Method for species-independent measurement of complement activation in animals
View Patent ↗A method for species-independent measurement of complement (C) activation in animals. The method comprises taking samples in the range of 3-100 microliter of anticoagulated blood, plasma or serum of an animal (specimen), mixing the specimen with a specificity converting protein matrix (SCM), mixing to the specimen/SCM mixture an activator of the C system (Act), incubating the specimen/SCM/Act mixture at a temperature between 36° C. to 38° C. for a time of 5-120 min and determining the production of one or more human proteins by ELISA or other analytical methods.
1. A method for species-independent quantitative measurement of a level of C3 or C4 in non-human animals, comprising:
a) mixing 3-100 microliters of a specimen comprising anticoagulated blood, plasma, or serum of a non-human animal with a specificity converting protein matrix (SCM) to form a specimen/SCM mixture, wherein the SCM is derived from a human serum and the non-human animal is selected from the group consisting of a bovine, a chicken, a goat, a guinea pig, a horse, a mouse, a pig, a rabbit, a rat, a sheep, a turkey, and a dog;
b) mixing the specimen/SCM mixture and an activator of the C system (Act) to form a specimen/SCM/Act mixture, wherein the activator is zymosan or heat aggregated gamma globulin (HAGG);
c) incubating the specimen/SCM/Act mixture at a temperature between 36° C. to 38° C. for a time of between about 5-120 minutes;
d) measuring an amount of human SC5b-9 by a human-specific ELISA in the specimen/SCM/Act mixture, wherein the measuring the amount of human SC5b-9 is proportional to the level of the non-human animal C3 or C4 in the specimen;
e) measuring known amounts of the non-human animal C3 or C4 by the human-specific SC5b-9 ELISA and generating a calibration formula; and
f) quantifying the amount of non-human animal C3 or C4 in the specimen by correlating the measured amount of human SC5b-9 from step (d) to the calibration formula;
wherein the measuring of human SC5B-9 is species-independent.
2. A method according to claim 1 , wherein in step a) the mixing of the specimen and the specificity converting protein matrix (SCM) is done at a low specimen/SCM ratio of 1:9 to 4:6.
3. The method according to claim 1 , wherein the SCM in step a) is either C3-depleted normal human serum (C3depl-NHS) or lyophilized C3depl-NHS (lyoC3depl-NHS) or C4-depleted normal human serum (C4depl-NHS) or lyophilized C4-depleted normal human serum (lyoC4depl-NHS).
4. The method according to claim 1 , wherein the SCM in step a) is supplemented with purified or recombinant human C5.
5. The method according to claim 1 , wherein the SCM in step a) is supplemented with purified or recombinant human C5, C6, C7 and C9.
6. The method according to claim 1 , wherein the SCM in step a) is a mixture of purified or recombinant human C5, C6, C7 and C9.
7. The method according to claim 1 , wherein in step b) the activator of the C system (Act) is zymosan in a concentration ranging from 0.1-10 mg/mL or HAGG in a concentration ranging from 0.1-20 mg/mL.
8. The method according to claim 1 , wherein in step c) the incubating of the specimen/SCM/Act mixture is done at 37° C. for 45-60 min with shaking.
9. The method according to claim 1 , wherein the Act is heat aggregated gamma globulin (HAGG).
10. The method according to claim 1 , wherein in step d) determining the production of one or more human proteins measures human SC5b-9 using a human CH50 kit.
11. The method of claim 1 , wherein the SCM added to the mixture contains C5-9 in excess such that the method quantitates only the levels of the non-human animal C3 or C4.
12. A method for species-independent quantitative measurement of C3 in animals, comprising:
a) obtaining a specimen comprising anticoagulated blood, plasma or serum of a non-human animal in an amount ranging from 3-100 microliters, wherein the specimen is obtained from a bovine, chicken, goat, guinea pig, horse, mouse, pig, rabbit, rat, sheep, turkey or dog;
b) mixing the specimen with a specificity converting protein matrix (SCM) to form a specimen/SCM mixture, wherein SCM is either C3-depleted normal human serum (C3depl-NHS) or lyophilized C3depl-NHS (lyoC3depl-NHS);
c) mixing the specimen/SCM mixture and an activator of the C system (Act) to form a specimen/SCM/Act mixture, wherein the Act is zymosan or heat aggregated gamma globulin (HAGG);
d) incubating the specimen/SCM/Act mixture at a temperature between 36° C. to 38° C. for a time of between about 5-120 minutes;
e) measuring an amount of human SC5b-9 by a human SC5b-9 ELISA, wherein the measuring the amount of human SC5b-9 is proportional to the level of the non-human animal C3 in the specimen,
f) measuring known amounts of the non-human animal C3 by the human-specific SC5b-9 ELISA and generating a calibration formula; and
g) quantifying the amount of non-human animal C3 in the specimen by correlating the measured amount of human SC5b-9 from step (e) to the calibration formula;
wherein the measuring of human SC5B-9 is species-independent.
13. The method according to claim 12 , wherein the SCM is C3-depleted normal human serum (C3depl-NHS).
14. The method according to claim 12 , wherein, in step a) the specimen is serum of a bovine.
15. The method of claim 12 , wherein the SCM added to the mixture contains C5-9 in excess such that the method quantitates only the levels of the non-human animal C3 or C4.