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Objectives: To review evidence that the immune system plays a role in controlling the spread of cancer and findings that perioperative pain relief improves immune status and health outcomes., Data Sources: Research studies and review articles pertaining to immunity, immune function, stress, and immune-suppressive nature of pain., Conclusions: Pain not only results in suffering but is a pathogen itself, capable of facilitating the progression of metastatic disease. Adequate pain relief decreases these risks., Implications for Nursing Practice: Adequate pain relief is not only a primary concern in caring for individuals in pain but may be a matter of physiologic necessity as further studies reveal the immune-suppressive nature of pain.

A recently introduced automated immunoassay which is based on kinetic interaction of microparticles in solution (Roche ONLINE), was evaluated for the detection of cocaine metabolite benzoylecgonine (BE) and opiates in human urine. Cross-reactivity for the opiates morphine (100%), codeine (88%), 6-monoacetylmorphine (88%), and morphine 3-glucuronide (72%) was assessed. Analytical recovery evaluated on blank urines spiked with 0, 250, 300, 350, and 500 micrograms/L of morphine and BE (n = 10), varied from 85.2 to 100.2% for opiates and from 81.4 to 93.1% for the cocaine metabolite. The within-day precision ranged from 1.4 to 4.7% for morphine and from 4.2 to 4.8% for BE. The repeatability of the standards over 1 month was 1.0-3.3% for opiates and 1.7-5.1% for BE, and thus allowing measurements to continue over 30 days without re-calibration. This method compared favourably with the SYVA EMIT d.a.u system and gas chromatography/mass spectroscopy (GC/MS) methods.

The new technique of molecular imprinting has increasingly been adopted by research laboratories worldwide during the last few years. We have studied the use of such imprints against drugs as artificial antibody-binding mimics in competitive radioimmuno-style binding assays. The recognition sites "molded" in the polymers mimic the binding sites of natural antibodies in their interactions with the target antigen. Binding constants are as low as 4.0 nmol/L for a small number of well-defined sites, and cross-reactivities are similar to or better than those observed with biological antibodies. In some cases, the polymers have been used to determine drug concentrations in human serum specimens.

This study deals with a novel role of morphine in the modulation of cellular responsiveness to immunostimulatory substances that, at first glance, appears to be in contrast to the well documented immunoinhibitory short-term effects of opiate alkaloids on cells simultaneously exposed to stimulatory molecules. Vertebrate and invertebrate immunocytes pre-exposed to morphine (10(-6) M) in vitro for at least 24 h prior to the administration of lipopolysaccharide (LPS; 1.0 micrograms/ml) or other immunoactivating substances have revealed a distinct enhancement of their responsiveness to these signals, e.g. monocytes exposed to LPS alone resulted in 21% activation, whereas the morphine pretreated level was at 40% (P < 0.01). Prolonged pretreatment with morphine of naive human monocytes had the same effect on their sensitivity to plasma from patients having undergone cardiopulmonary bypass (CPB) operations followed by a diffuse inflammatory response. These results suggest that endogenous opiates may participate, in more than one way, in re-establishing an organism's readiness to meet a new demand on its immune system. Additional support for the concept of a role of endogenous opiates in immunomodulation was obtained by the results of in vivo tests with experimentally induced stress in Mytilus. Following their stress-induced stimulation, these animals' immunocytes could be shown to become exposed for some time to a measurable rise in endogenous morphine-like material (9 pmol/ml increasing to 59). These immunocytes, like those preincubated with exogenous morphine, displayed a heightened sensitivity to stimulation by LPS (control 21.3 +/- 3.1% activation compared to 47.2 +/- 5.1) when the morphine levels dropped. The mechanism of this enhancement of responsiveness to immunostimulation following the prolonged exposure of immunocytes to morphine, and its relationship with the known short-term immunoinhibitory opiate effects on the immune system, remains to be ascertained.

