**3. Representative results**

After incubation, the ACP activity is calculated after measuring the diameter using the vernier caliper or taking the ACP dish in photo and measure each surface well by *Image J®* (**Figure 1**). Area surfaces are calculated by the formula S = π\*r2 ,

**107**

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

with r = d/2. (r: radius, d: diameter, s: surface). The same procedure is applied to the different NHP dilutions included to establish the calibration curve. This is because diluting NHP means that all ACP components are diluted equally. As example of interpretation, the more the ring is small the more the effectiveness of Eculizumab is good. The absence of reaction in a well signifies a complete ACP blockage, either

*ACP dishes for different sera from patients and pooled normal plasmas NHP. (A) Rings size of well 1 and 9 are found in a complete ACP blockade by Eculizumab or total deficiency of one of the ACP proteins. The remaining wells show different plasmatic levels of ACP. (B) Sera tested in this ACP dish are within normal* 

1.Chicken erythrocytes (CE) conserved in Alsever's solution.

5.AP buffer is GVB containing 5 mM Mg and 5 mM ethylene glycol bis[βaminoethylether]N,N′-tetraacetic acid (EGTA, E4378 Sigma).

7.Barbitone buffer is made by mixing 0.1 M solutions of sodium barbitone and barbituric acid to obtain the target pH and adjusting volume to obtain required

1.Suitable Petri dishes or glass plates. Size depends on the number of samples. Volumes can be adjusted so that the final depth of the gel is ~1–1.5 mm.

2. 56°C incubator and dishes/plate warmer (water bath can also be used).

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

by a drug or by a pathological process.

*range but vary according to the clinical presentation.*

2.Phosphate-buffered saline (PBS).

3.Veronal buffered saline (VBS).

4.Gelatin veronal buffer (GVB).

final molarity.

3.46°C water bath.

**3.2 Equipment**

6.N-saline is 9 g NaCl dissolved in 1 L H2O.

**3.1 Materials**

**Figure 1.**

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

#### **Figure 1.**

*Biochemical Testing - Clinical correlation and Diagnosis*

b.Wash twice in ACP buffer.

supernatant at 412 nm.

level tray. Mix carefully and quickly.

with a pipette tip or gloved finger.

standards are needed on each dish.

tion of % normal activity in test samples.

well.

rature.

1 cm apart.

but this is optional.

**3. Representative results**

blood stored in Alsever's solution.

a.Under aseptic conditions, remove 100 μl packed CE from stock chicken

c.Resuspend in the same buffer to the required concentration for assay by 2.1 mL of ACP buffer and put the cell suspension in a water bath at a temperature of 46°C. The concentration of CE can be calculated by lysing 0.1 mL of the stock CE in 2.9 mL H2O and measuring the absorbance of the

3.Melt 2% agarose stock (most conveniently in a microwave oven although immersion in boiling water will suffice) and, using a warm pipet, pipet 12.25 mL aliquots into universal containers (one for each gel), keep at 56°C.

4.Add the 9.8 mL warmed 1×ACP (56°C) to each bottle of melted agarose. Mix

5.To ease pouring the gel, place the warmed Petri dish (or rectangular plate) on

6.Pour the mixture evenly onto the level plate. The mixture should go to the mid edges of the Petri dish/rectangular plate. Remove bubbles by touching them

7.Cool plate to 4°C, punch holes using a Pasteur pipette upside down at least

8.Fill wells with a measured serum volumes (30 μL). Include a normal human plasma (NHP) standard, and NHP diluted 1/2 and 1/4 on each individual dish/ plate. The size of rings depends on factors such as gel thickness, and NHP

37°C. Incubate at 37°C for 1–2 h. Incubate either overnight at 22°C and 2 h at 37°C.

lysis can be read after photography, or after making direct photographic prints,

10. Measure the diameters of the rings of lysis and calculate the areas. Areas of

11. Standard curve: A crude standard curve is drawn by plotting % concentration NHP *vs.* area of lysis (diameter squared can also be used). This allows calcula-

After incubation, the ACP activity is calculated after measuring the diameter using the vernier caliper or taking the ACP dish in photo and measure each surface well by *Image J®* (**Figure 1**). Area surfaces are calculated by the formula S = π\*r2

,

9.Incubate overnight at 4°C, examine Petri dish before transferring to

Transfer one bottle with diluted agarose to 45°C and allow to cool to this tempe

**106**

*ACP dishes for different sera from patients and pooled normal plasmas NHP. (A) Rings size of well 1 and 9 are found in a complete ACP blockade by Eculizumab or total deficiency of one of the ACP proteins. The remaining wells show different plasmatic levels of ACP. (B) Sera tested in this ACP dish are within normal range but vary according to the clinical presentation.*

with r = d/2. (r: radius, d: diameter, s: surface). The same procedure is applied to the different NHP dilutions included to establish the calibration curve. This is because diluting NHP means that all ACP components are diluted equally. As example of interpretation, the more the ring is small the more the effectiveness of Eculizumab is good. The absence of reaction in a well signifies a complete ACP blockage, either by a drug or by a pathological process.
