Journal of Immunological Methods 7 (1975) 1-24 © North-Holland Publishing Company

Review DRUG

IMMUNOASSAYS*

Vincent P. B U T L E R , Jr. Department of Medicine, College of Physicians & Surgeons, Columbia University, New York, N.Y. 10032, U.S.A.

Received 17 September 1974,

accepted 30 October 1974

Because of their specificity, sensitivity, rapidity and convenience, drug-specific immunoassays are now widely used both in clinical and investigative laboratories. Drug immunoassays are of clinical value in the determination of appropriate dosage schedules with certain drugs and in the documentation of recent ingestion of other drugs, particularly certain drugs of abuse. Immunoassays have already been of investigative value in studies of the bioavailability, absorption, metabolic degradation and excretion of several drugs. This review deals with the synthesis and characterization of drug protein conjugates, immunization with drug-protein conjugates, the detection and immunological characterization of drug-specific antibodies, the use of drug-specific antibodies in the development of immunoassays, general applications of drug immunoassays and, finally, individual descriptions of currently available drug immunoassay methods. 1. I n t r o d u c t i o n The past decade has been a c c o m p a n i e d by significant advances in the capacity to measure drug concentrations, at the picomolar level, in tissues and biological fluids (Vesell and Passananti, 1971). These advances have resulted, in large measure, f r o m the r e f i n e m e n t of quantitative s p e c t r o f l u o r o m e t r i c (Udenfriend, 1962; Trevor et al., 1971), gas c h r o m a t o g r a p h i c (Gudzinowicz, 1967; H a m m a r et al., 1969; Wilkinson, 1971 ; Hawks, 1974) and mass spectrometric ( H a m m a r et ah, 1969; J e n d e n and Cho, 1973; H o m i n g et al., 1973; Hawks, 1974) methods for the sensitive and specific d e t e r m i n a t i o n of drug c o n c e n t r a t i o n s in specimens obtained from man and experimental animals. Most recently, drug-specific antibodies have been used w i t h increasing f r e q u e n c y in the d e v e l o p m e n t o f rapid, sensitive, specific and convenient i m m t m o l o g i c assay procedures for the m e a s u r e m e n t o f serum and urine concentra*Presented at a symposium entitled 'Drug Assays at Sub-Microgram Levels' conducted by the American Society for Pharmacology and Experimental Therapeutics at East Lansing, Michigan, 23 August 1973. This work was supported by research grants from the U.S.P.H.S. (HL 10608), the American Heart Association (72-853), the New York Heart Association and the Burroughs Wellcome Co. Dr. Butler is the recipient of an Irma T. Hirschl Career Scientist Award.

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KP. Butler, Jr., Drug immunoassays

tions of various compounds of pharmacologic interest (Butler, Jr., 1973; Spector, 1973; Bidanset, 1974; Marks et al., 1974; Mul6 et al., 1974b). It is the purpose of this review to describe the production of antibodies to drugs, the detection and immunologic characterization of such antibodies, their use in the development of specific immunoassays and, finally, the application of these antibodies in clinical and experimental studies.

2. Production of antibodies to drugs 2.1. General principles

Most drugs are relatively small compounds, with molecular weights less than 1000. Such molecules are not ordinarily immunogenic but, as delineated in the classic studies of Landsteiner (1945), these low molecular weight substances will, when conjugated as haptens to protein or polypeptide carriers, elicit the production of antibodies capable of reacting specifically with the free, unconjugated hapten. 2.2. Choice o f a carrier

Ahnost any protein (including homologous plasma proteins) may be used as a carrier for an haptenically coupled drug. Serum albumins of various species have been most commonly used as such carriers for a number of reasons: their ready availability, low cost, high degree of immunogenicity, excellent solubility, and relative resistance to denaturation by the organic solvents and somewhat rigorous chemical conditions employed in sonre conjugation procedures. The functional groups in albumins or other protein carriers to which haptenic drugs or drug derivatives may be conjugated include: free amino groups (e-amino of lysine and NH2terminal residues); free carboxyl groups (aspartic acid, glutamic acid and COOHterminal residues); and phenolic (tyrosine), sulfhydryl (cysteine), imidazo (histidine), indolyl (tryptophan) and guanidino (arginine) functions. Drugs and drug derivatives have most frequently been coupled to the amino, carboxyl or phenolic groups of protein carriers (Beiser et al., 1968; Parker, 1972; Butler, Jr. and Beiser, 1973 ; Erlanger, 1973). 2.3. CTu~ice (~ta con/ugation method

The method selected for conjugation of the haptenic drug or drug derivative to a carrier must employ chemical conditions which do not cause significant structural alterations of the hapten and which do not produce sufficient denaturation of the carrier protein to render it insoluble. A number of such relatively gentle methods have been described for coupling small molecules to protein carriers via amino, carboxyl or hydroxyl groups in drugs or chemically related derivatives (Beiser et al.,

V.P. Butler, Jr., Drug immunoassays

3

1968; Parker, 1972; Butler, Jr. and Beiser, 1973; Erlanger, 1973; Marks et al., 1974). For example, aliphatic amines can be coupled to carriers using water-soluble carbodiimides (Goodfriend et al., 1964), bifunctional diisocyanates (Haber et al., 1965a), or by conversion to p-nitrobenzoylamides followed by reduction to the p-aminobenzoyl derivative which, upon diazotization, can be coupled to the tyrosine residues of protein (Anderer, 1963). Aromatic amines can be diazotized and coupled directly to carriers (Hamburger, 1966). Drugs or derivatives with free carboxyl groups can be coupled to amino groups of proteins either by the mixed anhydride (Vaughan, Jr. and Osato, 1952; Erlanger et al., 1957) or carbodiimide (Goodfriend et al., 1964) method. Numerous methods have been developed for coupling compounds with free hydroxyl groups to carrier proteins. The hydroxyl groups of steroids react with succinic acid to form the hemisuccinate (Erlanger et at., 1957) which can then be coupled to a protein via its carboxyl group, using the mixed anhydride or carbodiimide methods, as described above. Phenols have been converted to active reagents by reaction with diazotized p-aminobenzoic acid (Weliky and Weetall, 1965); such p-aminobenzoate derivatives, like hemisuccinate derivatives, can then be coupled to the amino groups of protein via their functional carboxyl group. Compounds with vicinal hydroxyl groups can conveniently be coupled to amino groups of protein carriers by the periodate oxidation method (Khym, 1963; Erlanger and Beiser, 1964; Butler, Jr. and Chen 1967). These and other conjugation methods have been reviewed in detail recently (Beiser et al., 1968; Parker, 1972; Butler, Jr. and Beiser, 1973; Erlanger, 1973; Marks et al., 1974). 2.4. Characterization o f drug pro tein con/ugates