A key component of an implant that can be triggered by external morphine to release naltrexone is an inactivated enzyme that can be activated by morphine and which can then rapidly remove a protective coating surrounding a bioerodible polymer containing dispersed naltrexone. In this article we describe a lipase that has been conjugated with O3-carboxymethylmorphine, morphine-beta-3-glucuronide and O3-carboxypropylmorphine. The enzyme conjugate was then inactivated by complexation with affinity-purified goat polyclonal antimorphine antibodies. Antibody lipase interactions were measured by pH Stat and ELISA techniques. Affinity constants of the antibodies determined by radioimmunoassay using tritium-labeled morphine were 4.10 x 10(6), 3.18 x 10(6) and 3.38 x 10(7), respectively. While a concentration of 10(-5)M morphine was required to restore lipase activity, it is likely that a combination of correct morphine tether and correct affinity-purified antibody can increase sensitivity to the desired 10(-8)10(-9)M morphine level. Thus, a functioning device can almost certainly be constructed. However, it is unlikely that reactivation times of 1-2 h necessary for clinical usefulness in treatment of narcotic addiction can be achieved.

The purpose of this study was to examine the possible influence of the phases of the menstrual cycle on dermal reactivity to skin-prick testing. We studied 15 atopic, menstruating women with seasonal rhinoconjunctivitis and/or asthma, with known sensitivity to olive and parietaria (mean age 25.2 years) and 15 non-atopic, healthy, female controls (mean age 24.7 years). Skin-prick tests with histamine, morphine, and in the atopic group with parietaria/and/or olive, were repeated three times during the same menstrual cycle, corresponding to bleeding (day 1-4), midcycle (day 12-16) and the late progesterone phase (day 24-28). None of the patients had received oral antihistamines or exogenous hormones for at least 1 month prior to testing. Results indicate a significant increase in weal-and-flare size to histamine, morphine, and parietaria on days 12-16 of the cycle, corresponding to ovulation and peak oestrogen levels. This was observed in both atopic and non-atopic women. Differences in skin reactivity to histamine and morphine between the groups were not significant. Therefore, in women, the phase of the menstrual cycle is another factor that may influence skin-test results.

Different kinds of single, multiple, acute or chronic administrations of morphinized animal models were established, with which a series of experiments in both in vivo and in vitro systems as well as molecular levels were pharmacologically designed to investigate the psychoneuroimmunological effects of morphine (Mor) and the immunoprotection of Ganoderma polysaccharides peptide (GPP) in Mor-dependent mice. It was first discovered that both c-myb and c-myc mRNA expression in splenocytes of repetitive Mor-treated mice were detected to be significantly decreased, and that GPP could induce restoration of several immunologic parameters depressed by Mor treatment to or even beyond normal levels. This provides the experimental animal evidence that immune response modifiers such as GPP could be of potential application in controlling abuse of opiates-induced immunodeficiency.

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten-dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5-hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm x 20 microns column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5.10(-8) M, about 10x more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.

The use of routine nonspecific immunoassays to detect or quantitate opiates in biological fluids raises the question of the relevance of such immunoassays in the investigation of opiate overdose disposition. We investigated the plasma disposition of morphine in 13 patients intoxicated by the intravascular (i.v.) (n = 5) or oral routes (n = 8) using both a highly morphine-specific antibody radioimmunoassay (RIA) and a nonspecific morphine RIA. Both RIAs showed a first-order elimination rate after i.v. intoxication (apparent plasma terminal half-life ranged from 2.9 to 4.7 hours for unchanged morphine and from 3.2 to 4.9 hours for total opiates) and a persistent opiate concentration with rebound after oral ingestion, suggesting a slow release of opiates from the gastrointestinal tract, in dealers and bodypackers. Moreover, i.v. and oral kinetic data were similar for the two RIAs, except for the ratio between total and unchanged morphine concentrations. The nonspecific morphine assay gave a threefold to 16-fold higher concentration than the specific morphine assay but with parallel kinetics for all patients. We conclude that the current, routine nonspecific morphine immunoassays could be a valuable analytical tool for investigating opiate toxicokinetics.

