Various assays/methods can be used to mesasure activities of complement pathway molecules and activaiton of the complement system. (see US Patent No. 6,087,120 and Newell, J Lab Cin Med, 100:437-44, 1982). The two most commonly used techniques are hemolytic assays and immunological assays.

Definitions/General Principals

(1) EGTA blocks the classical C pathway because it chelates Ca++ and thus inactivates C1 (Platts-Mills, J. Immunology, 113(1), 1974).

(2) Amplifcation loops of the AP: The AP pathway involves an amplifcaiton loop utilizing C3b producec by the CP and lectin pathways. Some molecules of C3b generated by the CP C3 convertase are funneled into the AP. Surface bound C3b binds Factor B to yield C3bB, which becomes a substrate for Factor D of the AP. Factor D is a serine esterase that cleaves the Ba fragment, leaving C3bBb bound to the surface of the target cell. C3bBb is stablized by proeprdin, forming C3bBbP, which acts as the AP C3 convertase. This C3 convertase participates in an amplifcaiton loop to cleave many C3 molecules, resulting in the deposition of C3b on the target cell. Some of these C3b bind back to C3bBb to form C3bBb3b, the AP C5 convertase which cleaves C5 into C5a and C5b. C5b binds to the surface of the cell to initiate the formation of MAC. (Fung, 2006/0140939).

Antibody sensitized sRBCs only activate the CP. They do not, by themselves, activate the AP. However, in the presence of sufficient NHS (e.g., 7-10%) activated CP will utilize the amplication loop of the AP. (Bansal, US13/646,286).

(3) Calcium/magnesium: The CP is a calcium/magnesium dependent cascade whereas the AP is a magnesium dependent cascade. With the lectinpathway, Ca++ dependent binding of MBL to a mannan coated surface triggers activaiton of C3. C2 is a single chain plasma protein that is specific for the CP and LP. Membrane bound C4b expresses a binding site which, in the presence of Mg++, binds the proenzyme C2 near its amino termus and present it for cleavage by C1s (for the CP) or MASP-2 (for the LP) to yeild a 30 kD amino terminal fragment, C2b, and 70 kD carboxy terminal fragment, C2a.  (Fung WO01/70818).

Hemolytic Assays/techniques

Hemolytic Assays measure the functional capacity of the entire pathway.

Erythrocyte Lysis Assays: 

Erythrocyte lysis assays are based on the formation of a terminal complement complex on the surface of rabbit red blood cells (rRBC). As a reuslt of the formation of this complex, the rRBCs are lysed. The progressive decrease in light scatter at 700 nm is a direct measure of erythrocyte lysis.  

Assays for the Alternative Pathway (AP)

Rabbit erythrocyte hemolysis:

It is well established that rabbit erythrocytes specifically activate the complement AP, with a resulting lysis of the cells by the C5b-9 complex (Bansal, US 2005/0107319)

Alternative Pathway Isolated from the Classical Pathway: 

It is accepted that AP activation is dependent on Mg++ but not Ca++ (Platts-Mills, J. Immunology, 113(1), 1974). AP activation in Mg++ ions without calcium ions guarantees only the AP activaiton (Bansal, 13/646286). 

—-Rabbits RBCs in 10% NHS/Mg2+:EGTA: Introducing rabbit Erythrocytes (rRBC) into 10% human serum (with Mg2+/EGTA) represent the introduction of a foreign cell surface which initiates the AP cascade. AP activation in Mg++ ions without calcium ions (EGTA is used to chelate the calcium ions to prevent CP activation)guarantees only AP activation. Rabbit RCs can be used to activate the AP in 10% NHS in the presence of Mg2+ in the absence of Ca2+ and in the presence of sufficient NHS concentration . Because the CP requires the presence of Ca2+, the CP will not be active under these conditions. 

Example (anti-properdin): To determine the effects of a MoAb to propderin on alternative pathway (AP) function, increasing amounts of the antibody are added to human serum that is incubated with rabbit erythrocytes in buffer containin EGTA and Mg++. The cells cause AP activation which results in C5b-9 formation and consequent lysis of the cells. The extent of erythrocyte lysis can be monitored by examining the amount of light scattered by intact red blood cells (Polhill, 1978). Human serum + rabbit erythrocytes in buffer containing EGTA and Mg++. These cells cause AP activation, which results in C5b-9 formation and consequent lysis of the cells. (Bansal, US 13/646286)

—-Rabbit erythrocytes + 7.5% NHS + GVB-Mg+++-EGTA: Song (US2010/0263061) disclose incubation of rabbit erythrocytes with 7.5% NHS in GVB with or withoug anti-P antibodies or EDTA. Song shows properdin mAbs which dose dependently inhibited complement mediated lysis of the rabbit red blood cells. Anti-P antibodies and EDTA were used as positive controls for inhibition (EDTA blocks CP complement).