Landsteiner (1945) and Erlanger (1973) have found that the incorporation of too much, as well as too little, hapten into a hapten-protein conjugate may lead to a poor antibody response; in their experience, 10 haptenic groups per molecule of carrier seemed optimal when serum albumin was used as the carrier. Hence, before one immunizes experimental animals with a newly synthesized drug-protein conjugate, it is usually desirable to determine the number of drug molecules one has conjugated to the protein (or polypeptide) carrier employed. If the haptenic group has an absorption spectrum which can allow one to differentiate it from the protein carrier, the ratio between the molar extinction coefficient (Little and Donahue, 1968) of the conjugate and that of the hapten at an appropriate wavelength can be used to calculate the molar incorporation of hapten onto protein carrier. However, as Erlanger (1973) has pointed out, even if there is overlap in spectra between hapten and carrier, reasonably accurate estimates of molar incorporation of hapten can be made by determining differences in molar extinction coefficients between conjugate and carrier, and then comparing the difference with the molar extinction coefficient of the hapten (Erlanger et al., 1957; Butler, Jr. et al., 1962; Smith et al., 1970; Tigelaar et al., 1973). The extent of

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V.P. Butler, Jr., Drug imrnunoassays

haptenic incorporation into a hapten-protein conjugate can also be determined by various chemical analyses of conjugates (Goodfriend et al., 1964; Dumasia et al., 1973), extent of incorporation of radiolabelled drug into conjugates (Lewis et al., 1972; Cheng et al., 1973; Chung et al., 1973; Cook et al., 1973; Mahon et al., 1973), or by the decrease in free amino groups of carrier, following conjugation of a hapten to carrier by a method which utilizes the amino groups of the carrier (Erlanger et al., 1957; Erlanger, 1973). 2.5. I m m u n i z a t i o n

Ordinarily, drug protein conjugates are suspended in complete Freund's adjuvant mixture (Freund et al., 1948) at a final concentration of 1 mg/ml or less; usually 1 mg or less is injected into experimental animals, most often rabbits or guinea pigs, at intervals of 1 week or more. Longer periods of time ( 8 - 1 6 months, in some instances) than are employed for protein antigens may be required to obtain anti-hapten antibody of optimal titer, specificity, and affinity (Smith et al., 1970; Jaffe et al., 1971). Vaitukaitis et al. (1971) have recently described a method which employs a small, divided primary immunizing dose together with Bordetella pertussis vaccine and which may be particularly useful when the quantity of hapten or conjugate is limited. These and other practical problems connected with raising antisera for use in immunoassays have been reviewed recently(Chase, 1967; Hum and Landon, 1971; Parker, 1972; Hum, 1974). 2. 6. Detection o,f drug-specti/i'c antibodies

Since most animals immunized with drug protein conjugates form antibodies with specificity for the carrier protein, the method chosen for detection of antidrug antibodies must be one in which antibodies specific for the carrier will not also interact. The simplest and most direct methods for the detection of drug-specific antibodies without interference by carrier-specific antibodies involve the direct demonstration of binding of radioactively labeled drugs or drug derivatives by antibody. Such binding of radiolabeled drug by antibody can be demonstrated directly by equilibrium dialysis (Eisen, 1964) or indirectly by one of the many methods now available to separate antibody-bound drug from unbound drug; currently popular methods include the dextran-coated charcoal technique (Herbert et al., 1968) gel or membrane filtration (Haber et al., 1965b; Van Vunakis and Levine, 1974), electrophoresis (Yalow and Berson, 1964), and coprecipitation of drug with antibody by the so-called 'double antibody' method (Morgan and Lazarow, 1963; Van Vunakis and Levine, 1974). Since many drugs are bound to a significant degree by normal serum proteins (especially in undiluted serum), it is important to ascertain that binding of radiolabeled drug is not observed with appropriate dilutions of control sera from non-immunized animals and from animals immunized with unrelated antigens.

V.P. Butler, Jr., Drug immunoassays

5

In the absence of radiolabeled drug, anti-drug antibodies can be demonstrated by other methods, including hemagglutination of drug-erythrocyte conjugates (Adler, 1974) and inactivation of drug-bacteriophage conjugates (Mfikelfi, 1966; Dray et al., 1972). It is also possible to employ classic precipitin, complement fixation or passive hemagglutination methods to demonstrate the interaction of anti-drug antibody with conjugates in which the hapten is attached to a carrier antigenically unrelated to the carrier used for immunization; in this latter instance, it is particularly important to ascertain that the interaction with antiserum is specifically inhibited by free, unconjugated drug (Butler, Jr. and Beiser, 1973). When antibodies to a carrier do interfere with the detection of anti-drug antibodies, such antibodies can usually be removed by prior absorption of antiserum with unconjugated carrier protein or with an appropriate insoluble immunoadsorbent containing the carrier protein (Butler, Jr. and Beiser, 1973). In the instance of one drug-albumin conjugate prepared by the carbodiimide method, native albumin did not completely remove anti-carrier antibody, but albumin polymerized with carbodiimide effectively removed the interfering antibodies (Adler and Liu, 1971). If one wishes to avoid the formation of anti-protein antibodies almost completely, one may take advantage of the fact that, while haptens coupled to homologous albumins are immunogenic, such conjugates usually do not elicit significant production of antibodies to the carrier (Butler, Jr. and Beiser, 1973). 2.7. Spec~li'city o f antibodies to drugs

The specificity of antibodies for a drug is usually determined by comparing the capacity of the non-radiolabeled drug and various structurally related compounds to interfere with the interaction between drug and antibody as measured by one of the methods described above. It is particularly important to ascertain that related compounds in serum, urine or tissue do not interact before the antibody is used in the development of an immunoassay (Parker, 1972; Butler, Jr. and Beiser, 1973). For example, antibodies to digitalis glycosides react with steroid hormones (Butler, Jr. and Chen, 1967); therefore, it was necessary to demonstrate that concentrations of steroid hormones encountered in human sera did not inhibit the binding of [3H]digoxin by anti-digoxin antibodies before the radioimmunoassay method could be used clinically to measure serum digoxin concentrations (Smith et al., 1969, 1970). It is important to recognize that antibodies to a given drug will usually react with metabolites of that drug. It may occasionally be possible to remove some of the antibodies which cross-react in this manner. It should be remembered, however, that absolute specificity of antibodies for a given molecule will rarely, if ever, be observed (Beiser et al., 1968). Cross-reactivity with drug metabolites may not represent a major disadvantage if, in practice, serum concentrations of 'immunoreactive' drug correlate well with values obtained by other methods and with the clinical state of the patients studied. For example, such a correlation does exist in the case

