Bacterial Antigens Kit (Wright, Tube and Rapid Tests)

Summary of Wright Test

Brucellosis is one of the most widespread zoonosis in the world and occurs mainly in farmers, slaughterhouse workers, and veterinarians via direct or indirect contact with infected animals or their products. The clinical symptoms of human brucellosis are nonspecific, such as fever, headache, chills, and sweating. Diagnosis and treatment of brucellosis requires laboratory tests. Although the serum tube agglutination test (SAT) is the gold standard, it is laborious and time-consuming. It also requires a number of reagents. 

Principles of Tube & Rapid Tests

Packaging Contents

Tube Wright Antigen Kit: 6 vials tube Wright Antigen (each vial contains 10 ml Antigen).

Rapid Wright Antigen Kit: 6 vials Rapid Wright Antigen (each vial contains 5 ml Antigen).

One vial containing polyvalent serum.

One vial containing negative serum.

Storage and Stability

Store at 2-8ºC. Do not freeze.

Antigen and controls are stable until the expiry date stated on the label.

Precautions

          •        Before use, allow reagents to reach room temperature (18-30°C).

          •        Do not use expired reagents.

          •        Use glassware washed and rinsed with distilled water or preferably                disposables.

          •        Use a new tip for each sample serum.

          •        Bacterial contamination of controls and specimens as well as freezing and thawing of the antigen may lead to false positive /negative results.

          •        The reagents in this kit contain chemical material. Do not allow to contact with skin or mucous membranes. 

Tube Test of Procedure

1. Take 10 Test tubes and label them 1 to 10.

2. Pipette 0.9 m of physiological serum to No.1.

3. Add 0.5 m of physiological serum to each of the remaining tubes (2-10).

4. Add 0.1 ml of serum sample to be tested to the tube No.1. Mix well.

5. Transfer 0.5 ml of the diluted serum from tube No.1 to tube No.2 and mix well.

6. Transfer 0.5 ml of the diluted serum from tube No.2 to tube No. 3 and mix well. Continue this serial dilution till tube No.9.

7. Discard 0.5 ml of the diluted serum from tube No. 9.

 8. Pipette 0.5 ml of physiological serum in tube No.10, which serves as a negative control.

9. Add 0.5 ml of appropriate Wright antigen suspensions to all the tubes and mix well.

10. Cover the tubes and incubate at 37°C for 24-48 hours.

11. Observe for agglutination macroscopically in each tube of the dilution series.

Reading of Tube Test

The titer of the serum is the reciprocal of the last dilution of the serum sample that gives a granular agglutination.

In negative reaction, the appearance of the suspension remains unchanged, which shows a typical swirl when the tube is flicked.

Accept Result: >1/80

Rapid Test of Procedure

          •        Before starting the experiment, bring all the reagents to room temperature, mix well, and homogenize.

          •        Using a pipette place 80 μl, 40 μl, 20 μl, 10 μl and 5 μl of the serum to be tested on 5 different circles on the glass slide. The corresponding titers obtained will be 1:20, 1:40, 1:80, 1:160, and 1:320 respectively.

          •        Place one drop of appropriate Rapid antigen suspensions to each circle.

          •        Mix contents of each circle uniformly over the entire circle with separate mixing sticks.

          •        Rotate the slide by rotating the hand. (Note that the dough is completely your hand and the use of a shaker is not recommended)

          •        Gently rock the slide back and forth, observe by using a light source for agglutination macroscopically at one minute against a white background.

Reading of Rapid Test

Agglutination is a positive test result. The titer of the serum corresponds to the visible agglutination in the test circle with the minimum amount of serum sample.

Note

          •        Do not dilute any of the reagents.

          •        Do not freeze any of the  reagents.

          •        In the Rapid test, the reactions obtained are roughly equivalent to those occurred in a tube test.

          •        Positive results obtained in the slide test should be confirmed with the tube test to establish whether the titers are diagnostically significant or not.

          •        Agglutinins are found in high proportion of normal individuals and titers less than 1:80 are doubtfully significant. A rising titer is more significant than a single high titer.

          •        False positive reactions may occur in sera of patients infected withPasteurella tularensisor vaccinated withVibrio cholerae.

          •        Cross-reactions betweenBrucellaantigens and other organisms such asYersinia enterocolitica,Escherichia coliandFrancisella tularensishave been reported.

          •        False positive results are likely if the test is read more than one minute after mixing on the slide test.

          •        Prozoning may sometimes be encountered in serum containing very high titers on slide test.

          •        Serological findings are not intended as a substitute for culture. An appropriate attempt should be made to recover and identify the etiologic organisms through various culture and biochemical tests.

          •        After usage, the antigen suspension should be immediately recapped and replaced at 2-8°C.

          •        Reagent vials that have leakage/ breakage problem should be discarded.

          •        It is recommended that results of the tests should be correlated with clinical findings to arrive at the final diagnosis.