A modified differential radioimmunoassay (RIA) technique for the measurement of morphine and its active metabolite, morphine-6-glucuronide (M6G), in plasma is described. Plasma samples were assayed following appropriate dilution, using a morphine specific antiserum and the results subtracted from those obtained with an antiserum which cross-reacts with both morphine and M6G. The sensitivity of measurement for morphine and M6G was 0.88 and 0.27 nmol l-1, respectively and inter-assay variation ranged from 3.4 to 11.0%. Recovery of morphine and M6G was quantitative over a range of concentrations (1-5000 nmol l-1). The presence of either M6G or morphine-3-glucuronide (M3G) did not affect the recovery of morphine. M6G was quantitatively recovered in the presence of morphine but high concentrations (> 1:20) of M3G caused some overestimation of M6G. Results obtained by differential RIA for both morphine and M6G correlated well with the results of HPLC analysis. The assay has been applied to the measurement of M6G in plasma following its administration to human volunteers.

An ELISA method for detection of specific antibodies to morphine-protein conjugate (MPC) in humans and animals was elaborated. In humans, antibodies could be detected predominantly as the IgM class of immunoglobulins. Elevated levels of such antibodies were found in about 50% of opiate addicts and morphine-treated rats. In the case of prolonged and regular drug use with the time since the last drug intake not exceeding 2 months, elevated levels of antibodies to MPC were detected more frequently (up to 75%). Antibodies to MPC, detected in opiate addicts, were specific to MPC, morphine and beta-endorphin. A similar approach was applied to the detection of antibodies to ephedrone (amphetamine-like derivative of ephedrine) in case of its chronic use.

The phenomenon of induction of antibodies to morphine in morphine-treated animals and opiate addicts was shown by means of ELISA. The reaction of the immune system to injections of a morphine solution had the characteristics of a primary immune response. Opiate addicts differed from normal controls in the level of antibodies to morphine of IgM class. Elevated levels of such antibodies could be detected in about 70-80% of addicts with regular longterm opiate use when the time since the last drug intake did not exceed 60 days. Antibodies to morphine could serve as indicators of chronic opiate use especially in periods long after the last drug intake.

Six murine monoclonal antibodies against morphine were produced using N-(4-aminobutyl)normorphine as a hapten. Most of the antibodies obtained distinguished the substituents at the 3 and 6 positions of morphine. This property of the antibodies led to a reduction in cross-reactivity with codeine, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) to negligible levels. However, one of the antibodies distinguished the substituent only at the 3 position of morphine, which cross-reacted with M-6-G, naloxone and naltrexone. In the competitive inhibition enzyme-linked immunosorbent assay, morphine was detected at concentrations as low as circa 100 pg/ml.

V region nucleotide sequences were determined by mRNA sequencing for 11 monoclonal anti-morphine antibodies with slightly different specificities for morphine-related opiates. The VH region nucleotide sequences of the antibodies MOR8, MOR33, MOR35, MOR44, MOR83, and MOR76 were classified into the VH-5 (7183) family, while the antibodies MOR39, MOR115, MOR131, MOR158 and MOR180 used VH-1 (J558) family genes. MOR39, MOR115 and MOR131 used the V lambda-1 gene for their L chain V region. MOR158 and MOR180 used the Vk-10 gene. MOR8, MOR33, OR35, MOR44, MOR76 and MOR83 used VK-21D. The antibody sets MOR158 and MOR180; MOR39 and MOR131; and MOR8, MOR33, MOR35, MOR44, MOR76 and MOR83 appeared to be somatic mutants derived from the same clones since they showed the same VH/VL usage and V(D)J recombination pattern. The pH-reactivity profiles for these antibodies revealed that the binding of morphine to the antibodies is highly dependent on the pH value of the assay solution, suggesting the importance of the electrostatic interaction between the positive charge of morphine and the negative charges at or near the combining sites. Direct UV-photoaffinity labeling with 3H-morphine was carried out in order to estimate the orientation of morphine in the combining sites. The H chains were preferentially labeled in MOR8, MOR33, MOR35, MOR76, and MOR83, whereas most of the crosslinked hapten was found in the L chains in MOR39, MOR115, MOR131, MOR158 and MOR180. Thus, these 11 antibodies were classified into two types in terms of reactivity in the photoaffinity labeling.

