CA1272193A - Divalent hapten derivatives, process for preparing and use of them - Google Patents
Divalent hapten derivatives, process for preparing and use of themInfo
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- CA1272193A CA1272193A CA000494628A CA494628A CA1272193A CA 1272193 A CA1272193 A CA 1272193A CA 000494628 A CA000494628 A CA 000494628A CA 494628 A CA494628 A CA 494628A CA 1272193 A CA1272193 A CA 1272193A
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- hapten
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Abstract
Abstract Divalent hapten derivatives used for the determination of drugs having the formula I
A-X-A
I
where A is a bonded hapten moiety and X is a bifunctional spacer having the formula II
(B)m-Y-(CH2)n-Z-(CH2)n-Y-(B)m II
where m is independently 0 or 1, B is (CH2)n', wherein n' is an integer from 1-4, or CO(CH2)n", wherein n" is an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R is hydrogen or an aliphatic radical; n is an integer from 1-10; and Z is an organic residue containing one or more hydrophilic atoms, and a process for preparing such derivatives.
A-X-A
I
where A is a bonded hapten moiety and X is a bifunctional spacer having the formula II
(B)m-Y-(CH2)n-Z-(CH2)n-Y-(B)m II
where m is independently 0 or 1, B is (CH2)n', wherein n' is an integer from 1-4, or CO(CH2)n", wherein n" is an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R is hydrogen or an aliphatic radical; n is an integer from 1-10; and Z is an organic residue containing one or more hydrophilic atoms, and a process for preparing such derivatives.
Description
3.~7~
Divalent Rapten Derivatives, Proces3 for Preparing and Use of Them _ The invention relates to a divalen~ derivative of a hapten of the formula A-X-~, in which derivative two haptens are connected to each other by a chemical spacer, and in which formula A is a hapten and X i5 a chemical spac~r, to a process for preparing such a divalent derivative as well as to the use of such a divalent derivative in an immuno-logic assay.
A hapten may be defined as a chemical composition of limited molecular weight (usually less than 1000) which in and of itself does not elici~ antibody formation when introduced into a host animal. However, when covalently bonded to a high molecular weight antigenic carrier, the resultant hapten-carrier conjugate can elicit in the host animal the formation of antibodies which recognize the hapten compositionO Examples of haptens to which antibodies have been raised in this fashion are numerous, including such classes of materials as therpeu~ic drugs, amino acids and metabolites, small peptides, steroidc and aromatic residues such as the dinitrophenyl moiety. Typical carriers are large polyvalent molecules such as proteins which are not native to the host animal. The methods for preparation of hapten-carrier conjugate~ are well known in the art and have been reviewed by B.F. Erlanger in Methods of Enzymo-l~gy, vol. 70, p. 84, l9U~.
A divalent hapten may be considered a~ a subclass of hapten-carrier conjugates. The carrier~ which may be of any molecular weight, i8 covalently bonded to the haptens by means of only two functional groups. The two functions may be different but they are generally the same. The carrier molecule also may~be of any molecular weight or of any molecular -~hape.
Divalent Rapten Derivatives, Proces3 for Preparing and Use of Them _ The invention relates to a divalen~ derivative of a hapten of the formula A-X-~, in which derivative two haptens are connected to each other by a chemical spacer, and in which formula A is a hapten and X i5 a chemical spac~r, to a process for preparing such a divalent derivative as well as to the use of such a divalent derivative in an immuno-logic assay.
A hapten may be defined as a chemical composition of limited molecular weight (usually less than 1000) which in and of itself does not elici~ antibody formation when introduced into a host animal. However, when covalently bonded to a high molecular weight antigenic carrier, the resultant hapten-carrier conjugate can elicit in the host animal the formation of antibodies which recognize the hapten compositionO Examples of haptens to which antibodies have been raised in this fashion are numerous, including such classes of materials as therpeu~ic drugs, amino acids and metabolites, small peptides, steroidc and aromatic residues such as the dinitrophenyl moiety. Typical carriers are large polyvalent molecules such as proteins which are not native to the host animal. The methods for preparation of hapten-carrier conjugate~ are well known in the art and have been reviewed by B.F. Erlanger in Methods of Enzymo-l~gy, vol. 70, p. 84, l9U~.
A divalent hapten may be considered a~ a subclass of hapten-carrier conjugates. The carrier~ which may be of any molecular weight, i8 covalently bonded to the haptens by means of only two functional groups. The two functions may be different but they are generally the same. The carrier molecule also may~be of any molecular weight or of any molecular -~hape.
2 - ~ 2~
The carriers disclosed herein function primarily to separate the two hapten moieties by a chemical spacer.
Because of their divalent character they can bridge antigen binding sites on adjacen antibody molecules direc~ed ~owards hapten, thus differentia~ing from free hapten.
The concept of divalent haptens for bridging adjacent antibody molecules was investigated by R.C~ Valentine and N.M. Green for a series of 2,4-dinitrophenylated poly-methylenediamlnes (J.Molecular Biol. 27, 615, 1967). It was found that a minimum of an eight carbon length spacer allowed bridging of antibodie~. Howevex, the water solubi-lity of the spacers described by Valentine and Green wa~
found to be very poor. A divalent hapten sy~tem which is more water soluble i5 needed and desired.
This invention r~lates to a divalent hapten d~rivative.
More particularly, this invention relates to a derivative wherein two hapten moieties are connected by means of a bifunctional space~ wherein the derivative has the for-mula I
A-XA
where A iR a bonded hapten moiety and X is a bifunctional spacer having the formula II
(B)~-y-(cH2)n-z-(cH2)n-y-(B)~ II
where m i8 lndependently O or 1, B is (CH2)n, wherein n' i~ an integer from 1 4, or CO(CH2)n., where$n n" i~ an integer from 2-4; Y i~ independently -CONH-, -MHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R i~ hydrogen or alkyl; n i8 an integer from 1-10 and n may be the same or a different in~eger on opposite ~ides of component Z; and Z i~ an organic re~idue containing one or more hydrophilic atoms.
, ....
~ 3 Thi~ invention relates to a divalent hapten derivative in which two hapten moieties are coupled by means of a bifunctional spacer having the formula A-X-A
where A is a radical of a hap~en and X i~ a bifunctional spacer having the formula II
(B)~-y-(cH2)n-z-(~H2)n-y-(B)~ II
where m is independently 0 or 1, B i~ (CH2)n~, wherein n~ i8 an integer from 1-4, or CO(CH2)n~, wherein n" i~ an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO , -O-, -S-, or -NR-, wherein R is hydrogen or alkyl; n i~ an in~eger from 1-10;
and n may be the same or a dif~erent in~eger on oppo3ite side~
of component Z; and Z is selected from the group con~isting of a C-C single bond, alXylene, -alkylene-O-alkylene-, alkylene-NH-alkylene, alkylPne-N-alkylene, ,,,,al~ylens alkyl -N N-~ alkylene 20 -N-H-alkylene-NH, -O-alkylene-O- , OH OH
-alkylene-CH-alkylene-, -alkylene-C~alkylene-, alkyl o -alkylene-S-alkylene-, and -alkylene-S-alkylene-, O O
where tha term alkyl means a straight or branched chain saturated hydrocarbon group havin~ 1 to 6 carbon atom~ and tha term alkylene re~ers to 8 bivalent radical of the branched or unbranched alkyl group it i~ derived from having valence bonds from two termlnal carbons th~reof.
The invention di3closed herein i~ a new class of divaletn haptens in which the carrier (herein simply .referred to as bifunctional spacer) is a homobifunc~ional, predomina~ely ~
~ .
linear organic molecule in which one or more heteroatoms have been substituted for carbon atoms. Examples of pre-ferred heteroatoms are oxygen and nitrogen. Incorporation of these heteroatoms imparts a significant increase in water solubility to the hapten conjugates over analogs in which they are absent. For hydrophobic haptens, this solubilizing feature of the spacer allows the constitution of the divalent haptens in aqueous buffers suitable for subsequent interaction with antibodies.
Haptens may be coupled to the bifunctional spacer by a variety of covalent bonds, but a particularly preferred method is formation of an amide bond. For the formation of an amide bond, the hapten must either contain a carboxyl function or be readily converted therto. An example of a suitable hapten which contains a carboxyl function is L-thyroxine. Examples of haptens to which a carboxyl group may be introduced include Theophylline, Phenytoin and Phenobarbital.
In those cases where the hapten to be coupled does not contain a carboxyl function, it may be introduced by a variety of methods known to those skilled in the art. For instance, those haptens which contain a nucleophilic function such as an amine, alcohol or thiol may be alky-lated with a halo-alkyl-carbocylic acid or reacted with succinic anydride to give a hemisuccinate.
