Phosphatase / chemistry
Binding Sites
Enzyme-Linked Immunosorbent Assay / methods*
Immunoglobulin G
Polylysine / chemistry
Polysorbates / chemistry*
Serum Albumin, Bovine / chemistry*
Chemical
Reg. No./Substance:
0/Immunoglobulin G; 0/Polysorbates; 0/Serum Albumin, Bovine; 25104-18-1/Polylysine; EC 3.1.3.1/Alkaline Phosphatase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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