Materials and Reagents

Pipette tips: 1,250 µl, 200 µl, 10 µl (Sapphire, Greiner Bio-One, catalog numbers: 750-257, 737-257, 771-257, respectively)
1.5 ml Eppendorf tubes (Greiner Bio-One, catalog number: 616261)
50 ml Falcon tubes (Cell Star, Greiner Bio-One, catalog number: 227261)
15 ml Falcon tubes (Cell Star, Greiner Bio-One, catalog number: 188271)
Membrane filter, 0.22 µm pore size (Millipore, MF-Millipore^TM, catalog number: GSWP04700)
Adhesive film for microplates, polyester film non-sterile (VWR, catalog number: 60941-062)
96-well U-bottom clear microplate (Greiner Bio-One, catalog number: 650101)
96-well F-bottom clear microplate (Greiner Bio-One, catalog number: 655101)
Reagents reservoirs (Pierce, ThermoFisher Scientific, catalog number: 15075)
Normal human serum pool (NHS), stored in aliquots at -80°C (in house prepared from freshly collected blood from healthy volunteers, n = 3)
Normal male mouse serum (NMS) stored at -80°C (in house prepared from freshly collected blood, n = 22). The complement lytic activity in C57BL/6 female mouse is significantly reduced compared to male (in our hands ~40-50%) due to higher production of androgen and estrogen hormones that lower levels of terminal pathway proteins C5 and C6 (Churchill et al., 1967; Kotimaa et al., 2016).
Normal human C5-depleted serum (C5D; C5 used here as an exemplar; this method can be used to deplete any other complement components from the serum), generated by passage of NHS (in house prepared from freshly collected blood from healthy volunteers, n = 3) over an immunodepletion column comprising an in-house anti-C5 mAb immobilised on sepharose (GE Healthcare, 1 ml HiTrap NHS-Activated HP, catalog number: 17-0716-01), stored in aliquots at -80°C.
HEPES (Fisher Chemicals, Fisher Scientific, catalog number: BP310-500)
Magnesium chloride (Acros Organics, Fisher Scientific, catalog number: 2232/0010)
Calcium chloride (Fisher Chemicals, Fisher Scientific, catalog number: C/1400/53)
Sodium chloride (Alfa Asear, Fisher Scientific, catalog number: 12314)
Sodium hydroxide (Macron Fine Chemicals, Fisher Scientific, catalog number: 7066-33)
Sheep Blood in Alsever's Solution (TCS Biosciences, catalog number: SB069) as a source of sheep erythrocytes (ShE); over time, the ShE will pellet at the bottom of the Nalgene bottle, and the pelleted ShE are harvested for assaying
Rabbit anti-ShE antiserum (Siemens Amboceptor, Cruinn Diagnostics, catalog number: ORLC25)
Mouse anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (ThermoFisher Scientific, Invitrogen, catalog number: 31213)
Alsever’s solution (Merk, Sigma-Aldrich, catalog number: A3551-1L)
Purified human C5, stored at -80°C, by-product of the C5D serum preparation (also available at Hycult Biotech, catalog number: HC2141)
HBS buffer (see Recipes)

Equipment

Pipettes (Eppendorf, model: Eppendorf Research^® plus, catalog numbers: 3123000918, 3125000060)
Incubator (Memmert 0135)
Shaking water bath (VWR, model: 462-0494)
Centrifuge (ThermoFisher Scientific, Heraeus megafuge 40R, TX-1000 rotor)
Microplate reader (Tecan, Infinite F50)
Analytical balance (A&D Company Limited, model: FZ-300i-EC)
Stirrer (Stuart, Bio-Cote)
pH meter (Jenway Scientific Laboratories Supplies SLS, model: RS232)

Software

GraphPad Prism (Graphpad Holdings, LLC)

Procedure

Note: All procedures are performed in the standard laboratory settings. All chemicals used are of analytical grade.

Serum preparation
Human and animal sera were prepared in-house from freshly collected blood. For human, blood was collected into glass bottles, clotted at room temperature (RT) for 1 h, and then placed on ice for 2 h for clot retraction prior to centrifugation (2,000 × g for 15 min at 4°C) and harvesting of serum. For mouse, blood was collected into Eppendorf tubes by tail bleeding, placed on ice immediately after harvest and clotted for 2 h on ice prior to serum harvest. Sera were filtered (via 0.22 µm filter) and stored in aliquots (100 or 500 µl) at -80°C.

Preparation of HBS buffer
HEPES-buffered saline (HBS; 0.01 M HEPES, 0.15 M NaCl, 135 µM CaCl₂, 1 mM MgCl₂, final pH 7.4) prepared as previously described (Zelek et al., 2018).