If amine functions or other reactive groups are present in the hapten, such as in the case of amino aceids, these must be masked by a protecting group before attempting condensation with the diamine bifunctional spacer. Pro-tecting groups may be chosen from any of those well known in the art as long as their subsequent removal after conjugation does not interfere with the integrity of the divalent hapten. Suitable protecting groups include t-butyloxycarbonyl, benzyloxycarbonyl and trifluoroacetyl, and suitable masking procedures are well known in the art.
z~2~3 The bifunctional spacer of this invention is chosen to have a length such that steric interactions b~tween immobilized antibodv particles are minimized. In addition, the bifunctional spacer is chosen to contain features which lend water solubility to the divalent conjugate, for example by inclusion of hydrophilic atoms, such as nitro-gen or oxygen. For this reason, component Z of the bi-functional spacer is chosen to be a groups such as -O-(CH2)4-0-, piperaz-1,4-diyl(1,4-disubstited pipera zine), or -NH-(CH2)4-NH-, to provide water solubility to the conjugate. This feature provides an advantage of the instant compositions over those known in the art (R.C.
Valentine and N.M. Green J.Mol.Biol. 27, 615, 1967).
Although is is not necessary for the practice of this invention that the bifunctional spacer be symmetrical in composition, it is preferred for convenience of synthesis.
Suitable bifunctional spacers are readily synthesized by methods well known in the art and many are commercially available.
The coupling of the hapten carboxylate to a diamine bifunctional spacer is performed by preactivation of the carboxylic acid group by any of a number of techniques well known in the art for amide bond formation. These include the use of condensing reagents such as carbodii-mides or carbonyldiimidazole, as well as pre-formed anydrides, acid chlorides, or active esters of the carboxylic acid. An unusual activated carboxyl function which is uniquely useful for theophylline is the cyclic lactam of the 8-butyric acid derivative (see example 5 herein).
The coupling of the activated hapten carboxylate to the diamine bifunctional spacer is performed with a minimum molar ratio of 2 haptens per bifunctional spacer, and preferably with a moderate excess of hapten moiety in order to drive the reation to completion. Coupling may be employed in a variety of solvents. Particularly preferred ~ ~ 27~3 solvents are those of the dipolar aprotic type, such as dimethylformamide or dimethylsulfoxide.
The final reaction mixture generally is composed ex~
clusively of divalent hapten conjugate and excess activated hapten carboxylate. Isolation of the divalent hapten is generally accomplished by selective precipi-tation or crystallization. In more difficult cases, chromatographic methods such as silica gel chromatography may be used to advantage.
It is anticipated that formation of divalent haptens can be the basis for affinity purification of polyclonal antibody mixtures. Typically, a polyclonal antibody mixture contains only a small fraction_of molecules which will bind specifically to the hapten of interest. In order to isolate this fraction, an affinity support containing immobilized hapten may be constructed. Construction of such supports is often complex and the performance thereof is subject to variables which are difficult to optimize ~i.e., hapten load and length of the "leash" connecting hapten to solid matrix). The techniques and associated problems of affinity purification of antibodies on the solid phase have been reviewed in Immunoadsorbents in Protein Purification, ed. E. Ruoslahti, University Press, Baltimore, p. 18 (1976). Alternately, small soluble com-plexes between divalent haptens and specific antibodies may be formed and the separation from extraneous anti-bodies may be accomplished by molecular sieving techniques well known to those skilled in the art. Following column separation the complexes are dissociated by methods well known in the art such as treatment with chaotropic salts or acidification. The free purified antibody is then obtained by means of a second gel filtration.
It is anticipated that such a procedure can be conducted as follows:
_ - 7 -Polyclonal Immunoglobulin G, 10 mg, of known titer in phosphobuffered saline (PBS) pH 7~4, is adjusted to a concentration of 1-10 mg/ml and treated with a slight excess of divalent hap~en. After incubation for 30-60 minutes at room temperature, the mixture is loaded onto a column of 200 cc Sephadex*G-200 gel equilibrated with PBS.
The column is eluted with P~S, collecting 1-5 ml fractions The complexes (MW ~ 300,000~ are excluded from the gel and appear at the void volume (VO) of the column whereas the non-specific antibodies appear as a later retained peak.
The peak fractions are located by absorbance at 280 nm, and pooled. The complexes are dissociated by treatmen~ for 5-10 minutes with an equal volume of 3M potassium thio-cyanate or lM propionic acid followed by gel filtration to remove salts and free divalent hapten The divalPnt hapten derivatives of this invention may also be used in turbidimetric drug assays. In recent times, particular interest ha been directed at a clinical assay method or diagnosis in which the pathological state or the pathological prognosis of a patient is assessed, or the medicament dose which is to be administered to the patient is determined, in such a manner that the content is the urine or blood of a physiologically active substance which has a low molecular weight and is in his body in the form of a hapten and its metabolites, or of a medicament which has been administered to the patient and its metabolites, is measured.
Examples of the haptens to be measured in carrying out this clinical assay method or diagnosis are the following:
thyroid hormones such as, for example, T3 and T4.
The medicaments which are to be mea~ured in this assay method or diagnosis are the following: medicaments who~e dose ought to be determined with extreme care and whose actions exhibit a relation to their concentration in the * denotes trade mark ~ .~
blood or urine, for example digitalis preparations, antibiotics, such as tetracycline, psychotropic agents, such as amphetamine, narcotics, such as morphine, blood coagulants and anticoagulants, in particular theophylline and phenobarbital.
The term "hapten" according to the invention is to be understood to be a physiologically active substance of low molecular weight which is present in the human body, and its metabolites, or a medicament which is administered to the human body, and its metàbolites, a substance of this type being incapable by itself of eliciting an antibody and being able to elicit an antibody when it is bound to a carrier substance which is itself an antigen, such as protein, poolysaccharide or glycoprotein.
The haptens are usually found in trace quantities, being present in blood or urine as complex-bound or conjugated forms with complicated compositions. For this reason, an elaborate and time-consuming method to determine and measure them is required.
A variety of methods can be used to measure these haptens, namely physicochemicxal methods, immunochemical methods or competitive protein-binding methods.
Immunochemical methods are now superior to physicochemical methods in respect of reaction specificity and sensitivity of measurement. A large number of immunochemical methods for the measurement of traces of haptens in the human body are known at present, for example agglutination inhibition methods, radioimmunoassay (abbreviated to RIA below) and enzyme immunoassay (called EIA below). In a known EIA
method, use is made of particles which are loaded with the antibody to the hapten which is to be measured and of a coupling oroduct of the hapten with a carrier substance (German Offenlegungsschrift 21 55 658).
~ ~7;;~
g The procedure for an agglutination inhibition method entails the use of a coupling product of a hapten, which is the same hapten as the hapten which is to be measured, and a carrier substance, such as a protein, a polysaccha-ride or a glycoprotein, bound to solid particles, for example blood cells or latex with a high molecular weight as the antigen. An antibody to the hapten which is to be measured is obtained from an antiserum which is obtained from mammals, for example guinea pigs, rabbits or sheep, which have been immunized with the coupling product of the hapten and the carrier subs`tance.
When the antibody is mixed with the fine particles onto which the hapten-carrier conjugates are coupled, then an agglutination reaction between these two components takes pla,ce. The agglutination reaction is inhibited when the hapten which is to be measured is present in the sample.
This method is an extremely straightforward method which is able to measure the hapten which is present in the blood or urine in the form of a complex or conjugate, without there being the necessity for complicated metho-dological measure, such as hydrolysis or chromatogrpahy.
The sensitivity of this method is about 100 mg/ml, even if blood cells are used as the particles. However, since most haptens which it is of interest to measure in the human body are present in quantities between 500 pg/ml and 50 ng/ml, they must be concentrated in order to carry out successful measurement.
A further object of the invention is therefore to develop a new immunochemical method for the measurement of haptens by inhibition of agglutination which exhibits higher sensitivity of measurement than the known method~
This object is achieved by a process wherin a hapten is immunochemically determined by means of agglutination inhibition which comprises contacting particles loaded with antibodies to the hapten to be determined, a low 2~
molecular compound carrying the hapten to be determined twice and the liquid containing the hapten to be deter-mined and determininy the amount of the hapten in the liquid from the reduction of turbidity caused by the hapten in the liquid.
It is the state of the art that an agglutination inhi-bition method (German Patent 27 43 445 C2) makes use of particles loaded with the antibody to the hapten and of a hapten coupled to a carrier~.
In order to carry out the invention, usually a particular volume of the body fluid or of the excretion which contain the hapten which is to be measured is removed and used directly as the assay solution or is diluted appropriately to various concentrations, whereupon the reaction between a specified volume of the assay solution and a specified volume of a suspension of particles loaded with the antibody is allowed to take place.
Then, a specified volume of a solution of the divalent hapten (dihapten) is added to this reaction mixture. After a particular reaction time, a nephelometric or turbidime-tric measurement is carried out. The evaluation makes use of a reference curve measured in parallel.
The antibodies of the hapten are obtained in a known manner, as follows: a hapten is coupled to a carrier substance. An antiserum is obtained from an animal which has been immunized by a routine method with the hapten coupled to the carrier as the antigen. The antibodies to the hapten are obtained in such a manner that the other antibodies, i.e. the antibodies to the carrier and/or the linking part of the carrier and the hapten are removed by absorption. Any desired materials can be used as carrier substances. Good results are obtained when the carrier substances have high antigenicity.
