Quaternary ammonium immunogenic conjugates and immunoassay reagents

• US 5,492,841 A
• Filed: 02/18/1994
• Issued: 02/20/1996
• Est. Priority Date: 02/18/1994
• Status: Expired due to Term

• Current Assignee: Siemens Healthcare Diagnostics Incorporated (Siemens AG)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Craig, Alan R.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US08/199,380
• Time in Patent Office: 732 Days
• Field of Search: 530/807, 530/404, 530/405, 530/389.8, 436/533, 436/534, 436/816, 435/7.94, 435/188
• US Class Current: 436/534
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/94 involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
• CPC Class Codes: Y10S 436/816 Alkaloids, amphetamines, and barbiturates
• CPC Class Codes: Y10S 530/807 Hapten conjugated with peptide or protein

Abstract

This invention relates to novel quaternary ammonium immunogenic conjugates and reporter reagents useful for eliciting antibodies and in immunoassays. Processes for preparing such quaternary ammonium immunogenic conjugates and their use in immunoassays and in eliciting antibodies are also disclosed.

Claims

1. A quaternary ammonium conjugate useful for eliciting antibodies to a non-quaternary ammonium hapten or in an immunoassay for determining the presence and/or amount in a test sample of a non-quaternary ammonium hapten, the quaternary ammonium conjugate comprising compounds of the formula:

space="preserve" listing-type="equation">((Q.sup.+ --L--Z).sub.x M) B.sup.-

wherein

Q⁺ is a quaternary ammonium group, cyclic or acyclic, derived by covalent attachment of a linker to a hapten selected from the group consisting of cocaine, methadone, methaqualone, propoxyphenes, phencyclidine, amphetamine, benzodiazepams, quinidine, procanimide, N-acetyl procanimide, and tricyclic amines;

L is a linker comprising from 0 to 20 carbon atoms and heteroatoms arranged in a straight or branched chain and/or containing ring structures, with no more than a total of 6 heteroatoms and with no more than two heteroatoms linked in sequence;

Q⁺ is linked to L at the tertiary amine group of the hapten;

--Z-- is a residue group selected from the group consisting of --C═

O, --CH═

, --N═

N--, --NH--, --NCH₃, --NH--S═

C, --SO₂ --, --O--C═

O, and --C═

O--NH--NH₂ --;

x is greater than or equal to 1;

M is a carrier selected from the group consisting of poly(amino)acids, carbohydrates, yeasts, polysaccharides and solid phase particles;

B^- is an anion; and

(Q⁺ --L--Z)_x -- is covalently bound to M.

2. The quaternary ammonium conjugate of claim 1 wherein the linker is selected from the group consisting of --CH₂ CO-- and --CH₂ COHCO--.

5. A solid phase immunoassay for detecting the presence and/or amount of a non-quaternary ammonium hapten comprising the steps of

(1) immobilizing the immunogenic conjugate of claim 1 or 5 on a solid phase;

(2) incubating (a) a sample containing an unknown concentration of a non-quaternary ammonium hapten with (b) an antibody raised against the quaternary ammonium conjugate of claims 1 or 5 and reactive with the corresponding non-quaternary ammonium hapten;

(3) incubating the mixture of the product of step (2) with the immobilized conjugate of step (1);

(4) separating liquid phase from the solid phase of step (3);

(5) detecting either the bound free antibody in the separated liquid phase;

(6) correlating the detection signal to the amount or presence of the non-quaternary ammonium hapten.

6. The quaternary ammonium conjugate of claim 1 wherein the carrier is a latex particle.

7. A particle agglutination immunoassay for determining the amount or presence of a non-quaternary ammonium hapten in a test sample which comprises:

(1) incubating a mixture of the quaternary ammonium conjugate of claim 10, the test sample, and an antibody prepared from the conjugate of claim 3;

(2) measuring increased particle size resulting from step (1); and

(3) correlating the increased particle size with the amount or presence of non-quaternary ammonium hapten in a test sample.

8. The solid phase immunoassay of claim 5 wherein the antibody incubated in step (2) is labeled with a tag selected from the group consisting of a radioisotope, a chemiluminescent compound, a fluorophore, or an enzyme.

9. The solid phase immunoassay of claim 5 wherein the incubating steps (2) and (3) occur simultaneously.

10. The particle agglutination immunoassay of claim 7 wherein the mixture is formed either by

(1) combining the quaternary ammonium conjugate of claim 6 with the test sample and then adding to that mixture, or

(2) combining the antibody with the test sample and then adding to that mixture the quaternary ammoniun conjugate.

3. A quaternary ammonium conjugate prepared by a process which comprises:

coupling a quaternary ammonium hapten to a carrier, the carrier selected from the group consisting of poly(amino)acids, carbohydrates, yeasts, polysaccharides, and solid phase particles, the quaternary ammonium hapten obtained by attaching a bifunctional linking group to a hapten selected from the group consisting of cocaine, methadone, methaqualone, propoxyphenes, phencyclidine, amphetamine, benzodiazepams, quinidine, procanimide, N-acetyl-procanimide, and tricyclic amines, the bifunctional linking group covalently bindable to the carrier and comprising from 0 to 20 carbon atoms and heteroatoms arranged in a straight or branched chain and/or containing ring structures, with no more than a total of 6 heteroatoms and no more than two heteroatoms linked in sequence.

4. The quaternary ammonium conjugate according to claim 3 wherein the bifunctional linking group comprises a reactive group selected frown the group consisting of --CO₂ H, --NH₂, --NCO, --CONHNH₂, --CNOR, --CHO, --Br, --I, --NCS, --OCOCl, --SO₂ Cl, --OCSCl, and --NHCH₃.