Veterinary Epidemiology and Zoonosis

Standard tube agglutination test

DIAGNOSIS OF BRUCELLOSIS BY STANDARD TUBE AGGLUTINATION TEST

• This test helps to assess the titre of the antibodies in the serum.
• The tubes used are called ‘Wasserman’s tubes’. ­

Materials required

Materials required	Quantity and purpose
Brucella abortusplain antigen	10mL
Test serum suspected for brucellosis	0.2mL
Known brucella positive and negative serum	Each 5mL
Wasserman's tubes	10 numbers
Rack for placing Wasserman's tubes	One
Normal saline	10mL
Syringe (2mL) and Tuberculin syringes with long needles	Adequate numbers
Water bath	To incubate the tubes

Procedure

• Place 10 numbers of Wasserman's tube in agglutination rack.
• Add 0.8mL of normal saline to the first tube, and 0.5mL of normal saline to the other remaining 9 tubes, using 2mL syringe.
• Add 0.02mL of serum to be tested in the first tube, using tuberculin syringe.
• Mix the saline and serum gently without formation of air bubbles. The titre in the first tube will be 1:5.
• Transfer 0.5mL of this mixture to the 2^nd tube and mix with 0.5mL of saline in this tube which results in 1:10 titre, and then transfer 0.5mL of the mixture to the3^rd tube.
• Continue the serial two-fold dilutions up to the 8^th tube (up to the dilution of 1:640). After mixing serum and saline in the 8^th tube, discard 0.5mL of mixture.
• Add 0.5mL of brucella plain antigen to each tube, using a separate 2mL syringe.
• Keep positive and negative control: Positive control in the 9^th tube with 0.5mL of saline and negative control in the 10^th tube with 0.5mL of bacterial suspension (antigen).
• Keep the tubes with the rack in a water bath at 56°C for 30 minutes or 37°C overnight.
• Observe the tubes for agglutination.

Figure: Standard tube agglutination test (STAT)

Interpretation of result

• · A titre is defined as the reciprocal of the highest dilution of serum, which produces a definite agglutination reaction (100%) and expressed as international units.
• · 100% agglutination: Agglutination at the bottom and clear supernatant.
• · 50% agglutination: Agglutination at the bottom, but supernatant is not clear.

Titre	Result
In disease free area
1 in 40 (40 IU) and above	Positive
1 in 20 (20 IU)	Suspicious
Below 1 in 20 (20 IU)	Negative
In endemic area
1 in 80 (80 IU) and above	Positive
1 in 40 (40 IU) and below	Negative

Note

• To avoid the discrepancies in the results paired sera samples should be tested.
• An increase in four fold titre of agglutination should be considered as a result of recent infection.
• To remove the possibilities of false positive results, heat inactivation test can be done. Serum will be heated at 56°C for 45 minutes.
• Brucellosis is a chronic disease, IgG - class of immunoglobulin can be seen in the serum.
• Hence, the serum is treated with 2-mercaptoethanol solution (0.46mL/100mL of saline) for destroying the IgM antibodies if present in trace before 24 hours to the test.