Keyhole limpet hemocyanin may be mentioned as an example.
The particles which are to be loaded are finely divided carriers such as are normally used for carrying out immunochemical agglutination reactions or agglutination inhibition reactions such as high molecular weight latex.
To load the latex particles, the antibody to the hapten, which has been obtained by the methods described above, is covalently bonded in accordance with European Patent Application 0 080 614 (USP 4,448,908 and Certificate of Correction).
The hapten which is coupled to the carrier and is used to elicit an antibody to the hapten, and the dihapten used as a developer in the method of measurement, can differ from one another at the point of coupling between the caxrier substance and the hapten.
Examples of haptens which are to be measured according to the invention are the medicaments mentioned below:
theophylline, phenobarbital, diphenylhydantoin, digoxin, primidone, valproic acid, carbamazepine, gentamycin, tobramycin and kanamycin.
Examples of other haptens to be measured according to the invention are thyroid hormones such as T3 and T4.
Compared with the conventional method, the invention provides extremely high sensitivity of measurement.
Only small quantities of the antibody to the hapten and the dihapten are necessary according to the invention. For example, the quantity used of a particular dihapten, namely ditheophylline, can be reduced to about 1/100 to 1/1,000 of the quantity necessary to carry out the con-ventional method.
According to the conventional method, German Patent 27 43 445 C2, for the measurement of haptens by inhibition of agglutination, a hapten coupled to a carrier is used.
As described in this patent, the sensitivity of measure-ment can be increased further if the hapten-carrier conjugate is bound to fine particles.
We have found, surprisingly, that a dimeric hapten derivative is more suitable for nephelometric measurement than is a hapten-carrier conjugate.
The advantage of the dimeric hapten derivative are that the measurement is more sensitive and can be carried out in a shorter measuring time. Another great advantage is that the elaborate synthesis of a hapten-carrier con-jugate, such as, for example, a hapten-apoferritin or hapten-albumin conjugate, is dispensed with. As is familiar to those skilled in the art, the protein-hapten conjugates mentioned can be prepared with only moderate yields, because of side reactions, and there are wide variations between batches. These derivatives are very heterogeneous in respect of molecular weight (protein-protein crosslinking) and the quantity of hapten loaded.
The preparation of the dimeric hapten derivatives is straightforward and the costs are favorable. Since no uncontrolled side reactions can occur, constancy between batches is ensured and can reliably be checked by means of analytical data tmass spectroscopic analysis, C-H-N ana-lysis, infrared and nuclear magnetic resonance spectra).
The following examples are illustrative of the invention but it is understood that the invention is not limited thereto.
~ ~7~33 -Example 1 Theophylline-8-butyric acid 25 g of diaminodimethyluracil hydrate and 50 g of glutaric anhydride were refluxed in 250 ml dimethylaniline for 3 h.
The mixture was cooled and added to 1.5 liters of water and 50 ml of concentrated sodium hydroxide solution. The resultant solution was extracted with 3000 ml of ether to remove dimethylaniline. The aqueous layer was diluted further with 1.5 liters of~water, then passed through a 1.5 liters bed of Dowe~ 2 anion exchange resin in the formate form. The bed was washed with 6 liters of water, then 4 liters of 0.1 M formic acid. The product was eluted with 0.2 M formic acid in a 1:1 mixture of water and acetone. The eluates containing product were pooled and evaporated under reduced pressure to a crystalline slurry, The crystals were collected, washed with cold water, and recrystallized from hot isopropanol to give 20 g of theophylline-8-butyric acid.
Example 2 bis-(Theophylline-8-butyryl)-1,6-hexanediamide Theophylline-8-butyric acid, 1.0 g, was dissolved in 10 ml of dimethylformamide. Tributylamine, 0.89 ml, was added and the solution was chilled to 5C. Isobutylchloroformate, 0.48 ml, was then added and the mixture was stirred for 10 minutes. Hexanediamine, 0.20 g, dissolved in 10 ml of dimethylformamide and chilled to 5C was then added. The reaction mixture was stirred for 30 minutes at 0-5C. A
heavy gelatinous precipitate formed which was filtered off and washed with dimethylformamide~ The product was dried in vacuum to yield 0.75 g of bis-(theophylline-8-butyryl)-1,6-hexanediamide.
* denotes trade mark ~2~ 3 Example 3 bis-(Theophylline-8-butyryl)-4,9-dioxa-1,12-dodecane-diamide The synthesis was performed as in example 2, substituting 0.40 ml of 4,9-dioxa 1,12-dodecanediamine for 1,6-hexane-diamine. The reaction mixture, a solution, was poured into 500 ml of ethyl ether while stirring. After refrigeration overnight (about 16 hours), the suspension was filtered.
The crude solid was recrystallized from methanol to give 0.4S g of bis-(theophylline-8-butyryl)-4,9-dioxa-1,12-dodecane-diamideO
Example 4 Solubility comparison for bis (theophylline-8-butyryl)-diamides The bis-(theophylline-8-butyryl)-diamide~ of 1,6-hexane-diamine and 4,9-dioxa-1,12-dodecanediamine (examples 2 and
The carriers disclosed herein function primarily to separate the two hapten moieties by a chemical spacer.
Because of their divalent character they can bridge antigen binding sites on adjacen antibody molecules direc~ed ~owards hapten, thus differentia~ing from free hapten.
The concept of divalent haptens for bridging adjacent antibody molecules was investigated by R.C~ Valentine and N.M. Green for a series of 2,4-dinitrophenylated poly-methylenediamlnes (J.Molecular Biol. 27, 615, 1967). It was found that a minimum of an eight carbon length spacer allowed bridging of antibodie~. Howevex, the water solubi-lity of the spacers described by Valentine and Green wa~
found to be very poor. A divalent hapten sy~tem which is more water soluble i5 needed and desired.
This invention r~lates to a divalent hapten d~rivative.
More particularly, this invention relates to a derivative wherein two hapten moieties are connected by means of a bifunctional space~ wherein the derivative has the for-mula I
A-XA
where A iR a bonded hapten moiety and X is a bifunctional spacer having the formula II
(B)~-y-(cH2)n-z-(cH2)n-y-(B)~ II
where m i8 lndependently O or 1, B is (CH2)n, wherein n' i~ an integer from 1 4, or CO(CH2)n., where$n n" i~ an integer from 2-4; Y i~ independently -CONH-, -MHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R i~ hydrogen or alkyl; n i8 an integer from 1-10 and n may be the same or a different in~eger on opposite ~ides of component Z; and Z i~ an organic re~idue containing one or more hydrophilic atoms.
, ....
~ 3 Thi~ invention relates to a divalent hapten derivative in which two hapten moieties are coupled by means of a bifunctional spacer having the formula A-X-A
where A is a radical of a hap~en and X i~ a bifunctional spacer having the formula II
(B)~-y-(cH2)n-z-(~H2)n-y-(B)~ II
where m is independently 0 or 1, B i~ (CH2)n~, wherein n~ i8 an integer from 1-4, or CO(CH2)n~, wherein n" i~ an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO , -O-, -S-, or -NR-, wherein R is hydrogen or alkyl; n i~ an in~eger from 1-10;
and n may be the same or a dif~erent in~eger on oppo3ite side~
of component Z; and Z is selected from the group con~isting of a C-C single bond, alXylene, -alkylene-O-alkylene-, alkylene-NH-alkylene, alkylPne-N-alkylene, ,,,,al~ylens alkyl -N N-~ alkylene 20 -N-H-alkylene-NH, -O-alkylene-O- , OH OH
-alkylene-CH-alkylene-, -alkylene-C~alkylene-, alkyl o -alkylene-S-alkylene-, and -alkylene-S-alkylene-, O O
where tha term alkyl means a straight or branched chain saturated hydrocarbon group havin~ 1 to 6 carbon atom~ and tha term alkylene re~ers to 8 bivalent radical of the branched or unbranched alkyl group it i~ derived from having valence bonds from two termlnal carbons th~reof.
The invention di3closed herein i~ a new class of divaletn haptens in which the carrier (herein simply .referred to as bifunctional spacer) is a homobifunc~ional, predomina~ely ~
~ .
linear organic molecule in which one or more heteroatoms have been substituted for carbon atoms. Examples of pre-ferred heteroatoms are oxygen and nitrogen. Incorporation of these heteroatoms imparts a significant increase in water solubility to the hapten conjugates over analogs in which they are absent. For hydrophobic haptens, this solubilizing feature of the spacer allows the constitution of the divalent haptens in aqueous buffers suitable for subsequent interaction with antibodies.
Haptens may be coupled to the bifunctional spacer by a variety of covalent bonds, but a particularly preferred method is formation of an amide bond. For the formation of an amide bond, the hapten must either contain a carboxyl function or be readily converted therto. An example of a suitable hapten which contains a carboxyl function is L-thyroxine. Examples of haptens to which a carboxyl group may be introduced include Theophylline, Phenytoin and Phenobarbital.
In those cases where the hapten to be coupled does not contain a carboxyl function, it may be introduced by a variety of methods known to those skilled in the art. For instance, those haptens which contain a nucleophilic function such as an amine, alcohol or thiol may be alky-lated with a halo-alkyl-carbocylic acid or reacted with succinic anydride to give a hemisuccinate.
