Immunology & Serology

LABORATORY

Activity 2
Serial Dilution and Determination of Antibody Titer

Objectives

At the end of the activity, the student should be able to:

1. Perform serial dilution of anti-serum.
2. Compute the dilution in the series of tubes.
3. Determine the antibody titer of the anti-serum.

Materials
Test tubes
0.9% saline
1000 ul micropipets and tips
100 ul micropipets and tips
Serologic centrifuge
RBC suspension (previously prepared)
Markers for labeling

Procedure
Serial Dilution:
1. Label four tubes as 1, 2, 3, and 4.
2. Place 8 ul of anti-D in tube 1.
3. Add 192 ul of NSS to tube 1 to achieve a total volume of 200 ul. Mix well by shaking the
tube.
4. To compute the dilution in this tube, the following formula is used:
𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑒𝑟𝑢𝑚
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 =
𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

8 𝑢𝑙 1
𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = =
200 𝑢𝑙 25

Therefore, the dilution in tube 1 is 1:25.

5. Prepare tubes 2, 3, and 4 for serial dilution. Place 100 ul of NSS in tubes 1,2 and 3.
6. Transfer 100 ul from tube 1 to tube 2. Mix well by shaking the tube.
7. To compute the present dilution, follow the formula above. To compute the final
dilution in this tube, use the formula below:
Immunology & Serology
LABORATORY

𝐹𝑖𝑛𝑎𝑙 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 = 𝑝𝑟𝑒𝑣𝑖𝑜𝑢𝑠 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑥 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

8. Transfer 100 ul of the diluted solution from tube 2 to tube 3. Compute the present and
final dilution for tube 3.
9. Transfer 100 ul of the diluted solution from tubes 3 to 4. Compute the present and final
dilution for tube 4.

Determination of Antibody Titer

1. Label 4 tubes as D1, D2, D3 and D4.
2. Transfer 50 ul from Tube 1 to D1, Tube 2 to D2, Tube 3 to D3, and Tube 4 to D4.
3. Add 50 ul of RBC suspension to tubes D1, D2, D3, and D4. (Mix RBC suspension well
before adding.)
4. Centrifuge the four tubes for one minute.
5. Observe for hemolysis and agglutination in each tube. Grade the hemolysis and/or
agglutination based on the following:
Agglutination:
Negative (0): Red cell button entirely resuspended and not visible agglutination
1+: Red cell button entirely resuspended with many small aggregates present
2+: Red cell button entirely resuspended with several medium sized aggregates present, clear
supernatant
3+: Red cell button entirely resuspended with few medium to large sized aggregates present,
clear supernatant
4+: Red cell button entirely resuspended with one large sized aggregate present, clear
supernatant
1w: very small agglutinates, turbid background
w+ or +/- : barely visible agglutination, turbid background
mf : mixture of agglutinated and unagglutinated red cells (mixed field)

Hemolysis:
H: complete hemolysis
PH: partial hemolysis, some red cells remain

6. Record your results.

7. Determine the last tube that showed hemolysis or agglutination.
Immunology & Serology
LABORATORY
8. The antibody titer is the reciprocal of the dilution of the last tube that showed a positive
result. Determine the antibody titer based on your results.

Output
Present the tubes for checking.
Antibody titer should be reported.
Immunology & Serology
LABORATORY
(Submit only this part of the protocol.)

NAME ___________________________________________ SECTION ______________

Activity 1
Serial Dilution and Antibody Titer

Results

Tube 1 2 3 4
Final Dilution 1:25

Show your computations:

Tube D1 D2 D3 D4
Final Dilution
(after adding
RBC suspension)
Result after
centrifugation

Show your computations:

Antibody titer: ____________

Immunology & Serology
LABORATORY
Laboratory Performance
Write here the specific tasks that you did in this activity.

Rubrics for Laboratory Performance

5 4 3 2 1
Timeliness Able to finish 1-5 6-10 minutes 11-15 More than 15
the activity minutes late minutes minutes late
on time late late
Materials Complete Incomplete
materials materials
Following All steps There There was a
procedures done once was a need to
with no need need to repeat more
to repeat any repeat a than 1 step
step step
Output Titer was Titer is Titer is Titer is
correctly incorrect incorrect due incorrect due
reported. due to to wrong to wrong
wrong computations computations
conversion OR wrong AND wrong
from reading of reading of
dilution. results. results.
Question Able to Unable to Unable to
and Answer answer all answer 1 answer all
questions question questions

Guide Questions:

1. Give two specific serologic tests that require the computation of titer.

2. What is the purpose of doing a serial dilution?