IVD

Bacterial Antigens Kit (Wright, 2ME Test)

 SUMMARY OF WRIGHT TEST

Principles of 2ME Test

This antigen is applied for diagnosis of anti-BrucellaIgG in 2ME Wright test, which is used for the differential diagnosis of acute and chronic Brucellosis. 2ME Wright test can be used if Wright test yields a positive result. This test is recommended when negative result of blood culture and lowBrucella-specific agglutinin titer in the serum are reported despite Brucellosis symptoms exist. In other words, this test determines serum IgG, which is the sign of chronic Brucellosis. In this method, 2ME existing in the buffer removes IgM while IgG remains active.

Packaging Contents

2ME Antigen: 3 vials of 2ME Antigen (each vial contains 10 ml)

3 vials of 2ME Buffer (each vial contains 10 ml)

1 vial containing polyvalent serum

1 vial containing negative serum

Storage and Stability

Store at 2-8°C. Do not freeze.

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

Procedure

Bring the test reagents and samples to room temperature.

Resuspend the antigen vial gently. Pipette the solution several times to obtain a homogenous mixture.

Dilution of Serums for Tube Test

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

2. Pipette 0.9 ml of 2ME Buffer in No.1.

3. Add 0.5 ml of 2ME Buffer 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 2ME Buffer in tube No.10, which serves as a negative control.

9. Add 0.5 ml of appropriate 2ME antigen suspensions to all the tubes and mix well.

10. Incubate in an incubator at 37°C for 24 to 48 hours after sealing the tubes with parafilm.

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

Reading

          •        Without disturbing their contents, examine the tubes against a black background, by placing a light source above and behind the tubes.

          •        First verify that there is no agglutination in the titration control tube.

          •        Several types of agglutinations can be seen:

          •        Pancake-like agglutination in the bottom of the tube with a clear supernatant (+++).

          •        Clearly visible agglutination in a slightly cloudy fluid (++).

          •        Agglutination visible only on examination with an agglutinoscope (+).

          •        The absence of agglutination indicates a negative result (-).

          •        If the test is positive, the antibody titer in the test serum corresponds to the highest dilution yielding a degree of cloudiness similar to that seen in the titration control tube.

          •        A titre equal to or greater than 1/80 (120 IU/ml) indicates active brucellosis: titres are usually higher than these cut-off levels.

          •        Lower titres (1/40 or even 1/20) indicate suspicion of brucellosis and the serologic test should be repeated several days after.

          •        During incubation, it is better to close the mouth of the tubes with parafilm tape or adhesive tape to prevent evaporation inside the tubes.

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. Preferably, use disposable tubes.

          •        Use a new tip for each sample.

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

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