If amine functions or other reactive groups are present in the hapten, such as in the case of amino aceids, these must be masked by a protecting group before attempting condensation with the diamine bifunctional spacer. Pro-tecting groups may be chosen from any of those well known in the art as long as their subsequent removal after conjugation does not interfere with the integrity of the divalent hapten. Suitable protecting groups include t-butyloxycarbonyl, benzyloxycarbonyl and trifluoroacetyl, and suitable masking procedures are well known in the art.
z~2~3 The bifunctional spacer of this invention is chosen to have a length such that steric interactions b~tween immobilized antibodv particles are minimized. In addition, the bifunctional spacer is chosen to contain features which lend water solubility to the divalent conjugate, for example by inclusion of hydrophilic atoms, such as nitro-gen or oxygen. For this reason, component Z of the bi-functional spacer is chosen to be a groups such as -O-(CH2)4-0-, piperaz-1,4-diyl(1,4-disubstited pipera zine), or -NH-(CH2)4-NH-, to provide water solubility to the conjugate. This feature provides an advantage of the instant compositions over those known in the art (R.C.
Valentine and N.M. Green J.Mol.Biol. 27, 615, 1967).
Although is is not necessary for the practice of this invention that the bifunctional spacer be symmetrical in composition, it is preferred for convenience of synthesis.
Suitable bifunctional spacers are readily synthesized by methods well known in the art and many are commercially available.
The coupling of the hapten carboxylate to a diamine bifunctional spacer is performed by preactivation of the carboxylic acid group by any of a number of techniques well known in the art for amide bond formation. These include the use of condensing reagents such as carbodii-mides or carbonyldiimidazole, as well as pre-formed anydrides, acid chlorides, or active esters of the carboxylic acid. An unusual activated carboxyl function which is uniquely useful for theophylline is the cyclic lactam of the 8-butyric acid derivative (see example 5 herein).
The coupling of the activated hapten carboxylate to the diamine bifunctional spacer is performed with a minimum molar ratio of 2 haptens per bifunctional spacer, and preferably with a moderate excess of hapten moiety in order to drive the reation to completion. Coupling may be employed in a variety of solvents. Particularly preferred ~ ~ 27~3 solvents are those of the dipolar aprotic type, such as dimethylformamide or dimethylsulfoxide.
The final reaction mixture generally is composed ex~
clusively of divalent hapten conjugate and excess activated hapten carboxylate. Isolation of the divalent hapten is generally accomplished by selective precipi-tation or crystallization. In more difficult cases, chromatographic methods such as silica gel chromatography may be used to advantage.
It is anticipated that formation of divalent haptens can be the basis for affinity purification of polyclonal antibody mixtures. Typically, a polyclonal antibody mixture contains only a small fraction_of molecules which will bind specifically to the hapten of interest. In order to isolate this fraction, an affinity support containing immobilized hapten may be constructed. Construction of such supports is often complex and the performance thereof is subject to variables which are difficult to optimize ~i.e., hapten load and length of the "leash" connecting hapten to solid matrix). The techniques and associated problems of affinity purification of antibodies on the solid phase have been reviewed in Immunoadsorbents in Protein Purification, ed. E. Ruoslahti, University Press, Baltimore, p. 18 (1976). Alternately, small soluble com-plexes between divalent haptens and specific antibodies may be formed and the separation from extraneous anti-bodies may be accomplished by molecular sieving techniques well known to those skilled in the art. Following column separation the complexes are dissociated by methods well known in the art such as treatment with chaotropic salts or acidification. The free purified antibody is then obtained by means of a second gel filtration.
It is anticipated that such a procedure can be conducted as follows:
_ - 7 -Polyclonal Immunoglobulin G, 10 mg, of known titer in phosphobuffered saline (PBS) pH 7~4, is adjusted to a concentration of 1-10 mg/ml and treated with a slight excess of divalent hap~en. After incubation for 30-60 minutes at room temperature, the mixture is loaded onto a column of 200 cc Sephadex*G-200 gel equilibrated with PBS.
The column is eluted with P~S, collecting 1-5 ml fractions The complexes (MW ~ 300,000~ are excluded from the gel and appear at the void volume (VO) of the column whereas the non-specific antibodies appear as a later retained peak.
The peak fractions are located by absorbance at 280 nm, and pooled. The complexes are dissociated by treatmen~ for 5-10 minutes with an equal volume of 3M potassium thio-cyanate or lM propionic acid followed by gel filtration to remove salts and free divalent hapten The divalPnt hapten derivatives of this invention may also be used in turbidimetric drug assays. In recent times, particular interest ha been directed at a clinical assay method or diagnosis in which the pathological state or the pathological prognosis of a patient is assessed, or the medicament dose which is to be administered to the patient is determined, in such a manner that the content is the urine or blood of a physiologically active substance which has a low molecular weight and is in his body in the form of a hapten and its metabolites, or of a medicament which has been administered to the patient and its metabolites, is measured.
Examples of the haptens to be measured in carrying out this clinical assay method or diagnosis are the following:
thyroid hormones such as, for example, T3 and T4.
The medicaments which are to be mea~ured in this assay method or diagnosis are the following: medicaments who~e dose ought to be determined with extreme care and whose actions exhibit a relation to their concentration in the * denotes trade mark ~ .~
blood or urine, for example digitalis preparations, antibiotics, such as tetracycline, psychotropic agents, such as amphetamine, narcotics, such as morphine, blood coagulants and anticoagulants, in particular theophylline and phenobarbital.
The term "hapten" according to the invention is to be understood to be a physiologically active substance of low molecular weight which is present in the human body, and its metabolites, or a medicament which is administered to the human body, and its metàbolites, a substance of this type being incapable by itself of eliciting an antibody and being able to elicit an antibody when it is bound to a carrier substance which is itself an antigen, such as protein, poolysaccharide or glycoprotein.
The haptens are usually found in trace quantities, being present in blood or urine as complex-bound or conjugated forms with complicated compositions. For this reason, an elaborate and time-consuming method to determine and measure them is required.
A variety of methods can be used to measure these haptens, namely physicochemicxal methods, immunochemical methods or competitive protein-binding methods.
Immunochemical methods are now superior to physicochemical methods in respect of reaction specificity and sensitivity of measurement. A large number of immunochemical methods for the measurement of traces of haptens in the human body are known at present, for example agglutination inhibition methods, radioimmunoassay (abbreviated to RIA below) and enzyme immunoassay (called EIA below). In a known EIA
method, use is made of particles which are loaded with the antibody to the hapten which is to be measured and of a coupling oroduct of the hapten with a carrier substance (German Offenlegungsschrift 21 55 658).
~ ~7;;~
g The procedure for an agglutination inhibition method entails the use of a coupling product of a hapten, which is the same hapten as the hapten which is to be measured, and a carrier substance, such as a protein, a polysaccha-ride or a glycoprotein, bound to solid particles, for example blood cells or latex with a high molecular weight as the antigen. An antibody to the hapten which is to be measured is obtained from an antiserum which is obtained from mammals, for example guinea pigs, rabbits or sheep, which have been immunized with the coupling product of the hapten and the carrier subs`tance.
When the antibody is mixed with the fine particles onto which the hapten-carrier conjugates are coupled, then an agglutination reaction between these two components takes pla,ce. The agglutination reaction is inhibited when the hapten which is to be measured is present in the sample.
This method is an extremely straightforward method which is able to measure the hapten which is present in the blood or urine in the form of a complex or conjugate, without there being the necessity for complicated metho-dological measure, such as hydrolysis or chromatogrpahy.
The sensitivity of this method is about 100 mg/ml, even if blood cells are used as the particles. However, since most haptens which it is of interest to measure in the human body are present in quantities between 500 pg/ml and 50 ng/ml, they must be concentrated in order to carry out successful measurement.
A further object of the invention is therefore to develop a new immunochemical method for the measurement of haptens by inhibition of agglutination which exhibits higher sensitivity of measurement than the known method~
This object is achieved by a process wherin a hapten is immunochemically determined by means of agglutination inhibition which comprises contacting particles loaded with antibodies to the hapten to be determined, a low 2~
molecular compound carrying the hapten to be determined twice and the liquid containing the hapten to be deter-mined and determininy the amount of the hapten in the liquid from the reduction of turbidity caused by the hapten in the liquid.
It is the state of the art that an agglutination inhi-bition method (German Patent 27 43 445 C2) makes use of particles loaded with the antibody to the hapten and of a hapten coupled to a carrier~.
In order to carry out the invention, usually a particular volume of the body fluid or of the excretion which contain the hapten which is to be measured is removed and used directly as the assay solution or is diluted appropriately to various concentrations, whereupon the reaction between a specified volume of the assay solution and a specified volume of a suspension of particles loaded with the antibody is allowed to take place.
Then, a specified volume of a solution of the divalent hapten (dihapten) is added to this reaction mixture. After a particular reaction time, a nephelometric or turbidime-tric measurement is carried out. The evaluation makes use of a reference curve measured in parallel.
