Complement fixation test

Lab 11-Immunology د.زينب عادل چـابك
3. 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.
Reincubated with antibody to human IgG labeled with an enzyme, eg. Horseradish peroxidase (for 30 min. at room temp.).
Wash step.
Added substrate solution to all wells, to know the enzymes activity that leads to development of a color.
Incubate for exactly 15 min. at room temp. in the dark and then estimating the color reaction in a spectrophotometer.
Add stop solution into all well and then read in ELISA reader.
The amount of unknown antibody (or Ag) bound is proportional to the enzyme activity. The titer of antibody (or Ag) in patient’s serum is the highest dilution of the serum that gives a positive color reaction.

6. Immunofluorescence test:
Fluorescent dyes, eg. fluorescein and rhodamine, can be covalently attached to antibody molecules and made visible UV light in the fluorescent microscope. Such labeled antibody can be used to identify antigens, eg. On the surface of bacteria (such as Streptococci and Treponemes), in cells in histological section, or in other specimens.
The immunofluorescence reaction is direct when known labeled antibody interact directly with unknown antigen (Used to detect antigen), and indirect when a two-stage process is used (Ag or Ab detection). For example, known antigen is attached to a slide, the patient’s serum (unlabeled) is added, and the preparation is washed; if the patient’s serum contains antibody against the antigen, it will remain fixed to it on the slide and can be detected on addition of a fluorescein dye-labeled antibody to human IgG and examination by UV microscopy. The indirect test is often more sensitive than direct immunofluorescence, because more labeled antibody adheres per antigenic site.