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V.P. Butler, Jr., Drug immunoassays

of digoxin; this correlation, however does not necessarily apply to other drugs because digoxin is somewhat unusual in that it is not extensively degraded in man, and in that several of its major metabolites are both pharmacologically and immunologically active (Butler, Jr., 1972). Antibodies to a given drug will often react with related drugs of the same class. but this cross-reactivity should not constitute a major problem clinically if it can be established with certainty that the patient is receiving a given drug and has not recently received a chemically related agent (Butler, Jr., 1972). If problems are encountered in obtaining antibodies of satisfactory specificity for use in an immunoassay procedure, it is important to remember that different individual animals may produce antibodies which vary greatly in specificity; one should, therefore, examure several antisera before selecting the one with optimal specificity. It also may be useful to recall that the specificity of anti-hapten antibodies appears to be directed primarily against that portion of the hapten molecule furthest from the site of conjugation to the carrier; antibodies of different specificity can usually be obtained if the hapten is coupled to the carrier via a different functional group (Parker, 1972; Butler, Jr. and Beiser, 1973; Erlanger, 1973). 2.8. Afji'nitv o f drug-specific antibodies Antiserum to a given drug usually contains a heterogeneous population of antidrug antibodies with different avidities or association constants. In general, antisera with high average intrinsic association constants are most useful in the development of immunoassay procedures of optimal sensitivity, rapidity and reproducibility (Parker, 1972). The determination of association constants is therefore useful in the selection of antisera for use in immunoassay work; it is, however, not essential since the avidity of antibodies may be interred from a variety of measurements of hapten-antibody interactions (Hunter, 1973). Recent studies have also called attention to the fact that dissociation constants of drug-antibody complexes may be important when the adsorbent used to separate free from antibody-bound drug in an immunoassay procedure competes with antibody for drug molecules which dissociate from antibody during the separation step of the immunoassay (Meade and Kleist, 1972; Smith and Haber, 1973). 2. 9. Titer o f drug specific-antibodies Titer is generally defined as the greatest dilution of antibody which will produce a given degree of binding of a stated amount of a drug (Hunter, 1973). The higher the tiler, the more determinations one can perform with a given volume of antiserum; for example, anti-digoxin sera frequently can be used at dilutions theoretically great enough to allow for the performance of 200,000 digoxin determinations with 1 ml of antiserum. Titer is, however, far less important than specificity and affinity in the choice of an antiserum for use in an immunoassay procedure; a

V.P. Butler, Jr., Drug immunoassays

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high-titered antiserum will be much less useful than a lower-titered serum with greater specificity or affinity.

3. lmmunoassay procedures 3.1. Principles

lmmunoassay methods for the measurement of drugs are based upon the ability of drugs to inhibit the reaction between drug-specific antibodies and the corresponding drug-carrier conjugate or the corresponding labeled hapten. Using principles delineated by Berson and Yalow, increasing concentrations of a known standard solution of the drug are incubated with constant predetermined amounts of drug-specific antibody and of drug-carrier conjugate, or of labeled drug, under conditions of antigen excess. A standard curve is then constructed, upon which decreasing amounts of drug antibody interaction can be shown to correspond with increasing concentrations of drug. If the biological fluid to be assayed (a) does not interfere with the drug antibody reaction, (b) does not degrade the drug, and (c) does not contain substances which crossreact significantly with the drug antibody, the concentration of the drug in that biological fluid can be determined from the degree to which it inhibits the reaction between antibody and drug carrier conjugate or labeled drug, when compared with a simultaneously performed standard curve (Yalow and Berson, 1964; Parker, 1972; Hunter, 1973 ; Ekins, 1974). 3.2. Methods

Inhibition of complement fixation (Hamburger, 1966) or of passive hemagglutination (Adler, 1974) have been used to measure drugs. However, because of their greater sensitivity, precision, rapidity and convenience, greater experience has been obtained with immunoassay methods which employ radioactively or physicochemically labeled drugs or drug derivatives. Until recently, most of the studies with labeled haptens employed radioactivelylabeled drugs or drug derivatives. Since a high specific activity is generally required for optimal sensitivity in radioimmunoassay procedures, 3H-labeled drugs have generally been more useful than 14C-labeled compounds. Since 3H and 14C are both beta-emitting isotopes, their use necessitates the employment of liquid scintillation counting techniques which are expensive and cumbersome, and which may be associated with problems of chemilluminescence and variable counting efficiency when biological fluids are studied (Butler, Jr., 1972; Smith and Haber, 1973). The development of methods which can be used for the radioiodination of drugs and drug derivatives (Oliver et al., 1968; Parker, 1972; Hunter, 1974; Van Vunakis and Levine, 1974) has permitted the use of simpler gamma-counting procedures and should permit the more rapid introduction of drug radioimmunoassays into general and clinical investigative use.

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v.P. Butler, Jr., Drug immunoassays

