Method for quantitative analysis for limonin

Abstract

Method for determining the amount of limonin in a sample by subjecting a mixture of the sample and a known amount of a labeled limonin derivative to reaction with limonin-specific antibodies, analyzing to determine the amount of reaction product of antibodies and labeled limonin derivative, and applying the result to a previously prepared correlation to determine the amount of limonin in that sample.

What is claimed as new and what is desired to secure by Letters Patent of the United States is.].  .Iadd.What is claimed is.Iaddend.: 
     
       1. A method for .[.the.]. quantitative analysis of limonin .Iadd.in a sample, .Iaddend.which .Iadd.method .Iaddend.comprises reacting a known amount of limonin-specific antibodies.[.,.]. with .[.a mixture of a known volume of.]. .Iadd.said .Iaddend.sample .[.containing an unknown amount of limonin.]. and .Iadd.with .Iaddend.a known amount of a .Iadd.readily-detectable, labeled .Iaddend.limonin derivative .[.labeled with an enzyme or with a radioactive isotope.]., determining the amount of labeled limonin-derivative which has reacted with said antibodies and .[.calculating.]. therefrom the .[.unknown.]. amount of limonin in said sample. .[.2. The method of claim 1 wherein said limonin-derivative is 
     
     
        limonin-7-(O-carboxymethyl) oxime..]. 3. The method of claim 1 wherein said limonin-derivative is enzyme labeled and is the reaction product of 
     
     
        limonin-7-(O-carboxymethyl) oxime and horseradish peroxidase. 4. The method of claim 1 wherein said limonin-derivative is labeled with radioactive isotope and is the reaction product of 
     
     
        limonin-7-(O-carboxymethyl) oxime and tyrosine methylester. 5. The method of claim 1 wherein said limonin-specific antibodies are produced by immunization of an animal with a limonin-7-(O-carboxymethyl) oxime 
     
     
        conjugate. 6. The method of claim 5 wherein said .[.protein is.]. 
     
     
        .Iadd.conjugate comprises .Iaddend.bovine serum albumin. 7. The method of claim 1 wherein said antibodies are coated on .[.the inside surface of a tube and the limonin-derivative is labeled with an enzyme.]. .Iadd.a solid support.Iaddend.. .Iadd.8. The method of claim 1 wherein the label of said labeled limonin derivative is indirectly bonded to said limonin molecule. .Iaddend. .Iadd.9. The method of claim 8 wherein said indirect bonding is effected by a bridging group attached to said limonin molecule and to said readily detectable label. .Iaddend. .Iadd.10. The method of claim 9 wherein said bridging group is the ##STR2## 
     
     
        group contained in aminooxyacetic acid. .Iaddend. .Iadd.11.  The method of claim 10 wherein said bridging group is attached to the limonin molecule by reaction with the limonin carbonyl oxygen is the 7 position. .Iaddend. .Iadd.12. The method described in claims 8 or 9 or 10 or 11 wherein said 
     
     
        label is an enzyme or is radioactive. .Iaddend. .Iadd.13.  A method for determining the content of limonin in a sample, which method comprises contacting both said sample or an aliquot thereof and a known amount of a readily detectable labeled limonin derivative with a predetermined quantity of limonin-specific antibodies, said contacting being under conditions effective to cause said antibodies to react with some of said limonin and said limonin derivative, determining the amount of labeled limonin derivative reacted, and thereby determining the limonin content of said sample. .Iaddend.