Detection of polymyxins
The present invention relates to antibodies for use in detecting polymyxins and tracers; and to a single-capture immunodetection method and kits, each utilising the antibodies of the invention, and disclosed tracers.
1. A polyclonal antibody raised against an immunogen having the structure
wherein Z is —CH 2 —CH(NH 2 )—C(O)—,
wherein the accm includes keyhole limpet haemocyanin activated with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate,
wherein the polyclonal antibody binds specifically to Colistin and Polymyxin B, wherein the polyclonal antibody has a cross-reactivity of 100% to Polymyxin B and 80% to Colistin, wherein the cross-reactivity is measured in the presence of a tracer; and
wherein the tracer is a conjugate of horseradish peroxidase activated with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate with a hapten having the structure:
wherein R is isobutyl; X 1 and X 3 are H; and X 2 is H—S—CH 2 —CH(NH 2 )—C(O)—.
2. The polyclonal antibody of claim 1 , wherein the polyclonal antibody binds Polymyxin B with an IC 50 of 0.931 ng/ml and binds Colistin with an IC 50 of 1.191 ng/ml.
3. A polyclonal antibody selected from the group consisting of RS2790, RS2801, and RS2830, wherein the polyclonal antibody is raised against an immunogen having the structure
wherein Z is —CH 2 —CH(NH 2 )—C(O)—,
wherein the accm includes keyhole limpet haemocyanin activated with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate,
wherein the polyclonal antibody has a relative % cross-reactivity to Polymyxin B and Colistin at different antibody concentrations and different tracer concentrations as follows:
Antibody RS2790
Antibody RS2801
Antibody RS2830
0.1
0.75
0.1
0.75
0.1
0.75
Tracer
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
mg/ml
dilution
Col
Poly
Col
Poly
Col
Poly
Col
Poly
Col
Poly
Col
Poly
1/50K
5
100
8
100
1/100K
23
100
59
100
1/500K
20
100
67
100
6
100
8
100
1/1000K
15
100
62
100
5
100
9
100
wherein the tracer is a conjugate of horseradish peroxidase activated with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate with a hapten having the structure:
wherein R is isobutyl; X 1 and X 3 are H; and X 2 is H—S—CH 2 —CH(NH 2 )—C(O)—.
4. A method for detecting or determining polymyxins in a sample, the method comprising the steps of contacting the sample with the polyclonal antibody of claim 1 , and detecting or determining any polymyxins in the sample bound to the polyclonal antibody.
5. The method of claim 4 , wherein the polymyxin is Polymyxin B and/or Colistin.
6. The method of claim 4 , wherein the polymyxin is Polymyxin B.
7. The method of claim 4 , wherein the polymyxin is Colistin.
8. The method of claim 4 , wherein the polymyxin is Polymyxin B and Colistin.
9. A method of making an antibody comprising:
administering to a host animal an immunogen having the structure
wherein Z is —CH 2 —CH(NH 2 )—C(O)—,
wherein the accm includes keyhole limpet haemocyanin activated with N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; and
purifying the resulting antibody.
10. The method of claim 9 , wherein the antibody is a polyclonal antibody.