CA2014233A1 - Polarization immunoassay for cannabinoids - Google Patents
Polarization immunoassay for cannabinoidsInfo
- Publication number
- CA2014233A1 CA2014233A1 CA002014233A CA2014233A CA2014233A1 CA 2014233 A1 CA2014233 A1 CA 2014233A1 CA 002014233 A CA002014233 A CA 002014233A CA 2014233 A CA2014233 A CA 2014233A CA 2014233 A1 CA2014233 A1 CA 2014233A1
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- Prior art keywords
- acid
- releasing agent
- accordance
- naphthalene
- dodecyl sulfate
- Prior art date
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/948—Sedatives, e.g. cannabinoids, barbiturates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- Anesthesiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
ABSTRACT
An antibody reagent for the quantitative or qualitative fluorescence polarization immunoassay of 11-nor-.DELTA.9-tetra-hydrocannabinol-9-carboxylic acid which comprises an antibody reagent and a releasing agent is described. A
urine sample to be tested in the fluorescence polarization immunoassay for 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxy-lic acid which comprises urine and a releasing agent is described. The reagent, sample, and processes of the invention allow for a quantitative or qualitative assay of 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid in a fluorescence polarization immunoassay.
An antibody reagent for the quantitative or qualitative fluorescence polarization immunoassay of 11-nor-.DELTA.9-tetra-hydrocannabinol-9-carboxylic acid which comprises an antibody reagent and a releasing agent is described. A
urine sample to be tested in the fluorescence polarization immunoassay for 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxy-lic acid which comprises urine and a releasing agent is described. The reagent, sample, and processes of the invention allow for a quantitative or qualitative assay of 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid in a fluorescence polarization immunoassay.
Description
h In testing for ~he drug of abuse, marijuana, a level of 100 nanograms of its metabolite ll-nor-~9-tetrahydrocanna-binol-9-carboxylic acid per milliliter of urine is often employed as the threshold for a positive test as specified in the NIDA (National Institute of Drug Abuse) Guidelines 1988. That is, where more than 100 nanogeams per milliliter of urine are detected the test for marijuana is determined to be positive: however, if below 103 nanograms per milliliter of urine are detected the test is determined to be negative. Accordingly, to prevent false negative test results, it is nece6sary to be able to recover all of the ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid, a major metabolite of marijuana from different types of urine samples. Some urine samples contain high concentrations of proteins such as, human serum albumin. When such human serum albumin is presen~, the ll-nor-~9-tetrahydro-cannabinol-9-carboxylic acid (~9-T~IC-9-COOH) binds to non-specific bindillq sites on human serum albu~in and other proteins, thereby preventing a quantitative formation of antibody complex and thus preventing a quantitative assay for the 11-nor-~9-tetrahydrocannabinol-9-car~oxylic acid present. This can, of course, result in a false negative test for a specific sample.
;It is an~object of the invention to provide for a ~quanti~ative~fluorescence polarization immunoassay of l;l-nor-~9-tetrahydrocannabinol-9-carboxylic acid in urine so~as to avoid fals~e negative test results~for this compound.; It is another~ ob ject of the invention to provide for;a qualitati~e fluor;esce~nce polariza~ion immunoassay of ll-nor-Q9-tetrahydrocannabinol-9-carboxylic acid in urine.
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The invention relates to a reagent for use in a fluorescence polarization immunoassay for ll-nor-~9-tetra-hydrocannabinol-9-carboxylic acid which comprises a releasing or blocking agent. The invention also relates to a urine sample to be ~ested in a fluorescence polarization immunoassay for ll-nor-Q9-tetrahydrocannabinol-9-carboxy-lic acid which is treated with a releasing or blocking agent.
Finally, the invention relates to a process for a quantitative or qualitative assay for ll-nor-h9-tetra-hydrocannabinol-9-carboxylic acid in a fluorescence polarization immunoassay which comprises either premixing an antibody reagent with a releasing or blocking agent; or peetreating the sample urine with a releasing or blocking agent-The antibody reagent of the invention allows for a quantitative or qualitative assay of ll-nor-~9-tetrahydro-cannabinol-9-carboxylic acid in a fluorescence polarization immunoassay. The pretreated sample urine of the invention allows for a quantitative assay of this analyte. Also, the processes of the invention allow for a quantitative assay of this analyte.
As used herein the terms "antibody" or "antiserum" or "ascites" denote a protein which is eaised by ~irst ; conjugating a hapten, in this case a tetrahydrocannabinol deriva~ive to a protein carrier and injecting the conjugate product into an animal. The resulting antibody to ll-nor-a9-tetrahydrocannabinol-9-carboxylic acid may be isolated by conventional well-known antibody isolation techniques. As used herein, the term "qualitative"
; fluorescence polarization immunoassay means an assay that detects only th~presence or absence of drug metabolite in a specimen and not the absolute amount. The term "ligand" or "analyte" refers to a molecule which binds to the antibody to form a ligand-antibody complexO In ~his case, the ligand 2 3 ~
is ll-nor-~9-tetrahydrocannabinol-9-carboxy]ic acid, a major metabolite of marijuana. The term "Sample" or ~specimen~ refers to urine. The term "blocking agent" or "releasing agent" denotes a chemical compound which when added to a sample or antibody reagent competes with the analyte for non-specific binding si~es which are usually on ~roteins present in the urine sample. The term "tracer" or ~label~ refers to a fluorescent compound which is added to the eeagent mixture in the fluorescence polarization immunoassay of the invention. The term "tracer reagent"
refers to the fluorescent compound which is reacted with the ligand or analyte to form the "tracer" or "label". An example of a label or tracer which may be used i6 the following compound, where fluorescein isothiocyanate has been coupled to ll-nor-a8-tetrahydro-cannabinol-9-carboxylic acid.
