Method for measuring IgG-mediated complement activation

1. A method for measuring immunoglobulin G-mediated complement activation, comprising the following steps:

a) preparing a sample A of immunoglobulin G (IgG) and a sample B comprising natural serum, said natural serum optionally being diluted in a dilution buffer;

b) mixing sample A with sample B at a ratio (amount of IgG in sample A in grams): (volume of natural serum in sample B in liters) of between 30 and 75, at a temperature of between 2° C. and 6° C., and subsequently incubating the resulting reaction mixture at a temperature of between 35° C. and 40° C. for a period of between 30 minutes and 2 hours;

c) cooling the reaction mixture obtained at the end of step b) to a temperature of between 0° C. and 4° C. in the presence of EDTA; and

d) measuring the amount of C5a fragment in the cooled reaction mixture obtained in c).

2. The method according to claim 1 , wherein the sample A:sample B ratio is between 35 and 70.

3. The method according to claim 1 , wherein the reaction mixture is incubated in step b) at a temperature of approximately 37° C., for a period of between 45 minutes and 1 hour.

4. The method according to claim 1 , wherein the EDTA in step c) is present at a concentration of between 50 mM and 200 mM.

5. The method according to claim 1 , wherein sample A comprises intravenous immunoglobulin G (IVIG).

6. The method according to claim 1 , wherein sample B comprises human serum of blood group AB+.

7. The method according to claim 1 , further comprising

e) comparing the amount of C5a fragment obtained in d) with the amount of C5a fragment obtained by the following process:

a′) preparing a sample of lipopolysaccharides (LPS) of E. coli serotype 0127:B8, purified by extraction with phenol, and a sample B comprising natural serum, said natural serum optionally being diluted in a dilution buffer;

b′) mixing the sample of LPS with sample B at a ratio (amount of LPS in grams):(volume of natural serum in B in liters) of between 30 and 75, at a temperature of between 2° C. and 6° C., and subsequently incubating the resulting reaction mixture at a temperature of between 35° C. and 40° C. for a period of between 30 minutes and 2 hours;

c′) cooling the reaction mixture obtained at the end of step b) to a temperature of between 0° C. and 4° C. in the presence of EDTA; and

d′) measuring the amount of C5a fragment in the cooled reaction mixture obtained in c).

8. The method according to claim 1 , further comprising

e) measuring the percentage complement activation, obtained by the following formula:

(test−negative control)/(maximum complement activation−negative control)×100

in which:

“test” is the amount of C5a fragment measured in the reaction mixture tested;

“negative control” is the amount of C5a fragment measured in the reaction mixture in which sample A is replaced with the dilution buffer;

“maximum complement activation” is the amount of C5a fragment measured in the reaction mixture in the method according to claim 1 in which sample A is replaced with lipopolysaccharides (LPS) of E. coli serotype 0127:B8, purified by extraction with phenol.

9. The method according to claim 7 , wherein the ratio (amount of LPS in grams):(volume of natural serum in sample B in liters) is between 6 and 15.

10. The method according to claim 1 , wherein step d) is carried out by an ELISA assay.

11. The method according to claim 2 , wherein the reaction mixture is incubated in step b) at a temperature of approximately 37° C., for a period of between 45 minutes and 1 hour.

12. The method according to claim 3 , wherein sample A comprises intravenous immunoglobulin G (IVIG).

13. The method according to claim 3 , sample B comprises human serum of blood group AB+.

14. The method according to claim 3 , further comprising

step e) of comparing the amount of C5a fragment obtained in d) with the amount of C5a fragment obtained by the following process:

a′) preparing a sample of lipopolysaccharides (LPS) of E. coli serotype 0127:B8, purified by extraction with phenol, and a sample B comprising natural serum, said natural serum optionally being diluted in a dilution buffer;

b′) mixing the sample of LPS with sample B at a ratio (amount of LPS in grams):(volume of natural serum in sample B in liters) of between 30 and 75, at a temperature of between 2° C. and 6° C., and subsequently incubating the resulting reaction mixture at a temperature of between 35° C. and 40° C. for a period of between 30 minutes and 2 hours;

c′) cooling the reaction mixture obtained at the end of step b) to a temperature of between 0° C. and 4° C. in the presence of EDTA; and

d′) measuring the amount of C5a fragment in the cooled reaction mixture obtained in c).

15. The method according to claim 3 , further comprising

step e) measuring the percentage complement activation, obtained by the following formula:

(test−negative control)/(maximum complement activation−negative control)×100

in which:

“test” is the amount of C5a fragment measured in the reaction mixture tested;

“negative control” is the amount of C5a fragment measured in the reaction mixture in which sample A is replaced with a dilution buffer;

“maximum complement activation” is the amount of C5a fragment measured in the reaction mixture in the method according to claim 3 in which sample A is replaced with lipopolysaccharides (LPS) of E. coli serotype 0127:B8, purified by extraction with phenol.

16. The method according to claim 8 , wherein the ratio (amount of LPS in grams):(volume of natural serum in sample B in liters) is between 6 and 15.

17. The method according to claim 1 , wherein the sample A:sample B ratio is between 45 and 60.

18. The method according to claim 7 , wherein the ratio (amount of LPS in grams):(volume of natural serum in sample B in liters) is between 8 and 13.