In the early 1970's, some papers appeared reporting an immune response to opiates in animals treated with morphine and in heroin addicts, but further researches failed to confirm these results in humans. The aim of the present work is investigating with a newly developed enzyme immunoassay the existence of specific antibodies to morphine in a group of opiate chronic users, controlled by means of the toxicological analysis of hair. Twenty five opiate addicts inpatients for detoxication treatments were investigated for the presence of morphine specific antibodies and for the morphine content in hair, as a marker of addiction to opiates. Antibodies to morphine were investigated using an original ELISA method using a morphine-human serum albumin conjugate immobilized into the wells of polystyrene microtiter plates. Morphine determinations in hair were accomplished by a radioimmunologic screening followed by HPLC confirmation of positive results. The group of opiate users, in which all the subjects resulted positive for morphine content in hair, showed in the ELISA test an average D OD% value significantly higher than the control group (p < 0.001); in particular, 16 out of 25 addicts could be classified positive for anti-morphine antibodies, which were identified as IgM. Inhibition studies demonstrated Ka's for morphine ranging from 10(4) to 10(10) M-1 and a high cross reactivity for codeine. The presence of circulating antibodies specific to morphine in chronic users of opiates is strongly supported by the present findings.

The paper demonstrates induction of antibodies to morphine and neurotransmitters (dopamine, norepinephrine, serotonin) in rats treated with intraperitoneal injections of morphine hydrochloride during 2 and 4 weeks in increasing doses. A contribution of such antibodies to the mechanisms of morphine tolerance and formation of physical dependence is considered.

Morphine suppresses humoral immune responses, causes thymic hypoplasia and suppresses NK (natural killer) activity in animal models. There is evidence that thymic hypoplasia and NK suppression are predominantly mediated by indirect mechanisms. The mechanism of morphine-induced humoral immunosuppression is less certain. Recent reports suggest that morphine and other opioids can directly act on cells of the immune system to suppress the generation of antibody-forming cells (AFC) in Mishell-Dutton cultures. The present study was designed to assess the roles of direct and indirect mechanisms in morphine-induced suppression of humoral immunity. Splenocytes from mice treated with morphine by s.c. implantation of a slow-release 75 mg pellet were dysfunctional in Mishell-Dutton cultures. Exposure to morphine in vivo for 12 or 24 hr caused significant suppression of the AFC production stimulated by sheep erythrocytes in Mishell-Dutton cultures. In contrast, direct addition of morphine or the kappa selective opioid agonist U50,488H to Mishell-Dutton cultures under a variety of conditions had little or no effect on AFC generation. These results indicate that suppression of humoral responses by morphine is not primarily mediated by direct action of morphine on the immune system. Suppression of AFC responses by administration of morphine in vivo was substantially blocked by treating mice with the glucocorticoid antagonist RU 38486, suggesting that glucocorticoids may be involved in the indirect mechanism by which morphine causes splenocyte dysfunction.

We recently demonstrated morphine-6-hemisuccinate-human serum albumin conjugate (M-6-HS-HSA)-specific IgG in serum from ethic narcotics-manufacturing workers. In this article, we present results of epicutaneous tests to opiate compounds and lung-function studies in these same workers. Thirty-nine workers, exposed to opiates, were evaluated for possible work-related changes in lung function and were administered a questionnaire concerning opiate exposure and health history in February 1988. In December 1988, 33 employees with occupational exposure to opiates, six other workers (New Jersey referent) employed at the same factory with minimal exposure to opiate compounds, and 17 nonexposed individuals from Cincinnati, Ohio, were subjected to epicutaneous threshold testing with a panel of six opiate compounds and nine common aeroallergens. In opiate-exposed workers, significantly lower epicutaneous threshold concentrations were detected (compared to New Jersey referent and Cincinnati control subjects) for dihydrocodeine (p less than 0.01), hydrocodone (p less than 0.05), codeine (p less than 0.01), and morphine (p less than 0.05). Significant associates existed among epicutaneous threshold concentrations between the agents tested; that is, individuals with a positive morphine skin test would generally have a positive codeine skin test, etc. Atopic status (positive cutaneous test results to two or more of nine common aeroallergens) was not significantly associated (p greater than 0.05) with positive opiate skin sensitivity. Although the mean cross-shift decrements in FEV1 for all workers were nonsignificant, five opiate-exposed individuals demonstrated cross-shift decrements in FEV1 of greater than 10%. Daily maximum-minus-minimum changes in workweek PEFR (PEFRmax-min) were significantly reduced for Monday through Thursday (p less than 0.05) compared to PEFRmax-min changes during a nonwork, nonexposure 3-day weekend. Ten exposed workers demonstrated daily PEFRmax-min changes of greater than 20%, suggesting acute airway obstruction. Increased cutaneous reactivity to opiate compounds among opiate-exposed workers may reflect development of pharmacologic hyperresponsiveness to opiate compounds.