Many methods are available for the separation of free, unbound radiolabeled drug from antibody-bound radioactivity in the development of radioimmunoassay procedures (Hunter and Ganguli, 1971; Parker, 1972; Hunter, 1973; Ratcliffe, 1974). These include electrophoresis, gel filtration, adsorption systems (e.g., dextran-coated charcoal, membrane filtration), solvent and salt precipitation systems, solid-phase antibody procedures and the double antibody method. Because of its convenience and rapidity, the dextran-coated charcoal method of Herbert et al. (1968) has been widely used in drug radioimmunoassay procedures. It involves the almost instantaneous adsorption of non-antibody-bound drug onto charcoal particles and its rapid centrifugal separation from antibody-bound radioactivity; thus, it is not useful in instances wherein radiolabeled drugs or drug derivatives are not effectively adsorbed to the charcoal nor in instances in which the dissociation constant of a drug-antibody complex is great enough to cause a temporally related variability in results as dissociated drug is progressively adsorbed to charcoal as a function of the duration of the charcoal incubation step in the immunoassay procedure (Smith and Haber, 1973). Because of such problems with adsorption methods, many newly developed radioimmunoassay procedures have employed either the double antibody method or a solid phase antibody procedure. Theoretically, 'immunoradiometric' assay procedures employing radioiodinated antibody (Woodhead et al., 1974) may also be used, but extensive experience has not yet been obtained with such procedures in the assay of drugs. To eliminate some of the technical problems associated with the use of radiolabeled drugs in clinical laboratories or in automated equipment, physicochemically-labeled drugs have recently been employed in the development of non-isotopic immunoassay procedures. For example, spin-labeled drugs have been used in an immunoassay procedure which takes advantage of the fact that the mobility of free radicals of spin-labeled drugs in the free, unbound state differs from their mobility when bound to specific antibody, as measured in an electron spin resonance spectrometer. This technique has proved useful in screening biological fluids in the detection of opiates and other drugs of abuse (Leute et al., 1972a, b; Schneider et al., 1974). Another, perhaps more useful method, is based on the fact that anti-drug antibodies will inactivate drug-enzyme conjugates in a reproducible manner which is readily quantifiable by simple spectrophotometric assays of enzymatic activity; increasing quantities of drug will reduce the degree of enzyme inactivation in a reproducible and predictable manner which can be used as the basis for a simple immunoassay procedure (Engvall and Perlmann, 1971; Rubenstein et al., 1972; Schneider et al., 1973, 1974). 3.3. Problems in application o f drug immunoassay methods to the study o f biological Jluids

It is most convenient when an immunoassay procedure can be carried out with untreated serum, plasma or other biological fluid. In some instances, however, prior

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9

treatment or separatory procedures may be necessary to inactivate substances which degrade the drug being studied. In other cases, prior extractions or separatory procedures may be necessary to concentrate a drug, to remove it from a normal binding site on a plasma protein in the test serum, or to separate the drug from certain of its metabolites or from other structurally related compounds also capable of inhibiting the drug-antibody interaction (Parker, 1972; Butler, Jr. and Beiser, 1973). In radioimmunoassay procedures, the presence of a radioisotope (administered for diagnostic or therapeutic purposes) in a patient's serum constitutes a potential source of error, if the presence of the isotope is not known to the laboratory; in radioimmunoassay procedures employing liquid scintillation counting methods, chemilluminescence produced by urine and certain sera (especially from azotemic patients) may also interfere with assay results. Neither of these latter sources of error in radioimmunoassay procedures, however, should cause a problem if proper control procedures are carried out (Butler, Jr., 1971; Smith and Haber, 1973). 3.4. Stability o f reagents Although antibodies are stable for many years if properly stored in concentrated form, deterioration of antisera may occur if appropriate precautions are not taken. Deterioration of radiolabeled drugs or drug derivatives also may occur with time; this is particularly true in the case of radioiodinated derivatives of drugs (Kirkham and Hunter, 1971). Similarly, great care must be exercised in the preparation and storage of the drug standards used in the construction of standard immunoassay curves because results in unknown specimens will be calculated on the basis of values obtained with these standards of predetermined, known concentration. (Bangham and Cotes, 1974).

4. General applications of drug immunoassays The development of radioimmunoassay methods as well as of other new assay methods of great sensitivity and specificity has added greatly to our knowledge of the pharmacokinetics (absorption, compartmental distribution, degradation and excretion) of many important drugs and their metabolites (Levy and Gibaldi, 1972). With the increasing availability of information in this area, nmch has been learned about previously obscure processes which contribute greatly to the longrecognized but hitherto poorly understood individual variability in drug dosage requirements and in susceptibility to the toxic effects of drugs (Azarnoff, 1973; Butler, Jr. and Lindenbaum, 1975). In this regard, drug assay methods of many types have made it possible to study drug drug interactions (Solomon et al., 1971) and to identify clinically important differences in the biologic availability of different preparations of the same drug (Lindenbaum et al., 1971 ;Brodie and Heller,

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V.P. Butler, Jr., Drug immunoassays

1972: American Pharmaceutical Association, 1973), in the genetic capacity to metabolize certain drugs (La Du, 1972; Vesell, 1972) and in the serum protein binding (Davison, 1971) and renal excretion (Bennett et al., 1973) of other important drugs. Since, in many instances, immunoassay procedures can be carried out on microliter volumes of unextracted serum, such methods often offer significant advantages over some of the other new techniques for the measurement of drugs at the submicrogram level. In addition, their convenience and rapidity are major advantages in the clinical laboratory where the rapid analysis of large numbers of specimens is highly desirable in the evaluation of drug therapy and in the prompt determination of appropriate dosage schedules for individual patients being treated with a given drug. However, as with all drug assay procedures, drug immunoassay methods will yield useful information only if it can be established, experimentally or clinically, that the drug concentrations in the specimens being assayed bear some relationship to a pharmacological effect of, or the clinical response to, that drug (Brodie and Reid, 1971 ; Vesell and Passananti, 1971 ; Koch-Weser, 1972). In this latter connection, the time at which the specimen is best obtained with respect to the last dose of the drug is quite important and must be established before the immunoassay procedure can be used properly (Butler, Jr., 1972; Koch-Weser, 1972).

5. Specific drug immunoassays Table 1 lists drugs to which antibodies have been elicited, but for which immunoassay procedures have not been described, lmmunoassays have been described for the drugs listed in table 2. These immunoassays will be described individually in this section. 5.1. Analgesic drugs

An analogue of fentanyl, carboxyfentanyl, has been synthesized and conjugated to bovine y-globulin by the carbodiimide method. Rabbits immunized with carboxyfentanyl-protein conjugates formed antibodies capable of binding [3H]fentanyl, as assessed by the ammonium sulfate precipitation method. Unlabeled fentanyl and its metabolites are capable of inhibiting the binding of [3H] fentanyl by antibody, suggesting that this antibody will be useful in the development of a radioimmunoassay method for the measurement of fentanyl in biological fluids and tissues (Henderson et al., 1974). 5.2. Antibiotics

The nitro group of chloramphenicol has been reduced to form the amine, which could then be diazotized and coupled to protein carriers. Rabbits immunized with

V.P. Butler, Jr., Drug immunoassays Table 1 Drug antibodies not associated with immunoassays. Name of drug

Reference

Acetylsalicylic acid Arsenicals Hydralazine Methotrexate 1-Phenylalanine mustard Procaine amide Sulfonamides

Wicher et al., 1968 Singer, 1942 Friedman and Heine, 1963 Jaton and Ungar-Waron, 1967 Burke et al., 1966 Russell and Ziff, 1968 Wedum, 1942

Table 2 Available drug immunoassays.

