The document describes a method to measure antibody titers against thyroglobulin in patient serum samples using passive agglutination. Serial dilutions of control and patient serum samples were incubated with turkey red blood cells coated with thyroglobulin. Agglutination reactions indicated the presence and titer level of anti-thyroglobulin antibodies. The results showed that patient samples A and B tested positive with titers of 1280 and 320, respectively, while patient C tested negative, indicating no autoimmune thyroid disease.

2. Agglutination
Objective:
Measure the titer of antibody against thyroglobulin for different patient serum sample
by using passive agglutination.
Method:
1. In microtiter plate, 100uL and 25uL of diluents were added to well 1 and well 2-
12 respectively for each row that uses.
2. 25uL of control or patient serum samples is added to the 1st well of each row
and mixed depend on the flowing:
- Positive control serum in row A.
- Negative control serum in row B.
- Patient serum A in row C.
- Patient serum B in row D.

3. - Patient serum C in row E.
3. 25uL of diluted sample from the 1st well of each row was transferred to the 2nd
well of each row, mixture was mixed and then 25uL of it well discard.
4. 25uL from well 1 is transferred to well 3, mixture was mixed and 25uL of it was
transferred to well 4. The step was repeated to other wells respectively to achieve serial
dilution. Final 25uL from well 12 was discarded.
5. 25uL of control cells (Turkey red cell without human thyroglobulin) was added to
well 2 on each row and 25uL of test cells (Turkey red cells coated with human
thyroglobulin) was added to well 3-12 on each row.
6. Finally, the content of the plate was mixed on a plate shaker and then the plate
was incubated at room temperature for forty minutes.
7. After 40 minutes the plate was read and the titer is record.
Results:
In microtiter plate, Columns indicates by numbers and row in letters. In the table below
the result in each well indicate + (positive reaction), -(negative reaction) and Dil. (sample
diluents).Titter is the maximum dilution factor that gives agglutination. Figure1 indicates
the titter of each well in each row. As the table show positive control serum has
agglutination and the titter is 640. Patient serums A and B indicate positive agglutination
result with titer 1280 and 320 respectively. Patient C serum has a negative test.
Figure 1

4. Sample Titer
1 2 3 4 5 6 7 8 9 10 11 12
A Positive control serum Dil. - + + + + + + + + - - 640
B Negative control serum Dil. - - - - - - - - - - - No
C Patient serum A Dil. - + + + + + + + + - - 1280
D Patient serum B Dil. - + + + + + + - - - - 320
E Patient serum C Dil. - - - - - - - - - - - No
Discussion:
As the result show, positive control serum has agglutination due to the reaction
between antibody in the sample and antigen in test cell. The agglutination show positive
untie the well 9. After this point the reaction was negative due to depletion of antibody
during dilution. Of course negative control serum shows no agglutination because it's
not have antibody react with antigen in test cell. Patient serums A has agglutination
with titter higher than positive control serum which means that the patient has a lot of
antibody in hisher body and therefore suffer for sever autoimmune thyroid disease.
Patient serum B has titter lower than positive control serum's titter which means that
the patient has fewer antibodies in hisher body, so the suffering of disease is less
severe than patient A. Patient serum C show no reaction which means that the patient
not have the antibody in her/his blood that will interact with antigen in test cell.

5. Well 2 in each row have negative reaction which make us sure that there is no non-
specific reaction of patient serum with the peripheral Turkey red cell which does not
have antigen.
Conclusion:
Patient A and B are suffering for autoimmune thyroid disease with more severity in
patient A. patient C is free from the disease.