The structural features of mAb directed against the opiate morphine were analyzed by using competitive ligand analog-binding studies, examination of the V region amino acid sequence, and computer-aided molecular modeling of the fragment V region. The antibody response in BALB/c mice to morphine is relatively restricted, in that all of the mAb examined in this study contained the same lambda L chain and very similar H chain V regions. A three-dimensional model of the antimorphine-binding site was constructed by using computational and graphic display techniques. Each of the six complementary-determining regions was constructed by using fragment replacement methods employing canonical loop conformations of known "parent" structures. Experimental competitive ligand-binding data and theoretical modeling suggest that a charged glutamate residue at position H:50 and aromatic side chains of residues H:33W, H:47W, H:58F, H:95W, H:101iY, and L:91W are key features in ionic and hydrophobic interactions with the ligand. This study represents the first use of theoretical and experimental modeling techniques to describe the Ag-binding site of a mouse fragment V region containing a lambda L chain.

Fv fragments of four monoclonal antibodies specific for morphine binding have been produced from their divalent IgG forms by papain digestion using the classic procedure for Fab formation. The binding characteristics of one of the Fv fragments have been determined relative to the intact antibody by equilibrium dialysis. Its dissociation constant is a factor of five lower than the IgG. Previous work had resulted in the sequences of each the chains for the four Fv fragments. The light chains are all from the highly homologous lambda subclass while the gamma heavy chains are closely related except for their CDR regions. In this work optical molar extinction coefficients are predicted from amino acid sequences for each of the fragments. It is found that they differ significantly from each other and from the commonly used value for intact IgG. Detailed comparisons between our results and those reported previously on the molecular masses of Fv-derived light and heavy chains and hapten-Fv dissociation constants are given based on analytical gel electrophoresis and electroblotting experiments using dye and immunovisualization techniques. Isoelectric focusing experiments have been performed and the pIs obtained are compared to those predicted theoretically from the chain sequences. Gel filtration column chromatography, acrylamide gel electrophoresis and equilibrium dialysis experiments are consistent with significant aggregation of the Fv fragments in neutral solution with accompanying inactivation of the binding site. Comparison of sequences for the Fv light and heavy chains are made with those which have been proposed to be important for chain dimer association and for canonical hypervariable regions. This methods of Fv production is not regarded as a general one. However, it may be an approach which is general to lambda chain containing antibodies.

Subjects who experience life-threatening anaphylactic reactions to neuromuscular blocking drugs frequently have serum IgE antibodies that react with substituted ammonium groups on the drugs. Failure to detect drug-reactive antibodies may be due to the nature of the drug-solid support used for testing sera. With this in mind, solid phases of some selected compounds containing substituted ammonium groups, in particular triethylamine and morphine, were prepared and used to screen sera in an attempt to increase the frequency of detection of IgE antibodies complementary to tertiary and/or quaternary ammonium groups. For subjects who experienced an anaphylactic reaction to succinylcholine or gallamine, use of the supplementary assays increased the frequency of detection from 83 to 100%. For d-tubocurarine and alcuronium, detections increased from 92 to 100% and from 67 to 88%, respectively. Molecular models revealed a clear structural similarity between the conformations of the trialkylammonium groups on one face of the molecules of morphine and d-tubocurarine.

