Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP₅₀ in vitro Assay

Materials and Reagents

1. Pipette tips (Axygen Scientific, catalog number: T-200-Y)
2. 1.5 ml flip-topped tubes (Axygen Scientific, catalog number: MCT-150-C)
3. Plastic film (Greiner multiwell plate sealer, Sigma-Aldrich, catalog number: A5596)
4. Borosilicate glass beads, 5 mm (Sigma-Aldrich, catalog number: Z143944)
5. 96-well flat bottomed plates (Costar, catalog number: 3596) 
6. Barbitone complement diluent tablets (Oxoid, catalog number: BR0016)
7. NaOH pellets (UNIVAR, catalog number: A482)
8. Concentrated HCl (ANALAR, catalog number: 101256J)
9. Human serum (HuS), non-heat inactivated, store at -20 °C (collected from discard blood following routine clinical venesection in accordance with Southern Adelaide Local Health Network, Human Ethics Research Committee approval #343.16-HREC/16/SAC/306)
10. Rabbit blood, freshly collected (under Flinders University Animal Welfare Committee approval to use scavenge material)
11. Purified FH, generated in-house from human blood and stored at -20 °C [Diluted to 1 µg/µl in vehicle prior to use (Cabezas et al., 2018)]
12. Vehicle = corresponding carrier for the test samples for example DMEM (Hyclone, catalog number: SH30022.01)
13. Sodium chloride (NaCl) (Santa Cruz, catalog number: sc-203274A)
14. Gelatin solution (2% solution) (Sigma-Aldrich, catalog number: G1393)
15. EGTA (Sigma-Aldrich, catalog number: E3889)
16. Veronal buffer (see Recipes)
17. AP buffer (see Recipes)
18. Saline (see Recipes)
19. 10 N NaOH (see Recipes)
    

Equipment

1. Pipettes (Thermo Fisher Scientific, model: Finnpipette F1)
2. 50-100 ml conical flask/Erlenmeyer flask (Thermo Fisher Scientific, catalog number: KIM 265520-1)
3. Oven (Labnet mini incubator)
4. Hemocytometer (Counting chamber) (Hawksley, catalog number: AC1000)
5. Plate centrifuge (Heraeus Sepatech Omnifuge 2.0 RS)
6. Microplate reader (multimode detector) (Beckman Coulter, model: DTX880)
   

Procedure

1. Preparation of rabbit RBC
   Note: All procedures are performed on a standard laboratory bench.
   
   1. Collect rabbit blood into a 50-100 ml flask containing a layer of glass beads and gently swirl to allow a clot to form. This should occur within 10 min and be complete by approximately 20 min.
   2. Decant the defibrinated blood. Allow the cells to settle under gravity for 2-5 min. Discard supernatant and wash in approximately 25 ml AP buffer. The starting solution will be red, then becomes clear upon washing. Once the final cell-free supernatant is clear (usually following 3-4 washes) the cells are ready for use. Store at 4 °C until use (maximum 1-2 months).
   3. Prior to use, re-wash RBC in approximately 25 ml AP buffer.
   4. Enumerate the cells using a hemocytometer and adjust to 5 x 10⁷ cells/ml with AP buffer.
   
   
2. Assay method
   
   1. Dilute HuS, 2 in 5 with AP buffer to chelate calcium and inactivate the CP and LP, resulting in a 40% HuS stock. For instance; add 720 μl of AP buffer to 480 μl of HuS in a 1.5 ml tube. 
   2. Incubate on ice, 15 min.
   3. In 1.5 ml flip-topped tubes, mix 40% HuS with the test substance and AP buffer in a total volume of 100 μl. Include final concentrations of HuS at 0, 5% and 10% HuS for each test sample. Include 0, 5% and 10% HuS alone, controls. Include 0, 5% and 10% HuS with 50 µg purified FH, as a negative regulator (inhibition) control. Volume should be reconstituted to 100 μl with an appropriate vehicle, i.e., the media corresponding to the test sample, such as DMEM or RPMI. Sample set up is shown in Table 1.
      Note: Each test sample is assayed with a final concentration of HuS at 5% and 10% to ensure activity is neither saturated nor at the limit of detection so that both activators and inhibitors of the AP can be measured.
       
      Table 1. Tube components for test samples and controls
      
      
      
   4. Add 50 μl of each sample mix from Step B3 into a 96-well plate, in duplicate or triplicate if desired.
   5. Add 50 μl of the RBC suspension prepared as above in Procedure A. Include a total hemolysis control (50 μl cells + 50 μl of water). An example of sample setup is shown in Figure 2A.
      