The antibodies of the hapten are obtained in a known manner, as follows: a hapten is coupled to a carrier substance. An antiserum is obtained from an animal which has been immunized by a routine method with the hapten coupled to the carrier as the antigen. The antibodies to the hapten are obtained in such a manner that the other antibodies, i.e. the antibodies to the carrier and/or the linking part of the carrier and the hapten are removed by absorption. Any desired materials can be used as carrier substances. Good results are obtained when the carrier substances have high antigenicity.
Keyhole limpet hemocyanin may be mentioned as an example.
The particles which are to be loaded are finely divided carriers such as are normally used for carrying out immunochemical agglutination reactions or agglutination inhibition reactions such as high molecular weight latex.
To load the latex particles, the antibody to the hapten, which has been obtained by the methods described above, is covalently bonded in accordance with European Patent Application 0 080 614 (USP 4,448,908 and Certificate of Correction).
The hapten which is coupled to the carrier and is used to elicit an antibody to the hapten, and the dihapten used as a developer in the method of measurement, can differ from one another at the point of coupling between the caxrier substance and the hapten.
Examples of haptens which are to be measured according to the invention are the medicaments mentioned below:
theophylline, phenobarbital, diphenylhydantoin, digoxin, primidone, valproic acid, carbamazepine, gentamycin, tobramycin and kanamycin.
Examples of other haptens to be measured according to the invention are thyroid hormones such as T3 and T4.
Compared with the conventional method, the invention provides extremely high sensitivity of measurement.
Only small quantities of the antibody to the hapten and the dihapten are necessary according to the invention. For example, the quantity used of a particular dihapten, namely ditheophylline, can be reduced to about 1/100 to 1/1,000 of the quantity necessary to carry out the con-ventional method.
According to the conventional method, German Patent 27 43 445 C2, for the measurement of haptens by inhibition of agglutination, a hapten coupled to a carrier is used.
As described in this patent, the sensitivity of measure-ment can be increased further if the hapten-carrier conjugate is bound to fine particles.
We have found, surprisingly, that a dimeric hapten derivative is more suitable for nephelometric measurement than is a hapten-carrier conjugate.
The advantage of the dimeric hapten derivative are that the measurement is more sensitive and can be carried out in a shorter measuring time. Another great advantage is that the elaborate synthesis of a hapten-carrier con-jugate, such as, for example, a hapten-apoferritin or hapten-albumin conjugate, is dispensed with. As is familiar to those skilled in the art, the protein-hapten conjugates mentioned can be prepared with only moderate yields, because of side reactions, and there are wide variations between batches. These derivatives are very heterogeneous in respect of molecular weight (protein-protein crosslinking) and the quantity of hapten loaded.
The preparation of the dimeric hapten derivatives is straightforward and the costs are favorable. Since no uncontrolled side reactions can occur, constancy between batches is ensured and can reliably be checked by means of analytical data tmass spectroscopic analysis, C-H-N ana-lysis, infrared and nuclear magnetic resonance spectra).
The following examples are illustrative of the invention but it is understood that the invention is not limited thereto.
~ ~7~33 -Example 1 Theophylline-8-butyric acid 25 g of diaminodimethyluracil hydrate and 50 g of glutaric anhydride were refluxed in 250 ml dimethylaniline for 3 h.
The mixture was cooled and added to 1.5 liters of water and 50 ml of concentrated sodium hydroxide solution. The resultant solution was extracted with 3000 ml of ether to remove dimethylaniline. The aqueous layer was diluted further with 1.5 liters of~water, then passed through a 1.5 liters bed of Dowe~ 2 anion exchange resin in the formate form. The bed was washed with 6 liters of water, then 4 liters of 0.1 M formic acid. The product was eluted with 0.2 M formic acid in a 1:1 mixture of water and acetone. The eluates containing product were pooled and evaporated under reduced pressure to a crystalline slurry, The crystals were collected, washed with cold water, and recrystallized from hot isopropanol to give 20 g of theophylline-8-butyric acid.
Example 2 bis-(Theophylline-8-butyryl)-1,6-hexanediamide Theophylline-8-butyric acid, 1.0 g, was dissolved in 10 ml of dimethylformamide. Tributylamine, 0.89 ml, was added and the solution was chilled to 5C. Isobutylchloroformate, 0.48 ml, was then added and the mixture was stirred for 10 minutes. Hexanediamine, 0.20 g, dissolved in 10 ml of dimethylformamide and chilled to 5C was then added. The reaction mixture was stirred for 30 minutes at 0-5C. A
heavy gelatinous precipitate formed which was filtered off and washed with dimethylformamide~ The product was dried in vacuum to yield 0.75 g of bis-(theophylline-8-butyryl)-1,6-hexanediamide.
* denotes trade mark ~2~ 3 Example 3 bis-(Theophylline-8-butyryl)-4,9-dioxa-1,12-dodecane-diamide The synthesis was performed as in example 2, substituting 0.40 ml of 4,9-dioxa 1,12-dodecanediamine for 1,6-hexane-diamine. The reaction mixture, a solution, was poured into 500 ml of ethyl ether while stirring. After refrigeration overnight (about 16 hours), the suspension was filtered.
The crude solid was recrystallized from methanol to give 0.4S g of bis-(theophylline-8-butyryl)-4,9-dioxa-1,12-dodecane-diamideO
Example 4 Solubility comparison for bis (theophylline-8-butyryl)-diamides The bis-(theophylline-8-butyryl)-diamide~ of 1,6-hexane-diamine and 4,9-dioxa-1,12-dodecanediamine (examples 2 and
3) were each dissolved in dimethylsulfoxide. ~he 4,9-dioxa-1,12-dode~anediamide dissolved readily to give a solution of 10 mg/0.5 ml at room temperature. This solutîon when diluted into 2 ml of 50 ~M aqueous sodium phasphate buffer (pH 7.5) ~ave a clear, stable solution. on the other hand, the 1,6-hexanediamide re~uired heating to obtain a ~olution of lO mg/2 ml in dimethylsulfoxide and upon dilution of this solution into a two-fold volume of pH 7.5 phosphate buffer, immediate heavy precipitation of divalent hapten was noted.
.~1 ~Z7~3 Example S
Theophylline-8-butyric acid lactam Theophylline-8-butyric acid, 24 g (example 1), was dis-solved in 500 ml of acetic anhydride with heating. The mixture was gently refluxed for 30 minutes, then filtered through a preheated funnel to remove insoluble material.
The product crystallized upon cooling. The product was collected, washed with ether and hexane then dired in vacuum at 50C to give 20 g of theophylline-8-butyric acid lactam.
Example 6 bis-(Theophylline-8-butyryl)-N,N-bis-3-aminopropyl pipera-zine diamide Theophylline-8-butyric acid lactam (example 5), 0.5 g was suspended in 5 ml dimethylformamide. Bis-(3-aminopropyl)-piperazine, 0.2 ml, and tributylamine, 0.48 ml, were added with stirring. A solution was initially obtained but after 15 minutes, a heavy gelatinous precipitate formed. The reaction mixture was diluted with 5 mi dimethylformamide and stirred overnight. The precipitate was filtered off and washed with dimethylformamide. Drying gave 0.6 g of bis-(theophylline-8-butyryl)-N,N-bis-3-aminopropyl pipera-zine.
Example 7 Phenobarbital-N-propionic acid Phenobarbital, 93 g, and 3-bromopropionic acid, 74 g, were added to 200 ml of water. With mixing, 48 g of sodium .
~Z~3 hydroxide was then added. The mixture was heated on a steam bath with occasional swirling for 1.5 hour. At this time, an additional 16 g of sodium hydroxide was added and heating was continued for another 1.5 hour. The solution was diluted to 1.6 liters with water then adjusted to pH
6.5 with concentrated hydrochloric acid. A precipitate of unreacted phanobarbital formed. The mixture was extracted with ethyl acetate, 400 ml, three ~imes to remove most of the phenobarbital. The remaining aqueous solution was adjusted to pH 4.5 with concentrated hydrochloric acid to give a milky suspension. The suspension was extracted twice with 400 ml ethyl acetate. The combined extract containing product was washed with water followed by saturated sodium chloride solution. The ex~ract was dried over magnesium sulfate, filtered and evaporated to an oil.
The oil was crystallized by dissolving in acetone and adding hexane to turbidity. The product was collected and dried to yield 20 g of phenobarbital-N-propionic acid.
Example 8 bis-(Phenobarbital-N-propionyl~-4,9-dioxa-1,12-dodecane-diamide Phenobarbital-N-propionic acid, 1.27 g, was dissolved in S
ml dimethyl~ormamide. While stirring at room temperature, 0.61 g of carbonyldiimidazole dissolved in 5 ml of di-methylformamides was added dropwise. After 30 minutes, 0.40 ml of 4,9-dioxa-1,12-dodecanediamine in S ml of dimethylformamide was added dropwise. A~ter addition was complete, the reaction was stirred at room temperature for 2 hours. The solvent wa~ evaporated on high vacuum and the residue triturated with hexane to give a solid. The resultant crude product was dissolved in chloroform/metha-nol (9:1), 10 ml, and applied to a column of silica gel.
