ESTIMATION OF BRUCELLACAPT
Introduction: The high variety of clinical manifestations of the human brucellosis makes difficult the diagnosis. Direct agglutination, Rose Bengal test, Coombs test and ELISA are the most widely used techniques for the serologic diagnosis of brucellosis. While in acute forms, all these techniques are sensitive enough, only the Coombs test can be used in more evolved forms to discard serologically a brucellosis diagnosis. Besides, the increase of titers in serum as measured by the Coombs test may indicate a reactivation of the disease. In this test, incomplete non-agglutinating antibodies are detected by adding, after a washing step that removes immunoglobulins not specific for Brucella, an anti-human immunoglobulin serum that aids the reaction to take place. Yet, since it is tiresome to perform, it is not carried out routinely in many laboratories, leading to a considerable number of not diagnosed cases.
Brucellacapt is performed in an easy and direct way. A good correlation has been attained between titers in the Coombs test and in the BRUCELLACAPT®, thus showing a great sensitivity and Specificity.
Principle: The test consists of U-bottom well-strips coated with anti-human immunoglobulins. After addition and dilution of serum, the antigen is added and strips are incubated for 24 hours until agglutination takes place. This assay allows the detection of both agglutinating and incomplete antibodies which only could be measured by means of the Coombs test
Requirement:
1. Serum sample
2. Vercelli Brucellacapt Plate
3. Vercelli Brucellacapt Serum Diluents
4. Vercelli Brucellacapt Positive Control
5. Vercelli Brucellacapt Negative Control
6. Micropipettes (10 - 50 μl) .
7. Bacterial Suspension
8. Micro Plate Shaker
9. Adherent tape
10. Incubator
Procedure:
A: Screening Procedure
1. Prepare 1:160 tube dilution of serum sample
(10 µl serum + 1600 µl serum diluents)
2. Add 50 µl Serum diluents into wells 2 and 3
3. Add 100 µl of 1:160 serum dilution (from tube dilution to well no. 1)
4. Transfer 50 µl of 1:160 serum dilution ( from well no. 1) to well no. 2 – 1:320
5. Transfer 50C of 1:320 serum dilution (from well no. 2) to well no. 3 – 1:640
6. Remove 50 µl from well no. 3
7. Add 50 µl Brucella antigen (Suspension ) to all wells
8. Carefully shake the plate
9. Seal with adherent tape and incubate for 24 hours at 37ºC, in a humid chamber protected from light exposure.
10. Read results:
Row | Titer |
1 | 1:160 |
2 | 1:320 |
3 | 1:640 |
B: Assay Procedure
1. Take Vercelli Brucellacapt Plate and labeling
2. Add 50 μl to all wells of Vercelli Brucellacapt Serum Diluents
3. Add 150 μl to ‘A’ wells of Vercelli Brucellacapt Serum Diluents
4. Add 10 μl of serum to ‘A’ well
5. Transfer 50 μl from ‘’A’’ to ‘’H’’ wells
6. Make doubling dilutions with 50 μl of each well from A to H.
7. Add 50 μl to all wells of Bacterial Suspension
8. Carefully shake the plate
9. Seal with adherent tape and incubate for 24 hours at 37ºC, in a humid chamber protected from light exposure.
10. Read results taking into account that titers will be:
Row | Titer |
A | 1:40 |
B | 1:80 |
C | 1:160 |
D | 1:320 |
E | 1:640 |
F | 1:1280 |
G | 1:2560 |
H | 1:5120 |
Or (Another procedure after screening results – 1:1280)
a. Prepare 1:1280 tube dilution of serum sample (10 µl serum + 1270 serum diluents)
b. Take micro wells and label A, B, C ,D
c. Add 100 µl micro well –A (diluted sample from a)
d. Add 50 µl serum diluents B, C, D
e. Transfer 50 μl from ‘’A’’ to ‘’D’’ wells
f. Add 50 μl to all wells of Bacterial Suspension
Row | Titer |
A | 1:1280 |
B | 1:2560 |
C | 1:5120 |
D | 1:10240 |
VIGOROUSLY SHAKE THE BACTERIAL SUSPENSION BEFORE USE. CARRY OUT INCUBATION OF THE TEST IN A HUMID CHAMBER PROTECTED FROM LIGHT EXPOSURE.
Interpretation of results:
The test is positive if a net covering the whole well surface appears. A button of bacteria in the center of the well indicates a negative result. Titers higher than 1/320 suggest brucellosis, although they should be evaluated together with other clinical evidences and the seroprevalence of the disease in the area before giving a diagnosis. In endemic areas, titers <1/320 are often encountered. In the case of positive titers of 1/5120, it is advisable to test further dilutions of the serum, particularly if BRUCELLACAPT® is going to be used for the follow-up of the disease in these patients.
Recommendations and Precautions:
1) For in vitro diagnosis use only. For professional use only.
2) Use kit components only. Do not mix components from different kits or manufacturers. Only the serum dilution is compatible with the equivalents in other VIRCELL BRUCELLACAPT lots.
3) Clean pipette tips must be used for every assay step. Use only clean, preferably disposable aterial.
4) Do not use in the event of damage to the package.
5) Never pipette by mouth.
6) The controls in this kit include substances of animal and human origin. Although the human serum controls of this kit have been tested and found negative for Hepatitis B Surface Antigen (HBsAg), Hepatitis C antibodies and Human Immunodeficiency Virus antibodies, control sera and patient specimens should be handled as potentially infectious. Bacterial suspension contains inactivated B. abortus antigen. Nevertheless, they should be considered potentially infectious and handled with care. No present method can offer complete assurance that these or other infectious agents are absent. All material should be handled and disposed as potentially infectious. Observe the local regulations for clinical waste disposal.
7) Bacterial suspension contains formaldehyde (concentration <1%). Avoid contact with skin, eyes and mucosae.
8) The glass elements contained in this kit could cause physical damage in the event of break. Handle with care.
9) Use only protocols described in this insert. Incubation temperatures and humidity conditions other than specified may give erroneous results.
10) Do not leave the reagents at room temperature longer than absolutely necessary.
11) Incubations have to be realized in the wells covered with the adherent tape included in this kit. The use of other tape than the specified may give erroneous results.
12) It is necessary to vigorously shake the bacterial suspension 3 immediately before use.
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