Use of alpha-N,N-bis[carboxymethyl]lysine-modified peroxidase in immunoassays.
Horseradish peroxidase was activated by periodate oxidation of the carbohydrate moiety and then modified by the covalent attachment of alpha-N,N-bis[carboxyethyl]lysine (CM-Lys) by reductive alkylation using sodium cyanoborohydride. The resultant CM-Lys peroxidase was charged with nickel ions and then used as a specific labeling reagent for histidine-tagged recombinant proteins. This labeling method was effective for proteins that are soluble or insoluble in the absence of chaotropic agents. The labeled proteins were very effective in direct sandwich enzyme-linked immunosorbent assay for detecting antibodies against the protein in sera as demonstrated by assays for antibodies to such diverse viral proteins as hepatitis B surface and core proteins, hepatitis C core and helicase protein (NS3), and retroviral core proteins.
Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond 23298-0614, USA.
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Jin
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Wei
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Gomez
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Datta
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Birkett
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Peterson
D000478:Alkylation; D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D002614:Chelating Agents; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005069:Evaluation Studies as Topic; D006508:Hepatitis Antibodies / Q000032:analysis; D006639:Histidine; D006735:Horseradish Peroxidase; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007202:Indicators and Reagents; D008239:Lysine / Q000031:analogs & derivatives*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000276:immunology; D014764:Viral Proteins / Q000276:immunology
C097388:2-N,N-bis(carboxymethyl)lysine; D000914:Antibodies, Viral; D002614:Chelating Agents; D006508:Hepatitis Antibodies; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; D006735:Horseradish Peroxidase; D008239:Lysine