CA1160218A - Valproate conjugation using dicarbonyls - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

?-substituted derivatives of valproic acid are provided for conjugation to antigenic compositions, particularly poly(amino acids).
and enzymes. The antigenic conjugates are employed for the production of antibodies, which find particular use in immunoassays for the determination of valproate, while the enzyme conjugate finds use in a homogeneous enzyme immunoassay for the determination of valproate.
The compounds are synthesized by alkylating val-proate at the tertiary carbon atom by an aliphatic chain with a terminal double bond which is cleaved to provide an acid or aldehyde group.

Description

.

36~2-134 ¦ VALPRC)ATE CC~NJUG~T I ON US I NG D I CARBOI`IYLS

BACKGROUND OF T_ E I NVENT I ON
l Field of the !nvention 5 1 Valproate (sodium di-n-propylacetate) is recog-nized as an important antiepileptic drug. Its use as an anticonvulsant is beneficial due to its low toxicity. A
minimum level of 50~g/ml in serum is required for thera-l peutic efficacy; however, this level is often difficult 10 1 to maintain due to individual difFerences in serum ab-sorption and metabolism. It is therefore essential to monitor the serum levels regularly in order to insure that a minimum therapeutic level of valproate is being l maintained.
15 1 Of the current assay procedures for valproate, i vapor phase chromatography analysis is the most widely used. Known techniques have various inadequacies in being uneconomical, slow and/or requiring trained clini-cians to perform the assay properly. It is therefore de-sirable to provide a simple and rapid procedure for determining valproate levels in serum or other physiological fluids, which provides reproducible values and is specific for valproate.
The simple structure of vaproic acid -- a saturated branched aliphatic acid -- makes it one of the smallest and simplest drugs to be considered as a hapten-protein antigen for production of antibodies. As such, >

~ `Z18 > 2 > in the design of any hapten derivatives of valproic acid, 'o be Ua~u i n the coi-,; uyc~ . i ol~l io pr u Le i ns l or che pre-Iparation of antigenic conjugates, there is little guid-ance as to the effect of structural modification on the specificity of antibodies.
Descriptlon of the Prior Art Matsumoto et al.~ "Mass Spectrometry in Drug Metabolism" by Frigero and Ghesalberli, Plenum Publishing l Corp., N.Y., N.Y. (1977) and Kuhara ~ Matsumoto, 10 ¦Biomedical Mass Spectrometry~ 1, 291 (1974) teach that valproic acid is metabolized in humans via oxidation predominantly at the positions ~ and w to the carboxylic acid, and is eventually excreted in urine as the free l acids or via glucuronide formation. U.S. Patent No.
15 ¦ 3,817,837 describes a homogeneous enzyme immunoassay technique for the determination of a wide variety of l drugs.

> SUMMARY OF_ I NVENTI ON
,~, C ~ r: t h ~ t j r ~., r ~ r r rl I r _ i p I O ~ e ~ f ~ ~ ~ r ~ g novel ~-s~bstituted valproic acid derivatives and their conjugates to antigenic materials, in particular poly(amino acids) and enzymes. The antigenic conjugates are employed for the production of antibodies for use in immunoassays. The enzyme conjugates are employed as reagents for the determinat;on of valproate in immuno-assays. The antibodies and enzyme conjugates are pro-vided in combination in kits to be used for the rapid andaccurate determination of valproate in physiological fluids, e.g. serum.

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> DESCR I PT I ON OF THE SPEC I F I C EMBOD I MENTS
Compounds of the present invention are diacids, monoacid aldehydes, cyclic anhydrides and, as conjug3tes to poly(amino acids), amides formed from the cyclic anhydride or ester of derivatives of valproic acid or amines formed from the monoacid aldehyde derivatives of valproic acid.
Novel compounds are provided having at the ~-position of valproic acid, a linking group having at least one spacer carbon atom and an active functionality for forming a covalent bond to antigens to provide con-jugates for preparing antibodies to valproate and to enzymes for formin~ reagents for valproate determination.
The linking group is normally an alkylene bonded to a carbonyl as the active functionality.
For the most part, the compounds of the in-vention will have the following formula:
r R~cO mwl ~(CH3CH2CH2)2C ¦ Z

wherein:
R is an aliphatic group, preferably alkylene, of from l to 6, usually 1 to 4 carbon atoms, and pref-erably l to 3 carbon atoms, having O to 1 site of ali-phatic unsaturation, normally ethylenic. R is preferably saturated, and usually polymethylene;
when m and n are both 1, W and W1 may be taken together with Z to form a cyclic anhydride;
otherwise when W and W1 are not taken to~ether, W1 is hydroxyl;

