Enzyme immunoassay for the quantification of mithramycin using beta-D-galactosidase as a label.

A sensitive enzyme immunoassay for mithramycin (MTM) has been developed by using antibody induced in rabbits, beta-D-galactosidase-labeled MTM, and a double-antibody separation technique, which allowed us to measure accurately as little as 100 pg of MTM per assay tube. MTM-antibody was produced against MTM-bovine serum albumin conjugate prepared by the use of diazotized p-aminobenzoic acid as a cross-linker. The beta-D-galactosidase-labeled MTM conjugate was similarly prepared by a geometric m-isomer of diazotized aminobenzoic acid. This enzyme immunoassay was specific to MTM and showed a very slight cross-reactivity with MTM analogues, chromomycin A3 (5.6%) and olivomycin (2.4%), but no cross-reactivity with drugs commonly used with MTM in combination chemotherapy for cancer treatment. The values of MTM concentrations detected by this assay were comparable to those detected by the high-pressure liquid chromatography method. However, the enzyme immunoassay method was 100 times more sensitive in detecting MTM in lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats during 6 h after i.v. administration of MTM in a single dose of 2.0 mg/kg. Since MTM has long been used against a variety of human cancers, the enzyme immunoassay of the drug will be a valuable new tool in clinical pharmacological studies.

Enzyme-linked immunosorbent assay for the quantification of actinomycin D using beta-D-galactosidase as a label.

An enzyme-linked immunosorbent assay (ELISA) for actinomycin D (AMD) has been developed, which allowed us to measure accurately as little as 50 pg of AMD per assay well. Anti-AMD sera was obtained by immunizing rabbits with an AMD derivative, 7-aminoactinomycin D (7AMD), conjugated with mercaptosuccinyl bovine serum albumin via N-maleoylaminobutyric acid chloride as a coupling agent. An enzyme marker was similarly prepared by coupling 7AMD with beta-D-galactosidase (EC 3.2.1.23) via N-maleoylaminobutyric acid. The ELISA with anti-AMD immunoglobulin G fraction as a solid phase and 7-aminoactino-mycin beta-D-galactosidase conjugate was specific to AMD as well as 7 AMD and showed 30% cross-reaction with actinomycin V, while no cross-reactivity was seen with drugs commonly used with AMD in combination chemotherapy for cancer treatment. The sensitivity of the ELISA was about 1000 times higher than high performance liquid chromatography in detecting AMD in lower concentrations. Using this assay, drug levels were easily measured in the blood and urine of rats following administration of AMD in a single dose of 0.25 mg/kg i.v. These results indicate that the ELISA provides a nonradioactive, inexpensive, sensitive, and rapid method applicable for pharmacological analyses of the drug.

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent.

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.

The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a coupling agent in the preparation of enzyme-antibody conjugates.

The present study was undertaken to develop a novel method for the enzyme labeling of antibodies. Goat anti-rabbit IgG was used as a prototype and coupled to beta-D-galactosidase (Gal) with a new heterobifunctional cross-linking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM). The antibody was first azo-coupled with DPEM to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were purified by DEAE-cellulose column chromatography, being completely separated from non-reacted antibodies but remaining mixed with free Gal, and showing approximately 30% enzyme activity bound to antibody. This method is simple, reproducible and so mild that almost full enzyme and antibody activity can be retained. The conjugates were used as a label in an immunoassay and were able to detect first antibody at concentrations as low as 2 ng/tube. Furthermore, the present method was compared with the method using N-(gamma-maleimidobutyryloxy)succinimide (GMBS), and it was found that both conjugates produced comparable results.

A highly sensitive enzyme-linked immunosorbent assay for etoposide using beta-D-galactosidase as a label.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for etoposide (EP) was developed, which is capable of accurately measuring as little as 40 pg EP/ml. Anti-EP sera were obtained by immunizing rabbits with EP conjugated with mercaptosuccinyl bovine serum albumin (MS.BSA) using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling EP with beta-D-galactosidase (beta-Gal; EC 3.2.23) via DPEM. This ELISA was specific for EP and showed a very slight cross-reactivity with its major metabolite, cis-hydroxy acid of EP (0.91%), but none with 4'-demethylepipodophyllotoxin and drugs commonly used with EP in combination chemotherapy for cancer treatment. The values for EP concentration detected by this assay were comparable with those detected by the high-performance liquid chromatography (HPLC) method. However, the ELISA was about 1,250 times more sensitive in detecting EP at lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats for 7 h after i.v. administration of EP at a single dose of 3 mg/kg. Due to its sensitivity and specificity for EP, the ELISA should prove to be a valuable new tool for use in clinical pharmacological studies.

