Author Fred Lewis

Introduction

One of the most widely used antigenic preparations from schistosomes is SEA (soluble egg antigen). The crude extract obtained from the mature eggs is isolated from the tissues of the definitive host. The use of SEA has been critical in dissecting immunologically-driven responses to the eggs in an active infection.

Techniques for SEA isolation were first described by Boros and Warren (1970). As with any crude extract of a multicellular organism, it consists of a bewildering array of components, such as proteins, glycoproteins, polysaccharides and glycolipids.

Equipment

Hand-held Potter-Elvehjem glass homogenizer (15 ml capacity)

Refrigerated centrifuge

Ultracentrifuge

Dissecting microscope

Materials and reagents

Phosphate-buffered saline

10-ml disposable syringe with 0.2 µm filter

Procedure

·         Beginning with the purified egg collection as described in the final stage of the SOP “Collection of eggs from tissues (section on S. mansoni & S. haematobium)”, suspend the eggs in 4°C PBS at a concentration of 100,000 eggs/ml.

·         Homogenize the eggs on ice using the pre-chilled homogenizer, using a tight pestle.  During the process, which may take 10-15 minutes (depending on the number of plunging cycles and the force used), withdraw a small sample and examine using a dissecting microscope. Make a crude determination of the percentage of eggs that have been broken apart. Both empty egg shells and intact eggs are easily distinguished.

·         When approximately 95% (or more) of the eggs have been disrupted, centrifuge the crude mixture at 4°C at 200 x g for 20 minutes.

·         Collect the supernate, and ultracentrifuge it for 90 minutes at 100,000 x g at 4°C.

·         The supernate can be sterilized by passing it through a 0.2 µm filter.

 

Follow-up comments/recommendations

The use of SEA has been important in experimental immunology studies for its strong Th-2 polarizing activity. Buffers other than PBS may also be used at the discretion of the investigator. Sometimes a protease inhibitor (e.g., leupeptin, at 10 µg/ml) is also used in the extraction buffer.

 

References

Boros, D.L. and Warren, K.S. 1970. Delayed hypersensitivity granuloma formation and dermal reaction induced and elicited by a soluble factor isolated from Schistosoma mansoni eggs. Journal of Experimental Medicine. 132: 488-507.

 

Tucker, M. S., Karunaratne, L. B., Lewis, F. A., Frietas, T. C., and Liang, Y-S. 2013. Schistosomiasis, in Current Protocols in Immunology 19.1.1-19.1.57, John Wiley and Sons, Inc., (R. Coico, Ed).  Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142735.im1901s103.