> 5 > ¦ when m is o, w is a methylene bonded to amino groups of z and z is a poly(amino acid) which is anti-genic or an enzyme;
¦ when m is 1, w is a bond and Z is hydrogen, hydroxy, alkoxy of from 1 to 6 carbon atoms, preferably 1 to 3 carbon atoms, or an activating oxy group tG form an activated ester capable of amide formation in an aqueous medium, e.g. N-oxy succinimide and p-nitrophenoxy; and n is 1 when Z is other than poly(amino acid) and i 5 otherwise one to the molecular weight of Z divided by 500, more usually divided by 1000, and frequently divided by 1500, generally ranging ~rom about 1 to 500, preferably from about 10 to 100, when Z is an antigen, and from 1 to 30, more usually 2 to 20, and preferably from about 2 to 16, when Z is an enzyme.
For those compounds where n is one, the com-pounds will have the following formula:
r R(cO)mlw2l ~(CH3CH2CH2)2C J Z

Wherein:
R is as defined previously, usually poly-methylene of 1 to 3 carbon atoms when zl j5 other than hydrogen and 2 to 6 carbon atoms when Zl is hydrogen;
ml j5 1;
W2 and W3 may be taken together with Z1 to form a cyclic anhydride;
otherwise w2 is a bond to Z1 and W3 is hydroxyl; and ~ l > z1 is hydrogen, hydroxyl, alkoxy of from 1 to 6 carbon atoms, more usuallY from 1 to 3 carbon a-toms.
~particularly methyl and ethyl, an oxy group forming an activated ester which readily reacts with the amine groups o~ poly(amino acids) under mild conditions in an aqueous medium, such as N-oxy succinimide or p-nitrophenyl.
When n is at least one and Z is a poly(amino acid), the compounds for the most part will have the formula:

~ R~Co)m2W4 1 ~ (CH3cH2cH2)2c J z2 wherein:
R has been defined previously, usually poly-methylene of from 1 to 3 carbon atoms when W4 is a bond or of from 2 to 5 carbon atoms when W4 is methylene;
Z2 j5 a poly(amino acid) which is either anti-genic or an enzyme;
m2 is 0 or 1; when m2 is 0, W4 i 5 methylene bonded to Z2 through amino nitrogen, and when m2 is 1, W4 is a bond to Z2 to form amide linkages;
n2 is at teast l, usually greater than l;
when Z2 j5 an antigen, n2 will normally be at least 2, and not greater than the molecular weight of Z2 divided by 500, usually not greater than the molecular weight of Z2 divided by 1000, and preferably not greater than the molecular weight of Z2 divided by 1500, gen-erally ranging from about 2 to 500; when Z2 j5 an enzyme, n2 will be at least 1, usually not greater than 30, more >

