Title:
Isothiocyanate and thiourea derivatives of benzoyl ecgonine conjugated to polypeptides
United States Patent 3917582
Abstract:
Novel benzoyl ecgonine derivatives are provided having an isothiocyanate group for conjugation to polypeptides and proteins to provide derivatives as reagents in immunoassays or for preparation of reagents for immunoassays. Particularly, the isothiocyanate is conjugated to enzymes or antigenic polypeptides or proteins. The antigenic polypeptides or proteins are employed for preparing antibodies to benzoyl ecgonine or cocaine, while the conjugated enzymes are used in immunoassays, where the enzyme is the detector molecule. The enzyme conjugate is found to have a high degree of sensitivity for the detection of benzoyl ecgonine, a cocaine metabolite.
Inventors:
Soffer, Michael J. (Redwood City, CA)
Schneider, Richard S. (Sunnyvale, CA)
Schneider, Richard S. (Sunnyvale, CA)
Application Number:
05/369658
Publication Date:
11/04/1975
Filing Date:
06/13/1973
View Patent Images:
Export Citation:
Assignee:
Syva Corporation (Palo Alto, CA)
Primary Class:
Other Classes:
436/543, 435/188, 435/7.920, 530/345, 546/131, 435/961, 546/130, 530/404, 436/822, 530/389.800, 436/547, 435/7.900, 435/964, 530/806
International Classes:
C07D451/12; C07D451/00; C08H1/00; C07G7/00; C07G7/02
Field of Search:
260/112R,121,292,293.54,78A
Primary Examiner:
Schain, Howard E.
Claims:
What is claimed is
1. A conjugated antigenic poly(amino acid): ##SPC6##
2. A conjugated poly(amino acid) according to claim 1, wherein R is hydrogen, n' is in the range of 1 to 200, and PP has a molecular weight in the range of 12,000 to 1 million.
3. A conjugated poly(amino acid) according to claim 2, wherein PP is bovine serum albumin.
4. An antibody prepared in response to a conjugate according to claim 1.
5. An antibody prepared in response to a conjugate according to claim 3.
1. A conjugated antigenic poly(amino acid): ##SPC6##
2. A conjugated poly(amino acid) according to claim 1, wherein R is hydrogen, n' is in the range of 1 to 200, and PP has a molecular weight in the range of 12,000 to 1 million.
3. A conjugated poly(amino acid) according to claim 2, wherein PP is bovine serum albumin.
4. An antibody prepared in response to a conjugate according to claim 1.
5. An antibody prepared in response to a conjugate according to claim 3.
Description:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The use of cocaine for other than medicinal uses has become of increasing concern. Cocaine's availability has increased with concomitant efforts to police its use and prevent its distribution from unauthorized sources. In addition, in clinics providing drug therapy, it is important that the therapist be aware whether the person being treated has terminated or is continuing the use of drugs.
There has been continuing efforts to find reliable and rapid ways for detecting the use of drugs. It is also necessary that any method be able to distinguish cocaine or its metabolite from other drugs. Where urine is tested, it is found that cocaine is metabolized to benzoyl ecgonine. Therefore, in order to detect the use of cocaine, it is necessary to detect the presence of benzoyl ecgonine.
2. Description of the Prior Art
A number of immunoassay systems suggest themselves as useful for the detection of cocaine or its metabolites. Radioimmunoassay has been reported as useful in the detection of opiate alkaloids. See U.S. Pat. No. 3,709,868. U.S. Pat. No. 3,690,834 discloses a system using a free radical detector for immunoassays. U.S. Pat. application Ser. No. 143,609, filed May 14, 1971, now abandoned discloses an immunoassay technique employing enzymes, sold under the trademark "EMIT", by Syva Corporation. Odell, Competitive Protein Binding, Blackwell Scientific Publications, Oxford, 1971, discloses a number of conjugated haptens to proteins, see particularly Chapter 2, beginning at page 25. A wide variety of functionalities are disclosed as useful for conjugation.
SUMMARY OF THE INVENTION
Novel isothiocyanate modified benzoyl ecgonine compounds are provided for conjugation with poly(amino acids). Where anti-genic poly(amino acids) are employed, the product is useful in preparing antibodies, which show excellent specificity and sensitivity to benzoyl ecgonine, a cocaine metabolite. With conjugation to an enzyme, the enzyme conjugate is useful in immunoassays for the determination of benzoyl ecgonine or cocaine.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The compounds of this invention are thiocarbamoyl derivatives of benzoyl ecgonine or its methyl ester, as well as the antibodies prepared from the antigenic conjugates of the benzoyl ecgonine derivative with an antigenic poly(amino acid).
For the most part, the compounds of this invention will have the following formula: ##SPC1##
Wherein:
R is hydrogen or methyl, usually hydrogen;
α is hydrogen or may be taken together with β to form a bond between the nitrogen and the carbon atoms to which they are attached;
β may be taken together with α to form a bond, as indicated above, or is a poly(amino acid) residue to which at least one benzoyl ecgonine is conjugated; and
n is the number of benzoyl ecgonine groups conjugated to the poly(amino acid) and on the average will be at least 1 and not more than the molecular weight of β divided by 500, more usually not more than the molecular weight of β divided by 2000, and usually not exceeding 250.
The thiocarbamoyl group may be meta or para, usually para.
When α and β are taken together, the compound will have the following formula: ##SPC2##
wherein:
R is hydrogen or methyl, usually hydrogen, and the isothiocyanate group is either meta or para, normally para.
The isothiocyanate compound is prepared by esterifying the appropriate ecgonine derivative with the appropriate nitrobenzoic acid. The ester may then be reduced to the aminobenzoate, conveniently employing catalytic hydrogenation. The amino compound may then be derivatized to the isothiocyanate using thiophosgene. The amino compound is combined with the thiophosgene under mild conditions.
Conjugation of the isothiocyanate to a poly(amino acid) is carried out by combining the appropriate ratio of the isothiocyanate to the poly(amino acid) under mild conditions and at a constant mildly basic pH, normally in the range of about 8 to 9.
Of particular interest for the use of the subject compound is the conjugation to an amino group, which is part of a poly(amino acid) structure. By poly(amino acid) is intended both polypeptides and proteins. One group of poly(amino acids) is antigenic, so that by conjugation of the isothiocyanate to the antigenic poly(amino acid) a product is obtained which can be used in the formation of antibodies to benzoyl ecgonine. A narrow class of poly(amino acids), which can also be used as antigens, though not normally be used as such, are enzymes which are employed as the detector in an immunoassay system.
Polypeptides usually encompass from about 2 to 100 amino acid units (usually less than about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associated by covalent or non-covalent bonds. Subunits are normally of from 100 to 300 amino acid groups (approximately 10,000 to 35,000 molecular weight). For the purposes of this invention, polypeptide is intended to include individual polypeptide units or polypeptides which are subunits of proteins, whether composed solely of polypeptide units or polypeptide units in combination with other functional groups, such as porphyrins, as in hemoglobin or cytochrome oxidase.
