COMPLEMENT

COMPLEMENT: A term is originally used to refer to the heat labile factor in the serum. Complement is a set of serum proteins that undergo a sequential series of cleavage and association reactions in response to antibody-antigen complexes. If these complexes occur on a cell surface, complement reactions can cause cell lysis. The lysis of erythrocytes is particularly easy to detect visually, due to the release of hemoglobin, and this lysis forms the principle for a type of immunoassay. Antibody-antigen complexes in solution cause complement reactions to occur without cell lysis. Since the complement reactions are stoichiometric and the products short-lived, these non-cell-associated complexes deplete a portion of the complement proteins, a process termed “complement fixation.” This depletion reduces the lysis observed when the complement is subsequently added to antibody-coated erythrocytes.

COMPLEMENT SYSTEM:
The complement system is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks the pathogen’s cell membrane. It is part of the innate immune system, which is not adaptable and does not change over the course of an individual’s lifetime. The complement system can be recruited and brought into action by antibodies generated by the adaptive immune system.

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The complement system consists of a number of small proteins found in the blood, in general synthesized by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an amplifying cascade of further cleavages. The end result of this complement activation or complement fixation cascade is stimulation of phagocytes to clear foreign and damaged material, inflammation to attract additional phagocytes, and activation of the cell-killing membrane attack complex. Over 30 proteins and protein fragments make up the complement system, including serum proteins, and cell membrane receptors. They account for about 10% of the globulin fraction of blood serum.
Three biochemical pathways activate the complement system: the classical complement pathway, the alternative complement pathway, and the lectin pathway.

Classical pathway:
Although at least 21 different serum proteins have thus far been identified as part of the classical complement pathway, one can look at it as a pathway that is primarily initiated by either IgG or IgM binding (not IgE or IgA) to an antigen and involves 11 major serum protein components. IgG and IgM are classes of antibody molecules, as mentioned previously, one of the major defenses against microbes is the immune defenses’ production of antibody  molecules against that microbe. The “tips” of the antibody (the Fab portion have shapes that are complementary to epitopes- portions of microbial proteins and glycoproteins found on the surface of the microbe. The Fc portion of IgG and IgM can activate the classical complement pathway by enabling the first enzyme in the pathway, C1, to assemble.

The reactions are as follows:
Typically to activate the classical complement pathway, IgG or IgM is made in response to an antigen. The Fab   portion of IgG (2 molecules) or IgM (1 molecule) reacts with epitopes of that antigen. A protein called C1q first binds to the Fc   portion of antigen-bound IgG or IgM after which C1r and C1s attach to form C1, the first enzyme of the pathway
The activated C1   now enzymatically cleaves C4 into C4a and C4b. The C4b then binds to adjacent proteins and carbohydrates on the surface of the antigen and then binds C2. The activated C1 cleaves C2 into C2a and C2b forming C4b2a, the C3 convertase. Now the the classical complement pathway is activated. C3 convertase can now cleave hundreds of molecules of C3 into C3a and C3b.

Some molecules of C3b bind to C4b2a, the C3 convertase, to form C4b2a3b, a C5 convertase that cleaves C5 into C5a and C5b.

C5b binds to the surface of the target cell and subsequently binds C6, C7, C8, and a number of monomers of C9 to form C5b6789, the Membrane Attack Complex (MAC).

ALTERNATIVE PATHWAY:
The Alternative pathway (AP) is one of the three pathways of the complement system. In contrast to the other two pathways the AP is not triggered by antibodies or specific structures on the microorganisms. Complement component C3b present on the surface of foreign cells can bind another serum protein called Complement factor B (Factor B) to form a complex. C3b binding exposes a site on Factor B that serves as a substrate for enzymatically active serum protein called Complement factor D (Factor D). Factor D cleaves Factor B, releasing a small fragment Factor Ba and the larger fragment Factor Bb. Complement component C3b and Factor Bb form the enzymatically active complex C5 convertase (C3bBb). Subsequently, Complement factor Properdin (also called Factor P) binds to C5 convertase (C3bBb) and stabilizes it. The C5 convertase (C3bBb) complex has C3 convertase activity and thus is analogous to the C3 convertase (C2aC4b) complex in the classical pathway. C5 convertase (C3bBb) make a complex with C3b, which exhibits C5 convertase activity, analogous to the C5 convertase (C2aC4bC3b) complex in the classical pathway.
Regulatory molecule of the alternative pathway is a Factor I. Factor I cleaves C3b. CD46 molecule, complement regulatory protein (MCP) is a cofactor for Factor I -mediated degradation of C3b. Complement factor H (Factor H) accelerates the destructive action of Factor I. CD55 molecule decay accelerating factor for complement (DAF) also regulates the complement system activity by accelerating the decay of the C3/C5-convertase activity of the alternative pathway.

Smaller fragments resulting from complement cleavage, Complement components C3a and C5a, called anaphylatoxins, bind to receptors (CR3aR and CR5aR) on the surface of mast cells and blood basophils and induce degranulation upon release of histamine and other biologically active mediators.

LECTIN PATHWAY:
The lectin pathway (at the top, in the middle): activation of complement via the lectin pathway is mediated by pattern recognition proteins (PRPs). There are five distinct PRPs: mannan-binding lectin (MBL), collectin-11 (CL-11), ficolin-1, ficolin-2, and ficolin-3. Each of them complexes with MBL-associated serine proteases (MASPs), of which there are three forms (MASP1, MASP2, and MASP3) and non-enzymatic proteins (MAP1 and sMAP). Activation, for example, begins with MBL, which binds mannose on bacteria with the help of immunoglobulin A (IgA) and damaged endothelium. MBL is homologous to C1q and starts a cascade with help of MASPs. In further reactions, the lectin pathway is almost identical to the classic pathway to create C3 and C5 convertase. There are indications that MASPs in some cases can directly activate C3.

