Brucella Total
Determination of the Antibodies Associated
To The Brucellosis
PRINCIPLE:
The Antibodies associated to the infection by
Brucellas, in the presence of inactivared
bacteria in the suspension, give a visible flocculation.
The colour of the suspension facilitates the reading
Reagent composition
Bacterial Suspension
Inactivated Brucella abortus and melitensis
Preservatives and Stabilizers
According to the present laws the kit does not
contain substances classified as dangerous
Storage and Stability of Reagents
Store the kit at 2 - 8°C.
The reagent, is stable until the stated expiration date
if stored tightly closed and refrigerated
Preparation and Stability of Working solution
Bacterial suspension liquid and ready to use
Gently mix the suspension before use
Bring reagent at Room Temperature before use
Samples
Fresh serum
Serum may be stored at 2 - 8°C for 6 days
Safety Precautions
For in vitro diagnostic use only
Do not pipette by mouth
Exercise the normal precautions required for
handling laboratory reagents
Reagents
Tube 1
Saline Solution
Sample
900 μl
100 μl
Bacterial
Suspension
TITRE
1: 20
Tube 2
Tube 3
........
Tube 7
500 μl
500 μl
........
500 μl
500 μl
500 μl
........
500 μl
from Tube 1 from Tube 2
from Tube 6
discard 500 μl from the Tube 7
500 μl
500 μl
500 μl
........
500 μl
1: 40
1: 80
........
Suspension
Control
500 μl
500 μl
1: 1280
Gently shake the tubes and incubate at 37°C for 16 /18
hours or at 22°C for 2 days
Results and Interpretation
After incubation and away from vibrations, it is
possible execute a first reading:
Presence of a definite coloured button on tube bottom indicates negative test
Presence of smooth mat of flocculation on tube bottom indicates positive test
A non uniform flocculation on tube - bottom and a
button in the middle indicates faint
positive test
Then shake gently all tubes beginning from Tube 8
(Suspension Control) and in
succession 7, 6, 5, 4, 3, 2, 1.
Compare each suspension appearance with Tube 8
Negative:
Absence of flocculation
Positive:
Presence of strong flocculation
The serum titre will be the highest dilution still giving
a flocculation
*Qualitative method
1. Place one drop of Positive control (available as
Brucel Positive Control) onto the reaction circle of
glass slide.
2. Place ~ 80µl of saline onto the next reaction circle
of the glass slide.
3. Place ~ 80µl of patient serum to be tested onto
the next reaction circle.
4. Add one drop of the appropriate Brucel antigen
suspensions in each of the above circle.
(Containing positive control, saline, and the
patient serum to be tested.)
5. Mix Contents of each circle uniformly over the
entire circle with separate mixing sticks.
6. Gently rock the slide back and forth, observe for
agglutination macroscopically, at one minute
against white background.
*Semi-quantitative method
1. Using a pipette place 80µl, 40µl, 20µl, 10µl, and
5µl of patient serum to be tested on 5 different
circles on the glass slide. The corresponding titres
obtained will be 1:20, 1:40, 1:80,1:160 and 1:320
respectively.
2. Place one drop of appropriate Brucel antigen
suspension to each circle.
3. Mix contents of each circle uniformly over the
entire circle with separate mixing sticks.
4. Gently rock the slide back and forth, observe for
agglutination macroscopically at one minute
against a white background.
Reference values
It is appropriate to consider:
Negative
Titres till to 1: 40
Dubious
Titres from 1: 80 to 1:160
Positive
Titres over 1: 320
A significant increase of the titre, in subsequent
days, indicates infection
Performance Characteristics
A. SENSIBILITY
In presence of elevated titres of antibodies, could
occur a prozone effect with absence of flocculation
at lowest dilutions and presence of flocculation at
higher dilutions
B. SPECIFICITY
Comparison between this method and another
commercial one, utilizing 50 samples, showed a
specificity of 100 %
BioScope
Another Method
*Procedure
Prepare 7 serum dilutions as follows:
Brucella abortus
+
Brucella melitensis
+
Brucella abortus and +
melitensis
TOTAL
+
12
7
2
0
0
0
TOTAL
12
7
2
0
21
29
29
29
50
Note
A final diagnosis should not be made on the basis of
a single test result but should be correlated with
other tests and clinical data
For in vitro diagnostic use only.
The following symbols are used on labels
For in vitro diagnostic use
Use by (last day of the month)
Temperature limitation