Potentiation of cytotoxicity of mitomycin C by a polyacetylenic alcohol, panaxytriol.

Polyacetylenic alcohol, panaxytriol, which was isolated from Panax ginseng C. A. Meyer, has antiproliferative activity against several kinds of tumor cells. In this paper, the effect of panaxytriol on the cytotoxicity of mitomycin C (MMC) against a human gastric carcinoma cell line, MK-1, was investigated. The combination of a subthreshold concentration of MMC and panaxytriol produced a significant cytotoxic effect, which indicates that the effects of panaxytriol and MMC are synergistic. A synergistic effect was observed when MK-1 cells were treated with the mixture of MMC and panaxytriol or treated with MMC followed by panaxytriol. In contrast, when MK-1 cells were exposed to panaxytriol and then to MMC, only an additive effect was induced. With the aim of finding a possible mechanism, the effect of panaxytriol on the accumulation of MMC into the MK-1 cells was examined. Cellular concentrations of MMC were measured by high-performance liquid chromatography (HPLC). When MK-1 cells were treated with a mixture of panaxytriol and MMC or first with MMC and then with panaxytriol, the cellular level of MMC was significantly higher than that in MK-1 cells treated with MMC alone, but no significantly increased accumulation was found when MK-1 cells were treated with panaxytriol followed by MMC. These results suggest that synergistic effects of panaxytriol and MMC may be induced by acceleration of the effect of MMC on cellular accumulation by panaxytriol. In addition, they suggest that the enhanced accumulation of MMC in MK-1 cells treated with panaxytriol can probably be attributed to the decreased fluidity of the cell membrane caused by panaxytriol.

A possible mechanism for the cytotoxicity of a polyacetylenic alcohol, panaxytriol: inhibition of mitochondrial respiration.

A polyacetylenic alcohol, panaxytriol, isolated from Panax ginseng C. A. Meyer inhibits both tumor cell growth in vitro and the growth of B16 melanoma transplanted into mice. Our preliminary studies indicated that panaxytriol localizes to the mitochondria in human breast carcinoma cells (Breast M25-SF). This study focused on the effects of panaxytriol on mitochondrial structures and function in Breast M25-SF. The results indicate that panaxytriol rapidly inhibits cellular respiration and disrupts cellular energy balance in Breast M25-SF. At concentrations between 11.3 and 180 microM, panaxytriol causes a dose-dependent inhibition of the conversion of the tetrazolium (MTT assay) by mitochondrial dehydrogenase within 2 h. A 1-h treatment with 180 microM panaxytriol causes a significant loss of rhodamine-123 from cells with mitochondria prestained with rhodamine-123 (by flow cytometry). Specific toxic changes were observed by electron microscopy in the mitochondria of Breast M25-SF within 1 h after treatment with more than 180 microM panaxytriol. These data indicate that 180 microM panxytriol rapidly disrupts cellular energy balance and respiration in Breast M25-SF and suggest that panaxytriol may lower cellular ATP concentrations. After treatment with 180 microM panaxytriol, cellular ATP levels were 40% of those in control cells after 1 h. ATP depletion preceded the loss of cellular viability. Neither ATP depletion nor cytolysis was found in human erythrocytes that have no mitochondria. Thus, ATP depletion resulting from a direct inhibition of mitochondrial respiration is a critical early event in the cytotoxicity of panaxytriol.

[An evaluation of tumor markers and risk factors in mass screening for various cancers].

The borderline values of tumor markers and the clarification of risk factors for cancer are problems to be solved in the development of mass screening tests for various cancers. A tiny abnormality may be overlooked in an evaluation based upon traditional reference standards obtained by surveying healthy subjects. Both negative and positive values are required in mass screening for various cancers. That is, cut-off values should minimize the incidence of false negatives and false positives in the screening test. A modified combination assay using tumor markers and risk factors was used to screen 967 healthy subjects over age 50 for nonspecific cancers and the results were analyzed. Positive rates based on ordinary cut-off values for each tumor marker were about 6 to 7% in CEA, CA19-9 and BFP, 2.7% in SPAN-1, 1.3% in PAP, and less than 1% in AFP, SCC, CA125 and NCC -ST-439. For pepsinogen, a risk factor of various cancers especially in the digestive tract, the cut-off value was determined as the mean-SD in 967 healthy subjects over 50 years old. That is, the cut-of value for pepsinogen I was 18.7 ng/ml. A lower value was found in 149 subjects (15.4%). The cut-off value for pepsinogen II was 7.4 ng/ml and a lower value was found in 227 subjects (23.5%). The cut-off value for the pepsinogen I/II ratio was 1.2 and 108 subjects showed lower values (11.2%). Positive cases were referred to specialists in various fields to detect the specific suspected cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

[Relationship between antiproliferative activity of acetylenic alcohol, panaxydol, and its affinity for target cell membrane].