The column was eluted with chloroform followed by chloro-form/methanol mixtures ranging from 9:1 to 5:5. The product which eluted with 9:1 mixture was homogeneous by thin layer chromatography (TLC). The fractions were pooled and evaporated to an oil. Vacuum drying gave an amorphous solid, 0.65 g, of bis-(phenobarbital-N-propionyl)-4,9-dioxa-1,12-dodecanediamide.
Example 9 Trifluoroacetyl-L-thyroxine L-thyroxine, sodium salt, 10 g, was added with stirring to a chilled mixture of 150 ml ethyl acetate and 40 ml tri-fluoroactic acid. To the resultant solution, 10 ml of tri-fluoroacetic anhydride was added dropwise. After addition was complete, the reaction was stirred for 2 hours at 0-2C. The reaction mixture was then poured into 200 ml ice-cold sodium chloride solution. The mixture was diluted with 100 ml ethyl acetate and the phases were separated.
The ethyl acetate phase was washed with 1 M sodium bisul-fate followed by saturated sodium chloride solution. The solution was then dried over magnesium sulfate, filtered and concentrated to a small volume. Dilution with ether followed by low boiling petroleum ether gave a flocculent precipitate. The precipitate was collected and dried to give 8.2 g of product. TLC showed a minor second spot for thyroxine. Purification by silica gel chromatography in chloroform/methanol (90:10) gave 4.3 g of trifluoroacetyl-L-thyroxine.
Example 10 bis-(trifluoroacetyl-L-thyroxyl)-4,9-dioxa-1,12-dodecane-diamide ~ ~:7~3 Trifluoroacetyl-L-thyroxine (example 8), 1.23 g , was dissolved in 8 ml thetrahydrofuran and the solution was chilled to 2C in an ice/acetone bath. N-methylmorpholine, 0.16 ml, was added followed by isobutylchloroformate, 0.18 ml. After stirring for 7 minutes, a solution of 0.15 ml
.~1 ~Z7~3 Example S
Theophylline-8-butyric acid lactam Theophylline-8-butyric acid, 24 g (example 1), was dis-solved in 500 ml of acetic anhydride with heating. The mixture was gently refluxed for 30 minutes, then filtered through a preheated funnel to remove insoluble material.
The product crystallized upon cooling. The product was collected, washed with ether and hexane then dired in vacuum at 50C to give 20 g of theophylline-8-butyric acid lactam.
Example 6 bis-(Theophylline-8-butyryl)-N,N-bis-3-aminopropyl pipera-zine diamide Theophylline-8-butyric acid lactam (example 5), 0.5 g was suspended in 5 ml dimethylformamide. Bis-(3-aminopropyl)-piperazine, 0.2 ml, and tributylamine, 0.48 ml, were added with stirring. A solution was initially obtained but after 15 minutes, a heavy gelatinous precipitate formed. The reaction mixture was diluted with 5 mi dimethylformamide and stirred overnight. The precipitate was filtered off and washed with dimethylformamide. Drying gave 0.6 g of bis-(theophylline-8-butyryl)-N,N-bis-3-aminopropyl pipera-zine.
Example 7 Phenobarbital-N-propionic acid Phenobarbital, 93 g, and 3-bromopropionic acid, 74 g, were added to 200 ml of water. With mixing, 48 g of sodium .
~Z~3 hydroxide was then added. The mixture was heated on a steam bath with occasional swirling for 1.5 hour. At this time, an additional 16 g of sodium hydroxide was added and heating was continued for another 1.5 hour. The solution was diluted to 1.6 liters with water then adjusted to pH
6.5 with concentrated hydrochloric acid. A precipitate of unreacted phanobarbital formed. The mixture was extracted with ethyl acetate, 400 ml, three ~imes to remove most of the phenobarbital. The remaining aqueous solution was adjusted to pH 4.5 with concentrated hydrochloric acid to give a milky suspension. The suspension was extracted twice with 400 ml ethyl acetate. The combined extract containing product was washed with water followed by saturated sodium chloride solution. The ex~ract was dried over magnesium sulfate, filtered and evaporated to an oil.
The oil was crystallized by dissolving in acetone and adding hexane to turbidity. The product was collected and dried to yield 20 g of phenobarbital-N-propionic acid.
Example 8 bis-(Phenobarbital-N-propionyl~-4,9-dioxa-1,12-dodecane-diamide Phenobarbital-N-propionic acid, 1.27 g, was dissolved in S
ml dimethyl~ormamide. While stirring at room temperature, 0.61 g of carbonyldiimidazole dissolved in 5 ml of di-methylformamides was added dropwise. After 30 minutes, 0.40 ml of 4,9-dioxa-1,12-dodecanediamine in S ml of dimethylformamide was added dropwise. A~ter addition was complete, the reaction was stirred at room temperature for 2 hours. The solvent wa~ evaporated on high vacuum and the residue triturated with hexane to give a solid. The resultant crude product was dissolved in chloroform/metha-nol (9:1), 10 ml, and applied to a column of silica gel.
The column was eluted with chloroform followed by chloro-form/methanol mixtures ranging from 9:1 to 5:5. The product which eluted with 9:1 mixture was homogeneous by thin layer chromatography (TLC). The fractions were pooled and evaporated to an oil. Vacuum drying gave an amorphous solid, 0.65 g, of bis-(phenobarbital-N-propionyl)-4,9-dioxa-1,12-dodecanediamide.
Example 9 Trifluoroacetyl-L-thyroxine L-thyroxine, sodium salt, 10 g, was added with stirring to a chilled mixture of 150 ml ethyl acetate and 40 ml tri-fluoroactic acid. To the resultant solution, 10 ml of tri-fluoroacetic anhydride was added dropwise. After addition was complete, the reaction was stirred for 2 hours at 0-2C. The reaction mixture was then poured into 200 ml ice-cold sodium chloride solution. The mixture was diluted with 100 ml ethyl acetate and the phases were separated.
The ethyl acetate phase was washed with 1 M sodium bisul-fate followed by saturated sodium chloride solution. The solution was then dried over magnesium sulfate, filtered and concentrated to a small volume. Dilution with ether followed by low boiling petroleum ether gave a flocculent precipitate. The precipitate was collected and dried to give 8.2 g of product. TLC showed a minor second spot for thyroxine. Purification by silica gel chromatography in chloroform/methanol (90:10) gave 4.3 g of trifluoroacetyl-L-thyroxine.
Example 10 bis-(trifluoroacetyl-L-thyroxyl)-4,9-dioxa-1,12-dodecane-diamide ~ ~:7~3 Trifluoroacetyl-L-thyroxine (example 8), 1.23 g , was dissolved in 8 ml thetrahydrofuran and the solution was chilled to 2C in an ice/acetone bath. N-methylmorpholine, 0.16 ml, was added followed by isobutylchloroformate, 0.18 ml. After stirring for 7 minutes, a solution of 0.15 ml
4,9-dioxa 1,12-dodecanediamine in 2 ml tetrahydrofuran was added dropwise over 5 minutes. The mixture was stirred at 0C for 30 minutes then warmed up to room temperature. The reaction was filtered to remove salts and the filtrate evaporated to an oil. The oil was redissolved in ethyl acetate and the solution was washed with aqueous 1 M
sodium bisulfate followed by 0.5 M sodium chloride. The ethyl acetate solution was dried over magnesium sulfate, filtered and concentrated to an oil. The oil was crystal-lized from ethyl acetate/hexane to obtain 0.6 g of bis-(trifluoroacetyl-L-thyroxyl)-4,9-dioxa-1,12-dodecane-diamide from two crops.
Example 11 bis-(L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide, dihydro-chloride salt bis-(trifluoroacetyl-L-thyroxyl)-4,9-diocadodecane-1,12-diamide (example 8A), 0.34 g, was dissolved in 3 ml methanol. 1 M sodium hydroxide was added with stirring and the reaction was allowed to go at room temperature for 40 hours. The reaction mixture was diluted into 20 ml of cold l M hydrochloric acid to givP a fine suspension of product.
The suspension was centrifuged and the pellet was dis-solved in ethanol. The ethanol solution was evaporated to a solid residue of crude product. The product was re-crystallized from methanol/ether to obtain 0.18 g of bis-(L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide, dihydro-chloride salt.
~ ~2~L~3 Example 12 bis-(N-succinyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide A solution of 2.0 g succinic anhydride in 20 ml pyridine was added dropwise to a chilled solution of 2.0 g 4,9-dioxa-1,12-dodecanediamine in 20 ml pyridine. ~he reaction mixture was stored at 4C for 18 hours then evaporated to a solid residue. The residue was dissolved in 30 ml water and passed through a bed of 30 ml Dowex 50 cation-exchange resin in the H~ form. The bed was then washed with 60 ml water. The effluent was concentrated to about half-volume to give colorless crystals of bis-succinyl-4,9-dioxa-1,12-dodecanediamide which were collected and dried. The yield was 2.18 g.