Analgesics Fentanyl

Antibiotics Chloramphenicol Gentamicin Penicillin

Anticonvulsants Diphenylhydantoin

Cardiac glycosides Acetyl strophanthidin Digitoxin Digoxin Ouabain Proscillaridin CNS stimulants Amphetamine Benzoyl ecgonine (cocaine metabolite) Methamphetamine

Hallucinogenic drugs Lysergic acid diethylamide (LSD) Mescaline Tetrahydrocannabinol

Opiates Methadone Morphine

Ora'l hypoglycemic agents Glibenclamid

Sedatives and tranquillizers Barbital Chlorpromazine Cyclazocine Diazepam and N-desmethyldiazepam Glutethimide Pentazocine Pentobarbital Phenobarbital

Skeletal muscle relaxants d-Tubocurarine Synthetic steroids Estrogens Diethylstilbestrol Glucoeortieoid drugs Dexamethasone Methylprednisolone Prednisolone

Progestins Medroxyprogesterone acetate Norethisterone Norgestrel

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V.P. Butler, Jr., Drug immunoas'savs

chloramphenicol bovine y-globulin conjugates formed antibodies which fixed complement in the presence of chloramphenicol rabbit serum albumin. The capacity of unconjugated chloramphenicol to inhibit this complement fixation reaction enabled the development of an immunoassay method for the measurement of nanogram quantities of the antibiotic in HeLa cell nuclei and E. coli ribosomes (Hamburger, 1966). Rabbits immunized with gentamicin protein conjugates prepared by the carbodiinaide method formed antibodies capable of binding [aH]gentamicin as determined by the double antibody or dextran-coated charcoal method. The ability of the unlabeled antibiotic to inhibit this binding has formed the basis for the development of radioimmunoassay methods for the measurement of gentamicin in the sera of patients receiving this drug (Lewis et al., 1972; Mahon et al., 1973). Penicilloyl- bovine ")'-globulin conjugates have been prepared and injected into rabbits, causing the formation of anti-penicilloyl antibodies. These antibodies cause lysis of penicilloyl-coated erythrocytes in the presence of complement. The ability of penicillin G to inhibit this passive immune hemolytic reaction has enabled the development of an imnmnoassay for the measurement of penicillin G in human serum (Wiedermann et al., 1972). Similar antibodies have been used, together with 3 sS.labeled penicillins and penicillin derivatives in the development of penicillin radioimnmnoassay methods (Karchmer et al., 1972). 5.3. Antic 2 ng digoxin per ml, or > 25 ng digitoxin per ml) often are associated with clinical evidence of digitalis toxicity, while lower serum levels (0.5-2 ng digoxin per ml, or 5-25 ng digitoxin per ml) are usually found in non-toxic adult patients who exhibit a satisfactory therapeutic response to digitalis. Lower serum digitalis levels are often accompanied by an unsatisfactory therapeutic response to digitalis therapy. A knowledge of the serum digitalis concentration is particularly helpful in the determination of appropriate digitalis dosage schedules in patients with an inadequate therapeutic response to the drug, in patients whose ability to excrete digoxin decreases with impaired renal function (including elderly patients whose ability to excrete digoxin decreases with the age-related decrease in glomerular filtration rate), patients whose history of recent digitalis ingestion is uncertain, and in patients with suspected digitalis intoxication (Butler, Jr., 1972; Smith, 1972b; Butler and Lindenbaum, 1975; Smith, 1975). Serum and urinary digitalis radioimmunoassay methods have been used extensively in studies of the biological availability (Lindenbaum et al., 1971; Preibisz et al., 1974), intestinal absorption (Heizer et al., 1971; Huffman and Azarnoff, 1972) metabolism and excretion (Selden and Smith, 1972; Selden et al., 1973; Finkelstein et al., 1975) of cardiac glycosides.

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v.P. Butler, Jr., Drug immunoassays

5.5. Central nervous system stimulants

N-(4-Aminobutyl) methamphetamine has been synthesized and then conjugated to bovine serum albumin by the carbodiimide method. Rabbits immunized with the resulting hapten-albumin conjugates formed antibodies capable of binding [3H] amphetamine as determined by the ammonium sulfate method. Unlabeled amphetamine and methamphetamine, respectively, could be detected and measured by their abilities to inhibit the binding of [3HI amphetamine by these antibodies (Cheng et al., 1973). Amphetamines have also been measured with an enzymelinked immunoassay system (Schneider et al., 1974). An enzyme-linked immunoassay has been described for the detection, in urine, of benzoyl ecgonine, the major urinary metabilite of cocaine found in individuals with a history of recent cocaine abuse (Schneider et al., 1974). 5.6. Hallucinogenic drugs

D-Lysergic acid has been coupled to protein and synthetic polypeptide carriers by the carbodiimide method (Van Vunakis et al., 1971 ; Loeffler and Pierce, 1973; Lopatin and Voss, 1974); alternatively, D-lysergic acid diethylamide (LSD) has been conjugated to human serum albumin by a formaldehyde coupling reaction (Taunton-Rigby et al., 1973). Animals immunized with these conjugates formed antibodies capable of binding [3H] LSD (Loeffler and Pierce, 1973; Lopatin and Voss, 1974; Van Vunakis and Levine, 1974) or 12 Si.labele d LSD-polypeptide (Van Vunakis et al., 1971; Taunton-Rigby et al., 1973; Van Vunakis and Levine, 1974). Recently, anti-LSD antibodies have been employed in the development of double antibody (Taunton-Rigby et al., 1973) and coated charcoal (Loeffler and Pierce, 1973) radioimmunoassay methods for the detection and measurement of LSD in biological fluids. Mescaline-polyglutamic acid conjugates, prepared by the carbodiimide method, have, after electrostatic complexing with methylated bovine serum albumin, been injected into rabbits. Following absorption of antibodies to polyglutamic acid, antisera from these rabbits reacted with mescaline, as shown by the fact that mescaline was an effective inhibitor of the complement fixation reaction between these antisera and the mescaline-polyglutamic acid conjugate (Van Vunakis et al., 1969). Mescaline-specific antibodies have recently been used together with an 1 2 s I-labeled mescaline derivative in the development of a sensitive double-antibody radioimmunoassay method for the measurement of mescaline (Van Vunakis and Levine, 1974). Anti-mescaline antibodies have also been studied recently with a method which employs [ 14C] mescaline (Schnoll et al., 1973). Tetrahydrocannabinol is the principal pharmacologically active constituent of cannabis. Aa.Tetrahydrocannabinol has been coupled via its phenolic function to diazotized para-aminobenzoic acid and the resulting derivative has been conjugated to keyhole limpet hemocyanin by the carbodiimide method. Rabbits immunized