The first group of poly(amino acid) materials which will be considered are the antigenic poly(amino acids). These may be joined to the isothiocyanate group through an amino group. The thiourea product, which may include some thiourethane, can be used for the formation of antibodies to cocaine metabolites. The poly(amino acid) materials which may be used vary widely, normally being from about 1,000 to 10 million molecular weight, more usually from 12,000 to 1 million molecular weight, and most frequently from about 50,000 to 500,000 molecular weight.
With most conventional poly(amino acids), there will not be more than about one benzoyl ecgonine or derivative group per 1,500 molecular weight of poly(amino acid), usually not more than one group per 2,000 molecular weight. There will be at least one group per 500,000 molecular weight, usually at least one per 50,000 molecular weight. With intermediate molecular weight antigens (50,000 to 1 million), the number of benzoyl ecgonine or derivative groups will generally be from about 2 to 250, usually from 10 to 100.
With low molecular weight antigens, 1,000 to 5,000 molecular weight, the number of benzoyl ecgonine or derivative groups will be in the range of 1 to 10, usually in the range of 2 to 5. Therefore, there may be as many as one benzoyl ecgonine or derivative group per 500 molecular weight of poly(amino acid).
Usually, the number of groups bonded to the poly(amino acid) will be related to the available amino groups, e.g., the number of lysines present. While the benzoyl ecgonine or derivative group may be bonded through the isothiocyanate to hydroxyl or mercaptan groups, which are present in the polypeptide, for the most part the bonding will be to amino and, therefore, the compounds are described as thioureas. However, thionoesters may also be present.
Amino acids present in poly(amino acids) which have free amino groups for bonding to the isothiocyanate modified benzoyl ecgonine or derivative thereof, include lysine, arginine, histidine, etc. The hydroxylated and mercpatan substituted amino acids include serine, cystene and threonine.
Various protein types may be employed as the antigenic material. These types include albumin, serum proteins, e.g., globulins, ocular lens proteins, lipoproteins, etc. Illustrative proteins include bovine γ-globulin, etc. Small natural polypeptides which are immunogenic, such as gramicidin may also be employed. Various synthetic polypeptides may also be employed, such as polymers of lysine, glutamic acid, phenylalanine, tyrosine, etc., either by themselves or in combination. Of particular interest is polylysine or a combination of lysine and glutamic acid. Any synthetic polypeptide must contain a sufficient number of active groups, as for example, amino groups provided by lysine.
The second group of poly(amino acids) are the enzymes to which the isothiocyanate derivative may be conjugated. As indicated, the benzoyl ecgonine derivative modified enzyme is useful for immunoassays. The immunoassay technique will follow in more detail.
Various enzymes may be used such as oxidoreductases, hydrolases, lyases, and the like. These enzymes include esterases, amidases, phosphorylases, carbohydrases, oxidases, reductases and the like. Of particular interest are such enzymes as lysozyme, amylase, dehydrogenases, particularly malate dehydrogenase, lactate dehydrogenase, mannitol-1-phosphate dehydrogenase, and glucose 6-phosphate dehydrogenasee, β-glucuronidase, cellulase, and phospho-lipase, particularly phospholipase C. The enzymes will usually have molecular weights in the range of about 1 × 10 4 to 6 × 10 5 , more usually in the range of about 1.2 × 10 4 to 3 × 10 5 .
There will usually be at least one benzoyl ecgonine or derivative group per enzyme molecule and usually not more than one group per 1,500 molecular weight, usually not more than one group for 2,000 molecular weight. Usually, there will be at least one benzoyl ecgonine or derivative group per 50,000 molecular weight, and more usually at least one group per 30,000 molecular weight. The modified enzyme will retain on the average at least 10%, more usually at least 30% of the original activity of the unmodified enzyme.
Where the benzoyl ecgonine or derivative is bonded to a poly(amino acid), there need be only one benzoyl ecgonine or derivative group, but usually there will be at least two groups. With the enzymes, the number of benzoyl ecgonine or derivative groups will generally be of from 1 to 30, more usually 2 to 25. Usually, there will be at least two, more usually at least three groups per enzyme, when the enzyme is randomly substituted with the benzoyl ecgonine or derivative groups, and preferably not more than 16 groups.
The substituted poly(amino acid)s will, for the most part, have the following formula: ##SPC3##
wherein:
R is hydrogen or methyl, usually hydrogen;
the thiocarbamoyl group is either meta or para, usually para;
Pp is a poly(amino acid), which has been extensively described previously; and
n' is the number of benzoyl ecgonine groups bonded to the poly(amino acid), the various ranges having been indicated previously.
Instead of an enzyme, a stable free radical may be employed as the functionality for detection in the immunoassay. These stable free radicals are cyclic nitroxides having the nitrogen of the nitroxide as an annular member and from 0 to 1 of the heteroatoms, i.e., oxygen and nitrogen, as annular members.
The molecules bonded to the isothiocyanate will normally be of from 8 to 16 carbon atoms, usually of from 8 to 12 carbon atoms. The functionality for linking to the ecgonine derivative may be bonded directly to an annular carbon atom or bonded to an annular carbon atom through an aliphatic chain of from 1 to 4 carbon atoms, usually of from 1 to 2 carbon atoms. The molecules may have from 0 to 2 sites of ethylenic unsaturation, more usually from 0 to 1 site of ethylenic unsaturation.
For the most part, the stable nitroxide functionalities which are employed will have the following formula: ##SPC4##
wherein:
X is a divalent aliphatic radical, having from 0 to 1 site of aliphatic unsaturation, usually aliphatically saturated of from 1 to 6 carbon atoms, usually from 2 to 3 carbon atoms being annular atoms;
A is lower alkyl (1 to 6, usually one to 3 carbon atoms), preferably methyl; and
Y is of the following formula: ##SPC5##
wherein:
R has been defined previously and the thiocarbamoyl group is meta or para, usually para.
For the most part, the cyclic nitroxides are pyrrolidine or piperidine derivatives.
Illustrative spin label compounds include:
N-(2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl) N'-(4'-(3"-ecgoninyl benzoate) thiourea;
N-(2,2,5,5-tetramethylpyrrolidinyl-1-oxyl-3-methyl)
N'-(4'-(3"-ecgoninyl benzoate) thiourea; and
N-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl)
N'-(4'-(3"-ecgoninyl benzoate) thiourea.
ANTIBODIES
The preparation of antibodies specific for haptenic materials is a well-established practice. A thorough description of the procedure may be found in Williams et al, Methods in Immunology and Immunochemistry, Academic Press, New York and London, 1967, pages 197 to 385, particularly that portion beginning at page 197 and ending at page 254.
For preparation of antibodies to haptens, a hapten is conjugated to an antigenic material such as a polypeptide or protein, although polysaccharides, particularly containing aminosugars, can also be used.
The particular manner in which the hapten is bonded to the antigenic material will depend on the functionalities which are available on the haptenic material and the antigenic material, the number of haptenic groups to be conjugated to the antigenic material, and the like. Groups which find use include carboxy groups, which may be activated by employing the mixed carbonic acid anhydride or carbodiimide, imidates, diazo groups, α-haloketones, and the like. Numerous procedures for the conjugation of a wide variety of haptens have been developed and published.