COOH ~ d N C~NH ~ ~ o The term "labelled ligand-antibody complex" denotes a ligand-antibody complex to which a tracer, has been chemically bound. The term "standard" refers to an aqueous solution of an accurately known analyte concentration. The term "span" denotes an indication of the difference between the net fluorescence millipolarization at the pvint where the maximum and~the minimum amount of tracer is bound to antibody.
As used herein the "non-specific binding sites" denotes ; binding sites f~r ll-nor-a9-tetrahydrocannabinol-9-car-boxylic acid, and other tetrahydrocannabinol metabolites other than the specific binding sites on ~he antibody raised for this llgand. Exemplary of such non-specific binding ~ Q ~ 3 ~
sites are those found in ~he protein human serum albumin found in human urine.
As used herein the term "w~v"% refecs to a ratio of weight to volume expressed as a percen~. l.Ow/v% of salicylic acid, foL example, indicates l gram of salicylic acid to lOOml of solution. 0.005 w/v% ~f ANS indicates 0.005 grams of ANS to lOOml of solution. Unless otherwise indicated, all concentrations herein are given in w/v%.
The invention relates to an antibody reagent for conducting a fluorescence polarization immunoassay for ~l-nor-~9-tetrahydrocannabinol-9-carboxylic acid and other tetrahydrocannibinol metabolites in urine which comprises an 15 antibody and a releasing agent or blocking agent. Releasing agents or blocking agents are known in the art and have been employed in serum assay6 but have not been employed in urine assays.
Releasing agent i6 added to the sa~ple or antibody reagent usually in excass of the amount needed to bind to ; all the si~es in a clinical specimen of excessively high protein content. In this amount, the releasing agent ~- ~ competes successfully with the analyte foL non-specific 25 binding sites usually on proteins present in the sample.
The net result is that the non-specific binding sites are - -effectively filled with the releasing agent and the analyte of interest is unable to bind and is now free in solution and available to~be assayed. The releasing agent is usually hydrophilic and should be present in an amount sufficient to allow for approximately total recovery of ll-nor-d9-tetrahydrocannabinol-9-ca~boxylic acid but not in such an amount so as to destroy the~ activity of ~he antibody. For the releasing a~ent to be effective, ie needs to have a high : ~: : ~ :
affinity for the non-specific binding sites.
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Releasing agents which ma~ be employed in the invention are selected from the group consisting of l-anilinonaphtha-lene-8-sulfonic acid (ANS); and 1,1'-bi(4-anilino)naphtha-lene-5,5'-disulfonic acid (bis-ANS); 2-anilinonaphtha-lene-6-sulfonic acid(2.6-ANS); lithium dodecyl sulfate (LDS); sodium dodecyl sulfate (SDS): ammonium dodecyl sulfate; cholic acid: benzoic acid, salicylic acid;
COOH CO~H COOH COOH
,~,OH ~¢~OH ~,OH ,~OH
Witconate P10-59; an-l mixtures thereof. Witconate P10-59 is a surfactant which is made by the Witco Corporation, 5ZO
Madison Avenue, N.Y., N.Y. 10022. Preferred releasing agents of the invention are bis-ANS, ANS, and LDS.
As used herein l-anilinonaphthalene-8-sulfonic acid is synonymous with 8-anilino-1-naphthalenesulfonic acid. Both names refer to the compound whose abbre~iation is A~S.
1,1 ' -bi (4-anilino)na~hthalene-5,5'-disulfonic acid is 25 biS-ANS, Salicylic acid is COOH
: 30 ~,O~
; In the tables, salicylic acid is referred to salicylate, and cholic acid as ~holate. The compounds:
2~ L~2~3 ~ 6 --COOH COOH COC)H COOH
~r HO3S ~ I ~J~ Cl ~f H3CO
which are also releasing agents are salicylic acid de~ivatives.
Salts of dodecyl sulfates, such as the sodium, lithium, and ammonium salts pointed out above are also releasing agents.
The antibody used in the reagent is raised by conventional means, from an animal such as a goat.
A conventional antibody reagent is made by adding the antibody thus eaised, to an aqueous buffer. 5uch aqueous buffers are known in the art. The~buffer us~d should be capable of controlling~the pH in range of 7 to 8.5 and it should ~have a pKa of 7 to 8.5.
An aqueous buffer may c~ontain O.l molar phosphate buffer such as to allow for a pH of about ~.O to abou~ 8.5 (the buffer may be selected from the group consisting of TRIS
which is~ (HOCH2)3C-NHz,~ Bis-TRIS which is I0CH2c~2)2wc~(cH20H)3, ~and~Phosphate buffered ~saline);~about~0.0l w/~% of bovine gamma globulin; about 0.2 w/v%~sodium azide. ~ ~
The antibody reagent of the invention ma~ be made by ;adding~releasi~n~ or blocking agent ~o a~con~entional ; ; 35 antibody reag;ent for fluorescence polarization immunoassays.
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A sufficient amount of releasing or blocking agent is added to allow for approximately total recovery of ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid bu~ no~
to destroy the ability of the antibody to bind to the hapten or tracer. Preferred is to use an amount of releasing agent in terms of moles per liter which is in excess of the amount of protein present in moles per liter in the reagent mixture. The amount of releasing agent required for use in a particular assay will vary depending upon sample size, sample maturity. the amount of antibody present, and the amount of labeled ligand derivative present. The amount of releasing agent required preferably is that amount which will allow for a deteemination of at least 85% and preferably about 95-100% of ll-nor-Q9-tetrahydro-cannabinol-9-carboxylic acid present in a sample.
An elevated quantity of an interfering protein, for example human serum albumin, that may be present in a urine sample is 0.05w/v%. Accordingly, 0.05w/v% human serum aIbumin was added to the urine in the following examples.
In the tables below, the COBAS BIO FP~ instrument was used for making fluorescence polarization readings.