V.P. Butler, Jr., Drug irnmunoassays

15

with these hapten-protein conjugates formed antibodies capable of binding unconjugated tetrahydrocannabinol, as determined by a fluorescence quenching method (Grant et al., 1972). Using another method, originally described by Erlanger and his colleagues (Erlanger et al., 1957), the chlorocarbonate derivative of tetrahydrocannabinol has been prepared with phosgene and coupled to bovine serum albumin by the Schotten-Baumann reaction. A sheep immunized with the tetrahydrocannabinol-protein conjugate formed antibodies capable of binding [ 3H] tetrahydrocannabinol, as detected by a charcoal adsorption method (Teale et al., 1974). Unlabeled tetrahydrocannabinol inhibits the binding of the tritiated or radioiodinated tetrahydrocannabinol or tetrahydrocannabinol derivatives by antibody, suggesting that these reagents can be used in the development of radioimmunoassay methods for the detection and measurement of tetrahydrocannabinol in biological fluids (Gross and Soares, 1974; Teale et al., 1974; Van Vunakis and Levine, 1974). 5.7. Opiates

Morphine has been converted to 3-O-carboxymethylmorphine by reaction of the free base with sodium-3-chloroacetate in absolute ethanol. Rabbits immunized with 3O-carboxymethylmorphine-protein conjugates prepared by the carbodiimide method formed antibodies capable of binding [3H] dihydromorphine as detected by the ammonium sulfate precipitation method (Spector and Parker, 1970). After absorption of carrier-specific antibodies with carbodiimide-treated protein carrier, these antibodies were capable of agglutinating sheep erythrocytes coated with carboxymethylmorphine-protein conjugates (Adler and Liu, 1971). Antibodies to a carboxymethylmorphine-polylysine conjugate (injected in the form of an electrostatic complex with succinylated hemocyanin) fixed complement in the presence of carboxymethylmorphine-polylysine; these antibodies also bound an 12 Si.labeled carboxymethylmorphine-synthetic polypeptide conjugate, as detected by the double antibody method (Van Vunakis et al., 1972). Morphine has also been rendered immunogenic in another manner by synthesis of morphine-3-hemisuccinate and the preparation of morphine-3-hemisuccinyl bovine serum albumin by the mixed anhydride method; rabbits immunized with this conjugate formed antibodies capable of binding [14C] morphine as determined by the ammonium sulfate precipitation method (Wainer et al., 1972). In every one of the above instances, morphine and related opiates inhibited the reaction between antibody and morphine or a morphine derivative. This ability of morphine to inhibit the binding of morphine or its derivatives by antibody has enabled the development of radioimmunoassays (Spector and Parker, 1970; Spector, 1971; Gershman et al., 1972; Van Vunakis et al., 1972; Catlin et al., 1973b; Spector and Seidner, 1974; Van Vunakis and Levine, 1974), hemagglutination inhibition immunoassays (Adler and Liu, 1971; Adler et al., 1972; Catlin et al., 1973a; Adler, 1974), spin-labeled immunoassays (Leute et al., 1972a,b; Schneider et al., 1974)and enzyme-linked immunoassays (Rubenstein et al., 1972; Schneider et al., 1973, 1974) for the measurement of morphine in

16

V.P. Butler, Jr., Druk~irnmunoassays

serum, urine, saliva and other biological fluids. These assay methods have been utilized in studies of the metabolic disposition of morphine in experimental animals and in man (Spector, 1971; Spector and Vesell, 1971). Antibodies to morphine usually crossreact extensively with other opiates (Spector and Parker, 1970; Spector, 1971 ; Van Vunakis et al., 1972; Wainer et al., 1972; Leute et al., 1972a; Schneider et al., 1973; Spector et al., 1973; Brattin and Sunshine, 1974; Catlin, 1974; Mule and Bastos, 1974; Mule et al., 1974a; Spector and Seidner, 1974). Thus selected antisera with broad ranges of reactivity may be employed in the development of immunoassays designed to detect the presence of any of the commonly used opiates in the serum or urine of individuals suspected of drug abuse (Leute et al., t972a; Catlin et al., 1973a,b; Schneider et al., 1973, 1974; Bidanset, 1974; Brattin and Sunshine, 1974; Catlin, 1974; Mul6 and Bastos, 1974; Mule et al., 1974a,b). An analogue of methadone, 4-dimethylamino-2,2-diphenylvaleric acid, has been conjugated to bovine serum albumin by the carbodiimide method. Antisera from rabbits immunized with these hapten-albumin conjugates were shown to be capable of binding [ 14C] methadone by the ammonium sulfate method. Rabbit antisera also contained carrier-specific antibodies which could be removed by absorption with carbodiimide-treated bovine albumin. After such absorption the anti-hapten antibodies were capable of agglutinating red cells coated with the hapten-albumin conjugate. Methadone effectively inhibited the hemagglutination of these cells by antibody, enabling the development of a simple, rapid hemagglutination inhibition immunoassay capable of detecting methadone in concentrations as low as 1 ng/ml in human urine. The assay has sufficient specificity that heroin used simultaneously with methadone does not interfere in the test (Liu and Adler, 1973). An enzymelinked immunoassay for methadone has also been reported (Schneider et al., 1974). The availability of methadone immunoassays has facilitated urinary methadone detection which is so important in the assessment of patient compliance in methadone maintenance programs (Liu and Adler, 1973; Schneider et al., 1974). 5.8. Oral hypoglycemic agents

A hemisuccinate derivative of a glibenclamid metabolite has been coupled to bovine serum albumin by the mixed anhydride method. Rabbits immunized with the resulting conjugate formed antibodies capable of binding [14C] glibenclamid, as determined by the dextran-coated charcoal method. Glibenclamid and its metabolic derivative used in the aforementioned hapten-protein synthesis inhibited the binding of [14C] glibenclamid by antibody (Glogner et al., 1973). 5.9. Sedatives and tranquillizers

To elicit barbiturate-specific antibodies, 5-allyl-5-(1-p-nitrophenyloxycarbonylisopropyl) barbituric acid was synthesized and coupled to bovine gamma globulin.