Assays for the Classical Pathway (CP)

The classical CP is typically triggered by immune complexes, for example, an antibody boudn to a foreign particle and thus requires prior exposure to that particle for the generation of specific antibody. (Gupta-Bansal (US 6,333,034). The CP is a calcium/magnesium dependent cascade. C1, the first enzyme complex in the cascade, is a pentamolecular complex consisting of C1q, 2 C1r molcules and 2 C1s molecules. This complex binds to an antigen-antibody complext through the C1q domain to initiate the cascase. (Fung, US 2005/1096394)

CH50 Assay:

The complement system is a group of proteins that wehn activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Inidividual ocmmplement components can be quantified; however this does not provide any informaiton as to the activity of the pathway. The CH50 is a screening assay for the activaiton of the CP and it is sensitie to the reduciton, absence and/or inactivity of any component of the pathway. The CH50 tests the funciotnal capability of serum complement components of the CP to lyse sheep red blood cells pre-coated with rabbit anti-sheep red blood cell antibody (haemolysis). When antibody-coated SRBC are incubated with test serum, the classical pathway of coplement is activated and haemolysis results. If a complement component is absent, the CH50 level will be zerio; if one or mroe components are decreased, the CH50 will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the sueprnatant at 540 nm. The amount of complement acitvity is determiend by examining teh capacity of various dilutions of test serum to lyse antibody coated SRBC. (Costabile “Measuring the 50% haemolytic complement (CH50) activity of serum” J Visualized experiments, 2010). 

Classical Pathway isolated from alternative pathway: 

—-Antibody Sensitized Sheep cells in 1% NHS/Ca2+/Mg2+: To measure the functional capacity of the classical pathway, sheep red blood cells coated with hemolysin (rabbit IgG to sheep red blood cells) are used as target cells (sensitized cells). These ag-Ab complexes activate the classical pathway and result in lysis of the target cells when the components are functional and present in adequate concentration. To determine functional capacity of the alternative pathway, rabbit red blood cells are used as the target cell (see US Patent No. 6,087,120). The antibody sensitized sheep cells are used as an activator in 1% normal human serum in the presence of Ca2+/Mg2+. The calcium ionsare required for activation of the CP for the initial trigger of the C1q/C1r/s complexes. CP will not occur in the basence of the calcium ions. Mg2+ is required for AP activation. However, in 1% normal human serum containing Ca2+/Mg2+, only the CP proceeds to completion. Without the requisite levels of NHS which is 10%, the AP pathway will not have a significant presence.  (Bansal, US 13/646286) 

Classical Pathway activation of the Alternative Pathway Via way of the Amplication Loop of the AP

—-Antibody sensitized sheep erythrocytes + 7.5 NHS + GVB-Mg++: Antibody sensitized sheep erythrocytes were incubated with 7.5 NHS in GVB-Mg++ buffer with or without anti-P antibodies or EDTA. Antibody sensitized sheep erythrocytes are another well established assay for the classical pathway complement activation.  Cells completely lysed by hypotonic shock were aslo sued as a control (100% lysis). The degree of lysis was determined by hemoglobin release using a spectrophotometer. Polyclonal anti-P antibody had no effect on human complement mediated lysis of the antibody sensitized sheep erythrocytes. In contrast, EDTA inhibited human complement mediated lysis and was used as a positive control. (Song, 2010/0263061).  Antibody sensitized sRBCs only activate CP. They do not, by themselves, activate AP. However, in the presence of sufficient NHS, activated CP will utilize the amplifcation loop of the AP. (Bansal, US 13/646,286).

—-Antibody sensitized sheep cells in in 10% NHS/Ca2+/Mg2+: For CP and AP action, antibody sensitized sheep red blood cells are used as an activator in 10% ca2+/Mg2+ in human human serum. The difference between the assay above for the CP only is that NHS is 10%. The Ca2+/Mg2+ provides the level of Mg2+ required for AP activation and allows for both CP and AP to be active. Antibody sensitized sRBCs only activate the CP. They do not by themselves activate the AP. However, in the presence of sufficient NHS, activated CP will utilize the amplification loop of the AP. Under these conditions, the C3b produced vial the CP can feed into the AP causing amplificaiton of the AP loop. In other words, the AP has been activated by the CP. (Bansal, US 13/646286)

Assays for the Lectin pathway

 Assays for the lectin pathway include measuring the functional activity of lectin-MASP complext through activation of added exogenous C4 or measuring endogenous activation of C4 after blocking of CP and AP using a high ionic strenght dilution buffer (1M NaCl) Palarasah (US2010/0196927).