In a process of the invention, urine sample is added to antibody reagent containing the releasing reagent and background fluorescence intensity readings are made. A
fluorescent ~racer, such as the following fluorescein derivative OH
~ ~ h o~~ NH--C-NH~O
~ ~ 35 ~ ~ OH
.~
is then added and after mixing, fluorescence intensity readings are made and fluorescence polarization is calculated and analyte concentration is determined by reference to a standard calibra~ion curve.
In the tables below, the above procedures were used to determine the percent recovery of ll-nor-a9-tetrahydro-cannabinol-9-carboxylic acid from urine samples to whish were added 0.05w~v~ human serum albu~in and ~00 nanograms/
milliliter of 11-nor-~9-tetrahydrocannabinol-9-carboxylic acid. Where no releasing agent is used, ~as shown in Tables 1 and 2) less than half of ll-nor-~9-tetrahydrocanna-binol-9-carboxylic acid was recovered.
Prior to each reading, the antisera employed is diluted. For example, in Table 1 when 0.2w/v% salicylic acid i6 employed as the blocking agent, one volume of anti~era is dissolved in Antibody Bu~fer so as to make up a total final volume that was approximately 3,600 times larger.
The tracer which is employed in the examples given herein i8 a tetrahydrocannabinol derivative having the chemical formula ~
COOH ~ /) ' =9-NH~o 30 ~ ~ 0 0 0 It is described in ~uropean Patent Application 276,732, filed January 2ff, 198~.
:~
This com~ound is mixed to give a final concentration of 3 x 10 7 moles per liter into a solution having the , .. .
following composition: 0.1 molar ACES
~H2NCOCH2NHCHzCH2SO~H), buffer ~H 8; O.Olw/v%
bovine gamma globulin, O.lw/v% sodium azide.
TABLE_l Releasing agents premixed with Antibody Reagent. Recovery of ~9-THC-9-COOH~ from urine and mP Spans between Standards.
Blocking mP Span mP Span SampleaReCo~ery Agent w/v% 0-50 nq/ml 0-100 nq/ml Size ~ 9-THC-s-cooH*
None 46 119 14 49 0.2 Salicylate 33 103 - l.4 100 0.0005 LDS 40 108 14100 0.01 ANS 28 106 14 90 0.0005 bis-ANS 39 116 14 100 aUrine samples contained 0.05w/v% Human Serum Albumin and 100 ng/ml a9-THC-9-COOH.
; ~9-THC-9-COOH refers to ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid.
As can be seen from Table 1 just above, Salicylate, LDS, ANS, and bis-AWS are all effective as blocking agents in the ~fluorescence polarization immunoassay of the invention.
Alternatively, releasing or blocking agent may be directly mixed with the~urine sample prior to conducting the fluorescence polarization immunoassay. The amount of releasing agent employed is sufficient to allow for approximately total recovery of ll-nor-~9-tetrahydrocanna-binol-9-carboxylic acid but not such an amount as to destroy the activity of the antibody. In such a case, antisera reagent as de~cribed above is prepared without the releasing agent being added. Then urine sample and releasing agent are mixed in a sample cup and added to the antibody reagent. Background fluorescent intensity readings are made. Tracer as described above is added and after mixing, fluorescent intensity readings are made and the fluorescence polarization is calculated and th0 analyte concentration determined from a standard calibration curve.
A specific example where releasing agent is directly mixed with the urine sample prior ~o conducting the fluorescence polarization immunoassay is as follows: in Table 2 below, in the case where 0.005w/v% bis-ANS is used as the releasing agent, about 40~1 of urine and 120~1 of o~oosw/v% bis-ANS are added to a sample cup. 42~1 of this mixture (which is equivalent to 14~1 of urine) ace added ~o the antibody reagent and the background intensity readings are made. Accordingly, 14~1 of urine are entered in the column labelled "sample Size" in Table Z below.
In a like manner~ similar urine samples may be pretreated with a releasing agent of the invention. The fluorescent tracer is added as described above, and the resulting solution is mixed. The fluorescence polarization of this mixture is then determined.
Using the just above described process, and employing the above described tracer, and antibody reagent, the readings which appear in Table 2 below were made.
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Releasing Agentæ used in Sample Pretreatment Procedure.
Recovery of Q9-THC-ll-9-COOH from urine and 5mP Spans between Stanclards.
Releasing mP Span mP Span Sample aRecovery A~ent w/v% 0-50 nq/mL O-lOO nq/mL 5ize ~ G9-THC-9-COOH
None 46 119 14 49 0.005 LDS 53 136 14 86 4 Salicylate 44 117 14 g6 4 Salicylate~ 41 125 14 96 0.002 LDS
t5 4 Salicylate~ 46 123 14 94 0.2 cholate 0.1 ANS 46 116 13.3 92 Q.005 bis-ANS 49 142 14 100 a Urine samples contained 0.05 w/v% human and 100 ng/ml ~9-THC-9-COOH.
As can be seen from Table Z jus~ above, LDS; Salicylate;
Salicylate and LDS; Sallcylate and cholate; ANS: and bis-ANS
are all effective as reIeasing agents at the specified concentrations. It can be seen that a releasing agent may also be a combination of chemical compounds that alone are releasing agents. It can also be seen from Table 2 above , tha~ where no releasing agent is employed in the 30 fluorescence~polarization immunoas&ay, less than 50%
recovery~of desired~ nor-~9-tetrahydrocannabinol-9-carboxylic acid Is achieved.
:
The concent~atlon of~re;leasing agent was optimized for 35 assay performance and for recovery of ll-nor-~9-tetra-hydrocannabinol-9-carboxylic acid. In this connection, it i8 noted ~hat ii too mgch releasing agent i8 employed, then ' ` .