According to the International Narcotics Control Board, over 45,000 kg of morphine and 54,000 kg of codeine were ethically manufactured in 1986 at three facilities in the United States. Little information exists about possible adverse health effects associated with workplace exposure to opiate compounds in this industry. Because there are no specific federal standards for workplace exposure to narcotic dusts, exposure-control defaults to the nuisance dust standard (10 mg/m3, as an 8 hr time-weighted average). Narcotics manufacturing workers were evaluated for anti-morphine IgG before and 10 mo. after the implementation of an improved respiratory protection program (RPP). Significantly elevated IgG levels were measured before the improved RPP (P less than 0.005). After the improved RPP, a significant reduction was observed (P less than 0.001), suggesting that specific antibody levels could be used as biomarkers of exposure. Inhibition studies showed that the antibodies were specifically directed against morphine with some cross reactivity with morphine derivatives. Preliminary results are also shown which indicate that similar anti-morphine antibodies are present in the sera of intravenous heroin abusers. Elevated levels (P less than 0.05) of anti-morphine antibodies were detected in sera from heroin abusers, providing evidence that similar antibodies may be produced from non-occupational exposure to opiates. These finding have potentially far-reaching implications for addiction research and drug testing.

Antibodies that specifically recognize and bind opiate receptors were developed by the anti-idiotypic method. Antimorphine antibodies were generated in rabbits and showed selective binding to various opiate ligands. Guinea pigs were immunized with antimorphine antibodies to produce anti-idiotypic antibodies that cross-reacted with opiate receptors. Guinea pig antiserum inhibited binding of naloxone (0.6 nmol) to mouse brain homogenate. In isolated mouse vas deferens, contraction of electrically stimulated tissue was inhibited by the antiserum. The results indicate that anti-idiotypic antibodies which interact with opiate receptors were developed without isolating receptors.

Morphine antibody purified by affinity chromatography was used to develop a solid-phase radioimmunoassay for morphine in polystyrene tubes. The tubes are coated with an appropriate concentration of the purified antibody, rinsed three times with buffered saline, and stored at -15 degrees C. Using tritiated dihydromorphine, we determined competitive morphine binding by difference when the radioactivity in the assay supernates was measured after incubation (1 h, 37 degrees C). Five standard curves, with use of serum equivalents of morphine ranging from 0 to 6 mug/liter, were linear and had a mean correlation coefficient of 0.98. Uncer conditions of the assay, levorphanol was comparable to morphine in its inhibitory effect on binding of labeled dihydromorphine, whereas dextrorphan was essentially inactive. Morphine-3-glucuronide, a major metabolite, is 55-fold less inhibitory in terms of its capacity to displace the radiolabel. We believe that the sensitivity of the technique, coupled with the simplicity of nonseparatory sampling, renders the system suitable for rapid determination of morphine and related compounds in biological fluids.

The effects of prolonged morphine administration on immunologic reactivity against morphine was studied in a number of animal species: rabbit, monkey, guinea pig, rat, and cat. Some evidence for increased serum binding of 14C-labeled morphine was noted after morphine treatment in all test species, with the rabbit the best responder and the cat showing little or no response. In addition to measurements on serum binding of 14C-labeled morphine, other methods (measurement of serum binding of 14C-labeled codeine and methadone, competitive inhibition tests, radial immunodiffusion, and passive hemagglutination) were used for one or more of the species. Overall, results with these test methods have shown that prolonged morphine administration can result in immunologic responsiveness to morphine in animals.

A method has been devised for the preparation of sheep red blood cells sensitized by covalent morphine binding. The cells were used as a reagent in rosette formation and passive hemagglutination for revealing the immune response in rats and mice immunized with the morphine-BSA conjugate. It has been shown that hyperimmunization of the animals with the antigen mentioned together with the production of antimorphine antibodies, induces the formation of mononuclear spleen cells which bind morphine.