The antigenic conjugate may be injected in the fluid state; adsorbed to insoluble particles, such as alumina; or incorporated in matrix materials such as agar, calcium alginate, or Freund's adjuvants ("complete" or "incomplete", depending on whether mycobacteria are incorporated). The adsorbtion to various insoluble colloidal carriers is described in the aforementioned text, the carriers being illustrated by alumina, aluminum phosphate, blood charcoal and the like. Other materials include polyacrylamide gel, bentonite, and protein. As adjuvants, methylated bovine serum albumin and Freund's adjuvant find use. Complete Freund's adjuvant is a water-in-oil emulsion, using emulsion stabilizers such as lanolin, lanolin derivatives, e.g., Aquaphor, mannide mono-oleate and Arlacel A, available from Duke Laboratories, South Newark, Connecticut. The complete adjuvant is distinguished from the incomplete adjuvant by having mycobacteria, e.g., M.butyricum or M.tuberculosis. The adjuvants are commercially available from Difco Laboratories, Detroit, Michigan.
Immunization can be carried out in a variety of ways with a number of different animals. For the most part, for commercial production of antibodies, relatively large animals are employed, such as equine, bovine, porcine, canine, ovine, caprine, rodentia, rabbits and hares. Of particular interest are horses, goats, sheep and cows, that is, the larger domestic animals, as well as rabbits.
The antigenic material may be injected interperitoneally, intramuscularly, subcutaneously, and the like. When employing Freund's adjuvants, usually in combination with saline, the amount of antigen employed will vary depending on the particular antigenic material and the number and period of prior injections. Usually, about 0.1 to 5 mg of antigenic material will be employed per one ml of solution. The total amount of antigenic material and solution will depend on the size, nature and weight of the animal employed. The initial injection will normally be at a number of sites, aliquots of the composition being employed.
The first injections of antigen serve to load the animal, and a period of time is allowed to pass before booster injections are introduced, normally 2 to 8 weeks. Bleeding may occur after each injection, so as to follow the formation of the desired antibody. Depending on the animal, bleedings can be carried out via heart puncture, the carotid artery or external jugular vein. The bleeding will usually be carried out 4 to 14 days, preferably 6 to 8 days after an injection. The blood may then be combined with a small amount of sodium citrate, heparin or sodium EDTA, the mixture agitated and the erythrocytes settled by standing or centrifugation. The plasma is drawn off and combined with calcium chloride, with clotting resulting. If necessary, thrombin may be added to enhance clotting. After breaking up the clot, the clot is compressed and serum is withdrawn and filtered. Various other procedures are known and can be employed.
The serum can be treated in various ways, depending on its subsequent use. The serum may be fractionated by employing ethanol, Rivanol, neutral salts, such as ammonium sulfate or sodium sulfate, or the like. Usually, the product will be dialyzed after dissolution in a buffer, filtered and then isolated. Alternatively, the serum may be chromatographed on various modified cellulose columns, e.g., diethylaminoethylcellulose or carboxymethylcellulose. Various physical means may be employed to concentrate the desired antibodies.
Numerous preservatives can be employed to stabilize the antibodies and the antibodies will normally be stored at reduced temperatures.
The antibodies are primarily γ-globulin which are found to have a molecular weight of about 150,000. The antibodies will be specific for a particular spatial structure and polar-non-polar distribution. Varying structures deviating from an ideal structure will give different binding constants.
EXPERIMENTAL
The following examples are offered by way of illustration and not by way of limitation.
(All temperatures that are not otherwise indicated are in centigrade).
EXAMPLE I
Preparation of para-Aminobenzoylecgonine
A. Ecgonine hydrochloride (5.5g, 24.8 mmoles) was dissolved in 35ml of methanol (dried over 3-A Molecular sieves) and saturated with dry hydrogen chloride keeping the receiver cool by immersion in an ice bath. Upon saturation the receiver was heated to 40° for 0.5 hour and evaporated to dryness in vacuo. The white residue was stored at 0.05mm Hg over potassium hydroxide for 16 hours and then dissolved in the minimum amount of hot methanol to which 200ml of boiling acetone was quickly added. After cooling in ice and filtering, there was obtained 4.2g of white crystals, m.p. 214°-215° (lit. 214°-215°). Evaporation of the mother-liquor and repetition of the recrystallization yielded 0.8g m.p. 212°-214°. Total yield was 86.3% of theory.
B. To 20ml of cold saturated potassium carbonate solution in a 125ml separator funnel was added a solution of 5.0g (213 mmoles) ecgonine methyl ester hydrochloride in 5ml water. The aqueous mixture was extracted with 4 × 60ml of chloroform. The combined chloroform extracts were dried over anhydrous sodium carbonate and evaporated in vacuo. Pumping at 0.05mm Hg for 15 min. yielded 4.0g (93%) of TLC pure (20:1 CHCl 3 :MeOH) ecgonine methyl ester.
The 4.0g (20:1 mmoles) ecgonine methyl ester was dissolved in 50ml dry benzene and then 30ml benzene was distilled off. To the cooled distillation pot was added 3.65ml triethylamine and a solution of 3.72g freshly recrystallized p-nitrobenzoyl chloride in 5ml of dry benzene was added dropwise with cooling (ice bath) and agitation.
The resulting sludge was stirred at 40° for 1 hr under nitrogen. After cooling to room temperature the reaction mixture was taken up in 100ml of chloroform and washed with 3 × 20ml 5% aqueous sodium carbonate solution. The chloroform solution was dried over sodium carbonate, evaporated in vacuo and pumped (0.05mm Hg) on overnight to yield 5.7g (85.3%) of yellow oil [one spot on TLC (95/5, CHCl 3 /MeOH)] with same R f as known sample but having a slight odor of triethylamine. No further attempt at purification was made, and the product was used directly in the next step.
C. To a solution of 6.5g p-nitrococaine in 250ml absolute methanol was added 600mg 10% Pd/C under an N 2 blanket. The resulting mixture was hydrogenated at atmospheric pressure with rapid stirring and slight heating from the magnetic stirrer. After 0.5 hr. H 2 uptake ceased, [1.530 liters, calculated is 1.440 liters without correction for atmospheric pressure]. The catalyst was removed by suction filtration over a Celite pad in a fritted glass funnel (medium grade). The resulting clear solution was evaporated in vacuo to approximately 75ml and heated to dissolve crystals which formed and then allowed to cool to room temperature, followed by cooling in ice and filtering to give 4.0g white crystals, m.p. 188°-189°. The mother-liquor was concentrated to 3ml, cooled in ice and filtered. After washing the crystals with 6ml of cold methanol, there was obtained 1.2g powdery crystals, m.p. 185°-188°. Total yield 88%.
D. p-Aminococaine (2.08g) in 15ml of water was refluxed with rapid stirring under nitrogen for 6 hours. The solution was allowed to cool to room temperature and then cooled in ice and filtered. The crystals were washed with 5ml cold water and dried at 0.05mm Hg for 2 hours to yield 1.2g clear needlelike crystals, m.p. 287° (dec.). The compound slowly turns brown upon exposure to air and light. Recrystallization of 200mg from 2ml boiling water gave an analytically pure sample.
Calc, %: C, 63.14; H, 6.62; N, 920.
Found, %: C, 63.32; H, 6.62; N, 9.16.
