THE ISOLATION AND PURIFICATION OF. THE ANTIGEN.

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Determination of the haemolytic activity and the rate of haemolysis.

To determine the haemolytic activity, 2 ml of the above erythrocyte suspension were washed in saline and the red cells resuspended in 50 ml of saline. To 1 ml haemolysin solution in saline was added 0.5 ml of the latter suspension. The mixture was placed at 37°C and left, being shaken occasionally. The results of the experiments are given in the lowest amount of haemolysin that gave an absolutely clear solution after 4 hours.
The rate of haemolysis was measured according to the method of Bernheimer (1944). In this case 2 ml of the above erythrocyte suspension were suspended in 750 ml of buffered saline, after being washed. After establishment of the temperature, equal volumes of this erythrocyte suspension were mixed with a solution of the haemolysin in buffered saline (4~53 per cent (w/v) sodium chloride M/15 phosphate buffer, a buffer of pH 6 being generally used). The reaction mixtures were left at the temperature employed for the haemolysis and samples of 2 ml volume were taken from the mixtures at regular intervals and centrifuged immediately. The supernatant fluids were removed from the unlysed cells and the optical density measured in a Zeiss spectrophotometer at 410 ~ wavelength.
The degree of haemolysis is expressed as a percentage of complete haemolysis (which was obtained by freezing and thawing).

INTRODUCTION.
II. MATERIALS AND METHODS.
III. A PRELIMINARY STUDY OF THE CULTURES OF CL. CHAUVOEI STRAIN .
IV. THE ISOLATION AND PURIFICATION OF. THE ANTIGEN.
V. A CHEMICAL AND PHYSICO-CHEMICAL STUDY OF THE SOLUBLE ANTIGEN AND HAEMOLYSIN.
VI. A B IOCHEJYIICAL INVESTIGATION OF THE ANTIGEN AND HAEJVIOLYSIN.
VII. SOME SEROLOGICAL EXPERilfiliNTS IN RELATION TO THE STUDY OF THE SOLUBLE ANTIGEN AND HAill~OLYSIN OF CL. CHAUVOEI.

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