Detection of polymyxins

Patent metadata

Field	Value
Publication number	US-10620225-B2
Application number	US-201514873109-A
Country	US
Kind code	B2
Filing date	Oct 1, 2015
Priority date	Oct 1, 2014
Publication date	Apr 14, 2020
Grant date	Apr 14, 2020

Abstract

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.

First claim

The invention claimed is:

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.

Assignees

• Randox Laboratories Ltd

Inventors

• Benchikh Elouard
• Mcconnell Ivan
• Lowry Philip
• Fitzgerald Peter

Classifications

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K17/02

  Peptides being immobilised on, or in, an organic carrier · CPC title

• G01N33/9446 Primary

  Antibacterials · CPC title

• G01N33/535

  with enzyme label {or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates} · CPC title

• G01N2410/00

  Assays, e.g. immunoassays or enzyme assays, involving peptides of less than 20 animo acids · CPC title