Antigens Antibody Reaction

Complement Fixation Test

What is Complement System…..? The complement system helps or “complements” the ability of antibodies and phagocytic cells to clear pathogens. This system consists of 20 or more plasma proteins. They are generally synthesized by the liver, and normally circulating as inactive precursors (pro-proteins) that need stimulation. Antigen–antibody complexes cause activation of complement system.

Complement Fixation Test This test can be used to detect the presence of either specific antibody or specific antigen in a patient's serum.  It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods. Complement fixation is still used to diagnose some Viral, Fungal and Rickettsial diseases.

Steps There are two main steps i.e. 1. Complement fixation step and 2. Indicator step Complement fixation step Add antigen and complement (usually from guinea pig) to the patient’s serum. If the antigen matches the antibody present in patients serum, they will combine and use up the complement (A process known as Complement fixation).

Indicator step Add indicator system, consisting of "sensitized" red blood cells of sheep (i.e. red blood cells plus anti–red blood cell antibody). If the antibody matched the antigen in the first step, complement was fixed and so no complement is available to attach to the sensitized red blood cells. Therefore red blood cells remain unhemolyzed. This is a positive test, which indicates that the patient's serum had antibodies to that antigen and all the complement was used up when it was fixed by the original antigen-antibody complex.

If the antibody did not match the antigen in the first step, complement is free. So it attaches itself to the sensitized red blood cells and as a result they are lysed. This is a negative test, which indicates that the serum did not contain antibodies against the antigen added in the complement fixation step and complement was free.

Toxin Antitoxin Neutralization Tests In neutralization reactions, the harmful effects of a bacterial toxin or virus are eliminated by a specific antibody (antitoxin). An antitoxin is an antibody produced in response to a bacterial toxin or a virus that neutralizes the bacterial toxin and/or virus and therefore no harmful effects are produced.

Examples 1. In Vivo (Schick test) This test is used to determine whether or not a person is susceptible (not immune) to diphtheria toxin produced by Corynebacterium diphtheriae.

Procedure Inject 0.1ml of diphtheria toxin intradermally into the arm of a test person. If a person does not have enough antibodies to fight it off, the skin around the injection will become red and swollen, indicating a positive result. This swelling disappears after a few days. If the person has immunity, then little or no swelling and redness will occur, indicating a negative result.

Interpretation of Results: Positive: When the test results in a red necrotic area (Round wheel like) of 5-10 mm diameter. Pseudo-positive: When there is only a red colored inflammation and it disappears rapidly. Negative reaction: No redness.

2. In Vitro (Antistreptolysin “O” titration) Streptolysin O, is a hemolytic toxin produced by most strains of group A and many strains of groups C Streptococci. The O in the name stands for oxygen-labile. The main function of Streptolysin O is to cause hemolysis (the breaking of red blood cells) in particular, beta-hemolysis. Anti Streptolysin O is the antibody which is produced against Streptolysin O when a patient suffers from Streptococci infection.

ASO antibodies will be raised after infection with Streptococci. Levels greater than 200 units per milliliter are considered significant. Measurement of ASO can be useful diagnostically for Streptococcal infections e.g. Rheumatic fever Post streptococcal glomerulonephritis Scarlet fever

Test Procedure Make serial dilutions of patients serum as 1/100, 1/200, 1/400, 1/800, 1/1600 and 1/3200 in six labeled test tubes. After that add 0.1ml of Streptolysin O toxin to each tube and mix gentle shaking. Add 0.5 ml RBCs suspension to each tube. Centrifuge the tubes at 1000 g for 2 minutes and observe for haemolysis. Report as positive if titre above 200 units per milliliter indicates a significant infection by bacteria producing Streptolysin O toxin.