3 ~
- lZ -the antibody activity in the assay is diminished. I~ too little releasing agent is employed then quantitative recovery of ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid is not achieved~
It is preferred that releasing agellt be prasent in the antibody reagent in an amount sufficient to allow for recovery of at least about 85%, preferably 95-100% of the ll-nor-d9-tetrahydrocannabinol-9-carboxylic acid present in a sample~
Employing the procedures dsscribed above, there were obtained the data shown in Table 3 below which demonstrate the amount o~ releasing or blocking agent added to the antisera reagent needed for a 10-14 microliter urine sample. One skilled in the art will be able to determine the amount of releasing or blocking agent needed in accordance with the procedures herein and taking into account sample size, sample maturity, and the amount of antibody prssent.
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Releasing Agents Used When Premixed With the ~ntibody Reagent W~V%
Releasing Agent mP Span mP Span Sample %
~l 0-50_nq/ml0-100 ng~ml Size ~1 Recovery 0.2 Salicylate 33 103 14 100 0.2 Salicylate+ 40 108 14 100 0.0005 LDS
0.2 Salicylate~ 42 72 10 100 0.001 LDS
0.2 Salicylate 26 65 10 100 0.002 LDS
0.001 ANS 28 106 14 90 0.005 ANS 41 113 14 85 0.0005 bis~ANS 37 99 10 100 20 o.ooos bis-ANS 39 116 14 100 *Recovery of a9-T~C-9-COOH~from human urine containing 0.05w/v% human serum aLbumin.
Ab reagent means antibody~reagent.
The COBAS BIO FPTn instrument i6 employed collecting the da~a in this specification.
; 30 When the COBAS BIO FPm instrument is employed ana releasing agent is added directly ~o an amount of urine in a :: range of:abou~ 10~1 to about 17~1, the following are preferred concentrations in w~v% for the releasing agents ~: based on the total amount of releasing agent: about 0.02 to 35 0.5 for ANS about 0.003 to 0.1 for Bis-ANS, preferably about 0.005: about 3 to 10 for salicylic acid, preferably 4:
0.001 LDS to about 0.02 ~DS, preferably 0.005.
-- 1~
~ hen the COBAS BIO FP~ instrument is employed, and about 10~1 to about 14~1 of urine are tested and releasing agent is premixed with antibody reagent, the following are the preferred concentrations of releasing agent in w/v%: about 0.1 to 0.4, preferably about 0.2 salicylate about 0.2 salicylate with about 0.00Z LDS; about 0.0001 to about 0.002, preferably about 0.0005 Bis-ANS;
about 0.0001 to about 0.00Z, preferably about 0.0005 LDS
and about 0.005 to about 0.02 ANS, preferably about 0.01 ANS.
., .
;It is an~object of the invention to provide for a ~quanti~ative~fluorescence polarization immunoassay of l;l-nor-~9-tetrahydrocannabinol-9-carboxylic acid in urine so~as to avoid fals~e negative test results~for this compound.; It is another~ ob ject of the invention to provide for;a qualitati~e fluor;esce~nce polariza~ion immunoassay of ll-nor-Q9-tetrahydrocannabinol-9-carboxylic acid in urine.
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Klt/23.Z.gO
~: ' , ' . ' ~
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The invention relates to a reagent for use in a fluorescence polarization immunoassay for ll-nor-~9-tetra-hydrocannabinol-9-carboxylic acid which comprises a releasing or blocking agent. The invention also relates to a urine sample to be ~ested in a fluorescence polarization immunoassay for ll-nor-Q9-tetrahydrocannabinol-9-carboxy-lic acid which is treated with a releasing or blocking agent.
Finally, the invention relates to a process for a quantitative or qualitative assay for ll-nor-h9-tetra-hydrocannabinol-9-carboxylic acid in a fluorescence polarization immunoassay which comprises either premixing an antibody reagent with a releasing or blocking agent; or peetreating the sample urine with a releasing or blocking agent-The antibody reagent of the invention allows for a quantitative or qualitative assay of ll-nor-~9-tetrahydro-cannabinol-9-carboxylic acid in a fluorescence polarization immunoassay. The pretreated sample urine of the invention allows for a quantitative assay of this analyte. Also, the processes of the invention allow for a quantitative assay of this analyte.
As used herein the terms "antibody" or "antiserum" or "ascites" denote a protein which is eaised by ~irst ; conjugating a hapten, in this case a tetrahydrocannabinol deriva~ive to a protein carrier and injecting the conjugate product into an animal. The resulting antibody to ll-nor-a9-tetrahydrocannabinol-9-carboxylic acid may be isolated by conventional well-known antibody isolation techniques. As used herein, the term "qualitative"
; fluorescence polarization immunoassay means an assay that detects only th~presence or absence of drug metabolite in a specimen and not the absolute amount. The term "ligand" or "analyte" refers to a molecule which binds to the antibody to form a ligand-antibody complexO In ~his case, the ligand 2 3 ~
is ll-nor-~9-tetrahydrocannabinol-9-carboxy]ic acid, a major metabolite of marijuana. The term "Sample" or ~specimen~ refers to urine. The term "blocking agent" or "releasing agent" denotes a chemical compound which when added to a sample or antibody reagent competes with the analyte for non-specific binding si~es which are usually on ~roteins present in the urine sample. The term "tracer" or ~label~ refers to a fluorescent compound which is added to the eeagent mixture in the fluorescence polarization immunoassay of the invention. The term "tracer reagent"
refers to the fluorescent compound which is reacted with the ligand or analyte to form the "tracer" or "label". An example of a label or tracer which may be used i6 the following compound, where fluorescein isothiocyanate has been coupled to ll-nor-a8-tetrahydro-cannabinol-9-carboxylic acid.
COOH ~ d N C~NH ~ ~ o The term "labelled ligand-antibody complex" denotes a ligand-antibody complex to which a tracer, has been chemically bound. The term "standard" refers to an aqueous solution of an accurately known analyte concentration. The term "span" denotes an indication of the difference between the net fluorescence millipolarization at the pvint where the maximum and~the minimum amount of tracer is bound to antibody.