Acetylenic alcohol, panaxydol, isolated from Panax ginseng shows a significant growth inhibitory effect against various types of cultured cell lines. Its anti-proliferative effect is highly specific for malignant cells, but varies by cell lines. In the present study, the relationship between cellular sensitivity to panaxydol and the affinity of panaxydol for target cells was studied. Panaxydol was conjugated to bovine serum albumin (BSA). Panaxydol-BSA was first incubated with sensitive cells, MK-1 cells, or resistant cells, HeLa cells, and then FITC-labeled anti-BSA antibody was added. The percentage of labeled cells and relative mean of fluorescence were determined by flow cytometry. The results indicate that the sensitivity of target cells against panaxydol is partly prescribed by its affinity for target cells.

Three-dimensional bulk fermiology of CeRu2Ge2 in the paramagnetic phase by soft x-ray hnu-dependent (700-860 eV) ARPES.

By virtue of the soft x-ray angle-resolved photoelectron spectroscopy, the three-dimensional bulk fermiology has been successfully performed for a strongly correlated Ce compound, ferromagnet CeRu2Ge2 in the paramagnetic phase. A clear difference of the Fermi surface topology from either band calculation or de Haas-van Alphen results in the ferromagnetic phase is observed and interpreted by considering the difference of the 4f contribution to the Fermi surfaces in the paramagnetic phase.

Coexistence of strongly mixed-valence and heavy-fermion character in SmOs4Sb12 studied by soft- and hard-X-ray spectroscopy.

Sm-based heavy-fermion compound SmOs4Sb12 has been investigated by soft x-ray (hnu=1070-1600 eV) and hard x-ray (HX; hnu=7932 eV) spectroscopy. The HX photoemission spectroscopy clearly demonstrates that the strongly mixed-valence state and the heavy-fermion state coexist in the bulk. It is found that the Sm valence decreases below 100 K, indicating that the Kondo coherence develops with approaching the proposed Kondo temperature. Our theoretical analyses suggest that the origin of the coexistence in SmOs4Sb12 is the coincidence of two conditions, namely, (i) the energy difference between Sm divalent and trivalent states is very small and (ii) the hybridization between Sm 4f and conduction electrons is weak.

The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a heterobifunctional agent in developing enzyme immunoassay for neurotensin.

A heterobifunctional crosslinking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) was newly synthesized and characterized to possess the maleimide group with a stability greater than that previously reported for N-(4-diazophenyl)maleimide. Using the peptide hormone neurotensin (NT) as a model hapten, DPEM was used in the conjugation reaction with bovine serum albumin (BSA) and with beta-D-galactosidase (beta-Gal) in developing an enzyme immunoassay (EIA) for NT. The NT-DPEM-BSA conjugate elicited anti-NT antibodies in rabbits and the NT-beta-Gal conjugate behaved as an enzyme marker of NT in the EIA. The EIA developed double antibody was reproducible and sensitive in detecting NT at concentrations as low as 30 fmol per tube. The specificity of anti-NT serum seems to be primarily toward the carboxy-terminal region of NT, showing cross-reactions with such NT fragments as NT2-13, NT8-13, and NT1-8 for 120, 22, and less than 0.1%, respectively. The utility of this assay was also demonstrated by measuring the NT immunoreactivity in several rat organs. DPEM could be useful for developing EIAs for other peptide hormones (even those which contain neither a free amino group nor a free carboxyl group), using the imidazole, phenolic, or indole group(s) of amino acids as a binding site for carrier proteins.

Enzyme-linked immunosorbent assay (ELISA) for luteinizing hormone releasing hormone (LH-RH) using a heterobifunctional cross-linking agent, N-[beta-(4-diazophenyl)ethyl]maleimide.

