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SERO-IMMUNOLOGICAL REACTION
AND BASIC PRINCIPLE
COMPLEMENT FIXATION
An important constituent of the blood which is essential for the lytic action of certain antibacterial bodies and also for the opsonizing action of antibodies is called complement. The origin of complement is not very well understood. But apparently, it is a mixture of globulin and mucoprotein and that by lability, we can say that it is protein in nature. Complement consists of four components, known as C'1, C'2, C'3 and C'4. It is believed that antibody serves only as a go-between for the complement and what connects the cells to the complement is called an "amboceptor." Thus, if complement is not present, no reaction will go to completion. The mechanism by which complement produces its effect is not known. Combination of complement with sensitized cell may be due to non-specific physio-chemical forces. Example of this is some sort of digestion may follow after its combination with sensitized cell.
Complement occurs in blood of various species and one of the best sources of complement for hemolytic systems is guinea pig serum. It's often found, however, that an animal's complement is weak in activating the lysis of the blood cells of its own species. Heating of complement to 56 C for 30 minutes destroys its activity. It is also inactivated by various reagents or treatments such as prolonged shaking, additions of various salts, acid or alkali and also upon exposure to ultraviolet rays and alpha particles. Splitting of complement signifies that as electrolytes are removed from the serum, the complementary activity is lost. It has been known for instance that calcium ions enhance complementary action, that salt and magnesium sulfate increases the hemolytic activity of complement. Hence, absence of magnesium and calcium ions prevents complement from combining with antigen-antibody reaction. Calcium and magnesium must be present in order to produce hemolysis.
Complement fixation is a process in which the complement becomes bound or "fixed" in anumerous variety of other antigen-antibody reactions leading to the removal of the free complement from the system. The process of chemical fixation can be divided into 3 steps:
E - Erythrocytes
A - Antibody
C'x - One or more complement components
E + A = EA
EA + C'x + CA = EAC'x
EAC'x + C'y + Mg = EAC'xC'y
The ability of antigen-antibody compounds to combine with complement depends upon the following factors: species origin of antibody and complement, the size of the antigen-antibody complex, complete precipitation should occur. Antigen and antiserum have the power of non-specifically removing or inactivating some complement and this is called anticomplementary action. The anticomplementary effect of the reagents used must be carefully tested by setting up controls. Here are the probable causes of anticomplementary action:
- Presence of particulate matters that absorb complement
- Old sera or those contaminated with bacteria or by soap, acid, alkali and so forth.
- Sera of syphilitic patients (especially when it is heated).
The hemolytic effects of complement are very much emphasized because lysis is the most convenient way of detecting and measuring complement activity. Roles of complement in immnune reactions: opsonizing activity, lysis of certain microorganisms, effects on the precipitin and agglutinin reactions and virus neutralization. Conditiions that affect complement fixation: temperature, time of incubation, electrolysis and pH (hydrogen ion concentration). Complement fixation test is valuable in the detection of syphilitic reagin. The test is done by adding standard cardiolipin antigen and guinea pig serum complement to the patient's serum. After considerable time, sheep cells plus hemolysin are added. If hemolysis occurs, free complement is present and the unknown contains no antibody. If no hemolysis, the complement has been bound by antigen-reagin complex and the unknown is positive. Also, complement fixation tests conducted with cardiolipin antigens are of values not only in the diagnosis of syphilis and other treponemal diseases but, possibly, in relation to their treatment as well as on the basis that persistently negative serum and cerebrospinal fluid (CSF) reactions may be acceptable as criteria of complete recovery. Moreover, it is now well-established that complement fixation tests conducted with virulent Treponema pallidum and the Reiter protein antigen are of proven diagnostic value in the diagnosis of viral, rickettsial, mycotic infections and in protozoal and helminthic infestments.
OLD LAB PROCEDURES
One very popular test in the annals of serological test is the "Wasserman." There are many modifications and they differ considerably in specificity, that is, freedom from false positive reactions, and sensitivity (the ability to detect small amounts of specific antibody). It is necessary then to refer to the test by the generally accepted name of the modification used. The two technics assigned are the Kolmer modification and the one-fifth volume Kolmer test using the Reiter Protein antigen (R.P.C.F. test). The former is widely used and is properly suited for the study of complement fixation tests in general and permits the detection of sources of error. The R.P.C.F. is similar to the Kolmer but uses smaller quantities of reagents and treponemal antigen. As a whole, complement fixation techniques are intricate, depending on the interaction of several labile components.
MODERN LAB PROCEDURES
RPR Card Test For Syphilis (Rapid Plasma Reagin)
FTA-ABS Test (Fluorescent Treponemal Antibody - Absorption)
NEXT REACTION: C-REACTIVE PROTEIN
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