Purified Protein Antigen from Brucella

Abstract

Several preparations have been employed in determining allergy to the brucella group.^1-5 We have tried to obtain a standard preparation which could be measured accurately, would be a good antigen and which could be easily prepared with the least possible denaturation. This has been made possible by following a modification of Seibert's method 6 for the preparation of P.P.D. (purified protein derivative from tuberculin).

Since the brucella organisms cannot be made to grow on synthetic media, as previously demonstrated by investigators, 7 we were unable to utilize a filtrate of the cultures as with the tuberculin products. 6 We decided to start with a solution of the cellular constituents of the brucella organisms and proceed to extract and purify the protein fraction. Care was taken in selecting our methods and materials to make them as simple and economical as possible. A known strain of Brucella abortus (strain No. 456, National Institute of Health, Washington, D. C.) was selected for our experiment.

Large amounts of Br. abortus are grown on standard nutrient agar pH 7 8 in Blake bottles for 72 hours at 37°C. Approximately 5 ml of saline with 0.5% phenol are added to each bottle and left for 30 minutes in coatact with the culture to loosen the cells. With sterile pipettes the phenolized saline containing the suspension of cells is removeld from the bottles and the surface of the agar thoroughly washed with more phenolized saline. The organisms are recovered by centrifuging at high speed; then they are crushed with sand in an electrical grinding apparatus for about 2 hours or more. The mixture of sand and broken cells is mixed with 2 liters of 0.02 N sodium carbonate and placed in the refrigerator overnight. Next morning the alkaline suspension is filtered, first through paper and then through a Berkefeld candle. To the filtrate we add 0.5% phenol to avoid bacterial contamination during the following steps of the procedure. The filtrate is then concentrated by ultrafiltration through alundum cups impregnated with 13% gun-cottonglacial acetic acid solution prepared according to the method described by Seibert. 6