EXAMPLE II
Preparation of para-isothiocyanato Benzoyl Ecgonine
Into two ml of 2N hCl under nitrogen was introduced 100mg (0.33 mmole) of para-aminobenzoyl ecgonine. To the solution was added 31μl (46mg) of thiophosgene and the heterogeneous mixture stirred vigorously under nitrogen at room temperature. After 10 minutes, the thiophosgene could no longer be observed. The product crystallized. The mixture was cooled in ice, filtered, and the filtrate washed with water. After drying the solid over phosphorous pentoxide and potassium hydroxide, 62mg was isolated. m.p. 257° (dec).
To the mother-liquor was added approximately 3ml of water, the solution cooled in ice and the precipitate collected. The second crop yielded 66mg. m.p. 257°.
Anal. % Calc'd for C 17 H 19 N 2 O 4 S Cl: C, 53.33;
H, 5.02; N, 7.32; S, 8.36; Cl, 9.27. % Fd:
C, 53.39; H, 5.14; N, 7.32; S, 8.41; Cl, 9.01.
EXAMPLE III
Conjugation of para-Isothiocyanatobenzoyl Ecgonine to Lysozyme
A solution of 60mg (25μ mole) lysozyme in 5.0ml water was cooled to 4°C and adjusted to pH 9.0 with 0.05M NaOH. A total of 99.5mg (25μ mole) p-isothiocyanatobenzoyl ecgonine was added in one portion to the alkaline protein solution. This conjugation was run on the pH-STAT at 4° with the machine maintaining the pH at 9.0 with 0.05M NaOH. (The reaction can also be performed by the manual addition of base using a pH meter to follow the course of the reaction). After 33/4 hours, the clear solution was adjusted to pH 9.5. Since no precipitation occurred, the pH was lowered to 7.0 with dilute HCl. The clear solution was dialyzed against water for 48 hours. The dialysate was immediately suitable for the assay of benzoyl ecgonine.
EXAMPLE IV
Conjugation of para-Isothiocyanatobenzoyl Ecgonine to Glucose-6-Phosphate Dehydrogenase (G-6-PDH)
To a 0.5ml solution of G-6-PDH (2mg protein/ml) in 0.05M phosphate buffer at pH 7.0 at 4° was added dropwise a solution of 1.4 mg para-isothiocyanatobenzoyl ecgonine in 0.5ml of 0.05M tris at pH 7.0. The reaction mixture was stirred at 4° at pH 7.0 for 30 minutes before the pH was adjusted to 8.6 by the addition of 0.05M sodium hydroxide. After 5 hours, the mixture was dialyzed against 2 liters 0.055M tris pH 7.9.
The resulting dialysate was assayed for enzyme activity in the presence of NAD and glucose-6-phosphate by measuring the increase in absorption at 340nm. That benzoyl ecgonine had been attached to the enzyme was shown by the change in enzyme activity in the presence and absence of benzoyl ecgonine antibodies.
______________________________________ Enzyme Rate Amount of Benzoyl Ecgonine ΔQD/min. at Antibody added 340nm ______________________________________ 123 none 80 50μl ______________________________________
The addition of 50μl of antibody results in a 34% inhibition of enzyme activity.
The procedure for the G-6-PDH assay is as follows:
The following reagents are employed: 0.1M NAD in H 2 O, pH adjusted to 5-6 with 0.05M NaOH; 0.066M glucose-6-phosphate (G-6-P) in 0.055M Tris-Cl, pH 7.9; 0.055M Tris-Cl buffer, pH 7.9 with 0.1% rabbit serum albumin.
Into a spectrophotometer cell is metered 20μl NAD, 50μl G-6-P, specified amount of antibody solution, 10μl of a 1:4 dilution of the enzyme dialysate and sufficient buffer to bring the total volume to 1ml. The optical density is read at 340nm for 5 minutes and the enzyme rate determined by averaging the rate between the second and fifth minute. The temperature is maintained at 37°.
To demonstrate the use of the subject compounds, immunoassays were carried out.
Assays
The immunoassay employing an enzyme as the detector is carried out as follows. With lysozyme, a bacterial suspension of M.luteus is employed, dissolving 0.2ml of a suspension of 300mg of the bacteria in 400ml of 0.025M, pH6,Tris-maleate buffer. First the bacterial suspension is introduced into the assay vessel. When testing a sample, 50μl of the sample is then introduced. This is followed by 50μl of antibody solution in 0.025M, pH6, Tris-maleate buffer and the transfer made quantitative by washing with 325μl of the same buffer solution. The benzoyl ecgonine conjugate to lysozyme (50μl) is then added to give a binding site to benzoyl ecgonine ratio of about 1:1.5 and 325μl of buffer used to insure quantative transfer.
The results are then read by observing the decrease in optical density at 436nm for 40 seconds at 36°. The results are reported in OD/min. In the subject assay, the antibody employed was obtained in response to a conjugate of para-diazabenzoyl ecgonine with bovine serum albumin. The binding constant was about 1 × 10 8 M - 1 . The concentration of antibody was 6.9 × 10 - 6 m, based on binding sites as determined employing a free radical assay technique with a cocaine spin label.
Following the procedure described above, a number of drugs were studied for cross reactivity in the benzoyl ecgonine assay. The following table indicates the results. The results are reported as the amount of cross reacting drug necessary to produce a response equivalent to 1.0 μg per ml benzoyl ecgonine.
______________________________________ DRUG EQUIVALENT TO 1.0 μg/ml. BENZOYL ECGONINE ______________________________________ Benzoyl Ecgonine 1.0 Ecgonine 4.5 Cocaine 22.0 Caffeine >1000.0 Morphine >1000.0 Secobarbital >1000.0 Phenobarbital >1000.0 Phenylpropanolamine >1000.0 Codeine >1000.0 Atropine >1000.0 Methamphetamine >1000.0 ______________________________________
Employing the benzoyl ecgonine conjugate to lysozyme in accordance with the previously described method, the assay was found to have excellent sensitivity and reproducibility of results in the range of 0.5 to 5 μg per ml concentration of benzoyl ecgonine. Ten urine samples were each spiked to levels of 1 μg per ml and 5 μg per ml of benzoyl ecgonine. Each sample was assayed in triplicate and the average value of each sample was used to calculate the experimental concentration and the percent recovery. The recovery observed contained some contribution of spiking error, operator error, and the urine variation. The average recovery of activity lies around 95%. The following tables indicate the results.
______________________________________ Samples Spiked to 1.0 μg/ml Benzoyl Ecgonine ______________________________________ Average Sample OD Units μg/ml % Recovery ______________________________________ 1 96 0.8 80 2 106 1.3 130 3 90 0.5 50 4 103 1.2 120 5 103 1.2 120 6 92 0.6 60 7 94 0.7 70 8 102 1.1 110 9 94 0.7 70 10 10 1.0 100 Mean 98.1 0.01 91 Recovery 90-106 .5-1.3 μg/ml 60-130% Range ______________________________________
Samples Spiked to 5.0 μg/ml Benzoyl Ecgonine ______________________________________ Average Sample OD Units μg/ml % Recovery ______________________________________ 1 147 5.2 104 2* 179 10.5* 210* 3 147 5.2 104 4 150 5.7 114 5* 173 10.0* 200* 6 142 4.2 84 7 144 4.5 90 8 147 5.2 104 9 128 3.6 72 10 152 6.0 120 Mean 151 6.01 μg/ml 120% Recovery 128-179 3.6-10.5 μg/ml 72-210% Range ______________________________________ *Samples No. 2 and No. 5 give an extraordinarily high recovery of benzoyl ecgonine. Since the blanks for these two samples are negligible and also since this magnitude of urine variation is beyond normal limits, some error in the spiking of these two samples must have occurred.