Preparation of Antibody-Sensitized Sheep Erythrocytes for Classical Pathway Assay (ShEA)
1. Pipette 400 μl of the settled ShE pellet (see Materials #18), and add to 20 ml HBS with gentle mixing.
2. Wash the ShE three times in 20 ml HBS by centrifugation (1,500 × g, 10 min, 4°C) and resuspension in 20 ml fresh HBS with gentle mixing. The supernatant, red in first wash, should be clear by the final wash, confirming that the cells are viable for use.
3. Resuspend the final pellet in 10 ml HBS containing Amboceptor (usually 1:2,000 dilution, although working dilution should be titrated for each new batch to optimally sensitise without causing agglutination) at 37°C; add the solution slowly with mixing. Incubate the mixture for 30 min at 37°C, shaking in a water bath.
4. Wash the sensitised ShE (ShEA) as above and resuspend the final pellet in 20 ml HBS. ShEA can be stored at 4°C for up to one week or in igloo in a cold room (0°C equivalent) for up to two weeks with ice replaced as necessary.

Calculating concentration of ShEA
1. Aliquot the ShEA suspension into wells of a 96-well U-bottomed plate (50 μl/well), and add 100 μl/well of ddH₂O (in triplicate).
2. Centrifuge plate (1,500 × g, 3 min, 4°C).
3. Transfer 100 μl of the supernatant into an F-bottom plate. The supernatant contains ddH₂O and haemoglobin (red in colour) released from the ShEA. All cells should be lysed; ghost cells are not visible to the naked eye.
4. Measure the absorbance of the supernatants at 405 nm using the plate reader (lysed cells release haemoglobin, which creates a measurable red colour readout); the absorbance of OD1 equates to 8.5 × 10⁷/ml original concentration.

Testing ShEA for sensitivity to lysis by human serum
1. In 96-well U-bottomed plate, prepare NHS serial dilutions (in triplicate; 10-0%) in HBS (50 μl/well), and add 50 μl of 2% ShEA (prepared as described above, see section C) and 50 μl of HBS (total volume per well = 150 µl) into each well.
2. Prepare controls (in triplicate):
  1. 0% lysis: 50 μl ShEA + 100 µl of HBS
  2. 100% lysis: 50 μl ShEA + 100 µl of ddH₂O
3. Seal the plate and incubate at 37°C for 30 min.
4. Centrifuge plate (1,500 × g, 3 min, 4°C).
5. Transfer 100 μl of supernatant into an F-bottom plate.
6. Measure the absorbance of the supernatants at 405 nm using the plate reader. Optimally sensitised ShEA will be fully lysed (100% compared to controls) by NHS at 10% and above, under these conditions.

Further sensitisation of ShEA for mouse classical Pathway Assay (ShEA-m)
1. Take 5 ml of the 2% ShEA in HBS.
2. Pellet the cells (centrifuge at 1,500 × g for 10 min, 4°C)
3. Add pre-warmed (at 37°C in the water bath) mouse anti-rabbit IgG at a final concentration of 25 μg/ml in 5 ml of HBS and resuspend the cells.
4. Incubate at 37°C in the shaking water bath for 30 min.
5. Wash the cells three times (in 20 ml HBS by centrifugation; 1,500 × g, 10 min at 4°C) to remove excess antibody, resuspend in 5 ml HBS; ShEA-m can be stored on ice for up to two weeks.
6. Test the ShEA-m for sensitivity to lysis by mouse serum using dilutions of NMS from 100 to 0% (100% equates to a final dilution of 33% in the assay). In a 96-well U-bottomed plate, prepare serial dilutions (in triplicate) between 100 and 0% NMS (50 µl/ well) in HBS and add 50 μl of 2% ShEA-m and 50 μl of HBS (total volume per well = 150 µl) into each well.
7. Prepare controls (in triplicate):
  1. 0% lysis: 50 μl ShEA-m + 100 µl of HBS
  2. 100% lysis: 50 μl ShEA-m + 100 µl of ddH₂O
8. Seal the plate and incubate at 37°C for 30 min.
9. Centrifuge plate (1,500 × g, 3 min, 4°C).
10. Transfer 100 μl of supernatant into an F-bottom plate.
11. Measure the absorbance of the supernatants at 405 nm using the plate reader. Optimally sensitised ShEA-m will be fully lysed (100% compared to controls) by NMS at 100% under these conditions.

Assay method – modified mouse CP CH50 assay
1. In a U-bottom plate, prepare a serial dilution series (in triplicate) of NMS (100-0%; 50 μl/well). Add 50 μl of 2% ShEA-m, followed by 50 μl of HBS (total volume per well, 150 μl; final serum concentration, 33-0%).
2. Prepare controls (in triplicate):
  1. 0% lysis: 50 μl ShEA-m + 100 µl of HBS
  2. 100% lysis: 50 μl ShEA-m + 100 µl of ddH₂O
3. Seal the plate and incubate at 37°C for 30 min.
4. Centrifuge the plate at 1,500 × g for 3 min at 4°C.
5. Using a 12-channel multipipette, transfer 100 μl supernatant from each well to an F-bottom plate, taking care not to disturb the pelleted unlysed ShEA-m.
6. Measure the absorbances at 405 nm using plate reader within 10 min.
7. Calculate haemolysis; refer to Figure 2 for example calculations.
  Please see the step-by-step assay workflow in Figures 3 and 4 below.
  