As used herein the "non-specific binding sites" denotes ; binding sites f~r ll-nor-a9-tetrahydrocannabinol-9-car-boxylic acid, and other tetrahydrocannabinol metabolites other than the specific binding sites on ~he antibody raised for this llgand. Exemplary of such non-specific binding ~ Q ~ 3 ~
sites are those found in ~he protein human serum albumin found in human urine.
As used herein the term "w~v"% refecs to a ratio of weight to volume expressed as a percen~. l.Ow/v% of salicylic acid, foL example, indicates l gram of salicylic acid to lOOml of solution. 0.005 w/v% ~f ANS indicates 0.005 grams of ANS to lOOml of solution. Unless otherwise indicated, all concentrations herein are given in w/v%.
The invention relates to an antibody reagent for conducting a fluorescence polarization immunoassay for ~l-nor-~9-tetrahydrocannabinol-9-carboxylic acid and other tetrahydrocannibinol metabolites in urine which comprises an 15 antibody and a releasing agent or blocking agent. Releasing agents or blocking agents are known in the art and have been employed in serum assay6 but have not been employed in urine assays.
Releasing agent i6 added to the sa~ple or antibody reagent usually in excass of the amount needed to bind to ; all the si~es in a clinical specimen of excessively high protein content. In this amount, the releasing agent ~- ~ competes successfully with the analyte foL non-specific 25 binding sites usually on proteins present in the sample.
The net result is that the non-specific binding sites are - -effectively filled with the releasing agent and the analyte of interest is unable to bind and is now free in solution and available to~be assayed. The releasing agent is usually hydrophilic and should be present in an amount sufficient to allow for approximately total recovery of ll-nor-d9-tetrahydrocannabinol-9-ca~boxylic acid but not in such an amount so as to destroy the~ activity of ~he antibody. For the releasing a~ent to be effective, ie needs to have a high : ~: : ~ :
affinity for the non-specific binding sites.
~: :
Releasing agents which ma~ be employed in the invention are selected from the group consisting of l-anilinonaphtha-lene-8-sulfonic acid (ANS); and 1,1'-bi(4-anilino)naphtha-lene-5,5'-disulfonic acid (bis-ANS); 2-anilinonaphtha-lene-6-sulfonic acid(2.6-ANS); lithium dodecyl sulfate (LDS); sodium dodecyl sulfate (SDS): ammonium dodecyl sulfate; cholic acid: benzoic acid, salicylic acid;
COOH CO~H COOH COOH
,~,OH ~¢~OH ~,OH ,~OH
Witconate P10-59; an-l mixtures thereof. Witconate P10-59 is a surfactant which is made by the Witco Corporation, 5ZO
Madison Avenue, N.Y., N.Y. 10022. Preferred releasing agents of the invention are bis-ANS, ANS, and LDS.
As used herein l-anilinonaphthalene-8-sulfonic acid is synonymous with 8-anilino-1-naphthalenesulfonic acid. Both names refer to the compound whose abbre~iation is A~S.
1,1 ' -bi (4-anilino)na~hthalene-5,5'-disulfonic acid is 25 biS-ANS, Salicylic acid is COOH
: 30 ~,O~
; In the tables, salicylic acid is referred to salicylate, and cholic acid as ~holate. The compounds:
2~ L~2~3 ~ 6 --COOH COOH COC)H COOH
~r HO3S ~ I ~J~ Cl ~f H3CO
which are also releasing agents are salicylic acid de~ivatives.
Salts of dodecyl sulfates, such as the sodium, lithium, and ammonium salts pointed out above are also releasing agents.
The antibody used in the reagent is raised by conventional means, from an animal such as a goat.
A conventional antibody reagent is made by adding the antibody thus eaised, to an aqueous buffer. 5uch aqueous buffers are known in the art. The~buffer us~d should be capable of controlling~the pH in range of 7 to 8.5 and it should ~have a pKa of 7 to 8.5.
An aqueous buffer may c~ontain O.l molar phosphate buffer such as to allow for a pH of about ~.O to abou~ 8.5 (the buffer may be selected from the group consisting of TRIS
which is~ (HOCH2)3C-NHz,~ Bis-TRIS which is I0CH2c~2)2wc~(cH20H)3, ~and~Phosphate buffered ~saline);~about~0.0l w/~% of bovine gamma globulin; about 0.2 w/v%~sodium azide. ~ ~
The antibody reagent of the invention ma~ be made by ;adding~releasi~n~ or blocking agent ~o a~con~entional ; ; 35 antibody reag;ent for fluorescence polarization immunoassays.
: ;
, ~, .
-:
2 ~
A sufficient amount of releasing or blocking agent is added to allow for approximately total recovery of ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid bu~ no~
to destroy the ability of the antibody to bind to the hapten or tracer. Preferred is to use an amount of releasing agent in terms of moles per liter which is in excess of the amount of protein present in moles per liter in the reagent mixture. The amount of releasing agent required for use in a particular assay will vary depending upon sample size, sample maturity. the amount of antibody present, and the amount of labeled ligand derivative present. The amount of releasing agent required preferably is that amount which will allow for a deteemination of at least 85% and preferably about 95-100% of ll-nor-Q9-tetrahydro-cannabinol-9-carboxylic acid present in a sample.
An elevated quantity of an interfering protein, for example human serum albumin, that may be present in a urine sample is 0.05w/v%. Accordingly, 0.05w/v% human serum aIbumin was added to the urine in the following examples.
In the tables below, the COBAS BIO FP~ instrument was used for making fluorescence polarization readings.
In a process of the invention, urine sample is added to antibody reagent containing the releasing reagent and background fluorescence intensity readings are made. A
fluorescent ~racer, such as the following fluorescein derivative OH
~ ~ h o~~ NH--C-NH~O
~ ~ 35 ~ ~ OH
.~
is then added and after mixing, fluorescence intensity readings are made and fluorescence polarization is calculated and analyte concentration is determined by reference to a standard calibra~ion curve.
