Precipitation ring test
Principle:
- The ring or interfacial test is a simple serological method that exemplifies the precipitin reaction in solution. This antigen-antibody reaction can be indicated by the formation of a visible precipitate, a flocculent or granular turbidity, in the test fluid. Antiserum is injected into a small-diameter test tube, and the antigen is then carefully added to form a discrete upper layer. After a period of incubation of up to 4 hours, a ring of precipitate is visible at the point of contact (interface) in the presence of the antigen-antibody reaction. The rate at which the visible ring forms relies on the concentration of antibodies in the serum and the concentration of the antigen.
- To determine the precipitation reaction, a series of dilutions of the antigen is used because both insufficient and excessive amounts of antigen will prohibit the formation of a visible precipitate. The insufficient amount of antigen gives rise to zone of antibody excess and excessive amounts of antigen will give rise to zone of antigen excess. The visible precipitate ring is formed at zone of equivalence.
Requirements:
- Saline (0.85%) NaCl
- Bovine globulin antiserum
- Normal bovine serum diluted to 1:25, 1:50, and 1:75 with physiological saline.
- 0.5ml pipettes
- Serological test tubes (8* 75mm)
- Incubator
- Serological test tube rack
Procedure:
- Label three serological test tubes according to the antigen dilution to be used (1:25, 1:50, and 1:75) and the fourth test tube as a saline control.
- Transfer 0.3 ml of each of the normal bovine serum dilutions into its appropriately labelled test tube by using a different 0.5-ml pipette.
- Transfer 0.3 ml of saline into the test tube labelled as control by using a clean 0.5-ml pipette.
- Carefully overlay all four test tubes with 0.3 ml of bovine globulin antiserum.
- Tilt the test tube to prevent mixing of the sera, and allow the antiserum to run down the side of the test tube.
- Incubate all test tubes for 30 minutes at 37°C.
- Examine all test tubes for the development of a ring of precipitation at the interface. Indicate the presence or absence of a ring in the Lab Report.
- Determine and record the antigen dilution that produced the greatest degree of precipitation; this is indicative of the optimal antibody: antigen ratio.
Observations and Results interpretation:
- Positive ring test: Observation of white ring at the junction of antigen and antibody reaction indicates positive result
- Negative test: absence of white ring formation