A more complete characterization is given for four previously reported anti-morphine monoclonal antibodies that bind the hapten with high affinity and to which anti-idiotypic antibodies have been raised that mimic opiates at receptor binding sites. The variable (V) region nucleotide sequences of the heavy (H) and light (L) chains of these murine antibodies have been determined by direct sequencing of the poly(A)+ mRNAs using specific oligonucleotide primers and dideoxynucleotide chain-termination, and the deduced amino acid sequences are compared. The primary sequences predicted for the VH segments of 10C3 and 11C7 antibodies are closely associated with the VHIIIB subgroup of mouse H-chain (80 to 82% homology), while those for the V-regions of 3B9 and 12D4 H-chains correlate well with the VHIIC subgroup (64 to 67% homology). The 11C7, 10C3, 3B9 and 12D4 hybridoma cell lines use JH1, JH2, JH3 and JH4 DNA segments, respectively. Since considerable variations in length and primary sequence in the CDR3 (complementarity determining region) peptides of all the H-chains are evident, conservation of the D-region structure does not appear to be necessary for effective hapten binding. However, sequence homologies of the CDR2 regions of all the antibodies indicate that residues Glu H-50, Ile H-51, Pro H-52a and Tyr H-59 are conserved, and that these segments may be more critically involved in binding than the other H and L-chain hypervariable regions. The marked VL sequence homology, greater than 93%, among the L-chains and consensus lambda sequence, suggests derivation from a similar or identical VL germ-line gene. The L-chain J-region peptides for all the antibodies are classified JL1 and no VL-JL junctional diversity was apparent. The antimorphine antibody L-chains are apparently generated by the joining of a specific J-gene segment to a single germ-line V-gene segment, and minor sequence variations are the result of somatic mutations within the coding region. The leader sequence for one of the H-chains was determined. The inhibition of morphine binding by phenoxybenzylation or iodination of the affinity-purified immunoglobulins indicates the involvement of a single tyrosyl residue within or close to the antibody-combining site for the opiate. This conclusion is supported by the sequence data and nuclear magnetic resonance measurements reported in the accompanying paper, in which the results are used to interpret nuclear magnetic resonance measurements on one of the ligand-antibody systems. The possible importance of additional contact amino acids, tryptophan, aspartic and/or glutamic acids, is discussed.

Twelve hybridoma cell lines producing MAbs against morphine were established by using morphine hemisuccinate-conjugated bovine serum albumin as an immunogen. The MAbs belonged to the IgG1 subclass with kappa- or lambda-chains. The association constants of the antibodies ranged from 4.6 x 10(8) to 4.7 x 10(10) (M-1). These antibodies revealed slightly different cross-reactivities with various agonistic opiates and antagonists. In general, the antibodies were strongly cross-reactive with the opiate agonists, codeine, ethylmorphine, dihydromorphine and dihydrocodeine, while their cross-reactivities with norcodeine and the opiate antagonists, naloxone and naltrexone, were weak. The cross-reactivities with dihydromorphinone, dihydrocodeinone, naloxone, naltrexone, dextromethorphan and homatropine varied from clone to clone. Interestingly, certain MAbs displayed weak but significant cross-reactivities with the synthetic opiate, meperidine. However, none of the antibodies was cross-reactive with the opioid peptides, beta-endorphin, Met-enkephalin, and D-Ala2-D-Leu5-enkephalinamide. Radioimmunoassay for morphine using one of the antibodies (MOR 131.5.13) was shown to be sufficiently sensitive (IC50 = 0.1 nM) for the purposes of forensic analysis of morphine. This set of monoclonal anti-opiate antibodies is assumed to be suitable for analyzing the structure-function relationship in the hapten-antibody interaction, since the antibodies revealed similar but not identical cross-reactivities with various morphine related compounds.

The effect of immunization of rats with the conjugated antigen morphine-bovine serum albumin synthetized by the authors on the analgetic activity of morphine hydrochloride has been investigated. Morphine binding antibodies were shown to appear in the blood of rats during immunization. The spleen demonstrated mononuclear cells also capable of morphine binding. It has been established that immunization of rats with the above synthetic antigen makes them tolerate the pharmacological effects of the narcotic. Morphine at a dose of 5 mg/kg is not capable of inducing an inherent increase in the threshold of pain sensitivity in immunized rats. Immunization also slightly diminishes the morphine effect at a dose of 20 mg/kg on the EEC. Prolonged daily administration of morphine does not remove morphine tolerance due to immunization.

