Immunoglobulins to which biotin was covalently linked were found in human sera for the first time in 1993.1) The prevalence of biotin-binding immunoglobulin was higher in patients with atopic dermatitis, other dermatitis, allergic disorders, and autoimmune disease. [1][2][3] Recently, biotin-binding IgG was detected in a multiwell microplate format for the first time. 4) We developed a quantitative assay for biotinbinding IgG without any purification of samples using F(ab') 2 anti-human IgG-coated multiwell microplates. 5) We showed that the ratio of biotin molecules to protein molecules (biotin-protein ratio) in biotinylated IgG in human sera affected the detection limit in this assay.5) The slope of doseresponse curve of the assay decreased as the biotin-protein ratio of standards decreased and the observed values of biotinylated IgG in human sera depended on the biotin-protein ratio of standards. Thus it was very important to measure the biotin-protein ratio of biotinylatd immunoglobulins which were used as standards in the assay.In this study, we investigated a synthesis method for biotinylated human immunoglobulins, how to determine the biotin-protein ratio of the biotinylated proteins, and their stability.
MATERIALS AND METHODS
MaterialsThe following materials were obtained from the sources as indicated: flat-bottomed multiwell microplates (Immulon I) from Dynatech Laboratories, Inc., U.S.A.; N-hydroxysuccinimidobiotin (NHS-biotin) and BCA Protein Assay Reagent kit from Pierce Chemical Co., Rockford, U.S.A.; biotin, horseradish peroxidase (HRP)-labeled streptavidin, dimethyl sulfoxide (DMSO), human IgG, human IgA, human IgM, and BSA from Sigma Chemical Co., St. Louis, U.S.A.; ELISA Color Reagent kit (Type O) from Sumitomo Bakelite Co., Ltd., Tokyo, Japan; Biotin Assay Medium ® from Nissui Pharmaceutical Co., Ltd., Tokyo, Japan; Microcon YM-3 from Millipore Co., Beldford, U.S.A. All other chemicals were of reagent grade or better. The water used was 17-Mohm grade.Biotinylation of IgA, IgM, IgG, and BSA Biotinylation of BSA was also examined, since biotinylated BSA was used for the quantitation of biotin in this study. Twenty-five microliters of NHS-biotin DMSO solution at various concentrations was added to 1 ml of 2 mg ml Ϫ1 IgA, IgM, IgG, or BSA in 50 mmol l Ϫ1 sodium bicarbonate/HCl buffer (pH 8.6). The mixture was gently stirred at room temperature for a fixed time. The reaction solution was dialyzed against 0.15 mol l
Ϫ1NaCl and finally against Dulbecco's phosphate buffered saline without Ca 2ϩ and Mg 2ϩ (PBS). The amounts of biotinylated IgA, IgM, IgG, and BSA were expressed as the amounts of IgA, IgM, IgG, and BSA, respectively, which were determined using a BCA Protein Assay kit with human IgG and BSA as standards for biotinylated IgA, IgM, and IgG and for BSA, respectively.
Quantitation of Biotin Using Biotinylated BSA-Coated Multiwell MicroplateThe principle of biotin quantitation was the same as has been previously reported. [6][7][8][9] We used the following procedure with a small amount of biot...
