Complement fixation test

Complement fixation test:

It tests for the presence of either specific antibody or specific antigen in a patient s serum. The reaction consists of the following two steps:
1. Antigen and antibody (one known and the other unknown) are mixed, and a measured amount of complement is added. If the antigen and antibody match, they will combine and use up “fix” the complement.
2. An indicator system, consisting of sensitized red blood cells (ex. Red blood cells plus anti-red blood cells antibody), is added. If the antibody matched the antigen in the first step, complement was fixed and less (or none) is available to attach to the sensitized red blood cells. The red blood cells remain unhemolyzed, the test is positive because the patient s serum had antibodies to that antigen. If the antibody did not match the antigen in the first step, complement is free to attach to the sensitized red blood cells and they are lysed, the test is negative. Patient’s serum must be heated to (56 °C) for 30 minutes to inactivate any human complement activity.

4. Enzyme-Linked Immunosorbent Assay (ELISA):
This method can be used for the quantitation of either antigens or antibodies in patient specimens. It is based on covalently linking an enzyme to a known antigen or antibody, reacting the enzyme-linked material with the patient’s specimen and then assaying for enzyme activity by adding the substrate of the enzyme. The method is nearly as sensitive as RIA (Radio-immuno assay) yet requires no special equipment or radioactive labels.

Assay procedure:
For measurement of antibody, known antigens are fixed to
a surface (eg. The bottom of small wells on a plastic plate).
Incubated with dilutions of the patient’s serum for 1hr. at 37°C.
Wash step.