The lysozyme benzoyl ecgonine conjugate employed in the subject assay is found to be stable for long periods of time when stored at 4°C. The enzyme retains a substantial proportion of the original activity after conjugation, so as to provide a high degree of activity in the assay. In addition, the enzyme conjugate is able to provide a substantial rate change when the amount of benzoyl ecgonine is varied from 0.5 to 5.0 μg/ml. Therefore, the subject conjugate provides an accurate, effective and rapid method for determining extremely small quantities of benzoyl ecgonine. Furthermore, the assay only requires 50μl or less of urine sample, so that extremely small amounts of benzoyl ecgonine are required for detection.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
1. Field of the Invention
The use of cocaine for other than medicinal uses has become of increasing concern. Cocaine's availability has increased with concomitant efforts to police its use and prevent its distribution from unauthorized sources. In addition, in clinics providing drug therapy, it is important that the therapist be aware whether the person being treated has terminated or is continuing the use of drugs.
There has been continuing efforts to find reliable and rapid ways for detecting the use of drugs. It is also necessary that any method be able to distinguish cocaine or its metabolite from other drugs. Where urine is tested, it is found that cocaine is metabolized to benzoyl ecgonine. Therefore, in order to detect the use of cocaine, it is necessary to detect the presence of benzoyl ecgonine.
2. Description of the Prior Art
A number of immunoassay systems suggest themselves as useful for the detection of cocaine or its metabolites. Radioimmunoassay has been reported as useful in the detection of opiate alkaloids. See U.S. Pat. No. 3,709,868. U.S. Pat. No. 3,690,834 discloses a system using a free radical detector for immunoassays. U.S. Pat. application Ser. No. 143,609, filed May 14, 1971, now abandoned discloses an immunoassay technique employing enzymes, sold under the trademark "EMIT", by Syva Corporation. Odell, Competitive Protein Binding, Blackwell Scientific Publications, Oxford, 1971, discloses a number of conjugated haptens to proteins, see particularly Chapter 2, beginning at page 25. A wide variety of functionalities are disclosed as useful for conjugation.
SUMMARY OF THE INVENTION
Novel isothiocyanate modified benzoyl ecgonine compounds are provided for conjugation with poly(amino acids). Where anti-genic poly(amino acids) are employed, the product is useful in preparing antibodies, which show excellent specificity and sensitivity to benzoyl ecgonine, a cocaine metabolite. With conjugation to an enzyme, the enzyme conjugate is useful in immunoassays for the determination of benzoyl ecgonine or cocaine.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The compounds of this invention are thiocarbamoyl derivatives of benzoyl ecgonine or its methyl ester, as well as the antibodies prepared from the antigenic conjugates of the benzoyl ecgonine derivative with an antigenic poly(amino acid).
For the most part, the compounds of this invention will have the following formula: ##SPC1##
Wherein:
R is hydrogen or methyl, usually hydrogen;
α is hydrogen or may be taken together with β to form a bond between the nitrogen and the carbon atoms to which they are attached;
β may be taken together with α to form a bond, as indicated above, or is a poly(amino acid) residue to which at least one benzoyl ecgonine is conjugated; and
n is the number of benzoyl ecgonine groups conjugated to the poly(amino acid) and on the average will be at least 1 and not more than the molecular weight of β divided by 500, more usually not more than the molecular weight of β divided by 2000, and usually not exceeding 250.
The thiocarbamoyl group may be meta or para, usually para.
When α and β are taken together, the compound will have the following formula: ##SPC2##
wherein:
R is hydrogen or methyl, usually hydrogen, and the isothiocyanate group is either meta or para, normally para.
The isothiocyanate compound is prepared by esterifying the appropriate ecgonine derivative with the appropriate nitrobenzoic acid. The ester may then be reduced to the aminobenzoate, conveniently employing catalytic hydrogenation. The amino compound may then be derivatized to the isothiocyanate using thiophosgene. The amino compound is combined with the thiophosgene under mild conditions.
Conjugation of the isothiocyanate to a poly(amino acid) is carried out by combining the appropriate ratio of the isothiocyanate to the poly(amino acid) under mild conditions and at a constant mildly basic pH, normally in the range of about 8 to 9.
Of particular interest for the use of the subject compound is the conjugation to an amino group, which is part of a poly(amino acid) structure. By poly(amino acid) is intended both polypeptides and proteins. One group of poly(amino acids) is antigenic, so that by conjugation of the isothiocyanate to the antigenic poly(amino acid) a product is obtained which can be used in the formation of antibodies to benzoyl ecgonine. A narrow class of poly(amino acids), which can also be used as antigens, though not normally be used as such, are enzymes which are employed as the detector in an immunoassay system.
Polypeptides usually encompass from about 2 to 100 amino acid units (usually less than about 12,000 molecular weight). Larger polypeptides are arbitrarily called proteins. Proteins are usually composed of from 1 to 20 polypeptide chains, called subunits, which are associated by covalent or non-covalent bonds. Subunits are normally of from 100 to 300 amino acid groups (approximately 10,000 to 35,000 molecular weight). For the purposes of this invention, polypeptide is intended to include individual polypeptide units or polypeptides which are subunits of proteins, whether composed solely of polypeptide units or polypeptide units in combination with other functional groups, such as porphyrins, as in hemoglobin or cytochrome oxidase.
The first group of poly(amino acid) materials which will be considered are the antigenic poly(amino acids). These may be joined to the isothiocyanate group through an amino group. The thiourea product, which may include some thiourethane, can be used for the formation of antibodies to cocaine metabolites. The poly(amino acid) materials which may be used vary widely, normally being from about 1,000 to 10 million molecular weight, more usually from 12,000 to 1 million molecular weight, and most frequently from about 50,000 to 500,000 molecular weight.
With most conventional poly(amino acids), there will not be more than about one benzoyl ecgonine or derivative group per 1,500 molecular weight of poly(amino acid), usually not more than one group per 2,000 molecular weight. There will be at least one group per 500,000 molecular weight, usually at least one per 50,000 molecular weight. With intermediate molecular weight antigens (50,000 to 1 million), the number of benzoyl ecgonine or derivative groups will generally be from about 2 to 250, usually from 10 to 100.
With low molecular weight antigens, 1,000 to 5,000 molecular weight, the number of benzoyl ecgonine or derivative groups will be in the range of 1 to 10, usually in the range of 2 to 5. Therefore, there may be as many as one benzoyl ecgonine or derivative group per 500 molecular weight of poly(amino acid).
Usually, the number of groups bonded to the poly(amino acid) will be related to the available amino groups, e.g., the number of lysines present. While the benzoyl ecgonine or derivative group may be bonded through the isothiocyanate to hydroxyl or mercaptan groups, which are present in the polypeptide, for the most part the bonding will be to amino and, therefore, the compounds are described as thioureas. However, thionoesters may also be present.
