**1. Introduction**

Complement system is the pillar of the immune system by its dual role in homeostasis and disease. It is the first line of the innate immunity and augments adaptive immunity. Indeed, complement acts as a rapid and efficient immune surveillance system that has distinct effects on healthy and altered host cells and foreign intruders through a complex cascade of proteases [1]. Activation of the pathway occurs through three primary pathways: classical, lectin, and alternative pathways. Instead to the other pathways and in addition to properdin as the initiating molecule, the alternative complement pathway (ACP) is activated via a low level of constitutive spontaneous hydrolysis of C3 in a process known as tick-over. Importantly, thanks to its amplification loop, ACP plays a major role for the final effect of initial specific activation of the classical and lectin complement pathways and contributed to 80–90% of any C5 activation regardless the initiating pathway [2]. Interestingly, ACP has been shown to play a particularly important role in preclinical disease models [3].


#### **Table 1.**

*Clinical presentations of ACP proteins acquired or genetic abnormalities.*

The ACP functional assessment constitutes an unmet need in medicine and applied research fields as the health valorization of bio-molecules extracted from nature. In clinical field it has at least nine applications in disease diagnosis and follow-up. For instance in therapy monitoring, it allows to screen patients responders to the complement blockers like Eculizumab, a patient with abolished activity means that he has no C5 mutation and is considered as eligible to this therapy. Furthermore, ACP activity makes possible to assess drug effectiveness at the plasmatic level [4]. Moreover, by evaluating ACP function we can predict and avoid immune-complex diseases flares and end organs damages as in systemic lupus erythematosus (SLE).

For the disease diagnosis, several international consensuses include functional hypocomplementemia and ACP abnormalities as a diagnostic criterion:


**105**

**2.3 Procedure**

*Semi-Solid Phase Assay for the Alternative Complement Pathway Activity Assessment (AP100)*

5.Hypocomplementemic hypersensitivity reactions to synthetic hemodialysis membrane at the origin of cardiovascular complication, the most frequent and

6.Several clinical presentations linked to ACP abnormalities as resumed in

7.ACP is particularly considered in sepsis, due to its uncontrolled amplification

The procedure is essentially the same as for the ACP by kinetic fluid phase assay except the concentration of AP buffer and the chicken erythrocyte density used to make the gels [11]. A value of 100% of plasma ACP function should be defined by the pooled normal human plasmas (NHP standard), prepared from a total of 100

Patient serum samples for the functional hemolytic assays need to be fresh, that is serum should be separated on the day of venepuncture and used the same day, or stored at −80°C. This is probably the single most difficult, yet important, step because if the cold chain is broken, the results become impossible to interpret correctly [12]. Whole blood, with ethylenediaminetetraacetic acid (EDTA) as the

Chicken red blood cells (CRBC) was collected in tubes containing Alsever or

1.Phosphate-buffered saline (PBS) contains: 137 mM NaCl, 2.7 mM KCl, 1.5 mM

3.Agarose: use an agarose that has a low melting point as plates are easier to pour.

1.Before starting, in a sterile tube put 9.8 mL of 1×ACP buffer at 56°C. Also let warming some Petri dish (or rectangular plate) in the 56°C incubator.

2.Preparation of chicken erythrocytes (CE) for ACP assay

KH2PO4, 8.1 mM Na2HPO. Sodium azide can be added if required.

2.Alternative complement pathway buffer (ACP buffer) is PBS containing 100 mM ethylene glycol-bis(β-aminoethyl ether)N,N′,N′-tetraacetic acid (EGTA) and 7 mM MgCl2; resulting in chelation of Ca2+, but not Mg2+, and providing additional Mg2+ [13]. This prevents complement activation via the classical pathway and facilitates complement activation via the ACP in agarose gels. ACP buffer/gelatin contains 1 g/L gelatin and is used when protein

20% (v/v) acid citrate dextrose (ACD) and stored at 4°C.

*DOI: http://dx.doi.org/10.5772/intechopen.81743*

**Table 1** [2].

**2. Protocol**

**2.1 Samples**

in sepsis conditions [10].

healthy individuals separate for each sex.

anticoagulant can also be used.

**2.2 Buffers and other reagents**

concentrations are low.

life-threatening complication in hemodialysis [9].

*Semi-Solid Phase Assay for the Alternative Complement Pathway Activity Assessment (AP100) DOI: http://dx.doi.org/10.5772/intechopen.81743*