> l > lusually in the range of about 2 to 20, and preferably in the ran3e of about 2 to 16.
The poly~amino acids) will generally range from labou-t 5000 molecular weight, having no upper molecular 5 Iweight limit, normally being not more that 10,000,000, ¦usually not more than about 600,000. There will usually ¦be different ranges, depending on whether an antigen or ¦an enzyme is involved, with antigens ranging from about 15000 to 107, usually from about 20,000 to 600,000, and 10 ¦more usually from about 25,000 to 250,000 molecular ¦weight; while enzymes will generally range from about ¦10,000 to 600,000, more usually from about 10,000 to 300,000 molecular weight. There will usually be at least l about one conjugate per 500,000 molecular weight, more usually at least one per 50,000 molecular weight. With intermediate molecular weight antigens (35,000 to 1,000,000), the number of conjugate groups will generally be from about 2 to 250, more usually from 10 to 100.
~ith lower molecular weight antigens, below 35,000, the number of conjugates will generally be in the range of from about 2 to ~0, usually in the range o~ 2 to 5.
Various protein types may be employed as the antigenic material. These types include albumins, serum proteins, e.~., globulins, ocular lens proteins, lipo-proteins, etc. Illustrat;ve proteins include bovineserum albumin. keyhole limpet hemocyanin, egg ovalbumin, bovine y-globulin, etc. Alternatively, synthetic poly(amino acids) rnay be prepared having a sufficient number of available amino groups, e.g., Iysines.
The enzymes can be varied widely, depending upon the rapidity with wh;ch one desires a result and the physiological fluid in which the valproate is to be measured. Primarily, the enzymes of choice, based on the I.U.B. classification are C!ass 1. Oxidoreductases and Class 3. Hydrolases. Particularly in Class 1, the >

~ l ~ z~

> ~enzymes of interest are dehydrogenases oF Class ~ more ~articularly 1. 1 . 1 and ] . ] . 99 ~n~ pProx i dasec, in C!ass 1.11. of the hydrolases, particularly Class 3.1, more l particularly 3.1.3 and Class 3.2, more particularly 5 1 3.2.1.
Illustrative dehydrogenases include malate dehydrogenase, glucose-6-phosphate dehydrogenase, and lactate dehydrogenase. Of the peroxidases, horse radish I peroxidase is illustrative. Of the hydrolases, alkaline, lO ¦ phosphatase, ~-galactosidase, ~-glucosidase and Iysozyme are illustrative.
Particularly preferred are those enzymes which employ nicotinamide adenine dinucleotide (NAD) or its l phosphate (NADP) as a cofactor, particularly the former.
Most perferred as the choice of enzyme is glucose-6-phosphate dehydrogenase.
In preparing the subject intermediates, the central (~) carbon atom in valproic acid furnishes one replaceable hydrogen for a site of alkylation. Carbanion 20 formation via the lithium salt allows for substitution of that hydrogen atom by an aliphatic chain with a terminal double bond, said double bond allowing for further con-version into an aldehyde or an acid group.
The synthetic scheme for preparing the subject 25 compounds is set forth in the following chart:

> ll (rlPr~)2CHC02H ~ YR~CH=CH2 ¦ a.
S

I (nPr)2c(co2H)R2cH=cH2 l / ~ b.
¦ / (nPr)2C(CO2H)R2CHO
10 1 / ¦ d.

I / (~nPr~2C(C02H)R2c~2)n3z4 l c.
15 ¦(npr)2c(co2H)R2co2H
j ¦ e.

l( ~nPr)2C(~O2H)R2 CO)n3 Z4 Y - halogen of atomic number 17-35 Z4 Hn3 - P Iy(amino acid~
R2 _ alkylene of 1 to 6 carbon atoms l n3 ~ 1 to ~he molecular weight of Z4 Hn3 divided by a. nBuLi or NaH
l b. o3,Zn 30 ¦ c. MnO4e d- Z4Hn3 NaCNBH3 e. where R2 is alkylene of 1 to 3 carbon atoms, (1) AC20 l (2) ZqHn3 35 l Z'~

The antigenic conjugates may be injected into a wide variety of vertebrates in accordance with conven-tional me-thods for the production of antibodies.
¦Usually~ the animals are bled periodically with -the 5 Isuccessive bleeds improving in titer and specificity and ¦then plateauing and diminishing in their specificity and ¦titer.
¦ As previously indicated, the antibodies and ¦enzyme reagents prepared in accordance with the subject 10 ¦invention find particular use in immunoassays for the ¦determination of valproate. A description of the method jfor carrying out the immunoassay, which is a ho~ogeneous ¦enzyme immunoassay, may be found in U.5. Patent No.
¦3,817,837. The method involves combining the enzyme 15 ¦conjugate, the unknown sample suspected of containing valproate, an antibody for valproate in an aqueous buf-fered mediu~ at temperatures in the range of about 10 to 50, more usually from about 20 to 40C, and determining l the enzyme activity as compared to the enzyme activity of 20 ¦ an assay medium having a known amount of valproate.