In the tables below, the above procedures were used to determine the percent recovery of ll-nor-a9-tetrahydro-cannabinol-9-carboxylic acid from urine samples to whish were added 0.05w~v~ human serum albu~in and ~00 nanograms/
milliliter of 11-nor-~9-tetrahydrocannabinol-9-carboxylic acid. Where no releasing agent is used, ~as shown in Tables 1 and 2) less than half of ll-nor-~9-tetrahydrocanna-binol-9-carboxylic acid was recovered.
Prior to each reading, the antisera employed is diluted. For example, in Table 1 when 0.2w/v% salicylic acid i6 employed as the blocking agent, one volume of anti~era is dissolved in Antibody Bu~fer so as to make up a total final volume that was approximately 3,600 times larger.
The tracer which is employed in the examples given herein i8 a tetrahydrocannabinol derivative having the chemical formula ~
COOH ~ /) ' =9-NH~o 30 ~ ~ 0 0 0 It is described in ~uropean Patent Application 276,732, filed January 2ff, 198~.
:~
This com~ound is mixed to give a final concentration of 3 x 10 7 moles per liter into a solution having the , .. .
following composition: 0.1 molar ACES
~H2NCOCH2NHCHzCH2SO~H), buffer ~H 8; O.Olw/v%
bovine gamma globulin, O.lw/v% sodium azide.
TABLE_l Releasing agents premixed with Antibody Reagent. Recovery of ~9-THC-9-COOH~ from urine and mP Spans between Standards.
Blocking mP Span mP Span SampleaReCo~ery Agent w/v% 0-50 nq/ml 0-100 nq/ml Size ~ 9-THC-s-cooH*
None 46 119 14 49 0.2 Salicylate 33 103 - l.4 100 0.0005 LDS 40 108 14100 0.01 ANS 28 106 14 90 0.0005 bis-ANS 39 116 14 100 aUrine samples contained 0.05w/v% Human Serum Albumin and 100 ng/ml a9-THC-9-COOH.
; ~9-THC-9-COOH refers to ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid.
As can be seen from Table 1 just above, Salicylate, LDS, ANS, and bis-AWS are all effective as blocking agents in the ~fluorescence polarization immunoassay of the invention.
Alternatively, releasing or blocking agent may be directly mixed with the~urine sample prior to conducting the fluorescence polarization immunoassay. The amount of releasing agent employed is sufficient to allow for approximately total recovery of ll-nor-~9-tetrahydrocanna-binol-9-carboxylic acid but not such an amount as to destroy the activity of the antibody. In such a case, antisera reagent as de~cribed above is prepared without the releasing agent being added. Then urine sample and releasing agent are mixed in a sample cup and added to the antibody reagent. Background fluorescent intensity readings are made. Tracer as described above is added and after mixing, fluorescent intensity readings are made and the fluorescence polarization is calculated and th0 analyte concentration determined from a standard calibration curve.
A specific example where releasing agent is directly mixed with the urine sample prior ~o conducting the fluorescence polarization immunoassay is as follows: in Table 2 below, in the case where 0.005w/v% bis-ANS is used as the releasing agent, about 40~1 of urine and 120~1 of o~oosw/v% bis-ANS are added to a sample cup. 42~1 of this mixture (which is equivalent to 14~1 of urine) ace added ~o the antibody reagent and the background intensity readings are made. Accordingly, 14~1 of urine are entered in the column labelled "sample Size" in Table Z below.
In a like manner~ similar urine samples may be pretreated with a releasing agent of the invention. The fluorescent tracer is added as described above, and the resulting solution is mixed. The fluorescence polarization of this mixture is then determined.
Using the just above described process, and employing the above described tracer, and antibody reagent, the readings which appear in Table 2 below were made.
~' :
:: _ .
Releasing Agentæ used in Sample Pretreatment Procedure.
Recovery of Q9-THC-ll-9-COOH from urine and 5mP Spans between Stanclards.
Releasing mP Span mP Span Sample aRecovery A~ent w/v% 0-50 nq/mL O-lOO nq/mL 5ize ~ G9-THC-9-COOH
None 46 119 14 49 0.005 LDS 53 136 14 86 4 Salicylate 44 117 14 g6 4 Salicylate~ 41 125 14 96 0.002 LDS
t5 4 Salicylate~ 46 123 14 94 0.2 cholate 0.1 ANS 46 116 13.3 92 Q.005 bis-ANS 49 142 14 100 a Urine samples contained 0.05 w/v% human and 100 ng/ml ~9-THC-9-COOH.
As can be seen from Table Z jus~ above, LDS; Salicylate;
Salicylate and LDS; Sallcylate and cholate; ANS: and bis-ANS
are all effective as reIeasing agents at the specified concentrations. It can be seen that a releasing agent may also be a combination of chemical compounds that alone are releasing agents. It can also be seen from Table 2 above , tha~ where no releasing agent is employed in the 30 fluorescence~polarization immunoas&ay, less than 50%
recovery~of desired~ nor-~9-tetrahydrocannabinol-9-carboxylic acid Is achieved.
:
The concent~atlon of~re;leasing agent was optimized for 35 assay performance and for recovery of ll-nor-~9-tetra-hydrocannabinol-9-carboxylic acid. In this connection, it i8 noted ~hat ii too mgch releasing agent i8 employed, then ' ` .
3 ~
- lZ -the antibody activity in the assay is diminished. I~ too little releasing agent is employed then quantitative recovery of ll-nor-~9-tetrahydrocannabinol-9-carboxylic acid is not achieved~
It is preferred that releasing agellt be prasent in the antibody reagent in an amount sufficient to allow for recovery of at least about 85%, preferably 95-100% of the ll-nor-d9-tetrahydrocannabinol-9-carboxylic acid present in a sample~
Employing the procedures dsscribed above, there were obtained the data shown in Table 3 below which demonstrate the amount o~ releasing or blocking agent added to the antisera reagent needed for a 10-14 microliter urine sample. One skilled in the art will be able to determine the amount of releasing or blocking agent needed in accordance with the procedures herein and taking into account sample size, sample maturity, and the amount of antibody prssent.
