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Complement and Primary Immunodeficiency Antibodies

Antibodies to detect various complement components and factors including Mannan-Binding Lectin (MBL).

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The complement system is part of the innate immune system and is made up of a number of small proteins synthesised by the liver.  Complement proteins normally circulate in the blood in an inactive form but once activated there is a cascade of cleavage and activation events that help antibodies and phagocytic cells clear pathogens from the body.

Complement proteins serve numerous roles in the immune system including:

  • Opsonosation – enhancing phagocytosis of antigens (C3b)
  • Chemotaxis – attracting macrophages and neutrophils (C4a, C2b, C5a)
  • Cell lysis – rupturing membranes of foreign cells (Membrane Attack Complex)
  • Clumping of antigen-bearing agents

There are three activation pathways that can initiate the complement cascade –ClassicalMannan-Binding Lectin (MBL), and Alternative.  Both the Classical and Lectin pathways begin with protease activation of C4 and C2 whilst the Alternative pathway is initiated by spontaneous hydrolysis of C3.

Clinical investigations of recurrent infections or unexplained inflammation may include complement assays to determine if there are deficiencies or abnormalities in the complement system. Total complement activity (CH50) may be measured to assess the integrity of the entire classical complement pathway and individual components such as C3C4C1-esterase inhibitor or MBL may be investigated when deficiencies are suspected.

 

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The complement cascade is an important part of the innate immune system and comprises a large number of plasma proteins that react with one another to opsonize pathogens and induce a series of inflammatory responses to fight infection.

BioPorto offer monoclonal antibodies targeting numerous proteins within the cascade. Common primary immunodeficiencies include C2, C4 and mannan-binding lectin (MBL) which instigates the lectin pathway of complement activation.

Classical Pathway

The Classical pathway typically requires antigen-antibody complexes for activation at C1 and is driven by a specific immune response.  The C1 complex is made up of one C1q subunit, two C1r subunits and two C1s subunits – C1qr2s2.  In its inactive state it is bound to C1-inhibitor (C1-INH) (also known as C1-esterase inhibitor).  A single IgM antigen complex (or multiple IgG complexes) can bind the C1q subunit and activate the C1 complex overcoming the C1-INH inhibition.  In some instances the C1 complex can be activated without an antibody complex, where the C1q subunit binds the pathogen surface directly.  

Binding of the C1q subunit to initiating complexes/pathogen produces a conformational change to the protein and results in activation of the C1r subunits.  C1r is a serine protease which once activated cleaves C1s (another serine protease).  The C1r2s2 then cleave C4 and C2 in order to assemble the classical pathway C3-convertase (C4bC2a).  C3-convertase then cleaves C3 into C3a and C3b the latter of which combines with the C3-convertase to form C5-convertase causing a cascade of subsequent cleavage and activation.  The terminal complement complex (TCC) consists of C5b-C9 and forms the Membrane Attack Complex (MAC).

Mannan-Binding Lectin Pathway

The Mannan-Binding Lectin (MBL) pathway is similar to the Classical pathway as after activation both proceed through the activation of C4 and C2 to form C3-convertase and the onward cascade.  However, the MBL pathway is not dependant upon antibody complexes and is a non-specific immune response. 

Mannan-binding lectin (also known as mannose-binding protein) is a member of the collectin familiy and initiates the complement cascade by binding to pathogen surfaces.  The MBL binds to terminal mannose, glucose or other sugar molecules on carbohydrate or glycoproteins components of microorganisms including bacteria such as salmonella and listeria.  Fungal pathogens such as Candida albicansand Cryptococcus neoformans and some viruses including Respiratory syncytial virus (RSV) are also bound by MBL.

Ficolins are soluble pattern recognition molecules that can also activate the complement cascase.  There are three human ficolins: L-ficolinH-ficolin and M-ficolin (also known and ficolin-2, -3 and -1)  which are structurally similar to collectins, such as MBL, and surfactantproteins A and D.  Ficolins can also initiate the MBL complement pathway (sometimes referred to as lectin pathway) via the MASP proteases. 

MBL is an important component of the innate immune system, serving as a pattern recognition molecule in the initiation of the complement cascaded and exerting other more subtle immunomodulatory effects.  Only normally oligomerised MBL is functional and capable of binding to pathogens and associating with MASPs.  Each subunit of the hexameric MBL is made up of three MBL monomers which join in a collagen helix.  The six subunits then join to form the functional MBL molecule.

MBL deficiency is the most common primary immunodeficiency with approximately 12% of the Caucasian population having insufficient levels of functional oligomerised MBL.   Such deficiency is associated with increased susceptibility to infections and greater disease severity and in aggressive antibiotic treatment can be required in high risk patients such as those on chemotherapy or immunosuppressants.

Alternative Pathway

The Alternative pathway is continually active at a low level as a result of spontaneous hydrolysis of C3 where the thioester bond is cleaved to form C3(H2O).  The conformational change permits binding of Factor B to the hydrolysed C3 and enables the serine protease Factor D to cleave Factor B.  The Ba fragment is released whilst the second Bb fragment remains bound the to the C3 complex forming C3(H2O)Bb which is also known as fluid-phase C3 convertase.  This is an unstable complex that can be stabilised by the binding of properdin (Factor P).

The alternative pathway C3-Convertase can cleave C3 to give C3a and C3b and binding of the latter to the C3-Convertases produces the alternative pathway C5-Convertase (C3bBbC3b) which continues the complement activation cascade and assembly of the membrane attack complex.

Hydrolysed C3 can bind to host and pathogen surfaces, but host cells can limit complement activation via endogenous complement regulatory proteins.  Formation of C3-Convertase can be prevented by the action ofFactor I, a protease that inhibits free C3b by cleaving the protein to give the inactive iC3b.  Such inhibition by Factor I also requires a C3b-binding cofactor such as Factor H, Complement Receptor 1 (CR1 or CD35) or Membrane Cofactor of Proteolysis (MCP or CD46).  CR1 and Decay accelerating factor (DAF or CD55) can also regulate the alternative pathway by competing with Factor B to bind the hydrolysed C3 and can even displace Factor B from the C3bBb complex.

Membrane Attack Complex

Activation of the complement cascade by any of the three pathways can result in the assembly of the membrane attack complex (MAC).  The MAC is initiated when C5-convertase cleaves C5 into C5a and C5b.  C5a is released and C5b is then bound by C6.  The C5bC6 complex is then bound by C7 which alters the protein configuration exposing a hydrophobic site on C7 that allows the C7 to insert into the phospholipid bilayer of the pathogen. 

Similarly, when C8 and C9 bind to the complex hydrophobic sites are exposed allowing insertion of these proteins into the bilayer.  C8 comprises tow proteins, C8-beta and C8-alpha-gamma, it is the latter that inserts into the lipid membrane and induces the polymerisation of C9 into a pore-forming structure.  Between 10 and 16 molecules of C9 will link to one another to form a ring structure in the membrane called the Membrane Attack Complex.  These transmembrane channels disrupt the phospholipid membrane of targeted cells, permitting free diffusion of molecules in and out of the cell.  If sufficient pores are formed within the cell membrane this will result in cell lysis and death.

A complement regulatory protein called Protectin or Membrane inhibitor of reactive lysis (MIRL or CD59) inhibits the membrane attack complex by binding C5b678 and preventing the binding and polymerisation of C9.  It is present on “self” cells to prevent damage by complement and some viruses such as HIV and human cytomegalovirus will incorporate the host CD59 into their own viral envelope to avoid lysis by MAC.

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