Polyanethol sulphonate (PAS) + activation of lectin pathway:

Palarasah (US2010/0196927) discloses a method of determining functional deficiencies in the lectin pathway by diluting a sample with polyanethol sulphonate (PAS) which blocks the CP and AP, activating the lectin pathway and then determining activation of one or more complement factors C3, C4 of one or mroe components of the C5-C9 complex.

Immunologic Assays/techniques: 

Immunologic assays use antibodies agaisnt the different epitopes of the various complement components (e.g., C3, C4, and C5) to detect split product of the complement components (e.g., C3a, C4a, C5a and the C5b-9 terminal complex).

Alternative Pathway: To accomplish this assay one immobilizes LPS onto microtiter wells to activate the AP in diluted serum samples. Can then measure AP components like C5b-9 (MAC).

Particular Complement Components

—-C3b:

Example: LPS + 10% NHS/Mg++: LPS is a specific activator of the AP.  (Bansal, US 13/646286) AP activation generates C3 and C3b as a result of C3 cleavage by the C3 convertase of the AP. AP is acivated in NHS by LPS under conditions that allow activation of the AP. This assay was used to demonstrate whether an anti-properdin antibody would inhibit the foramtion and deposition of C3b which initiates the start of AP. As a way of mechanims, activated and deposited C3b provides high affinity binding to properdin. Properdin-C3b complexes bind factor B and the complex is cleaved by factor D to generate PC3bBb, an AP C3 convertase. As the AP proceeds, C5b-9 complexes are formed and deposited. Polystyrene microtiter plate wells were coated with LPS. Normal human serum (NHS) at 10% in AP buffer was mixed with varying concentrations of an anti-properdin antibody and C3b was detected with rabbit anti-human C3c, properdin was detected with goat anti-human P, Bb was detected with goat anti-human factor Bb and C5b-9 was detected with HRPO-conjugated neo-anti-human C5b-9. The presence of C3b, p and Bb and MAC together was indicative of AP C3 convertase formation, and the antibody was shown to inhibit C3b formation. (Bansal, US8,664,362 and 13/583879; 14/195, 458). 

—-C5 protein and fragments: Methods for determining whether an antibody can block the generation or activity of the C5a and/or C5b active fragments of C5 or binding to complement component C4b or C3b are known in the art (US6355245). 

—-C5a: Methods for measuring C5a activity include chemotaxis assays, RIAs, or ELISas (Ward and Zvaifler (1971) J Clin Invest 50(3): 606-16). 

Classical Pathway:

–CH50 EIA:  The binding of the C1q component of C1 to immune complexes triggers the CP which results in a cascade of enzymatic and non-enzymatic reactions culminating in the formation of terminal complement complexes (TCC). Th CH50 EIA measures the hemolytic complement (CG50) in human serum and allowed detection of a deficiency of one or more complement components C1-C9. It is a tradtional method for measuring the CP. In this lytic assay, antibody sensitized sheep erythroytes (EA) are used to activate the CP and various dilutions of the test serum are used to determine the amount required to give 50% lysis. The assay uses a mAb to a unique neoantigen to caputre the TCC analytic.

Lectin Pathway: Mannan can be as a ligand and coated on plates and then serum is added. Detection of MBL, C4 and C3 can then be performed with mAbs against these components. As both the LP and the CP are calcium dependent and lead to activation of C4, one challenge in devising an assay to assess the functional characterization of the LP is distinguishing its activation from activation of the CP. Using manna, for example, to activate the LP is also likely to activate the CP via anti-annan Ab which is present in huan serum.

Roos (Mol Immunol. 2003; 39: 655-68) describe a lectin pathway assay which uses a mAb to C1q to inhibit CP (blocks CP but allows lectin activation as by mannan to proceed normally) and a mAb to factor D to inhibit AP.

Petersen (J Immunol Methods, 2001, 257: 107-16) escribe blocking CP and AP using a high ionic strenght dilution buffer (1M NaCL) and then measuring endogenous activation of C4. 

Palarasah (US 12/676283) disclose an in vitro method for determining functional deficiencies in the lectin pathway by diluting a sample with a polyanion, preferably a sulphated polyanion and even more preferably polyanethol sulphonate (PAS), which is an inhibitor of the AP and CP, but not the lectin pathway, then activating the lectin pathway and then determining the activation of one or more of the complement factors C3, C4 or one or more of the components of the C5-C9 complex such as with an antibody, wherein a lower level compared to a normal reference level is indicative of a functional deficiency in the lectin pathway.