  Figure 2. Example data of mouse CP CH50 assay utilising double sensitised ShEA-m. A. Assay plate setup and calculations; red colour indicates the signal intensity. B. CH50 plotted for NMS dilution series (100-0%). The dotted lines show the serum dose to give 50% ShEA lysis. NMS: normal mouse male serum.
  
  Figure 3. Workflow of the modified mouse CP CH50 assay
  
  Figure 4. Pictured step-by-step workflow of the modified mouse CP CH50 assay

Assay method – titration of mouse component-specific CH50 haemolytic assay
1. Prepare serial dilution series (5-0%; 50 μl/well) of NHS, NMS, C5D, and C5D supplemented with 25% NMS in a U-bottom plate (in triplicate).
2. Add 2% ShEA suspension in HBS (50 μl/well).
3. Add 50 μl/well HBS to NHS, C5D, and NMS (total volume per well: 150 μl; serum concentration: 1.7-0%).
4. Prepare controls (in triplicate):
  1. 0% lysis: 50 μl ShEA + 100 µl of HBS
  2. 100% lysis: 50 μl ShEA + 100 µl of ddH₂O
5. Seal the plate and incubate at 37°C for 30 min.
6. Stop the reaction by spinning down the plate at 1,500 × g for 3 min at 4°C.
7. Transfer 100 μl supernatant of each row to an F-bottom plate without disturbing the pelleted ShEA.
8. Measure the absorbances at 405 nm within 10 min using the plate reader; see Figure 5 for the example calculations.
  
  Figure 5. Example data of CP CH50 assay utilising human depleted sera and mouse serum (C5 as example). A. Assay plate setup and calculations; red colour indicates signal intensity. B. CH50 plotted for NMS (▼; 5-0%), human C5D (♦), C5D supplemented with NMS (25%; ▲), and NHS (●). The assay controls are included in the same assay. The dotted lines show the serum dose to give 50% ShEA lysis. NMS, normal male mouse serum; NHS, normal human serum; C5D, human C5 depleted serum.

Assay method – add back mouse component-specific CP CH50 haemolytic assay
1. Prepare serial dilution series (10-0%; 50 μl/well) of NHS, C5D + NMS (25% vol:vol), and C5D + human (75 μg/ml) in a U-bottom plate (in triplicate).
2. Add 2% ShEA suspension in HBS (50 μl/well).
3. Add 50 μl/well HBS (total volume per well, 150 μl; serum concentration, 3.33-0%)
4. Prepare controls (in triplicate):
  1. 0% lysis: 50 μl ShEA + 100 µl of HBS
  2. 100% lysis: 50 μl ShEA + 100 µl of ddH₂O
5. Seal the plate and incubate at 37°C for 30 min.
6. To stop the reaction, spin down the plate at 1,500 × g for 3 min at 4°C.
7. Transfer 100 μl supernatant of each row to an F-bottom plate without disturbing the pelleted ShEA.
8. Measure the absorbances at 405 nm within 10 min using the plate reader; see Figure 5 for the example calculations.

Data analysis

Confirm controls show respectively 0 (Background) and 100% (Maximum) lysis.
Calculate % Lysis in test sample as follows:
% Lysis = (Sample A₄₀₅ – Background A₄₀₅)/(Maximum A₄₀₅ – Background A₄₀₅) × 100%
See Figure 6 for the example calculations.

Figure 6. Example data of CP CH50 assay utilising human depleted sera and mouse serum. A. Assay plate setup and calculations; red colour indicates signal intensity. B. Haemolysis for samples with the highest concentration of serum (10%), including the assay controls. C. Calculating CH50. The graph shows a log-log plot of Y-axis % of lysis vs. X-axis % of diluted serum for a test sample. The broken line from the Y-axis intercepts this point on the hemolysis curve, and the intercept from this point to the X-axis permits the serum volume at 50% hemolysis to be read. The dotted lines show the serum dose to give 50% ShEA lysis. D. Data exported from GraphPad (EC50) and calculations to obtain CH50 values; the standard NHS sample measurement is designated a CH50 of 100 HU. CH50s of samples are calculated by applying the formula NHS EC50/Sample EC50 × 100.

Calculate SEM ± SD % lysis of test triplicates using GraphPad Prism.
Plot dilution curves and calculate CH50 (the amount of serum giving 50% hemolysis) for each test sample (see Figure 6D).
Determine the statistical significance of differences between samples (e.g., using the Student’s t-test (parametric) or one-way ANOVA/Tukey’s test) compared to the appropriate control using GraphPad Prism.