Luteinizing hormone-releasing hormone (LH-RH) was first labeled with an enzyme, beta-D-galactosidase (beta-Gal; EC 3.2.1.23), using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional cross-linking agent. An antigen was similarly prepared by coupling LH-RH to mercaptosuccinylated bovine serum albumin with DPEM and was used for the immunization of rabbits for antibodies against LH-RH. A new, simple enzyme-linked immunosorbent assay (ELISA) for LH-RH was developed by using the principle of direct competition between LH-RH and beta-Gal-labeled LH-RH for anti-LH-RH IgG which had been adsorbed to the plastic surface of microtiter plates. LH-RH concentrations lower than 50 pg/assay well were measurable reproducibly by the ELISA, the sensitivity of which was found to be about 6250 times greater than the corresponding high performance liquid chromatography (HPLC) procedure. The specificity of this ELISA seems to be primarily toward the C-terminal region of LH-RH, showing a cross-reaction with the LH-RH6-10 fragment to the same extent as with LH-RH, but no cross-reaction with the LH-RH1-3 and LH-RH4-6 fragments. Using this assay, LH-RH levels were easily measured in the blood and urine of rats following the administration of LH-RH in a single dose of 0.5 mg/kg i.v. The present, newly developed ELISA is a nonradioactive, inexpensive and rapid method, and might be useful for elucidating experimental hypothalamic-pituitary-gonad interactions.

Determination of topotecan by ELISA.

A highly sensitive ELISA for the determination of (s)-9-dimethylaminomethyl-10-hydroxy-camptothecin (topotecan) capable of measuring as low as 80 pg/ml was developed. Anti-topotecan antibody was obtained by immunizing rabbits with topotecan conjugated with bovine serum albumin using diazotized m-aminobenzoic acid as a cross-linker. Enzyme labeling of topotecan with beta-D-galactosidase was performed by utilizing another cross-linker, N-(4-diazophenyl)maleimide. The specificity of this ELISA appears to be primarily toward the lactone moiety of topotecan, showing a very slight cross-reactivity with the lactone opened-ring of topotecan. The values for the topotecan concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. These findings suggest that this ELISA can detect the natural amounts of the lactone form. Using this assay, drug levels were easily determined in the blood and urine of rats for 5 h after i.v. administration of topotecan at a single dose of 1 mg/kg. The sensitivity and specificity of the ELISA should provide a useful tool for developing pharmacokinetic and pharmacodynamic studies of topotecan.

ELISA for the quantification of pilsicainide.

A sensitive and specific ELISA for an antiarrhythmic drug, pilsicainide, was developed, which is capable of measuring as low as 1.6 ng/ml. Anti-pilsicainide antibody was obtained by immunizing rabbits with pilsicainide conjugated with bovine serum albumin using diazotized N-(3-amino-2,6-dimethylphenyl)-8-pyrrolizidinylacetamide (3-aminopilsicainide). Enzyme labeling of pilsicainide with beta-D-galactosidase was similarly performed using a diazotized 3-aminopilsicainide. Cross-reactivity data showed that the antibody well recognizes both the aromatic ring and the pyrrolizidine moieties, and thus specific enough to the structure of pilsicainide. The values for the pilsicainide concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. Moreover, the ELISA was about 30-fold more sensitive in detecting pilsicainide at lower concentrations. Using this assay, drug levels were easily measured in the serum of rabbits after oral administration of pilsicainide at a single dose of 1 mg/kg. The ELISA should be a valuable tool in therapeutic drug monitoring (TDM) and pharmacokinetic studies of pilsicainide.

Development of ELISAs for irinotecan and its active metabolite SN-38.

Two highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for the determination of 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin (irinotecan) and 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, were developed, which are capable of measuring as low as 16 and 160 pg of each drug/ml, respectively. Anti-irinotecan antibody was obtained by immunizing rabbits with irinotecan conjugated with mercaptosuccinyl bovine serum albumin (MS.BSA) using N-(4-diazophenyl)maleimide (DPM) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling irinotecan with horseradish peroxidase (HRP) via DPM. This ELISA for irinotecan was specific for irinotecan and showed almost no cross-reactivity with its active metabolite SN-38. Anti-SN-38 antibody was obtained by immunizing rabbits with SN-38 conjugated with BSA using the N-succinimidyl ester method. An enzyme marker was prepared by coupling SN-38 with HRP employing DPM. The ELISA for SN-38 was specific to SN-38 and showed a very slight cross-reactivity with irinotecan (0.08%). Using the 2 assays, we reconfirmed the rapid metabolite of irinotecan with rat serum. The 2 ELISAs may be a valuable tool for studies of the pharmacokinetics and pharmacodynamics of these drugs.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for antitumor polyacetylenic alcohol, panaxytriol.

