Titration is a semi-quantitative technique of measuring the concentration of an antibody in a serum. The titer of an antibody is usually determined by testing two-fold serial dilution of the serum against selected red cells.
SAMPLE: 4 to 5 ml clotted blood. REAGENTS: SALINE AGH Pooled Cells.
METHOD: Label a row of tubes according to serum dilution 1 to 10
Place 1 volume (0.1 ml) or 1 drop of saline in all tubes except the first.
Add 1 volume (0.1ml) or 1 drop of serum to tubes 1 and 2 so that the first tube contains neat serum (1:1) and 2nd tube has 1 volume of serum in volume of saline (1:2).
Using a clean pipette mix the contents of tube 2 (1 : 2 dilution) without forming any bubbles and transfer one volume of mixture to tube 3 (1:4).
Continue the same process through all dilutions, Remove I volume from last tube and save for use if further dilutions are required. Add 1 volume of 2-5% saline suspended appropriate red cells to each tube. Mix well and incubate at RT for 60 minutes (IgM antibodies) and centrifuge all tubes at 1000 rpm for 1 minute. Gently dislodge the cell button and record results using grades of agglutination reaction.
The last tube showing positive reaction is considered as the titer of the antibody. For detection of IgG antibodies: arrange a 2nd row of tubes with the same serial dilution. Incubate at 37o C. Centrifuge and remove supernatant, incubate at 37C for 45 minutes. Wash with saline thrice.
Arrange fresh tubes and add 1 drop of AHG and add the corresponding washed cells. Incubate at room temperature for 5 minutes, spin at 1000 rpm for 1 minute and look for clumping.
INTERPRETATION: If there is clumping in first row of test tubes, it indicates the presence of saline antibodies or IgM.
If there is clumping in the second row of test tubes in indicates the presence of IgG antibodies.