      
      Figure 2. Example data. A. Assay plate setup. Red = HuS alone controls; Blue = HS + FH inhibition controls; Green = test samples 1-8; Pink = 100% hemolysis; Purple = Blank control Grey = wells left empty. B. OD measurements. C. Example calculations.
      
      
   6. Seal plate with plastic film and incubate for 30 min in a 37 °C oven with gentle rocking every 10 min.
   7. Add 150 μl of ice-cold saline buffer to all wells except the 100% hemolysis control.
   8. Spin the plate, 1,000 x g, 5 min.
   9. Carefully remove 100 μl of the clarified supernatant with a pipette and transfer to a new flat-bottomed 96-well plate, taking care not to disturb the pelleted RBC.
   10. Read absorbance at 405 nm, yielding an example range of OD values shown in Figure 2B.
       
   Note: A multichannel pipette can be used for Steps B5, B7 and B9.

Data analysis

1. Average the OD₄₀₅ values for 100% lysis and blank.
2. For each well, calculate % lysis using the equation shown in Figure 2C.
3. Calculate average % lysis ± SD of test triplicates.
4. Confirm controls are acting appropriately: 0, 5% and 10% HuS alone (should show increasing lytic ability); HuS + FH (inhibition); 0% HuS + test (lytic ability of test sample alone).
5. Determine significance P < 0.05 (e.g., by Student's t-test [one variable] or one-way ANOVA/Tukey's test [multiple variables]), compared to the appropriate %HuS alone control using GraphPad Prism.
   

Recipes

1. Veronal buffer
   One barbitone complement dilutent tablet is dissolved in 100 ml of warm water and stored at room temperature
   Note: Veronal buffer, used to make AP buffer contains barbitone, which is a narcotic drug. Ensure buffers and reagents are stored in accordance with regulations.
2. AP buffer
   Veronal buffer plus 0.01 M EGTA and 0.1% (w/v) gelatin, pH 7.5
   

   Final con.	For 1 L
   0.01 M EGTA	3.80 g
   0.1% (w/v) gelatin	200 ml 2% gelatin solution

   1. Boil solution, by microwaving to solubilize gelatin
   2. Add EGTA and adjust the pH to 8-8.5 by dropwise addition of 10 N NaOH to dissolve EGTA. Once the solution is clear then add concentrated HCl to adjust final pH to 7.5
   3. Filter sterilize to prevent microbial growth
3. Saline 
   

   Final con.	For 1 L
   0.15 M NaCl	8.76 g

4. 10 N NaOH
   

   Final con.	For 1 L
   10 N	400 g

Note: All buffers are made up in MilliQ water.

Acknowledgments

Ms Cabezas-Falcon is supported by an Australian International Post-graduate Research Scholarship.

Competing interests

Authors have no conflicts of interest or competing interests.

Ethics

Human sera was collected in accordance with approval from the Southern Adelaide Clinical Human Research Ethics Committee (SA HREC EC00188), ORF number 343.16. Sheep blood was collected in accordance with the Flinders University Animal Welfare Committee approval to use scavenge material.

References

1. Blom, A. M., Volokhina, E. B., Fransson, V., Stromberg, P., Berghard, L., Viktorelius, M., Mollnes, T. E., Lopez-Trascasa, M., van den Heuvel, L. P., Goodship, T. H., Marchbank, K. J. and Okroj, M. (2014). A novel method for direct measurement of complement convertases activity in human serum. Clin Exp Immunol 178(1): 142-153. 
2. Cabezas, S., Bracho, G., Aloia, A. L., Adamson, P. J., Bonder, C. S., Smith, J. R., Gordon, D. L. and Carr, J. M. (2018). Dengue virus induces increased activity of the complement alternative pathway in infected cells. J Virol 92(14). 
3. Joiner, K. A., Hawiger, A. and Gelfand, J. A. (1983). A study of optimal reaction conditions for an assay of the human alternative complement pathway. Am J Clin Pathol 79(1): 65-72. 
4. Prohaszka, Z., Nilsson, B., Frazer-Abel, A. and Kirschfink, M. (2016). Complement analysis 2016: Clinical indications, laboratory diagnostics and quality control. Immunobiology 221(11): 1247-1258. 
5. Ricklin, D., Hajishengallis, G., Yang, K. and Lambris, J. D. (2010). Complement: a key system for immune surveillance and homeostasis. Nat Immunol 11(9): 785-797.