~¦ EXPEPIMENTAL
The follo~ing examples are offered by way of illustration and not by way of limitation.
l (All temperatures not otherwise indicated are 5 ICentigrade. All percents not otherwise indicated are by weight. Parts are by weight~ except when two liquids are combined and are then by volume. The following abbrevia-tions are employed: HOAc - acetic acid; DMF - dimethyl-formamide; THF - tetrahydrofuran.) Ex.lA. Preparation of 7-Carboxy-7-propy!-1-decene by the Alkylation of Valproic Acid with 6-Bromo-l-hexene l Under an argon blanket, sodium hydride (1~, 50~
¦oil~ 0.022mole) was washed twice with hexane and the 15 ¦washings decanted. To the washed NaH was added 50ml of ¦THF (freshly distilled from LiAlH4) and followed by the ¦dropwise addition of valproic acid (2.88g, 0.02~ole) in ¦20ml of THF. The rate of addition was adjusted according ¦to the amount of hydrogen evolved. The sodium salt was 20 ¦cooled in a salt ice bath to 0 and then diisopropylamine (29, 0.02mole, distilled over CaH2~ was added. The mix-¦ture was heated to 55 for 15 minutes, and then cooled to ¦room temperature in two hours. The solution was then l cooled to -1 and n butyllithium (lOml, 0.02mole) was 25 ¦ added while keeping the temperature between about 0-5.
After 15 minutes, the reaction mixture was warmed to 35 for 33 minutes and again cooled to 0. To the cold reaction mixture was added dropwise cold (0-2) 6-bromo-1-hexene (3.29, 0.02mole) in 20ml of THF. The resulting solution was cooled in an ice bath for 30 minutes, then maintained at 33 for an hour. NaBr precipitated and the reaction mixture was allowed to stir overnight at room temperature. A sample of the solution was taken and shaken with D2O; nmr showed no tertiary hydrogen present.
The reaction mixture was then cooled in an ice bath, 25ml .-~ ~ !L~ L8 >

> of water was added and the mixture was extracted with ¦50ml of ether. The ethereal solution had 50ml of 5%
K2Co3 added to it and then wa5 extracted wi-th 2x50ml of hexane. The basic aqueous solution in an ice bath was acidified with concentrated HCI to pHl, saturated with salt, and then extrac~ed with 2x5Qml of ether. The ethereal solution was washed with 3xlOml of brine, dried (MgS04) and concentrated to give 2.9g of an oil. Tlc on silica (10% methanol in chloroform) gave a Rf 0.76 vis-ualized by bromocresol green stain.
Ex.lB. Preparation of 6-Carboxy-6-propy!nonanoic Acid The 7-carboxy-7-propyl-1-decene (3.42g~
0.015mole) prepared in Example lA was d;ssolved in 75ml of acetone, 29 of NaHC03 added and was cooled in an ice bath. Potassium permanganate (9.5g, 0.06mole) was added over four hours. The mixture was left in the cold bath for an additional hour and then NaHS03(8g) was added alternately with 5N H2S04 to bring the pH to 2 and to 20 reduce the residual permanganate. After fiItering, the filtrate was concentrated to an oily residue which was extracted with lOOml ethyl acetate, washed by water and saturated brine, dried (MgS04), concentrated to give an oil weigh;ng 3.2g and finally distilled by molecular 25 distillation to give the pure product.
Ex.2 reparation of the Conju~ates of 6-Carboxy-6-e oeyInonanal with BgG and BSA
2A. Ozono!ys~s of 7-Carboxy-7-propyl-1-decene The unsaturated acid (Ex.lA.) was dissolved in 25ml of CH2C12 under an argon blanket and cooled to -45 with a dry-ice acetone bath. Ozone was introduced until a blue color persisted for 10 minutes. The reaction mixture was flushed with argon until colorless. Acetic acid (lOOml) and 50ml of ether with lOg of zinc dust were 35 ~adde~ an the mi~ture left at oon t-mperature overnight.

