Serum resulted from the hyper-immunizated rabbits with sheep hematies, which are used for the preparation of hemolitic system, necessary in the attachment reaction of the complement (RFC).
It is used for the preparation of the hemolitic system necessary in RFC.
- 1 phyal Hemolysin
- instructions for useMore info
Saline natural solution, pH 7 – 7,2; Adjustable micro-droppers, 2 – 1000 ml; Wasserman pipes; Baie marie of 37°C and 56°C; Centrifuge, 500 – 2000 rpm; Sheep hematies; Lyophylized Alexina.
Preparation of hemolitic system necessary for the titration of alexina
The hematies are washed 3 – 4 times in saline natural solution and are sedimented by centrifugation (1500 rpm – 15 minutes). The anti-sheep hemolitic serum, conditioned under lyophylized state (with the known title), is re-hydrated in one ml saline natural solution pH 7,2.
H = (4 X C) : T
H = necessary quantity of anti-sheep hemolytic serum
C= necessary volume of hemolitic system
T = title of hemolitic serum
Components of the hemolitic system: the quantity of sheep anti-hematic hemolitic serum calculated according to the above formula; 1,25 ml of the deposit of sheep hematies. It is completed up to 100 ml with saline natural solution.
The mix is incubated for 30 minutes at 37°C in Marie bath.
Titration of lyophylized alexina
Alexina, conditioned in lyophylized state, is re-hydrated in one ml natural saline solution. Alexina is diluted 1/10 in SF and it is distributed in reaction pipes, in increasing quantities, from 0,10 – 0,40 ml by a step of 0,02. The alexina titration diagram is shown in the table no. l.
TABLE NO. 1
|Saline natural solution||1,40||1,38||1,36||1,34||1,32||1,30||1,28||1,26||1,24||1,22||1,20||1,18||1,16||1,14||1,12||1,10||1,5|
|Hemolitic system (SH)||1||1||1||1||1||1||1||1||1||1||1||1||1||1||1||1||1|
The samples are incubated for 15 minutes at Marie bath of 37°C. The Alexic title is represented by the minimum quantity of alexina that determines the total lysis of hematies.
Establishment of the hemolitic title of the test hemolitic serum.
A suspension of 2,5% in SF is prepared from the deposit of hematies. The anti-sheep hemolitic serum is re-hydrated with 1 ml saline natural solution. There are issued series dilutions of hemolysine in the saline natural solution (1/500; 1/1000; 1/1500; 1/2000; 1/2500,1/3000……….1/10.000). The samples are incubated for 30 minutes at 56°C in Marie bath. It is taken 1 ml from each dilution (1/500; 1/1000; 1/1500; 1/2000; 1/2500, 1/3000……….1/10.000) and is distributed in the Wasserman pipes,and 0,5 ml of the hematies suspension 2,5% is added in each pipe. The samples are incubated for 30 minutes at 37°C in Marie bath, then it id added 0,5 ml alexina in each pipe that include 2 alexic units. It is incubated for 15 minutes at 37°C.
The title of hemolitic serum is represented by the maximum dilution able to produce total hemolysis and must be ≥ 1/1000. For the preparation of hemolitic system, the number of hemolysing doses used depends on the used RFC technique. The lyophylized product will be re-hydrated with 1 ml saline natural solution and will be used depending on its title and the number of hemolysing doses required by the used RFC technique.
The mixing or association of Hemolysin resulted from different series is not allowed. Once started, the test must be finished.
At 2 – 8°C, within the validity limit marked on the label.
Validity: 24 months from the conditioning date.
It is delivered in capsulated phials of 3 ml, containing 1 ml lyophylized product, the instructions of use are also introduced in the boxes. The dosing corresponds to the following hemolitic titles:
1000 doses = title 1/4000
PRODUCTS FROM THE CATEGORY: Reagents for other species