Preparation of globulin fraction

A crude fractionation of antisera can be carried out to remove most of the albumin. Either slowly add an equal volume of cold, saturated ammonium sulphate solution (adjusted to pH 7 with ammonia) at 4°C and stir for a further 30 min or dialyse overnight at 4°C against 50 volumes of 50% saturated ammonium sulphate.Centrifuge at 1200g for 20 min at 4°C and dissolve the precipitate in PBS-A and dialyse against PBS-A (three changes of 50 volumes) to remove the ammonium sulphate. 

Resuspend the ammonium sulphate pellet in 1 ml PBS pH 6.5 and apply to a 20 ml column of QAE Sephadex A-50 (Pharmacia) equilibrated with the same buffer, which is also used to elute the column. Elution of antibodies may be detected by ELISA or by polyacrylamide gel electrophoresis (Campbell, 1984). 

Conjugation of antisera with fluoroscein or rhodamine These are the two common fluorochromes and they are covalently linked to proteins by using activated forms (e.g. the isothiocyanates). 

Fluoroscein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) are commercially available from, for example, Sigma Chemical Co. (Appendix 3). 

More uniform reaction is obtained if the isothiocyanates are first absorbed onto Celite. FITC-Celite is available from Sigma Chemical Co. Ltd., but can easily prepared  

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