> 13 > ¦After fiItering the fiItrate was concentrated on a rotary evaporator to give 4.759 o-F aldehyde (6-Carboxy-~-propyl-¦nonanal). Tlc or, silica gel ~50% EtOAc/benze~e) gave R-f G.63 (one spot stained by both ceric sulfate and

2,4-~NP). A derivative of 2,4-DNP-hydrazone was pre-pared, mp. 146-148.
2B. Preparation of 6-Carboxy-6-propylnonanal-BSA Conjugate To 900mg of BSA in 50ml of phosphate buffer at pH7.2 was added sodium cyanoborohydride (mixed with tritiated NaCNBH3) and the 6-carboxy-6-propylnonanal prepared in Example 2A in 10ml of carbitol. The solution was mixed at room temperature and stirred at 4 over the weekend. The conjugate solution was dialyzed with 7x4 liter of water adjusted to pH9 with NHl,OH. Lyophiliza-tion gave 790mg of conjugate. Hapten number was esti-mated to be 17 by tritium label.
2C. Preparation of 6-Carboxy-6-propyl-nonanal-BgG Conju3ate BgG (9OOmg) and the 6-carboxy-6-propylnonanal (205mg) prepared in Example 2A were employed for conjugation as descr7bed in xample 2B. Lyophilization yielded 550mg of conjugate with a hapten number of 26.
Ex. 3 Preparation of 2-Propyl-2-butenylpentanoic Acid Into a dry 500ml three-necked flask under argon was placed 4g of NaH (50% oil, 0.087mole). The NaH was washed twice with 50ml hexane which was decanted and the clean NaH was flushed dry by argon. Freshly distilled THF (50ml) was added and cooled to 0-5. Valproic acid (2-propylpentansic acid, 11. 52gJ 0.08mole) in 50ml THF
was added dropwise to the NaH slurry over thirty minutes, followed by diisopropylamide (8ml). The mixture was warmed to 55 for 15 minutes and allowed to cool to room temperature in two hours~ then cooled to 2 in an ice bath. n-Butyllithium (40ml, 2M in hexane) was added via >~1 14 > ¦syringe àt a ~emperature between 5-10~. The resulting ¦mixture was warmed to 35 for 30 minutes and then cooled Ito 2. 4-Bromo-l-butene was added dropwise maintaining ¦the pot temperature between 10-20. The reaction mixture 5 ¦was warmed again to 35~ for 38 minutes and stirred at ¦room temperature overnight. The reaction mixture WâS
¦concentrated and added to 100ml water, acidified to pHl ¦with concentrated HCI and then extracted with 150ml each ¦of ether and ethyl acetate. The organic ~xtract was 10 ¦washed with 4x50ml water and 2x20ml saturated brine, ¦dried (magnesium sulfate), and concentrated to give a Iyellow liquid weighing 15g. Distillation gave lOg of a ¦colorless liquid, bp. 90/0.05mm Hg. Tlc on silica (1:1 ¦ether: hexane) gave Rf 0.63, stained by bromocresol lS Igreen.
¦EX. 4. Preparation of 2-Propyl-2-(?'-carboxyethyl~~
I pentanoic Acid and 2-Propyl-2-(carboxymethy12-¦ pentanoic Acid 1 2-Propyl-2-butenylpentanoic acid (3g, 20 ¦0.015mole) prepared in Example 3 was dissolved in lOOml of acetone and NaHCO3 (lg, 0.018mole~ was added. The ¦mixture was cooled in an ice bath, and then potassium permanganate (9.5g, 0.06mole) was added over 5 hours and l the mixture stirred for an additional hour at room temp-25 ¦ erature.` After being cooled in an ice bath the mixture was acidified to pH2 with 5N H2SO4 and then NaHSO3 (99, 0.096mole~ was added until the purple color of the permanganate disappeared. The mixture of colorless l aqueous solution and brown manganese dioxide was filtered 30 ¦ through Celite* The filtrate was concentrated to a small aqueous volume and extracted with 150ml each of ether and ethyl acetate, washed with brine, dried (MgSO4~ and concentrated to give a slightty colored liquid weighing l 3g. Tlc on silica with 10% methanol in chloroform gave 35 ¦ Rf 0.45.
> ¦ *Trade Mark ~ Z~