The intermediate was dissolved in 30 ml dimethylformamide along with 1.5 g N-hydroxysuccinimide. The solution was chilled and 2.45 g dicyclohexyl-carbodiimide in 15 ml dimethylformamide was added all at once. After 18 hours at 4C, the reaction mixture was filtered and the filtrate evaporated to an oil. The oil was crystallized from isopropanol and ether to give a crystalline product of 1.3 g bis-succinimido 0,N-succinyl-4,9-dioxa-1,12-dioxa-dodecanediamine. The latter active ester, 0.3 g, was dissolved in 2 ml dimethylformamide and added dropwise to a suspension of 0.89 g L-thyroxine, sodium salt in 4 ml dimethylformamide containing 0.11 ml N-methylmorpholine.
The reaction mixture was stirred for 72 hours. The re-sultant solution was evaporated to an oil. The oil was washed with a mixture of ethyl acetate and 1 M sodium bisulfate to give a solid which was collected by fil-tration, washed with water then methanol and dried to give 0.22 g of bis-(N-succinyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide.
7%~3 Example 13 Determination of theophylline The dihapten used was N,N'-di(theophylline-8-butyrylamino 3-propyl)piperazine.
The dihapten was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml, and then diluted 1:51,2000 in distilled water, with the addition of 4 % of human serum albumin. The diluted dihapt~en acts as a "developer" in the assay. A latex reagent was prepared by binding antiserum to theophylline onto polystyrene latex. The antitheophyl-line latex reagent was prepared as described in Canadian Patent No. 1,206,656. For the measurement, 10 ~1 of a sample containing theophylline, diluted 1:1000 in a 0.1 M
glycine/NaCl buffer, pH 8.0, were mixed in a cuvette with 10 ~1 of the developer dilution described above and with 20 ~1 of a phosphate-buffered sodium chloride solution containing 4.6 % of polyethylene glycol 6000 and with 10 ~1 of the antitheophylline latex reagent described above.
The mixture was incubated at room temperature for 12 min.
Then, a further 200 ~1 of a phosphate-buffered sodium chloride solution ~ontaining 4.6 % of polyethylene glycol 6000 were added, mixed in, and the light scattering was measured in a ~ehrinqwerke laser nephelometer.
An incubation curve as described in Fig. 1 was obtained with different quantities of theophylline added. The measuring range extended from about 1 to 40 ~g/ml theo-phylline.
For comparison, a measurement with an apoferritin de-veloper according to the state of the art was carried out.
The apoferritin-theophylline derivative was obtained from Kallestad (batch number: R 1). It was diluted 1:1,280 in aqueous human serum albumin solution and was used in this ,~
dilution as the developer in the assay. The mixture for the measurement was made up as described previously, ~ith the single exception that the apoferritin-theophylline derivative was used in place of the dihapten developer.
The inhibition curve (see Fig. 2) obtained with different quantities of theophylline added showed lower sensitivity, i.e. the lower detection limit was higher.
Example 14 Determination of phenobarbital The dihapten used was 1,12-di(phenobarbital-N-propionyl-amino)-4,9 dioxadodecane The dihapten was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml and then diluted 1:40,000 in aqueous 4 % strength human serum albumin solution. The diluted dihapten acts as a "developer" in the assay.
A latex reagent was prepared by binding antiserum to phenobarbital onto polystyrene latex. The antiphenobarbi-tal late7 raeagent was prepared as described in~ @æ~
Patent ~ 73Ø
For the measurement, 20 ~l of a sample containing pheno-barbital, diluted l:lO0 in a 0.1 M glycine/NaCl buffer, pH
8.0, were mixed in a cuvette with 20 ~1 of the developer dilution described above and with 40 ~1 of a phosphate-buffered sodium chloride solution containing 4.6 ~ of polyethylene glycol 6000 and with 20 ~1 of the antipheno-barbital latex reagent described above. The mixture was incubated at room temperature for 25 min. Then a further 200 ~l of a phosphate-buffered sodium chloride solution containing 4.6 ~ of polyethylene glycol 6000 were added, mixed in and the light scattering was measured in a ~.27;~ 33 Behringwerke laser nepholometer. An incubation curve as described in Fig. 3 was obtained with different quantities of phenobarbital added. The measurement range extended from about 2.5 to 80 ~g/ml phenobarbital.
For comparison, a measurement with an apoferritin deve-loper according to the state of the art was carried out.
The apoferxitin-phenobarbital derivative was obtained from Kallestad (batch number 021-Sl). It was diluted 1:750 in aqueous human serum albumin solution and was used in this dilution as the developer in the assay. The mixture for the measurement was made up as described previously, with the apoferritin-phenobarbital derivative being employed in place of the dihapten developer. A very flat incubation curve (see Fig. 4) resulted with different quantities of phenobarbital added, and this can not be used for satis-factorily reproducible measurement for this reason.
sodium bisulfate followed by 0.5 M sodium chloride. The ethyl acetate solution was dried over magnesium sulfate, filtered and concentrated to an oil. The oil was crystal-lized from ethyl acetate/hexane to obtain 0.6 g of bis-(trifluoroacetyl-L-thyroxyl)-4,9-dioxa-1,12-dodecane-diamide from two crops.
Example 11 bis-(L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide, dihydro-chloride salt bis-(trifluoroacetyl-L-thyroxyl)-4,9-diocadodecane-1,12-diamide (example 8A), 0.34 g, was dissolved in 3 ml methanol. 1 M sodium hydroxide was added with stirring and the reaction was allowed to go at room temperature for 40 hours. The reaction mixture was diluted into 20 ml of cold l M hydrochloric acid to givP a fine suspension of product.
The suspension was centrifuged and the pellet was dis-solved in ethanol. The ethanol solution was evaporated to a solid residue of crude product. The product was re-crystallized from methanol/ether to obtain 0.18 g of bis-(L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide, dihydro-chloride salt.
~ ~2~L~3 Example 12 bis-(N-succinyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide A solution of 2.0 g succinic anhydride in 20 ml pyridine was added dropwise to a chilled solution of 2.0 g 4,9-dioxa-1,12-dodecanediamine in 20 ml pyridine. ~he reaction mixture was stored at 4C for 18 hours then evaporated to a solid residue. The residue was dissolved in 30 ml water and passed through a bed of 30 ml Dowex 50 cation-exchange resin in the H~ form. The bed was then washed with 60 ml water. The effluent was concentrated to about half-volume to give colorless crystals of bis-succinyl-4,9-dioxa-1,12-dodecanediamide which were collected and dried. The yield was 2.18 g.
The intermediate was dissolved in 30 ml dimethylformamide along with 1.5 g N-hydroxysuccinimide. The solution was chilled and 2.45 g dicyclohexyl-carbodiimide in 15 ml dimethylformamide was added all at once. After 18 hours at 4C, the reaction mixture was filtered and the filtrate evaporated to an oil. The oil was crystallized from isopropanol and ether to give a crystalline product of 1.3 g bis-succinimido 0,N-succinyl-4,9-dioxa-1,12-dioxa-dodecanediamine. The latter active ester, 0.3 g, was dissolved in 2 ml dimethylformamide and added dropwise to a suspension of 0.89 g L-thyroxine, sodium salt in 4 ml dimethylformamide containing 0.11 ml N-methylmorpholine.
The reaction mixture was stirred for 72 hours. The re-sultant solution was evaporated to an oil. The oil was washed with a mixture of ethyl acetate and 1 M sodium bisulfate to give a solid which was collected by fil-tration, washed with water then methanol and dried to give 0.22 g of bis-(N-succinyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide.
7%~3 Example 13 Determination of theophylline The dihapten used was N,N'-di(theophylline-8-butyrylamino 3-propyl)piperazine.
The dihapten was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml, and then diluted 1:51,2000 in distilled water, with the addition of 4 % of human serum albumin. The diluted dihapt~en acts as a "developer" in the assay. A latex reagent was prepared by binding antiserum to theophylline onto polystyrene latex. The antitheophyl-line latex reagent was prepared as described in Canadian Patent No. 1,206,656. For the measurement, 10 ~1 of a sample containing theophylline, diluted 1:1000 in a 0.1 M
glycine/NaCl buffer, pH 8.0, were mixed in a cuvette with 10 ~1 of the developer dilution described above and with 20 ~1 of a phosphate-buffered sodium chloride solution containing 4.6 % of polyethylene glycol 6000 and with 10 ~1 of the antitheophylline latex reagent described above.
The mixture was incubated at room temperature for 12 min.
Then, a further 200 ~1 of a phosphate-buffered sodium chloride solution ~ontaining 4.6 % of polyethylene glycol 6000 were added, mixed in, and the light scattering was measured in a ~ehrinqwerke laser nephelometer.
An incubation curve as described in Fig. 1 was obtained with different quantities of theophylline added. The measuring range extended from about 1 to 40 ~g/ml theo-phylline.
For comparison, a measurement with an apoferritin de-veloper according to the state of the art was carried out.