V.P. Butler, Jr., Drug immunoassays

17

Rabbits immunized with the resulting conjugates formed antibodies capable of binding [ 14C] barbital or [ 14C] pentobarbital, enabling the development of sensitive and specific ammonium sulfate radioimmunoassay methods for the measurement of the corresponding unlabeled barbiturates. These methods have been used in studies of the plasma disappearance rate of these two drugs (Spector and Flynn, 1971; Flynn and Spector, 1972; Spector et al., 1973). Since metharbital does not displace [3HI phenobarbital from the anti-barbiturate antibodies while barbital is quite effective in competing with [3H]phenobarbital for binding sites on these antibodies, it has been possible to use these antibodies, together with [3H] phenobarbital, in studies of the hepatic conversion (involving N-demethylation) of metharbital to barbital in vitro (Flynn and Spector, 1974). In another study, a phenobarbital analogue has been conjugated to bovine serum albumin by the carbodiimide method. Rabbits immunized with the resulting conjugate formed antibodies capable of binding [3H]phenobarbital which have been used in the development of a sensitive and'specific radioimmunoassay method for the detection and measurement of unlabeled phenobarbital (Chung et al., 1973). Utilizing another approach, p-nitrophenobarbital has been synthesized, catalytically reduced to form p-aminophenobarbital, diazotized and coupled to acetylated bovine serum albumin; rabbits immunized with p-azophenobarbital-proteins formed phenobarbital-specific antibodies as demonstrated by a hemagglutination inhibition technique (Satoh et al., 1973). Diazotized derivatives of diazepam, and its N-demethylated metabolite, NdesmethyldiazelJam, have been synthesized and coupled to bovine serum albumin. Rabbits immunized with each of these conjugates formed antibodies specific for the corresponding compound. Using [14C] diazepam and the ammonium sulfate technique, sensitive and specific radioimmunoassay methods have been developed and used in studies of the plasma concentrations of diazepam and of N-desmethyldiazepam in humans given diazepam (Peskar and Spector, 1973a). A p-aminophenyl derivative of glutethimide, 2(p-aminophenyl)-2-ethylglutarimide, has been diazotized and coupled to bovine serum albumin. Rabbits immunized with the resulting conjugates formed anti-glutethimide antibodies, which have been used in the development of a hemagglutination-inhibition immunoassay method for the measurement of glutethimide in serum and urine from human subjects who have ingested this drug (Valentour et al., 1973). Azobenzoic acid and carboxymethyl derivatives of pentazocine have been coupled through their carboxyl groups to poly-L-lysine by the carbodiimide method. Rabbits immunized with each of these conjugates formed antibodies capable of binding [3H] pentazocine, as determined by an ammonium sulfate precipitation method. Animals immunized with the azobenzoic acid derivative formed antibodies with greater specificity for pentazocine than did rabbits immunized with the carboxymethyl derivative but antisera from anbnals immunized with either derivative could be used, together with [ 3H] pentazocine, in the immunological assay of pentazocine in plasma and urine (Williams and Pittman, 1974). Antibodies to cyclazo-

18

V.P. Butler, Jr., Drug immunoassays

cine have been raised in rabbits immunized with an azobenzoic acid-cyclazocine derivative coupled to poly-L-lysine by the carbodiimide technique; these antibodies have been used, together with [3HI cyclazocine, in the development of a radioimmunoassay for the unlabeled drug (Pittman and Williams, 1973). Chlorpromazine and its 8-hydroxy derivative have been coupled to protein carriers. Rabbits immunized with the drug protein conjugates formed antibodies which have been used, together with [3HI chlorpromazine, in the development of a radioimmunoassay method which has been applied to studies of the half-life of chlorpromazine in biological fluids (Kawashima and Spector, 1974). Perphenazine hemisuccinate and desdimethylchlorpromazine monoamide have been synthesized and coupled to bovine serum albumin by the mixed anhydride, and carbodiimide, methods, respectively. Rabbits immunized with each of these phenothiazine derivative-albumin conjugates formed antibodies capable of binding [14C]-chlorpromazine, as measured by the dextran-coated charcoal method (Shostak, 1974). 5.10. Skeletal muscle relaxants

d-Tubocurarine has been coupled via a diazonium linkage to diazotized paraaminobenzoic acid and the resulting compound was conjugated to bovine serum albumin by the carbodiimide method. Rabbits immunized with these hapten-protein conjugates formed antibodies capable of binding [3H]-d-tubocurarine, as demonstrated by the ammonium sulfate precipitation method. The ability of the unlabeled alkaloid to inhibit this binding has formed the basis for a radioimmunoassay procedure for the measurement of d-tubocurarine in serum and urine from human subjects and from experimental animals (Horowitz and Spector, 1973). 5.11. Synthetic steroid drugs

The methods originally described by Erlanger and his coworkers to conjugate steroid hormones to protein carriers (Erlanger et al., 1957; Beiser et al., 1968; Erlanger, 1973) have been utilized recently to couple synthetic steroid drugs to albumin carriers. The 21-hemisuccinate derivatives of dexamethasone (Dumasia et al., 1973; Hichens and Hogans, 1974) and of prednisolone (Colburn and Bullet, 1973a), the 1 lc~-hemisuccinate derivatives of norethisterone (Cameron et al., 1974) and of medroxyprogesterone acetate (Royer et al., 1974), and the 3-(O-carboxymethyl) oxime derivatives of medroxyprogesterone acetate (Cornette et al., 197t), of dexamethasone (Meikle et al., 1973), of methylprednisolone (Colburn and Buller, 1973b), of norethisterone, and of norgestrel (Warren and Fotherby, 1974) have been synthesized and coupled to bovine serum albumin, using the mixed anhydride, carbodiimide or carbonyldiimidazole (Axen, 1974) method. Animals immunized with these synthetic drug-protein conjugates have formed antibodies capable of binding the corresponding 3H-labeled drug or its 12 Si_labele d derivative. The antibodies have been used in the development of sensitive and specific double