' .
:
~ 30 :
~ .
., : .
.
.
: ~ ~ . ' ' ' ' : ,:
Releasing Agents Used When Premixed With the ~ntibody Reagent W~V%
Releasing Agent mP Span mP Span Sample %
~l 0-50_nq/ml0-100 ng~ml Size ~1 Recovery 0.2 Salicylate 33 103 14 100 0.2 Salicylate+ 40 108 14 100 0.0005 LDS
0.2 Salicylate~ 42 72 10 100 0.001 LDS
0.2 Salicylate 26 65 10 100 0.002 LDS
0.001 ANS 28 106 14 90 0.005 ANS 41 113 14 85 0.0005 bis~ANS 37 99 10 100 20 o.ooos bis-ANS 39 116 14 100 *Recovery of a9-T~C-9-COOH~from human urine containing 0.05w/v% human serum aLbumin.
Ab reagent means antibody~reagent.
The COBAS BIO FPTn instrument i6 employed collecting the da~a in this specification.
; 30 When the COBAS BIO FPm instrument is employed ana releasing agent is added directly ~o an amount of urine in a :: range of:abou~ 10~1 to about 17~1, the following are preferred concentrations in w~v% for the releasing agents ~: based on the total amount of releasing agent: about 0.02 to 35 0.5 for ANS about 0.003 to 0.1 for Bis-ANS, preferably about 0.005: about 3 to 10 for salicylic acid, preferably 4:
0.001 LDS to about 0.02 ~DS, preferably 0.005.
-- 1~
~ hen the COBAS BIO FP~ instrument is employed, and about 10~1 to about 14~1 of urine are tested and releasing agent is premixed with antibody reagent, the following are the preferred concentrations of releasing agent in w/v%: about 0.1 to 0.4, preferably about 0.2 salicylate about 0.2 salicylate with about 0.00Z LDS; about 0.0001 to about 0.002, preferably about 0.0005 Bis-ANS;
about 0.0001 to about 0.00Z, preferably about 0.0005 LDS
and about 0.005 to about 0.02 ANS, preferably about 0.01 ANS.
., .
Claims (22)
1. A reagent for use in fluorescence polarization immuno-assays comprising an antihody for 11-nor-.DELTA.9-tetrahydro-cannabinol-9-carboxylic acid; a releasing agent selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid,
2-anilinonaphthaline-6-sulfonic acid, lithium dodecyl sulfate, sodium dodecyl sulfate, ammonium dodecyl sulfate, cholic acid, benzoic acid, Witconate P10-59 and mixtures thereof; and aqueous buffer of pH in a range of about 7.0 to 8.5.
2. A reagent in accordance with claim 1 wherein the releasing agent is selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid; 1,1'-bi(4-anilino)-naphthalene-5,5'-disulfonic acid; and lithium dodecyl sulfate; and mixtures thereof.
2. A reagent in accordance with claim 1 wherein the releasing agent is selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid; 1,1'-bi(4-anilino)-naphthalene-5,5'-disulfonic acid; and lithium dodecyl sulfate; and mixtures thereof.
3. A reagent in accordance with claim 2 wherein the releasing agent is 1,1'-bi(4-anilino)naphthalene-5,5'--disulfonic acid.
4. A reagent in accordance with claim 1 which further comprises bovine gamma globulin and sodium azide.
5. A reagent in accordance with claim 5 wherein the bovine gamma globulin is present in 0.01w/v%: and sodium azide is present in 0.2w/v%.
6. A reagent in accordance with claim 1 wherein the releasing agent is present in an amount sufficient to allow for approximately total recovery of 11-nor-.DELTA.9-tetrahydro-cannabinol-9-carboxylic acid but not in such an amount so as to destroy the activity of the antibody.
7. A reagent in accordance with claim 6 wherein the releasing agent is present in an amount sufficient to allow for the recovery of at least about 95% of the 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid present in a urine sample.
8. A reagent in accordance with claim 7, wherein the releasing agent is present in an amount sufficient to allow for the recovery of at least about 85% of the 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid present in a urine sample.
9. A reagent in accordance with claim 1, comprising antisera from a goat; 0.1M phosphate buffer having a pH of about 8: 0.01w/v% bovine gamma globulin: 0.2w/v% sodium azide and a releasing agent.
10. A reagent in accordance with claim 9, wherein the releasing agent is 0.0005w/v% 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid.
11. An aqueous solution comprising urine and a releasing agent wherein the releasing agent is selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid, 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid, 2-anilinonaphthalene-6- sulfonic acid, lithium dodecyl sulfate, sodium dodecyl sulfate, ammonium dodecyl sulfate, cholic acid, benzoic acid, Witconate P10-59, and mixtures thereof.
12. An aqueous solution in accordance with claim 11 wherein the releasing agent is selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid; 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid; lithium dodecyl sulfate;
and mixtures thereof.
and mixtures thereof.
13. An aqueous solution in accordance with claim 12 wherein the releasing agent is 1,1'-bi(4-anilino)naphthalene-5.5'-disulfonic acid.
14. An aqueous solution in accordance with claim 11 wherein the releasing agent is present in an amount sufficient to allow for the recovery of at least about 95% of 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid in the urine.
15. An aqueous solution in accordance with claim 14, wherein the releasing agent is present in an amount sufficient to allow for the recovery of at least about 85% of 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxylic acid in the urine.
16. A fluorescence polarization immunoassay for 11-nor-.DELTA.9-tetrahydrocannabinol-9-carboxy1ic acid whlch is characterized by treating the urine sample to be assayed with a releasing agent 1-anilinonaphthalene-8-sulfonic acid;
1,1'-bi(4-anillno)naphthalene-5,5'-disulfonic acid;
2-anilinonaphthalene-6-sulfonic acid; lithium dodecyl sulfate; sodium dodecyl sulfate: ammonium dodecyl sulfate;
cholic acid; benzoic acid;
Witconate P10-59; and mixtures thereof.