The apoferritin-theophylline derivative was obtained from Kallestad (batch number: R 1). It was diluted 1:1,280 in aqueous human serum albumin solution and was used in this ,~
dilution as the developer in the assay. The mixture for the measurement was made up as described previously, ~ith the single exception that the apoferritin-theophylline derivative was used in place of the dihapten developer.
The inhibition curve (see Fig. 2) obtained with different quantities of theophylline added showed lower sensitivity, i.e. the lower detection limit was higher.
Example 14 Determination of phenobarbital The dihapten used was 1,12-di(phenobarbital-N-propionyl-amino)-4,9 dioxadodecane The dihapten was dissolved in dimethyl sulfoxide at a concentration of 1 mg/ml and then diluted 1:40,000 in aqueous 4 % strength human serum albumin solution. The diluted dihapten acts as a "developer" in the assay.
A latex reagent was prepared by binding antiserum to phenobarbital onto polystyrene latex. The antiphenobarbi-tal late7 raeagent was prepared as described in~ @æ~
Patent ~ 73Ø
For the measurement, 20 ~l of a sample containing pheno-barbital, diluted l:lO0 in a 0.1 M glycine/NaCl buffer, pH
8.0, were mixed in a cuvette with 20 ~1 of the developer dilution described above and with 40 ~1 of a phosphate-buffered sodium chloride solution containing 4.6 ~ of polyethylene glycol 6000 and with 20 ~1 of the antipheno-barbital latex reagent described above. The mixture was incubated at room temperature for 25 min. Then a further 200 ~l of a phosphate-buffered sodium chloride solution containing 4.6 ~ of polyethylene glycol 6000 were added, mixed in and the light scattering was measured in a ~.27;~ 33 Behringwerke laser nepholometer. An incubation curve as described in Fig. 3 was obtained with different quantities of phenobarbital added. The measurement range extended from about 2.5 to 80 ~g/ml phenobarbital.
For comparison, a measurement with an apoferritin deve-loper according to the state of the art was carried out.
The apoferxitin-phenobarbital derivative was obtained from Kallestad (batch number 021-Sl). It was diluted 1:750 in aqueous human serum albumin solution and was used in this dilution as the developer in the assay. The mixture for the measurement was made up as described previously, with the apoferritin-phenobarbital derivative being employed in place of the dihapten developer. A very flat incubation curve (see Fig. 4) resulted with different quantities of phenobarbital added, and this can not be used for satis-factorily reproducible measurement for this reason.
Claims (23)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A divalent hapten derivative comprising two hapten moieties connected by means of a bifunctional spacer having the formula I
A-X-A I
where A is a radical of a hapten and X is a bifunctional spacer having the formula II
(B)m-Y-(CH2)n-Z-(CH2)n-Y-(B)m II
where m is independently 0 or 1, B is (CH2)n', wherein n' is an integer from 1-4, or CO(CH2)n", wherein n" is an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R is hydrogen or alkyl; n is an integer from 1-10; and n may be the same or a different integer on opposite sides of component Z; and Z is selected from the group consisting of a C-C single bond, alkylene, -alkylene-O-alkylene-, alkylene-NH-alkylene, -N-H-alkylene-NH, -O-alkylene-O- , , , , and , where the term alkyl means a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms and the term alkylene refers to a bivalent radical of the branched or unbranched alkyl group it is derived from having valence bonds from two terminal carbons thereof.
A-X-A I
where A is a radical of a hapten and X is a bifunctional spacer having the formula II
(B)m-Y-(CH2)n-Z-(CH2)n-Y-(B)m II
where m is independently 0 or 1, B is (CH2)n', wherein n' is an integer from 1-4, or CO(CH2)n", wherein n" is an integer from 2-4; Y is independently -CONH-, -NHCO-, -OOC-, -COO-, -O-, -S-, or -NR-, wherein R is hydrogen or alkyl; n is an integer from 1-10; and n may be the same or a different integer on opposite sides of component Z; and Z is selected from the group consisting of a C-C single bond, alkylene, -alkylene-O-alkylene-, alkylene-NH-alkylene, -N-H-alkylene-NH, -O-alkylene-O- , , , , and , where the term alkyl means a straight or branched chain saturated hydrocarbon group having 1 to 6 carbon atoms and the term alkylene refers to a bivalent radical of the branched or unbranched alkyl group it is derived from having valence bonds from two terminal carbons thereof.
2. A divalent hapten derivative as claimed in claim 1 wherein R is an alkyl radical of 1 to 4 carbon atoms.
3. The divalent hapten derivative of claim 1 wherein said component Z is selected from the group consisting of -O-(CH2)4-O-, piperaz-1,4-diyl, and -NH-(CH2)4-NH.
4. The divalent hapten derivative of claim 1 wherein said bifunctional spacer is bis-(N-succinyl-4,9-dioxa-1,12-dodecanediamine).
5. The divalent hapten derivative of claim 1 wherein said bifunctional spacer is 4,9-dioxa-1,12-dodecanediamine.
6. The divalent hapten derivative of claim 1 wherein said bifunctional spacer is bis-(3-aminopropyl)piperazine.
7. The divalent hapten derivative of claim 1, 2 or 3 wherein said hapten is carbimazepine, primidone, phenobarbital, phenytoin, theophylline, gentamycin, tobramycin, amphetamine, diphenylhydantoin, kanamycin, digoxin or valproic acid.
8. The divalent hapten derivative of claim 1, 2 or 3, wherein said hapten is theophylline.
9. The divalent hapten derivative of claim 1, 2 or 3, wherein said hapten is L-thyroxine.
10. The divalent hapten derivative of claim 1, 2 or 3, wherein said hapten is Phenobarbital.
11. The divalent hapten derivative of claim 1, which comprises theophylline-8-butyric acid.
12. The divalent hapten derivative of claim 1, which comprises bis-(theophylline-8-butyryl)-4,9-dioxa-1,12-dodecanediamide.
13. The divalent hapten derivative of claim 1, which comprises bis-(theophylline-8-butyryl)-N,N-bis-3-aminopropyl piperazine diamide.
14. The divalent hapten derivative of claim 1, which comprises bis-(phenobarbital-N-propionyl)-4,9-dioxa-1,12-dodecanediamide.
15. The divalent hapten derivative of claim 1, which comprises bis-(trifluoroacetyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide.
16. The divalent hapten derivative of claim 1, which comprises bis-(L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide, dihydrochloride salt.
17. The divalent hapten derivative of claim 1, which comprises bis-(n-succinyl-L-thyroxyl)-4,9-dioxa-1,12-dodecanediamide.
18. A substrate for affinity purification of polyclonal antibody mixtures comprising the divalent hapten derivative of claim 1 bound to an insoluble carrier.
19. A substrate for a turbidimetric hapten assay comprising the divalent hapten derivative of claim 1 bound to an insoluble carrier.
20. Immunochemical method of determining a hapten by means of an inhibition of agglutination which comprises contacting a particle loaded with an antibody to the hapten to be measured, a divalent hapten derivative of claim 1 and a liquid in which that hapten is to be determined, measuring the reduction of turbidity caused by and depending on the concentration of the hapten in the liquid and determining therefrom the concentration of the hapten in the liquid.
21. The method as claimed in claim 20, wherein the particles carrying the antibody are composed of a high molecular weight latex.
22. The method as claimed in claim 20, wherein the antibody which is used is covalently bonded to a high molecular weight latex.
23. The method as claimed in claim 20, wherein the antibody which is used has been obtained by immunization of an animal with the coupling product of keyhold limpet hemocyanin and the particular hapten.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US67537484A | 1984-11-27 | 1984-11-27 | |
| US675,374 | 1984-11-27 | ||
| EP85105814A EP0183901B1 (en) | 1984-11-27 | 1985-05-11 | Bifunctional haptens, method for their production and their use |
| EP85.105814.9 | 1985-05-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1272193A true CA1272193A (en) | 1990-07-31 |
Family
ID=26096755
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000494628A Expired CA1272193A (en) | 1984-11-27 | 1985-11-05 | Divalent hapten derivatives, process for preparing and use of them |
Country Status (2)
| Country | Link |
|---|---|
| AU (1) | AU600432B2 (en) |
| CA (1) | CA1272193A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112794501B (en) * | 2020-12-29 | 2022-05-03 | 西北大学 | Method for removing boron ions in fracturing flow-back fluid |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU528246B2 (en) * | 1978-10-14 | 1983-04-21 | Teikoku Hormone Mfg. Co., Ltd. | Stable immunoassay reagent for hapten determination |
| AU6295180A (en) * | 1979-10-26 | 1981-04-30 | Dynasciences Corp. | Competitve binding assay for haptens + antigens |
| US4486344A (en) * | 1983-03-28 | 1984-12-04 | Miles Laboratories, Inc. | Urea-linked immunogens, antibodies, and preparative method |
-
1985
- 1985-09-18 AU AU47590/85A patent/AU600432B2/en not_active Ceased
- 1985-11-05 CA CA000494628A patent/CA1272193A/en not_active Expired
Also Published As
| Publication number | Publication date |
|---|---|
| AU600432B2 (en) | 1990-08-16 |
| AU4759085A (en) | 1986-06-05 |
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