Amino acids present in poly(amino acids) which have free amino groups for bonding to the isothiocyanate modified benzoyl ecgonine or derivative thereof, include lysine, arginine, histidine, etc. The hydroxylated and mercpatan substituted amino acids include serine, cystene and threonine.
Various protein types may be employed as the antigenic material. These types include albumin, serum proteins, e.g., globulins, ocular lens proteins, lipoproteins, etc. Illustrative proteins include bovine γ-globulin, etc. Small natural polypeptides which are immunogenic, such as gramicidin may also be employed. Various synthetic polypeptides may also be employed, such as polymers of lysine, glutamic acid, phenylalanine, tyrosine, etc., either by themselves or in combination. Of particular interest is polylysine or a combination of lysine and glutamic acid. Any synthetic polypeptide must contain a sufficient number of active groups, as for example, amino groups provided by lysine.
The second group of poly(amino acids) are the enzymes to which the isothiocyanate derivative may be conjugated. As indicated, the benzoyl ecgonine derivative modified enzyme is useful for immunoassays. The immunoassay technique will follow in more detail.
Various enzymes may be used such as oxidoreductases, hydrolases, lyases, and the like. These enzymes include esterases, amidases, phosphorylases, carbohydrases, oxidases, reductases and the like. Of particular interest are such enzymes as lysozyme, amylase, dehydrogenases, particularly malate dehydrogenase, lactate dehydrogenase, mannitol-1-phosphate dehydrogenase, and glucose 6-phosphate dehydrogenasee, β-glucuronidase, cellulase, and phospho-lipase, particularly phospholipase C. The enzymes will usually have molecular weights in the range of about 1 × 10 4 to 6 × 10 5 , more usually in the range of about 1.2 × 10 4 to 3 × 10 5 .
There will usually be at least one benzoyl ecgonine or derivative group per enzyme molecule and usually not more than one group per 1,500 molecular weight, usually not more than one group for 2,000 molecular weight. Usually, there will be at least one benzoyl ecgonine or derivative group per 50,000 molecular weight, and more usually at least one group per 30,000 molecular weight. The modified enzyme will retain on the average at least 10%, more usually at least 30% of the original activity of the unmodified enzyme.
Where the benzoyl ecgonine or derivative is bonded to a poly(amino acid), there need be only one benzoyl ecgonine or derivative group, but usually there will be at least two groups. With the enzymes, the number of benzoyl ecgonine or derivative groups will generally be of from 1 to 30, more usually 2 to 25. Usually, there will be at least two, more usually at least three groups per enzyme, when the enzyme is randomly substituted with the benzoyl ecgonine or derivative groups, and preferably not more than 16 groups.
The substituted poly(amino acid)s will, for the most part, have the following formula: ##SPC3##
wherein:
R is hydrogen or methyl, usually hydrogen;
the thiocarbamoyl group is either meta or para, usually para;
Pp is a poly(amino acid), which has been extensively described previously; and
n' is the number of benzoyl ecgonine groups bonded to the poly(amino acid), the various ranges having been indicated previously.
Instead of an enzyme, a stable free radical may be employed as the functionality for detection in the immunoassay. These stable free radicals are cyclic nitroxides having the nitrogen of the nitroxide as an annular member and from 0 to 1 of the heteroatoms, i.e., oxygen and nitrogen, as annular members.
The molecules bonded to the isothiocyanate will normally be of from 8 to 16 carbon atoms, usually of from 8 to 12 carbon atoms. The functionality for linking to the ecgonine derivative may be bonded directly to an annular carbon atom or bonded to an annular carbon atom through an aliphatic chain of from 1 to 4 carbon atoms, usually of from 1 to 2 carbon atoms. The molecules may have from 0 to 2 sites of ethylenic unsaturation, more usually from 0 to 1 site of ethylenic unsaturation.
For the most part, the stable nitroxide functionalities which are employed will have the following formula: ##SPC4##
wherein:
X is a divalent aliphatic radical, having from 0 to 1 site of aliphatic unsaturation, usually aliphatically saturated of from 1 to 6 carbon atoms, usually from 2 to 3 carbon atoms being annular atoms;
A is lower alkyl (1 to 6, usually one to 3 carbon atoms), preferably methyl; and
Y is of the following formula: ##SPC5##
wherein:
R has been defined previously and the thiocarbamoyl group is meta or para, usually para.
For the most part, the cyclic nitroxides are pyrrolidine or piperidine derivatives.
Illustrative spin label compounds include:
N-(2,2,5,5-tetramethylpyrrolidin-1-oxyl-3-yl) N'-(4'-(3"-ecgoninyl benzoate) thiourea;
N-(2,2,5,5-tetramethylpyrrolidinyl-1-oxyl-3-methyl)
N'-(4'-(3"-ecgoninyl benzoate) thiourea; and
N-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-yl)
N'-(4'-(3"-ecgoninyl benzoate) thiourea.
ANTIBODIES
The preparation of antibodies specific for haptenic materials is a well-established practice. A thorough description of the procedure may be found in Williams et al, Methods in Immunology and Immunochemistry, Academic Press, New York and London, 1967, pages 197 to 385, particularly that portion beginning at page 197 and ending at page 254.
For preparation of antibodies to haptens, a hapten is conjugated to an antigenic material such as a polypeptide or protein, although polysaccharides, particularly containing aminosugars, can also be used.
The particular manner in which the hapten is bonded to the antigenic material will depend on the functionalities which are available on the haptenic material and the antigenic material, the number of haptenic groups to be conjugated to the antigenic material, and the like. Groups which find use include carboxy groups, which may be activated by employing the mixed carbonic acid anhydride or carbodiimide, imidates, diazo groups, α-haloketones, and the like. Numerous procedures for the conjugation of a wide variety of haptens have been developed and published.
The antigenic conjugate may be injected in the fluid state; adsorbed to insoluble particles, such as alumina; or incorporated in matrix materials such as agar, calcium alginate, or Freund's adjuvants ("complete" or "incomplete", depending on whether mycobacteria are incorporated). The adsorbtion to various insoluble colloidal carriers is described in the aforementioned text, the carriers being illustrated by alumina, aluminum phosphate, blood charcoal and the like. Other materials include polyacrylamide gel, bentonite, and protein. As adjuvants, methylated bovine serum albumin and Freund's adjuvant find use. Complete Freund's adjuvant is a water-in-oil emulsion, using emulsion stabilizers such as lanolin, lanolin derivatives, e.g., Aquaphor, mannide mono-oleate and Arlacel A, available from Duke Laboratories, South Newark, Connecticut. The complete adjuvant is distinguished from the incomplete adjuvant by having mycobacteria, e.g., M.butyricum or M.tuberculosis. The adjuvants are commercially available from Difco Laboratories, Detroit, Michigan.
Immunization can be carried out in a variety of ways with a number of different animals. For the most part, for commercial production of antibodies, relatively large animals are employed, such as equine, bovine, porcine, canine, ovine, caprine, rodentia, rabbits and hares. Of particular interest are horses, goats, sheep and cows, that is, the larger domestic animals, as well as rabbits.