1,1'-bi(4-anillno)naphthalene-5,5'-disulfonic acid;
2-anilinonaphthalene-6-sulfonic acid; lithium dodecyl sulfate; sodium dodecyl sulfate: ammonium dodecyl sulfate;
cholic acid; benzoic acid;
Witconate P10-59; and mixtures thereof.
17. The process in accordance with claim 16 wherein the releasing agent is selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid; and 1,1'-bi(4-anilino) naphthalene-5,5'-disulfonic acid; and lithium dodecyl sulfate.
18. The process in accordance with claim 17 wherein the releasing agent is 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid.
19. A fluorescence polarization immunoassay for tetra-hydrocannabinol in urine, characterized by adding to the antibody reagent, a releasing agent selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid, 2-anilinonaphthaline-6-sulfonic acid, lithium dodecyl sulfate, sodium dodecyl sulfate, ammonium dodecyl sulfate, cholic acid, benzoic acid, Witconate P10-59, and mixtures thereof.
20. The process in accordance with claim 19 wherein the releasing agent is a 1-anilinonaphthalene-8-sulfonic acid;
1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid; and lithium dodecyl sulfate or a mixture thereof.
1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid; and lithium dodecyl sulfate or a mixture thereof.
21. The process in accordance with claim 20, wherein the releasing agent is a naphthalene sulfonic acid selected from the group consisting of 1-anilinonaphthalene-8-sulfonic acid, and 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid.
22. The process in accordance with claim 21 wherein the releasing agent is 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US33704089A | 1989-04-12 | 1989-04-12 | |
| US337,040 | 1989-04-12 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2014233A1 true CA2014233A1 (en) | 1990-10-12 |
Family
ID=23318846
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002014233A Abandoned CA2014233A1 (en) | 1989-04-12 | 1990-04-10 | Polarization immunoassay for cannabinoids |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0392332A3 (en) |
| JP (1) | JPH02293663A (en) |
| AU (1) | AU5316690A (en) |
| CA (1) | CA2014233A1 (en) |
| NO (1) | NO901649L (en) |
| ZA (1) | ZA902748B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159698A (en) * | 1996-07-18 | 2000-12-12 | Dade Behring Marburg Gmbh | Reagents for assays for mycophenolic acid |
| US6171801B1 (en) | 1996-07-18 | 2001-01-09 | Dade Behring Marburg Gmbh | Methods for releasing a ligand from a complex |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5292899A (en) * | 1991-11-27 | 1994-03-08 | Synthetic Technology Corporation | Synthesis of 11-nor-Δ-9-tetrahydrocannabinol-9-carboxylic acid glucuronide |
| US5474907A (en) * | 1994-03-25 | 1995-12-12 | Eastman Kodak Company | Multilayer analytical element for salicylate assay |
| JP3487643B2 (en) * | 1994-06-16 | 2004-01-19 | アボットジャパン株式会社 | Method for measuring specific IgM antibody and reagent therefor |
| JP2789306B2 (en) * | 1994-11-15 | 1998-08-20 | 株式会社第一ラジオアイソトープ研究所 | Immunological measurement method of insulin-like growth factor and kit for measuring insulin-like growth factor |
| IT1302564B1 (en) * | 1997-05-22 | 2000-09-29 | Hoffmann La Roche | POLLIZATION IMMUNOSAGE OF PERFECTED FLUORESCENCE. |
| EP2005170A1 (en) | 2006-03-24 | 2008-12-24 | Aokin Ag | Use of additives to lower the rate of a binding reaction |
| US8124359B2 (en) | 2006-03-24 | 2012-02-28 | Aokin Ag | Use of additives for the reduction of non-specific binding in assays |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4939264A (en) * | 1986-07-14 | 1990-07-03 | Abbott Laboratories | Immunoassay for opiate alkaloids and their metabolites; tracers, immunogens and antibodies |
| US4833073A (en) * | 1987-01-27 | 1989-05-23 | Hoffmann-La Roche Inc. | Immunoassay for tetrahydrocannabinol metabolites |
| DE3855490T2 (en) * | 1987-02-17 | 1997-03-27 | Abbott Lab | Fluorescence polarization immunoassay for tetrahydrocannabinoids |
| EP0283801A3 (en) * | 1987-03-27 | 1990-05-30 | Abbott Laboratories | Fluorescence polarization assay for cyclosporin a and metabolites and related immunogens and antibodies |
-
1990
- 1990-04-04 EP EP19900106444 patent/EP0392332A3/en not_active Withdrawn
- 1990-04-10 CA CA002014233A patent/CA2014233A1/en not_active Abandoned
- 1990-04-10 ZA ZA902748A patent/ZA902748B/en unknown
- 1990-04-11 JP JP2094201A patent/JPH02293663A/en active Pending
- 1990-04-11 AU AU53166/90A patent/AU5316690A/en not_active Abandoned
- 1990-04-11 NO NO90901649A patent/NO901649L/en unknown
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6159698A (en) * | 1996-07-18 | 2000-12-12 | Dade Behring Marburg Gmbh | Reagents for assays for mycophenolic acid |
| US6171801B1 (en) | 1996-07-18 | 2001-01-09 | Dade Behring Marburg Gmbh | Methods for releasing a ligand from a complex |
| US6887669B1 (en) | 1996-07-18 | 2005-05-03 | Dade Behring Marburg Gmbh | Reagents for assays for ligands |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02293663A (en) | 1990-12-04 |
| EP0392332A3 (en) | 1992-01-08 |
| AU5316690A (en) | 1990-10-18 |
| NO901649D0 (en) | 1990-04-11 |
| NO901649L (en) | 1990-10-15 |
| EP0392332A2 (en) | 1990-10-17 |
| ZA902748B (en) | 1990-12-28 |
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