COMPLEMENT FIXATION TEST:
The complement fixation test (CFT) was extensively used in syphilis serology after being introduced by Wasserman in 1909. It took a number of decades before the CFT was adapted for routine use in virology. CFT meet the following criteria; it is convenient and rapid to perform, the demand on equipment and reagents is small, and a large variety of test antigens are readily available. However, there is now a trend to replace the CFT with more direct, sensitive and rapid techniques, such as RIAs and EIAs. Although CFT is considered to be a relatively simple test, it is a very exacting procedure because 5 variables are involved. In essence the test consists of two antigen-antibody reactions, one of which is the indicator system. The first reaction, between a known virus antigen and a specific antibody takes place in the presence of a predetermined amount of complement. The complement is removed or “fixed” by the antigen-antibody complex. The second antigen-antibody reaction consists of reacting sheep RBCS with hemolysin. When this indicator system is added to the reactants, the sensitized RBCS will only lyse in the presence of free complement. The antigens used for CFT tend to be group antigens rather than type-specific antigens. In order for the CFT to be set up correctly, the optimal concentration of hemolytic serum, complement, and antigen should be determined by titration. The following is a protocol for setting a complement fixation test.

The complement fixation test is one of the major traditional tests for the demonstration of presence of specific antigens or antibodies. It requires a veritable zoo of reagents and numerous preparatory steps. There are almost as many versions as there have been users; the microtitre version developed at the Centers for Disease Control and Prevention (LBCF Test) includes rigorous controls and is commonly used.

The complement fixation test (CFT) does not depend on hem-agglutinating activity of the virus, but the antibodies must fix the complement, and the sera must be free of anticomplementary activity.  The test has been superseded, in many instances, by newer tests, such as enzyme immunoassay (EIA). The terminal components of the complement cascade, C789 (the membrane attack complex), can damage cell membranes in the presence of specific antibody, which fixes complement to the cell surface. In the CFT, erythrocytes are used as the target cell, because complement-induced leakiness of the membrane can be visualized or measured calorimetrically as an increase in free hemoglobin. In the presence of specific antibodies to an infectious agent, any complement in the system is bound, leaving no residual complement for reaction with antibodies to the erythrocytes. Thus, the presence of specific antibody is indicated by the absence of hemolysis.

MATERIALS AND REAGENTS:
Sheep erythrocytes suspension (5% suspension of washed sheep RBCs)
Hemolysin (rabbit anti-sheep red-cell antibody)
Guinea pig complement, free of antibodies to the agent of interest (Note: Guinea pig is the commonest source of fresh complement)
Barbital-buffered diluents
Plastic microtitre plate
Centrifuge adapter for microtitre plates
Water bath for incubation of plates
Color standards for judging hemolysis (prepared by lysing various concentrations of red cells)
PROCEDURE OF COMPLEMENT FIXATION TEST:
Complement Fixation Test (CFT) consists of two stage:
 First step (Complement fixation stage): a known antigen and inactivated patient’s serum are incubated with a standardized, limited amount of complement.

(Note: patient’s serum is heated at 56°C for 30 minutes to inactivate endogenous complement which may disturb the test calibration)
If the serum contains specific complement activating antibody, the complement will be activated or fixed by the antigen-antibody complex.

However, if there is no antibody in the patient’s serum, there will be no formation of antigen-antibody complex, thus complement will not be fixed but will remain free (In the indicator stage this complement will react with RBC coated with antibody to sheep RBC).

Second step (Indicator Stage): The second step detects whether complement has been utilized in the first step or not. This is done by adding the indicator system.

If the complement is fixed in the first step owing to the presence of antibody there will be no complement left to fix to the indicator system. There won’t be any lysis of RBCs.

However, if there is no specific antibody in the patient’s serum, there will be no antigen-antibody complex, therefore, complement will be present free or unfixed in the mixture. This unfixed complement will now react with the antibody- coated sheep RBCs to bring about their lysis.

OBSERVATION:
If hemolysis is observed: it indicates the absence of specific antibody in patient serum, so that complement has not been used which lysed the sensitized RBC giving hemolysis.

If no hemolysis is observed: it indicates that the patient serum contains antibody which reacts with Ag to form Ag-Ab complex and then fix complement .so that no complement is available to hemolyse sensitized RBC.

 RESULT INTERPRETATION:
Positive CFT: if no hemolysis is observed, it indicates positive complement fixation test. Antigen- antibody reaction and complement fixation occurs, so NO free complement is available to lyse the RBC.

Negative CFT: if hemolysis of RBC observed, it indicates negative complement test. NO complement fixation occurs, so the complement remain free and it hemolyse the RBC.

APPLICATIONS OF CFT:
CFT is used to detect and quantify antibody that does not agglutinate or precipitate with its antigen.

CFT can detect antibody at level less than 1 microgram per milliliter.

CFT is also used to detect antigen
CFT is Economical
CFT can be used to screen large numbers of viral or bacterial infection.

Limitations of CFT:
Less sensitive; it can not be used for immunity screening
Non- specific e.g. cross-reactivity between HSV and VZV
Time consuming
Labor intense