The antigenic material may be injected interperitoneally, intramuscularly, subcutaneously, and the like. When employing Freund's adjuvants, usually in combination with saline, the amount of antigen employed will vary depending on the particular antigenic material and the number and period of prior injections. Usually, about 0.1 to 5 mg of antigenic material will be employed per one ml of solution. The total amount of antigenic material and solution will depend on the size, nature and weight of the animal employed. The initial injection will normally be at a number of sites, aliquots of the composition being employed.
The first injections of antigen serve to load the animal, and a period of time is allowed to pass before booster injections are introduced, normally 2 to 8 weeks. Bleeding may occur after each injection, so as to follow the formation of the desired antibody. Depending on the animal, bleedings can be carried out via heart puncture, the carotid artery or external jugular vein. The bleeding will usually be carried out 4 to 14 days, preferably 6 to 8 days after an injection. The blood may then be combined with a small amount of sodium citrate, heparin or sodium EDTA, the mixture agitated and the erythrocytes settled by standing or centrifugation. The plasma is drawn off and combined with calcium chloride, with clotting resulting. If necessary, thrombin may be added to enhance clotting. After breaking up the clot, the clot is compressed and serum is withdrawn and filtered. Various other procedures are known and can be employed.
The serum can be treated in various ways, depending on its subsequent use. The serum may be fractionated by employing ethanol, Rivanol, neutral salts, such as ammonium sulfate or sodium sulfate, or the like. Usually, the product will be dialyzed after dissolution in a buffer, filtered and then isolated. Alternatively, the serum may be chromatographed on various modified cellulose columns, e.g., diethylaminoethylcellulose or carboxymethylcellulose. Various physical means may be employed to concentrate the desired antibodies.
Numerous preservatives can be employed to stabilize the antibodies and the antibodies will normally be stored at reduced temperatures.
The antibodies are primarily γ-globulin which are found to have a molecular weight of about 150,000. The antibodies will be specific for a particular spatial structure and polar-non-polar distribution. Varying structures deviating from an ideal structure will give different binding constants.
EXPERIMENTAL
The following examples are offered by way of illustration and not by way of limitation.
(All temperatures that are not otherwise indicated are in centigrade).
EXAMPLE I
Preparation of para-Aminobenzoylecgonine
A. Ecgonine hydrochloride (5.5g, 24.8 mmoles) was dissolved in 35ml of methanol (dried over 3-A Molecular sieves) and saturated with dry hydrogen chloride keeping the receiver cool by immersion in an ice bath. Upon saturation the receiver was heated to 40° for 0.5 hour and evaporated to dryness in vacuo. The white residue was stored at 0.05mm Hg over potassium hydroxide for 16 hours and then dissolved in the minimum amount of hot methanol to which 200ml of boiling acetone was quickly added. After cooling in ice and filtering, there was obtained 4.2g of white crystals, m.p. 214°-215° (lit. 214°-215°). Evaporation of the mother-liquor and repetition of the recrystallization yielded 0.8g m.p. 212°-214°. Total yield was 86.3% of theory.
B. To 20ml of cold saturated potassium carbonate solution in a 125ml separator funnel was added a solution of 5.0g (213 mmoles) ecgonine methyl ester hydrochloride in 5ml water. The aqueous mixture was extracted with 4 × 60ml of chloroform. The combined chloroform extracts were dried over anhydrous sodium carbonate and evaporated in vacuo. Pumping at 0.05mm Hg for 15 min. yielded 4.0g (93%) of TLC pure (20:1 CHCl 3 :MeOH) ecgonine methyl ester.
The 4.0g (20:1 mmoles) ecgonine methyl ester was dissolved in 50ml dry benzene and then 30ml benzene was distilled off. To the cooled distillation pot was added 3.65ml triethylamine and a solution of 3.72g freshly recrystallized p-nitrobenzoyl chloride in 5ml of dry benzene was added dropwise with cooling (ice bath) and agitation.
The resulting sludge was stirred at 40° for 1 hr under nitrogen. After cooling to room temperature the reaction mixture was taken up in 100ml of chloroform and washed with 3 × 20ml 5% aqueous sodium carbonate solution. The chloroform solution was dried over sodium carbonate, evaporated in vacuo and pumped (0.05mm Hg) on overnight to yield 5.7g (85.3%) of yellow oil [one spot on TLC (95/5, CHCl 3 /MeOH)] with same R f as known sample but having a slight odor of triethylamine. No further attempt at purification was made, and the product was used directly in the next step.
C. To a solution of 6.5g p-nitrococaine in 250ml absolute methanol was added 600mg 10% Pd/C under an N 2 blanket. The resulting mixture was hydrogenated at atmospheric pressure with rapid stirring and slight heating from the magnetic stirrer. After 0.5 hr. H 2 uptake ceased, [1.530 liters, calculated is 1.440 liters without correction for atmospheric pressure]. The catalyst was removed by suction filtration over a Celite pad in a fritted glass funnel (medium grade). The resulting clear solution was evaporated in vacuo to approximately 75ml and heated to dissolve crystals which formed and then allowed to cool to room temperature, followed by cooling in ice and filtering to give 4.0g white crystals, m.p. 188°-189°. The mother-liquor was concentrated to 3ml, cooled in ice and filtered. After washing the crystals with 6ml of cold methanol, there was obtained 1.2g powdery crystals, m.p. 185°-188°. Total yield 88%.
D. p-Aminococaine (2.08g) in 15ml of water was refluxed with rapid stirring under nitrogen for 6 hours. The solution was allowed to cool to room temperature and then cooled in ice and filtered. The crystals were washed with 5ml cold water and dried at 0.05mm Hg for 2 hours to yield 1.2g clear needlelike crystals, m.p. 287° (dec.). The compound slowly turns brown upon exposure to air and light. Recrystallization of 200mg from 2ml boiling water gave an analytically pure sample.
Calc, %: C, 63.14; H, 6.62; N, 920.
Found, %: C, 63.32; H, 6.62; N, 9.16.
EXAMPLE II
Preparation of para-isothiocyanato Benzoyl Ecgonine
Into two ml of 2N hCl under nitrogen was introduced 100mg (0.33 mmole) of para-aminobenzoyl ecgonine. To the solution was added 31μl (46mg) of thiophosgene and the heterogeneous mixture stirred vigorously under nitrogen at room temperature. After 10 minutes, the thiophosgene could no longer be observed. The product crystallized. The mixture was cooled in ice, filtered, and the filtrate washed with water. After drying the solid over phosphorous pentoxide and potassium hydroxide, 62mg was isolated. m.p. 257° (dec).
To the mother-liquor was added approximately 3ml of water, the solution cooled in ice and the precipitate collected. The second crop yielded 66mg. m.p. 257°.
Anal. % Calc'd for C 17 H 19 N 2 O 4 S Cl: C, 53.33;
H, 5.02; N, 7.32; S, 8.36; Cl, 9.27. % Fd:
C, 53.39; H, 5.14; N, 7.32; S, 8.41; Cl, 9.01.
