o-phthaldehyde fluorescence microassay for the determination of antibody concentration.
o-Phthaldehyde (OPA) reagent reacts with primary amines in aqueous solution at room temperature. When the reaction occurs in the presence of 2-mercaptoethanol, a bright blue fluorescence is produced. We investigated the application of the OPA reagent in the routine determination of monoclonal antibody concentration. The OPA reagent produced maximal fluorescence within 30 min and was stable for 2 h. Buffers containing primary amines gave high background fluorescence; however, the OPA reagent gave reliable results with the most commonly used antibody solutions. The OPA reagent provided a rapid and simple measure of protein concentration from 1 microgram to 1 mg and was readily adapted for use with a 96-well fluorescence reader.
Dana-Farber Cancer Institute, Brigham and Women's Hospital, Boston, MA 02115.
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D000588:Amines; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000302:isolation & purification; D004355:Drug Stability; D007070:Immunoglobulin A / Q000032:analysis; D007074:Immunoglobulin G / Q000032:analysis; D007075:Immunoglobulin M / Q000032:analysis; D007202:Indicators and Reagents; D008832:Microchemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012996:Solutions / Q000032:analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D009764:o-Phthalaldehyde
D000588:Amines; D000911:Antibodies, Monoclonal; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007202:Indicators and Reagents; D012996:Solutions; D009764:o-Phthalaldehyde