1. (REACCREDITED WITH B GRADE WITH A CGPA OF 2.71 IN THE II CYCLE BY NACC
AFFILIATED TO MANONMANIAM SUNDARANAR UNIVERSITY,THIRUNELVELI)
ALWARKURICHI -627 412 TAMILNADU INDIA.
POST GRADUATE & RESEARCH CENTER – DEPARTMENT OF MICROBIOLOGY
(GOVERMENT AIDED)
II SEM - CORE –IMMUNOLOGY
UNIT – III
COMPLEMENT FIXATION
SUBMITTED TO
THE GUIDE
DR.S.VISWANATHAN,PH.D
HEAD OF THE DEPARTMENT
SRI PARAMAKALYANI COLLEGE
ALWARKURICHI.
SUBMITTED BY
ESSAKIMUTHU.G
REG.NO : 20211231516109
I M.SC, MICROBIOLOGY
DATE: 30.04.2022
2. Definition
Complement fixation is one of the
most important and one of the
classical techniques for determining
antigen-antibody complexes present
in the testing sample.
3. Principle
When antigen and antibody interact with each other,
they form a complex called antigen-antibody (Ag-Ab )
complex. The complex then interacts with complement
protein and gets fixed with it. After fixing, the
complement degrades or gets cleaved into two
fragments i.e. smaller and larger fragments.
4. Positive test
Antibody in sample + Antigen (added) +
Complement → Ag-Ab Complex Fixed
with Complement
Complement fixed Ag-Ab + Indicator
System → No change (No hemolysis)
5. Negative test
Sample with no antibody + Antigen (added) +
Complement → Free Complement
Antigen (added) + Antibody in indicator system (On
RBC) → Ag-Ab complex
Ag-Ab complex + Complement → Fixed
Complement System → Hemolysis
6. Complement
Fixation Test Requirements
Samples such as serum or CSF (may or may not
contain the specific antigens or antibodies of interest)
Known complementary antigens based on the
component desired to be detected.
7. Complement Proteins
The native complement present in the sample is
inactivated. Complement obtained from the
serum of other organisms such as Guinea pig is
added to the sample during the test.
8. Indicator
System
Sheep erythrocytes or RBCs coated with
antibodies(mainly derived from Rabbit serum)
on the surface. These RBCs can also be called
sensitized RBCs.
9. Complement
Fixation Test Procedure
1.A serum sample is taken.
2.It is then heated at about 56 °C to remove the
complement proteins already present in the sample.
3.The serum is then adsorbed with washed sheep
RBC. It prevents interference in the test by anti-RBC
antibodies which are cross-reactive.
10. 4.Then the antigen and complement are added to the sample.
5.It is then subjected to incubation at a temperature of 37 °C for
30 minutes. It provides conditions and time for the formation of
the Ag-Ab complex.
6.And the indicator system is then added and the sample is
observed for change due to occurrence or non-occurrence of
hemolysis.
11.
12. Complement
Fixation Test Applications
•Wasserman’s test is one of the complement fixation tests for the
detection of syphilis. It is an antibody detection test.
•It can also be used for the detection of bacterial diseases
caused by Mycobacterium pneumoniae, Bordetella pertussis, etc.
•It can be used for the detection of viral infections, and fungal
infections such as Histoplasmosis, Cryptococcosis, etc.
13. Complement
Fixation Test Advantages
•Interpretation of the result after the test is easier.
•It can be used for the detection of a very small
number of antigen or antibody components in the
sample.
•It can be used for the detection of a variety of
infections.
•It has good sensitivity.
14. Complement
Fixation Test Limitations
•It is one of the old methods not used much in current
practices.
•It is slower and more complex in comparison to many
easier rapid detection tests being used currently.
•It is difficult to perform and arrange the reagents used for
it.
•Although it is one of the sensitive tests, it has less
sensitivity than tests such as ELISA.
15. References
1.Goldsby R.A., Kindt T.J., Osborne B.A., (1999) Kuby
Immunology, 4th edition, W.H.Freeman & Co Ltd.
2.Parija S.C., (2009), Textbook of Microbiology and Immunology,
2nd edition, Elsevier, a division of Reed Elsevier India Private
Limited
3.Jeffrey K., Complement Fixation Test Introductory Immunology
(Second Edition), 2019Miller, V. B. (1930). Tests for Syphilis: An
Explanation of the Wasserman Test. The American Journal of
Nursing, 30(6), 707–712.