> ¦ The relative proportions of 2-propyl-2-(2'-car-boxyethyl)pentanoîc acid and 2-propyl-2-carboxv-methylpentanoic acid were dependent on the control of the 'reaction time and temperature, the former being present in an amount equal to about 70-90~.
Ex. 5 Cyclization of ?-Propyl-2-(2'-carboxyethyl~-pentanoic Ac;d and 2-Propyl-2-carboxy methylpentanoic Acid To a mixture of the two diacids (3g) as pre-pared in Example 4 was added 10ml of distilled aceticanhydride under argon. The solution was then heated to 120 in two hours, kept at 120 for an additional 2 hours and then heated to 130. The solution was concentrated to dryness to give a crude product of 3g. Gas chroma-tography on S.E. 30 column at 185 separated the twocyclic anhyclrides, 2,2-dipropylglutaric anhydride and 2,2-dipropylsuccinic anhydride, in pure form.
Ex. 6 Conjugation of 2-Propyl-?-(2'-carboxyethyl2-pentanoic Acid to BSA (Bovine Serum Albumin) To a solution of BsA (300mg, 5xlO 4mmole) in 20ml of phosphate buffer was added the hapten, 2,2-dipro-pylglutaric anhydride as prepared in Example 5 (55mg, 0.25mmole in 200ml THF) in 4~1 portions over an hour.
The resulting clear solution was stirred at 4 overnight, after which it was dialyzed in 7x2 liter of water ad-justed to pH8 with phosphate buffer, followed by one time with deionized water. The dialysis took 5 days.
Lyophilization gave 320mg of conjugate having a hapten number of 4G as estimated by the amino group precipita-tion method.Ex.7. Conjugation_of 2-Propyl-2-(2'-carboxyethy!2-pentanoic Acid to Glucose-6-phQsphate Dehydrogenase(G6PDH) The 2,2-dipropylglutaric anhydride prepared in Example 5 ~4.Omg) was solubilized in 100~1 of DMF and I

~ 2~1~
>

> then added to a stirring solution of 2ml of G6PDH
(Beckman Lot HOI, 4.3mg/ml), 40mg of G6P disodium salt and 60mg of NADH. Carbitol (600~1) was then slowly added, the solution having a final pH of about 8.7-8.9.
The pH was mainta;ned during the reaction by addition of NaOH.
The enzyme was monitored in accordance with the enzyme assay to be described for determining % deactiva-tion. The % inhibition was determined by adding an excess of anti(valproate) and assaying the enzyme activ-ity according to the method described.
Table 1 shows the progress of the conjugation.

Sample No. Total Hapten(~l) ~ Deactivation % Inhibition .

3 8 8 15

4 16 18 30 6 36 35 58 .
7 36 36 60 . 5 2~ 9 49 . 45 70 The conjugate was worked up by chromatography over Sephadex*G-50M ~180ml column~, employing 0. 055M
tris-HCI buffer (pH8.1) as eluent, collecting 2.6ml samples (~1. 5% sample/bed volume ratio). FractiGns were pooled to give a total volume of 20.5ml.
In order to demonstrate the efficacy of com-pounds prepared in accordance with the subject invention, antibodies prepared from the conjugates described pre-viously and the enzyme conjugate were employed in > * Trade Mark ~ 21~
>