V.P. Butler, Jr., Drug immunoassays

19

antibody or dextran-coated charcoal radioimmunoassay methods capable of detecting picogram or nanogram quantities of unlabeled drug in biological fluids. In addition, the feasability of using antiestradiol-17/3 antibodies to detect diethylstilbestrol has been demonstrated (Gross and Grant, 1970). 5.12. Other compounds o f pharmacological importance Other substances of pharmacological interest for which immunoassays have been developed include cyclic AMP, cyclic GMP (Steiner, 1973), fibrinopeptide A (Nossel et al., 1974), genistein (Bauminger et al., 1969), insect hormones (Borst and O'Connor, 1972; Lauer et al., 1973), nicotine and its metabolites (Cernosek et al., 1973), normetanephrine (Peskar et al., 1972), paralytic shellfish poison (Johnson and Mulberry, 1966), peptide hormones (Berson and Yalow, 1967, 1972; Yalow, 1973; Bloom, 1974), plant hormones (Fuchs et al., 1971), prostaglandins (Jaffe and Parker, 1973 ; Levine, 1973), serotonin (Peskar and Spector, 1973b; Spector et al., 1973), steroid hormones (West et al., 1973; James and Jeffcoate, 1974), substance P (Powell et al., 1973), thyroid hormones (Mitsuma et al., 1972; Burke and Eastman, 1974), and vitamin A (Conrad and Wirtz, 1973). Other small molecules of pharmacological interest to which antibodies have been elicited, but for which immunoassays have not been described, include various carcinogens (Creech, 1952; Butler, Jr. and Beiser, 1973), DDT (Haas and Guardia, 1968; Centeno et al., 1970), folic acid (Jaton and Ungar-Waron, 1967; Ricker and Stollar, 1967), histamine (Davis and Meade, 1970), 5-hydroxyindole acetic acid (Ranadive and Sehon, 1967), pyridoxal (Ungar-Waron and Sela, 1966), and strychnine (Hooker and Boyd, 1940).

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Bidanset, J.H., 1974, J. Chromatog. Sci. 12, 293. Bloom, S.R., 1974, Brit. Med. Bull. 30, 62. Borst, D.W. and J.D. O'Connor, 1972, Science 178,418. Brattin, W.J. and 1. Sunshine, 1974, in: hnmunoassays for drugs subject to abuse, eds., S.J. Muld, I. Sunshine, M. Braudc and R.E. Willette (CRC Press, Cleveland), p. 107. Brodie, B.B. and W.M. Heller, eds., 1972, Bioavailability of drugs (S. Karger, Basel). Brodie, B.B. and W.D. Reid, 1971, m: Fundamentals of drug metabolism and drug disposition, eds. B.N. La Du, H.G. Mandel and E.L. Way (Williams and Williams, Baltimore), p. 328. Burke, C.W. and C.J. Eastman, 1974, Brit. Mcd. Bull. 30, 93. Burke, J.F., V.H. Mark, A.H. Soloway and S. Leskowitz, 1966, Cancer Res. 26, 1893. Butler, V.P., Jr., 1971, Lancet 1,186. Butler, V.P., Jr., 1972, Progr. Cardiov. Dis. 14, 57l. Butler. V.P., Jr., 1973, Metab. Clin. Exp. 22, 1145. Butler, V.P., Jr. and S.M. Beiser, 1973, Advan. Immunol. 17,255. Butler. V.P., Jr., S.M. Beiser, B.F. Erlanger, S.W. Tanelrbaum, S. Cohen and A. Bendich, 1962, Proc. Natl. Acad. Sci. U.S. 48, 1597. Butler, V.P., Jr. and J.P. Chen, 1967, Proc. Natl. Acad. Sci. U.S. 57, 71. Butler, V.P., Jr. and J. Lindenbaum, 1975, Amer. J. Med. 58, in press. Cameron, E.H.D., S.l'. Morris and B. Nieuweboer, 1974, J. t,;ndocrinol. 61, xxxix. Catlin, D.II., 1974, in: Immunoassays for drugs subject to abuse, eds., S.J. Muld, 1. Sunshine, M. Braude and R.E. Willette (CRC Press, Cleveland), p. 91. Catlin, D.If., F.L. Adler and C.-T. Liu, 1973a, Clin. lmmunol, hmnunopathol. 1,446. Catlin, D., R. Cleeland and E. Grunberg, 1973b, Clin. Chem. 19, 216. Centeno, E.R., W.J. Johnson and A.H. Sehon, 1970, hat. Arch. Allergy Appl. lmmunol. 37, 1. Cernosek, S.F., J.J. Langone, H.B. Gjika and H. van Vunakis, 1973, Federation Proc. 32, 51 lAbs. Chase, M.W., 1967, in: Methods in immunology and immunochemistry, Vol. I, eds. C.A. Williams and M.W. Chase (Academic Press, New York), p. 197. Cheng, L.T., S.Y. Kim, A. Chung and A. Castro, 1973, FEBS Letters 36, 339. Christensen, ll.D., J.A. Kepler and C.E. Cook, 1973, Pharmacologist 15, 197. Chung, A., S.Y. Kim, L.T. Cheng and A. Castro, 1973, Fxperientia 29,820. Colburn, W.A. and R.H. Buller, 1973a, Steroids 21,833. Colburn, W.A. and R.H. Buller, 1973b, Steroids 22,687. Conrad, D.H. and G.H. Wirtz, 1973, lmmunochemistry 10, 273. Cook, C.E., J.A. Kepler and H.D. Christensen, 1973, Res. Commun. Chem. Pathol. Pharmacol. 5, 767. Cornette, J.C., K.T. Kirton and G.W. Duncan, 1971, J. Clin. Fndocrinol. Metab. 33,459. Creech, H.J., 1952, Cancer Res. 12,557. Davis, T.R.A. and K.M. Meade, 1970, Nature 226, 360. Davison, C., 1971, in: Fundamentals of drug metabolism and drug disposition, eds. B.N. La Du, H.G. Mandel and E.L. Way (Williams and Wilkins, Baltimore), p. 63. Dray, F., E. Maron, S.A. Tillson and M. Sela, 1972, Anal. Biochcm. 50, 399. Dumasia, M.C., D.I. Cbapman, M.S. Moss and C. O'Connor, 1973, Biochem. J. 133,401. Eisen, tI.N., 1964, Methods Med. Res. 10, 106. Ekins, R.P., 1974, Brit. Med. Bull. 30, 3. Engvall, E. and P. Perhnann, 1971, Immunoehemistry 8, 871. Erlanger, B.F., 1973, Pharmacol. Rev. 25, 271. Erlanger, B.F. and S.M. Beiser, 1964, Proc. Natl. Acad. Sci. U.S. 52, 68. Erlanger, B.F., F. Borek, S.M. Beiser and S. Lieberman, 1957, J. Biol. Chem. 228, 713. Finkelstein, F.O., J.A. Goffinet, E.D. Hendler and J. Lindenbaum, 1975, Amer. J. Med., in press Flynn, E.J. and S. Spector. 1972, J. Pharmacol. Exptl. Ther. 181,547. Flynn, E.J. and S. Spector, 1974, J. Pharmacol. [£xptl. Ther. 189,550.

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