Notes

The TCS product is sheep blood diluted in Alsever’s solution. To prolong viability of erythrocytes, upon receiving, the cells are pelleted by centrifugation and resuspended in fresh Alsever’s solution to remove other blood products that can cause loss of viability on storage. Washed erythrocytes can be stored in Alsever’s solution (5% vol:vol) at 4°C for up to 3 months.
ShEA and ShEA-m can be stored in Alsever’s solution on ice for up to 2 weeks and for at least a week in 4°C refrigerator without loss of activity.

Recipes

HBS buffer

Reagent	Molarity	Mass
HEPES	0.01 M	2.4 g
NaCl	0.15 M	8.75 g
CaCl₂	135 µM	0.015 g
MgCl₂	1 mM	0.1 g
ddH₂O	n/a	Up to 1 L

Dissolve the salts in ~980 ml of ddH₂O.
Adjust pH of the solution to 7.4 with 5 M NaOH (prepared by dissolving 20 g NaOH in 100 ml of ddH₂O).
Measure the solution volume, add more ddH₂O, up to 1 L.
Re-measure pH (7.4).
Filter via 0.2 µm filter.

Acknowledgments

I would like to thank Professor Paul Morgan for his constructive criticism in preparation of this manuscript and for his ongoing support. Thank the members of the Complement Biology Group at Cardiff University for donating blood for the research. WMZ is supported by a Health and Care Research Wales Fellowship from Welsh Government. This protocol was inspired by our recent research published in Frontiers in Immunology (Zelek and Morgan, 2020).

Competing interests

I have no conflicts of interest or competing interests.

Ethics

Mouse serum was collected under UK Home Office regulations. Human serum was collected under Medical/Dental School Research Ethics Committee regulations; informed consent obtained.

References

Carpanini, S. M., Torvell, M. and Morgan, B. P. (2019). Therapeutic inhibition of the complement system in diseases of the central nervous system. Front Immunol 10: 362.
Churchill, W. H., Weintraub, R. M., Tibor, M. D. and Rapp, H. J. (1967). Mouse complement; the effect of sex hormones and castration on two the late acting components. J Exp Med 125(4): 657-672.
Kotimaa, J., Klar-Mohammad, N., Gueler, F., Schilders, G., Jansen, A. and Rutjes, H, et al. (2016). Sex matters: Systemic complement activity of female C57BL/6J and BALB/cJ mice is limited by serum terminal pathway components. Mol Immunol 76:13-21.
Mayer, M. M. (1967). Complement and complement fixation. Exp Immunochem 133-240.
Menny, A., Serna, M., Boyd, C. M., Gardner, S., Joseph, A. P. and Morgan, B. P., Topf, M., Brooks, N. J. and Bubeck, D. (2018). CryoEM reveals how the complement membrane attack complex ruptures lipid bilayers. Nat Commun 9(1): 5316.
Moleón, M. S., Merchant, M. E., Ortega, H. H. and Siroski, P. A. (2006). Genomic view of the evolution of the complement system. Innumogenetics 58(9): 701-713.
Morgan, B. P. and Harris, C. L. (2015). Complement, a target for therapy in inflammatory and degenerative diseases. Nat Rev Drug Discov 14(12): 857-877.
Podack, E. R. and Tschopp, J. (1984). Membrane attack by complement. Mol Immunol 21(7): 589-603.
Rice, C. E. and Boulanger, P. (1952). The interchangeability of the complement components of different animal species. J Immunol 68(2): 197-205.
Serna, M., Giles, J. L., Morgan, B. P. and Bubeck, D. (2016). Structural basis of complement membrane attack complex formation. Nat Commun 7: 10587.
Slodkowicz, G. and Goldman, N. (2020).Integrated structural and evolutionary analysis reveals common mechanisms underlying adaptive evolution in mammals. Proc Natl Acad Sci USA;117(11): 5977-5986.
Zelek, W. M. and Morgan, B. P. (2020). Monoclonal Antibodies Capable of Inhibiting Complement Downstream of C5 in Multiple Species. Front Immunol 11: 612402.
Zelek, W. M., Xie, L., Morgan, B. P. and Harris, C. L. (2019a). Compendium of current complement therapeutics.Mol Immunol 114:341-352.
Zelek, W. M., Taylor, P. R. and Morgan, B. P. (2019b). Development and characterization of novel anti-C5 monoclonal antibodies capable of inhibiting complement in multiple species. Immunology 157(4): 283-295.
Zelek, W. M., Harris, C. L. and Morgan, B. P. (2018). Extracting the barbs from complement assays: Identification and optimisation of a safe substitute for traditional buffers. Immunobiology 223(12): 744-749.