> a number of assays for valproate. In carrying out ~he assay, a GiIford 300N microsample spectrophotometer is employed with a Thermocuvette with a flow cell. All readings are made at 340mn. The following solutions are prepared as reagents for use in ~he assay.
Buffer: 0.055M tris-HCI pH8.1 (RT) Enzyme Conjugate: Buffer 0.9% NaCI
1.0% RSA, pH8.1 (RT) Sufficient enzyme conjugate to give a maximum rate of ~OD equal to 600-900 in the assay medium Assay buffer: Buffer 0.5% NaCI
0.01~ v/v Trito~ X-100, pH8.1(RT) Antibody Reagent: Buffer 1.0% RSA, G6P(Na) 0.066M, NAD 0.04M, pH5 (RT) Antivalproate optimized for assay (All % indicated are w/v g/ml. RSA-rabbit serum albumin).
The protocol employed for carrying out an assay is as follows: A sample, 50~1 is drawn up into a diluter and dispensed with 250~1 of the assay buffer into a one ml Croan cup. A 50~1 aliquot of the diluted sample is drawn up and dispensed with a 250~1 portion of assay buffer into a second Croan cup. Into the second Croan cup is introduced 50~1 of the antibody reagent with 250~1 of the assay buffer, followed by the addition of 50~1 of the enzyme reagent and 250~1 of the assay buffer. Immedi-ately after the enzyme addition, the entire sample i5 aspirated into the flow cell. After 10 seconds, a first reading is taken, ~ollowed by a second reading, after a 40 second interval from aspiration. The results are reported as the difference in absorbance X 2.667.
>
* Trade Mark .

>

> Sample Concentration of v~proate ~/ml ~OD
0 (5~1)'~ ~

max rate (1010) ¦ "lowest rate in assay with predetermined amount of antibody l ~"rate of enzyme in absence of antibody It is evident from the above results, that the compositions of the present invention provide for re-agents which can be used in a sensitive immunoassay for l valproic acid. Thus a rapid accurate method is provided 20 1 for the determination of valproic acid, which can be used in therapeutic dosage monitoring of patients to ensure that a therapeutic dosage is applied.
The antigenic conjugates o F the subject inven-l tion provide for the production of highly speci F i c anti-25 ¦ bodies. This result is obtained despite the fact thatthe tertiary carbon atom of valproate, adjacent the only polar group in the molecule is changed to a quaternary carbon atom, so as to enhance steric crowding at this l site and to increase the hydrophobicity about the 30 ¦ carboxyl group. In addition, the enzyme conjugates are able to compete with valproate for antibodies to provide a sensitive accurate assay.
¦ Although the foregoing invention has been described in some detail by way of illustration and > I

~> ~

> lexample for purposes of clarlty and understanding, it ~will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (5)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound useful for the preparation of antibodies specific for valproate having a short aliphatic chain substituted at the alpha position of valproic acid linking to a non-oxo-carbonyl group which is an amide group to an antigen poly(amino acid).

2. A compound of the formula:

wherein:
R is an aliphatic group of from 1 to 6 carbon atoms;
when n and m are each 1, W and W1 may be taken together with Z to form a cyclic anhydride;
otherwise when W and W1 are not taken together, W1 is hydroxyl;
when m is 0, W is a methylene bonded to Z through amino nitrogen, and Z is a poly(amino acid);
when m is 1, W is a bond to Z, and Z is a poly-(amino acid) wherein W is bonded to said poly(amino acid) to amino groups to form amide linkages; and n is in the range of one to the molecular weight of Z divided by 500.

3. A compound of the formula:

wherein:
R is an alkylene group of from 1 to 6 carbon atoms;
m is 0 or 1;
z is a poly(amino acid);
n2 is at least 1 and not greater than the molecular weight of Z2 divided by 500; and when m2 is 0, W4 is methylene bonded to amino groups of Z2 and when m2 is 1, W4 is a bond to amino groups of Z2 to form amide linkages.

4. Antibodies prepared in response to a poly(amino acid) according to Claim 3.

5. A method for determining the presence of valproate in a physiological fluid which comprises combining in an aqueous assay medium a sample of said physiological fluid, antibodies according to Claim 4, an enzyme conjugate according to Claim 3, wherein said poly(amino acid) is an enzyme, and determining the rate of transformation from NAD to NADH as compared to the rate determined with an assay medium having a known amount of valproate.