A new type of antitumor polyacetylenic alcohol, panaxytriol, was isolated from the roots of Panax ginseng C. A. Meyer. A highly sensitive enzyme-linked immunosorbent assay (ELISA) for the determination of panaxytriol was developed, which is capable of measuring as low as 25.6 pg/ml. Anti-panaxytriol antibody was obtained by immunizing rabbits with panaxytriol conjugated with bovine serum albumin using the N-succinimidyl ester method. An enzyme marker was similarly prepared by coupling panaxytriol with horseradish peroxidase. The specificity of this ELISA seems to be primarily toward both the glycol moiety and the diacetylene moiety of the panaxytriol, showing a slight cross-reaction with the other panaxytriol analogues which are structurally different only in C-9,10 positions, but no cross-reaction with the 1,2-decanediol or 3-nonyn-1-ol. The values for panaxytriol concentration detected by this assay were comparable with those detected by the gas chromatography method. The ELISA was about 5000 times more sensitive in detecting panaxytriol. Using this assay, panaxytriol levels were easily determined in the blood of rats. The ELISA may be a valuable tool for studies of the biological and pharmacological properties of the polyacetylenic alcohol, panaxytriol.

Isonicotinic acid hydrazide as an antigen.

Detection by in vitro serologic techniques of circulating antibody directed against isonicotinic acid hydrazide (INH, known as isoniazid) in humans has not been reported. In the past few months, however, sera from a patient with a recent history of isoniazid hypersensitivity of the immediate type have been studied, and reaginic antibodies (IgE) specific to INH were detected by means of an enzyme-linked allergosorbent test. Preliminary enzyme-linked allergosorbent tests also demonstrated that INH-specific IgE occurred in the serum of two of 150 patients with tuberculosis who had been treated with INH.

Screening of polyacetylenic alcohols in crude drugs using the ELISA for panaxytriol.

Polyacetylenic alcohols such as panaxytriol, panaxynol and panaxydol isolated from the roots of Panax ginseng C. A. MEYER have antiproliferative activity against various cultured tumor cells. Anti-panaxytriol antibody was obtained by immunizing rabbits with panaxytriol-bovine serum albumin conjugates. Although the antibody reactivity was directed mainly toward panaxytriol, there was a slight cross-reactivity with other polyacetylenic compounds. The antibody was, therefore, used for screening a large number of crude drugs for polyacetylenic compounds such as panaxytriol. Methanol-extracts from 31 crude drugs were examined. Significant reactivity was observed in 15 methanol-extracts from Aralieaceae, Compositae and Umbelliferae as reported by other investigators. Three out of the 15 crude drugs were selected for determination of the potent cross-reactive compounds. Four kinds of cross-reactive compounds were isolated by silica gel column chromatography, monitoring each fraction using the enzyme-linked immunosorbent assay (ELISA). Among them, panaxynol and heptadeca-1,8-diene-4,6-diyne-3,10-diol were identified from Saposhnikoviae Radix. Falcarindiol was newly identified from Peucedani Radix. A new polyacetylenic alcohol, 9,10-epoxy-16-hydroxy-octadeca-17-ene-12,14-diyne-1-al, was also isolated from Foeniculi Fructus. All these polyacetylenic alcohols inhibited the growth of a human gastric adenocarcinoma cell line, MK-1 cells, in a dose-dependent manner. These results indicate that the antibody against panaxytriol is an effective tool for "screening" antiproliferative polyacetylenic compounds.

The first specific antibody against cytotoxic polyacetylenic alcohol, panaxynol.

Antitumor polyacetylenic alcohol, panaxynol, was isolated and purified from a powder of the root of Panax ginseng C.A. Meyer. Panaxynol inhibited the growth of various kinds of cultured tumor cell lines in a dose-dependent manner. In this paper we demonstrated the first specific antibody production against panaxynol. Anti-panaxynol antibody was elicited in rabbits by immunization with panaxynol hemisuccinate-bovine serum albumin conjugate (panaxynol hemisuccinate-BSA conjugate). An enzyme immunoassay (EIA) for the determination of panaxynol was established using a double-antibody technique. The EIA was highly specific against panaxynol although the antibody showed a minimal cross-reactivity with other types of polyacetylenic alcohol, i.e. panaxydol (12.0%) and panaxytriol (0.77%). Panaxynol at a concentration as low as 6.4 ng/ml can be detected. Using this assay we reconfirmed the rapid consumption of panaxynol by target tumor cells in an in vitro-culture system. The anti-panaxynol antibody may be a valuable tool for studies of the biological properties of polyacetylenic compounds.