This report describes a radioimmunoassay for the simultaneous detection of morphine and barbiturates. Morphine and barbiturate antibodies, obtained from goats, were mixed with 125l-labeled antigens. By adjusting concentrations of the morphine and barbiturate antibodies and radiolabeled antigens, closely superimposed standard curves for the two drugs would be obtained. As a consequence, similar response curves were obtained for urine specimens containing morphine or barbiturates. Although concentrations as low as 25 mug/liter could be measured, to ensure against false positive reactions the test should be performed at the 100 mug/liter concentration. Unknown samples positive by the dual assay were confirmed by separately testing the specimens with the individual radioimmunoassay specific for morphine or barbiturate. Equivalency tests of urines positive for morphine, positive for barbiturates, or negative for both demonstrated complete correlation between the single and dual assays. The mixed reagent retained its sensitivity and specificity for at least three months when stored at 4 or 25 degrees C. The dual radioimmunoassay is a rapid, simple procedure that can be adapted to automated processes and that is suitable for large- and small-scale screening.

Data are presented which show that the increased binding of morphine by sera from rabbits which had been treated with morphine in an appropriate manner is mediated by immunoglobulins and is specific. These observations, together with findings reported in the preceding paper, suggest that morphine or one of its derivatives can evoke specific immune responses. It seems most likely that the response is evoked by a complex, formed in vivo, of a tissue or serum protein and morphine or one of its metabolites.

Animals immunized with hapten-protein conjugates subsequently circulate high concentrations of hapten bound by antibody. The levels of hapten detected are capable of significantly reducing antibody titer in the sera immunized animals. In the case of steroid-protein conjugates, the main source of increased plasma steroid concentration is the immunizing conjugate, although a contribution from increased host secretion may also occur. The results for rabbits immunized with digoxin-BSA indicate that the appearance of circulating digoxin followed the appearance of circulating antibody to digoxin. Appearance of digoxin in circulation appears to coincide with the operation of the immune response and may be related to macrophage activity. Similar conclusions are drawn from results obtained for circulating morphine in the serum of a sheep immunized with morphine-BSA. Injected hapten-protein antigens are probably processed by macrophage to produce low molecular weight haptenic fragments which are maintained in circulation for prolonged periods in the form of antibody-hapten complexes.

In order to examine the capacity of pharmacologically useful opiates to stimulate human mast cell secretion, subjects were skin tested with morphine, codeine, or meperidine hydrochloride. All three agents acted equipotently in eliciting positive immediate skin reactions from all subjects tested. Each agent demonstrated 10 mm of net whealing at 5 to 10 micrograms base (16.7 to 40.4 nmol) injected intradermally. The ability to elicit immediate skin test reactions with endogenous opioid peptides was examined with the use of dynorphin, [D-Ala, 2-D-Leu5] enkephalin, beta-endorphin, and morphiceptin . All four compounds induced wheal-and-flare reactions with the order of potency: dynorphin, greater than beta-endorphin, and greater than [D-Ala, 2-D-Leu5] enkephalin approximately equal to morphiceptin at dose ranges of 0.3 to 8.45 nmol. The inhibition of reactivity by hydroxyzine and the demonstration of mast cell degranulation by electron microscopy suggest that the immediate skin responses to opioid stimulation occur as a consequence of mast cell degranulation. Experiments with the opioid receptor antagonist, naloxone, suggest that both opioid and nonopioid receptors may be involved. These results imply that endogenous opioid peptides possibly may play a role in mast cell function and/or degradulation .

We describe a method for estimating the molar concentration of an unknown immunoreactive compound. The amount of antibody required to bind half of a standard is compared to the amount required to bind half of an equal number of immunoreactive equivalents of the unknown. We demonstrate the utility of the method using a morphine antibody affinity resin and compounds structurally related to morphine.

Specific antisera to morphine have been raised in response to immunization with a conjugate of N-carboxypropylnormorphine with bovine serum albumin (BSA). These antisera effectively distinguish changes in substituents at the 3 and 6 positions of the alkaloid, thus reducing cross-reactivity with codeine and morphine-3-glucuronide to negligible levels. The utility of these antisera has been illustrated by their application in radioimmunoassay procedures, along with similarly specific anti-codeine sera (Findlay et al., 1976) to a study of the biotransformation of codeine to morphine in the rat. After oral administration of codeine, serum levels of morphine were low, but significantly higher than codeine levels after 15 min., indicating rapid metabolism of codeine to morphine in this species.