CONJUGATION OF ANTI-IMMUNOGLOBULIN (M), ANTI IGMFLUOROCHROME DERIVATIVE (FITC, ISMOER 1)

Immunochemical methods, based on antigen-antibody interaction, have become the most important-tool in qualitative and quantitative plasma proteins determinations due to these high specifically and sensitivity. Immunoglobuline can be divided into five classes IgG, IgA, IgM, IgE and IgD. The normal human serum IgM is about 0.5-2.0 mg/ml. IgM is the dominant antibody formed early in the primary immune response to most antigens (immunogens). Its concentration in the circulation is high enough for it to be purified from normal serum.However,the preparation is facilitated by the use of serum from patients with waldenstromsmacroglobulinaemia .Because of the largesize (Mwt. 900,000) for IgM, gel filtaration is an important fractionation step in the preparation. Thechromatograghically purified IgM fraction antiserum is conjugated with Fluorescein-Isothiocyanate (FITC, Isomer 1). After conjugation, removing of unreacted (unconjugated) and reacted (conjugated) fluorochrome is carried out using gel filtration. The conjugation of anti-IgM-FITC proceededby nucleophile attack of the unprotonated ?- amino group of lysine to the fluorochroe, resulting in a thiourea bond in alkaline medium.