Document Detail


An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens.
MedLine Citation:
PMID:  10563786     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.
Authors:
J Naar; P Branaa; M Chinain; S Pauillac
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Bioconjugate chemistry     Volume:  10     ISSN:  1043-1802     ISO Abbreviation:  Bioconjug. Chem.     Publication Date:    1999 Nov-Dec
Date Detail:
Created Date:  2000-01-06     Completed Date:  2000-01-06     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  9010319     Medline TA:  Bioconjug Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1143-9     Citation Subset:  IM    
Affiliation:
Unité d'Océanographie Médicale, Institut de Recherches Médicales Louis Malardé, P.O Box 30, Papeete, Tahiti, French Polynesia.
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MeSH Terms
Descriptor/Qualifier:
Animals
Antibody Formation
Caseins / chemistry
Cholesterol / analysis,  chemistry*
Chromatography, High Pressure Liquid
Haptens / chemistry*
Hemocyanin / chemistry
Hydrogen-Ion Concentration
Hydroxylation
Immunization
Indicators and Reagents
Mice
Micelles
Ovalbumin / chemistry
Polylysine / chemistry
Proteins / chemistry*
Serum Albumin, Bovine / chemistry
Succinic Anhydrides / chemistry
Temperature
Chemical
Reg. No./Substance:
0/Caseins; 0/Haptens; 0/Indicators and Reagents; 0/Micelles; 0/Proteins; 0/Serum Albumin, Bovine; 0/Succinic Anhydrides; 108-30-5/succinic anhydride; 25104-18-1/Polylysine; 57-88-5/Cholesterol; 9006-59-1/Ovalbumin; 9013-72-3/Hemocyanin

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


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