Detecting or quantifying substances using labelling techniques

• US 4,478,817 A
• Filed: 11/14/1978
• Issued: 10/23/1984
• Est. Priority Date: 11/17/1977
• Status: Expired due to Term

• Current Assignee: Welsh National School of Medicine
• Original Assignee: Welsh National School of Medicine
• Inventors: Woodhead, James S., Campbell, Anthony K., Simpson, John S. A.
• Primary Examiner(s): Meyers, Albert T.
• Assistant Examiner(s): McCowin, Kathleen S.
• Application Number:
  US05/961,389
• Time in Patent Office: 2,170 Days
• Field of Search: 23/230 B, 424/1, 424/1.5, 424/7, 424/8, 424/12, 424/7.1, 250/302, 250/361 C, 250/373, 250/458, 435/7, 435/8, 436/547
• US Class Current: 435/6.14
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/553 Metal or metal coated
• CPC Class Codes: G01N 33/581 with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The invention relates to a method of detecting, analyzing, quantifying or locating a protein, antibody, antigen, hapten, hormone, metabolite, nucleic acid or steroid, in which the substance of interest is linked to a chemi-luminescent or bio-luminescent label. A luminescent reaction is then triggered by the addition of an oxidizing agent or a catalyst and the emitted light is observed in order to provide information about the substance. The invention employs a luminescent reagent which consists of antibodies labelled with a luminescent material such as luminol. The luminescent reagent can be used to quantify antigens in an immunological assay. The luminescent labelled antibodies selectively bind to the antigens and the amount of light emitted in a luminescent reaction gives an indication of the amount of antigens present. A luminescent labelled substance can also be reacted with an antibody or antigen labelled with a fluorescent material in order to carry out a homogeneous assay.

Claims

1. A method of detecting or quantifying a substance of biological interest in a body fluid or tissue, in which a labelled binding reagent comprising an antibody, or binding protein, binding specifically to the substance, is linked with a label comprising one or more components which can participate in a luminescent reaction, the labelled binding reagent being immunologically purified and such that it is stable and its properties are not significantly altered by the label, the substance is subsequently contacted with the labelled binding reagent, and a luminescent reaction is subsequently triggered, the emitted light being sensed or measured to provide information concerning the substance of interest.

2. A method according to claim 1, in which the reaction is triggered chemically, by means of a chemical reagent or catalyst.

3. A method according to claim 2, in which the luminescent reaction is triggered by an oxidising agent, a catalyst, or a change in pH.

4. A method according to claim 1, in which the reaction is triggered physically.

5. A method according to claim 1, in which the substance of interest, to which the labelling component is linked, is a protein, hapten, hormone, metabolite, nucleic acid, or steroid.

6. A method according to claim 1, constituting a protein binding assay, in which the binding protein or a standard substance is linked with said label.

7. A method according to claim 1, constituting a method of locating substances histologically.

8. A method according to claim 1, in which said label is a compound of the general formula:

##STR13## in which each of R₁, R₂ and R₃ is H, C₁ -C₁₀ optionally substituted alkyl or alkenyl, amino, substituted amino, carboxyl or hydroxyl, each of R₄ and R₅ is the same as R₁, R₂ or R₃, or is a diazo-linkage, a hemi-dicarboxylate linkage, an amide linkage, an organic acid halide, or an isothiocyanate group, with the proviso that at least one of R₁ to R₅ is amino or substituted amino.

9. A method according to claim 1, in which said label is a compound of the general formula:

##STR14## in which each of R₉, R₁₀ and R₁₁ is H, C₁ -C₁₀ optionally substituted alkyl or alkenyl, amino, substituted amino, carboxyl or hydroxyl, each of R₁₁ and R₁₂ is the same as R₉, R₁₀ or R₁₁, or is a diazo-linkage, a hemi-dicarboxylate linkage, an amide linkage, an organic acid halide, or an isothiocyanate group, with the proviso that at least one of R₉ to R₁₂ is amino or substituted amino.

10. A method according to claim 1, in which said label is a compound of the general formula:

##STR15## in which each of R₁₃ and R₁₄ is H, C₁ -C₁₀ optionally substituted alkyl or alkenyl, amino, substituted amino, carboxyl or or hydroxyl, or is a diazo-linkage, a hemi-dicarboxylate linkage, an amide linkage, an organic acid halide, or an isothiocyanate group, with the proviso that either R₁₃ or R₁₄ is amino or substituted amino.

11. A method according to claim 1, in which said label is a compound of the general formula:

##STR16## in which each of X₅,X₆ and X₇ is H, C₁ -C₁₀ optionally substituted alkyl or alkenyl, amino, substituted amino, carboxyl or hydroxyl, or is a diazo-linkage, a hemi-dicarboxylate linkage, an amide linkage, an organic acid halide, or an isothiocyanate group, with the proviso that at least one of X₅ to X₇ is amino or substituted amino, or is ##STR17##

12. A luminescent reagent comprising antibodies or binding proteins, labelled with one or more components which can participate in a luminescent reaction, and immunologically purified, for use in detecting or quantifying their specific antigens in biological tissues or fluids.

13. A reagent according to claim 12, in which the antibodies are sheep- (anti-rabbit IgG), sheep- (anti-guinea pig IgG), guinea pig IgG, sheep IgG, goat IgG and donkey IgG.

14. A luminescent reagent according to claim 12, in which at least 60% of the antibodies are capable of binding to corresponding antigens.

15. A method of preparing a luminescent reagent according to claim 12, in which a medium containing said antibodies is contacted with corresponding antigens to cause antibody-antigen association, said luminescent label being added before or after association occurs, and said labelled antibodies are subsequently dissociated from said antigens.

16. A method according to claim 15, in which said antigens are initially linked to a solid-phase immuno-adsorbent.

17. A method according to claim 16, in which said solid-phase immuno-adsorbent is used to purify said antibodies which have been previously labelled in solution by contacting with said label.

18. A method of carrying out an assay, in which a medium containing the substance of interest, is contacted with antibodies so that the antibodies selectively bind the substance, the product of the reaction is subsequently contacted with labelled antibodies according to claim 12, and after removal of unreacted labelled antibodies a luminescent reaction is triggered, the amount of substance of interest which is present being indicated by the amount of luminescence.

19. A method according to claim 18, in which said substance of interest is bound to said antibodies on a solid phase.

20. A method according to claim 19, in which said solid phase is cellulose powder or the wall of a reaction vessel.