Reagents and method for detecting polychlorinated biphenyls

• US 5,145,790 A
• Filed: 05/04/1990
• Issued: 09/08/1992
• Est. Priority Date: 05/04/1990
• Status: Expired due to Fees

• Current Assignee: Abbott Laboratories
• Original Assignee: Abbott Laboratories
• Inventors: Brashear, R. Jeffrey, Mattingly, Phillip G.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US07/519,039
• Time in Patent Office: 858 Days
• Field of Search: 435/7.92, 435/7.93, 435/962, 435/968, 435/7.9, 436/537, 436/546, 436/172, 436/800, 436/822, 436/825, 436/536
• US Class Current: 436/536
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10S 435/962 Prevention or removal of interfering materials or reactants or other treatment to enhance results, e.g. determining or preventing nonspecific binding
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/822 Identified hapten
• CPC Class Codes: Y10S 436/825 Pretreatment for removal of interfering factors from sample

Abstract

Reagents and an immunoassay for detecting the presence or amount of polychlorinated biphenyls in a test sample. The assay is performed by adding a known concentration of a tracer labeled with a detectable moiety and a known concentration of an analyte-specific antibody to a test sample to form a mixture, incubating the mixture to form labeled tracer-antibody and analyte-antibody complexes, and determining the presence or amount of tracer-antibody complexes formed as a measure of the presence or amount of analyte in the test sample. Reagents provided include tracers, immunogens and an additive compound useful in preventing non-specific binding of the polychlorinated biphenyls to proteins which may be present in the test sample. A kit for performing the assay also is provided.

Claims

1. A method for detecting the presence or amount of an analyte comprising polychlorinated biphenyl in a test sample, which method comprises the steps of:

(a) adding a known concentration of a tracer labeled with a detectable moiety and a known concentration of an analyte-specific antibody to the test sample to form a mixture,

wherein the tracer has the general structure of ##STR55## wherein A is a spacer group consisting of from 0 to 50 carbon atoms and heteroatoms, including not more than ten heteroatoms, arranged in a straight or branched chain, saturated or unsaturated, wherein

(i) not more than two heteroatoms are linked in the sequence --NH--A--M--Fl or --O--A--M--Fl,

(ii) A is not a single heteroatom, and

(iii) branching occurs only on carbon atoms;

M is a linking group selected from the group consisting of ##STR56## Fl is a detectable moiety; and

wherein independently x₁ =0-4 and x₂ =0-5, with the proviso that together x₁ and x₂ ≧

1;

(b) incubating said mixture for a time and under conditions sufficient to form labeled tracer-antibody and analyte-antibody complexes; and

(c) determining the presence or amount of tracer-antibody complexesformed as a measure of the presence or amount of analyte in the test sample.

2. The method according to claim 1 wherein said polychlorinated biphenyls comprise the following structure ##STR57## wherein independently x₁ =0-5 and x₂ =0-5, with the proviso that together x₁ and x₂ >

1[x=0-5].

3. The method according to claim 1 wherein said test sample is prepared by performing an extraction procedure prior to step (a).

4. The method according to claim 1 wherein the detectable moiety is selected from the group consisting of fluorescein and fluorescein derivatives.

5. The method according to claim 4 wherein said fluorescein derivatives are selected from the group consisting of fluorescein amine, carboxyfluorescein, α

-iodoacetamidofluorescein, 4'"'"'- aminomethylfluorescein, 4'"'"'-N-alkylaminomethylfluorescein, 5-aminomethylfluorescein, 6-aminomethylfluorescein, 2-4-dichloro-1,3,5-triazin-2-yl-aminofluorescein, 4-chloro-6-methoxy-1,3,5-triazin-2yl-aminofluorescein and fluorescein isothiocyanate.

6. The method according to claim 4 wherein said fluorescein derivative is aminomethylfluorescein.

7. The method according to claim 4 wherein said fluorescein derivative is 5-carboxyfluorescein.

8. The method according to claim 1 wherein said labeled antibody complexes formed are measured by a fluorescence polarization immunoassay wherein the complexes formed in step (b) are excited with polarized light and the polarization of the fluorescence emitted by free tracer and tracer-antibody complexes is measured.

9. The method according to claim 1 wherein step (a) further comprises adding to the test sample to prevent non-specific binding to proteins, a compound of the following structure ##STR58## wherein Y=O, R¹ =Cl, R² =H and J=CH₂ CO₂ H.

10. A kit for detecting the presence or amount of an analyte comprising polychlorinated biphenyl in a test sample which kit comprises

a. an analyte-specific antibody;

b. a tracer having the general structure of ##STR59## wherein A is a spacer group consisting of from 0 to 50 carbon atoms and heteroatoms, including not more than ten heteroatoms, arranged in a straight or branched chain, saturated or unsaturated, wherein

(i) not more than two heteroatoms are linked in the sequence --NH--A--M--Fl or --O--A--M--Fl,

(ii) A is not a single heteroatom, and

(iii) branching occurs only on carbon atoms;

M is a linking group selected from the group consisting of ##STR60## Fl is a detectable moiety;

independently x₁ =0-4 and x₂ =0-5, with the proviso that together x₁ and x₂ ≧

1; and

c. an additive compound which prevents non-specific binding of the analyte to proteins.

11. The kit according to claim 10 wherein the antibody of step (a) is polyclonal or monoclonal.

12. The kit according to claim 10 wherein the detectable moiety attached to the tracer is selected from the group consisting of fluorescein and fluorescein derivatives.

13. The kit according to claim 10 wherein said additive compound has the structure ##STR61## wherein Y=O, R¹ =Cl, R² =H and J=CH₂ CO₂ H.