Conjugates of Immunoglobulin G with Different Fluorochromes. I. Characterization by Anionic‐exchange Chromatography

The conjugation of purified IgG for 1 hour at room temperature and pH 9.5 was found to be a simple and reproducible method useful with FITC. MRITC. RBITC and RB200SC fluorochromes. Anionic‐exchange chromatography revealed some difference between conjugates with two batches of FITC and with two batches of MRITC from the same commercial source. Storage of FITC and MRITC as dry powders had only a minor deleterious effect. These fluorochromes as well as fresh solutions of RB200SC produced satisfactory conjugates; but reliable information about their properties could only be obtained from chromatographic analyses. High‐affinity reactions apparently gave rise to optimum conjugates and depended on addition of small amounts of fluoruchrome; increased amounts of fluorochrome favoured low‐affinity reactions with resulting over‐coupling. This was a problem mainly with FITC and RB200SC. RBITC as well as aged solutions of RB200SC produced undue reduction in the isoelectric point of IgG relatively to degree of coupling. Species differences in IgG were found to influence the reactivity of FITC and MRITC.

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