complement levels by hemolyticassay - (Jul/25/2001 )

I am teaching immunology for the first time. I would like to do one laboratory session in which the students detect complement levels using a hemolytic assay. The protocol I have found (Immunological techniques made easy, Cochet et al) uses a barbaturate buffer. The reagents needed are therefore DEA controlled and we (U of Redlands) does not have the license to buy such reagents. Does anyone have a protocol that does not use barbiturate as the buffer? Is there a reason I couldnt substitute a "normal" buffer like Tris or HEPES?

I do not know whether even after two years, this question is still active. However, I would like to point out that it is NOT necessary to use veronal buffer but a simple PBS pH 7.2 should be sufficient. I have used them and please see the following publications for details:

1- V.D.RAMANATHAN. Human alternative pathway assay. American Journal of Clinical Pathology.1983; 80: 412-3.

2- V.D.RAMANATHAN and U.Sengupta. In vitro inhibition of the human complement and coagulation systems by chloroquine. International Journal of Immunopharmacology. 1985; 7: 769-73.
3- V.D.RAMANATHAN, P.Sharma, G.Ramu and U.Sengupta. Reduced complement-mediated solubilization of immune complexes in leprosy patients. Clinical and Experimental Immunology. 1985; 60: 553-8.

I can be contacted at vdrnathan@yahoo.com if more details are needed.

V.D.Ramanathan, MB, PhD,
Dept. of Pathology,
Tuberculosis Research Centre,
Chennai - 600 034.