EXAMPLE III
Conjugation of para-Isothiocyanatobenzoyl Ecgonine to Lysozyme
A solution of 60mg (25μ mole) lysozyme in 5.0ml water was cooled to 4°C and adjusted to pH 9.0 with 0.05M NaOH. A total of 99.5mg (25μ mole) p-isothiocyanatobenzoyl ecgonine was added in one portion to the alkaline protein solution. This conjugation was run on the pH-STAT at 4° with the machine maintaining the pH at 9.0 with 0.05M NaOH. (The reaction can also be performed by the manual addition of base using a pH meter to follow the course of the reaction). After 33/4 hours, the clear solution was adjusted to pH 9.5. Since no precipitation occurred, the pH was lowered to 7.0 with dilute HCl. The clear solution was dialyzed against water for 48 hours. The dialysate was immediately suitable for the assay of benzoyl ecgonine.
EXAMPLE IV
Conjugation of para-Isothiocyanatobenzoyl Ecgonine to Glucose-6-Phosphate Dehydrogenase (G-6-PDH)
To a 0.5ml solution of G-6-PDH (2mg protein/ml) in 0.05M phosphate buffer at pH 7.0 at 4° was added dropwise a solution of 1.4 mg para-isothiocyanatobenzoyl ecgonine in 0.5ml of 0.05M tris at pH 7.0. The reaction mixture was stirred at 4° at pH 7.0 for 30 minutes before the pH was adjusted to 8.6 by the addition of 0.05M sodium hydroxide. After 5 hours, the mixture was dialyzed against 2 liters 0.055M tris pH 7.9.
The resulting dialysate was assayed for enzyme activity in the presence of NAD and glucose-6-phosphate by measuring the increase in absorption at 340nm. That benzoyl ecgonine had been attached to the enzyme was shown by the change in enzyme activity in the presence and absence of benzoyl ecgonine antibodies.
______________________________________ Enzyme Rate Amount of Benzoyl Ecgonine ΔQD/min. at Antibody added 340nm ______________________________________ 123 none 80 50μl ______________________________________
The addition of 50μl of antibody results in a 34% inhibition of enzyme activity.
The procedure for the G-6-PDH assay is as follows:
The following reagents are employed: 0.1M NAD in H 2 O, pH adjusted to 5-6 with 0.05M NaOH; 0.066M glucose-6-phosphate (G-6-P) in 0.055M Tris-Cl, pH 7.9; 0.055M Tris-Cl buffer, pH 7.9 with 0.1% rabbit serum albumin.
Into a spectrophotometer cell is metered 20μl NAD, 50μl G-6-P, specified amount of antibody solution, 10μl of a 1:4 dilution of the enzyme dialysate and sufficient buffer to bring the total volume to 1ml. The optical density is read at 340nm for 5 minutes and the enzyme rate determined by averaging the rate between the second and fifth minute. The temperature is maintained at 37°.
To demonstrate the use of the subject compounds, immunoassays were carried out.
Assays
The immunoassay employing an enzyme as the detector is carried out as follows. With lysozyme, a bacterial suspension of M.luteus is employed, dissolving 0.2ml of a suspension of 300mg of the bacteria in 400ml of 0.025M, pH6,Tris-maleate buffer. First the bacterial suspension is introduced into the assay vessel. When testing a sample, 50μl of the sample is then introduced. This is followed by 50μl of antibody solution in 0.025M, pH6, Tris-maleate buffer and the transfer made quantitative by washing with 325μl of the same buffer solution. The benzoyl ecgonine conjugate to lysozyme (50μl) is then added to give a binding site to benzoyl ecgonine ratio of about 1:1.5 and 325μl of buffer used to insure quantative transfer.
The results are then read by observing the decrease in optical density at 436nm for 40 seconds at 36°. The results are reported in OD/min. In the subject assay, the antibody employed was obtained in response to a conjugate of para-diazabenzoyl ecgonine with bovine serum albumin. The binding constant was about 1 × 10 8 M - 1 . The concentration of antibody was 6.9 × 10 - 6 m, based on binding sites as determined employing a free radical assay technique with a cocaine spin label.
Following the procedure described above, a number of drugs were studied for cross reactivity in the benzoyl ecgonine assay. The following table indicates the results. The results are reported as the amount of cross reacting drug necessary to produce a response equivalent to 1.0 μg per ml benzoyl ecgonine.
______________________________________ DRUG EQUIVALENT TO 1.0 μg/ml. BENZOYL ECGONINE ______________________________________ Benzoyl Ecgonine 1.0 Ecgonine 4.5 Cocaine 22.0 Caffeine >1000.0 Morphine >1000.0 Secobarbital >1000.0 Phenobarbital >1000.0 Phenylpropanolamine >1000.0 Codeine >1000.0 Atropine >1000.0 Methamphetamine >1000.0 ______________________________________
Employing the benzoyl ecgonine conjugate to lysozyme in accordance with the previously described method, the assay was found to have excellent sensitivity and reproducibility of results in the range of 0.5 to 5 μg per ml concentration of benzoyl ecgonine. Ten urine samples were each spiked to levels of 1 μg per ml and 5 μg per ml of benzoyl ecgonine. Each sample was assayed in triplicate and the average value of each sample was used to calculate the experimental concentration and the percent recovery. The recovery observed contained some contribution of spiking error, operator error, and the urine variation. The average recovery of activity lies around 95%. The following tables indicate the results.
______________________________________ Samples Spiked to 1.0 μg/ml Benzoyl Ecgonine ______________________________________ Average Sample OD Units μg/ml % Recovery ______________________________________ 1 96 0.8 80 2 106 1.3 130 3 90 0.5 50 4 103 1.2 120 5 103 1.2 120 6 92 0.6 60 7 94 0.7 70 8 102 1.1 110 9 94 0.7 70 10 10 1.0 100 Mean 98.1 0.01 91 Recovery 90-106 .5-1.3 μg/ml 60-130% Range ______________________________________
Samples Spiked to 5.0 μg/ml Benzoyl Ecgonine ______________________________________ Average Sample OD Units μg/ml % Recovery ______________________________________ 1 147 5.2 104 2* 179 10.5* 210* 3 147 5.2 104 4 150 5.7 114 5* 173 10.0* 200* 6 142 4.2 84 7 144 4.5 90 8 147 5.2 104 9 128 3.6 72 10 152 6.0 120 Mean 151 6.01 μg/ml 120% Recovery 128-179 3.6-10.5 μg/ml 72-210% Range ______________________________________ *Samples No. 2 and No. 5 give an extraordinarily high recovery of benzoyl ecgonine. Since the blanks for these two samples are negligible and also since this magnitude of urine variation is beyond normal limits, some error in the spiking of these two samples must have occurred.
The lysozyme benzoyl ecgonine conjugate employed in the subject assay is found to be stable for long periods of time when stored at 4°C. The enzyme retains a substantial proportion of the original activity after conjugation, so as to provide a high degree of activity in the assay. In addition, the enzyme conjugate is able to provide a substantial rate change when the amount of benzoyl ecgonine is varied from 0.5 to 5.0 μg/ml. Therefore, the subject conjugate provides an accurate, effective and rapid method for determining extremely small quantities of benzoyl ecgonine. Furthermore, the assay only requires 50μl or less of urine sample, so that extremely small amounts of benzoyl ecgonine are required for detection.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.