Sensitive Enzyme Immunoassay Method Research Articles

Cytidylate cyclase activity is stimulated via activation of a guanine nucleotide-binding protein

Cytidylate cyclase is a membrane-bound enzyme which catalyzes the biosynthesis of cytidine 3′, 5′-cyclic monophosphate (cCMP) from CTP. By using a sensitive and specific enzyme immunoassay method, we evaluated the participation of guanine nucleotide-binding protein (G-protein) in the regulation of cytidylate cyclase activity in rat brain and other tissues. AlF 4 −, an activator of G-proteins, effectively elevated the cyclase activity. The stimulation by GTPγS, a nonhydrolyzable GTP analogue, was also observed in time- and concentration-dependent manner during the preincubation and this effect was competitively inhibited by the addition of GDPβS. However, islet-activating protein and cholera toxin which affected adenylate cyclase activity had no effect on cytidylate cyclase activity. These results indicate that cytidylate cyclase is associated with a certain G-protein and its activity is regulated by the mode different from that of adenylate cyclase.

An enzyme immunoassay for the determination of taxol and taxanes in Taxus sp. tissues and human plasma

A rapid and sensitive indirect competitive inhibition enzyme immunoassay (CIEIA) method has been developed for quantitating taxanes in extracts of Taxus brevifolia Nutt. tissue and in human plasma. High titer rabbit polyclonal antibodies (pAb) were raised against a conjugate of 7-succinyltaxol and keyhole limpet hemocyanin. The presence of taxane analyte competitively inhibited the binding of the rabbit anti-taxane pAbs to a 7-succinyltaxol-bovine serum albumin solid phase coating antigen. The CIEIA detected taxol and cephalomannine in concentrations as low as 0.3 ng/ml (3.5 × 10 −4 μM), but did not detect baccatin III or 10-deacetylbaccatin III at concentrations below 1000 ng/ml (1.7 μM and 1.8 μM, respectively). This method is useful for estimating the taxane content of T. brevifolia extracts and may be useful for monitoring the taxol plasma level of taxol-treated patients.

Sensitive enzyme immunoassay for human Cu/Zn superoxide dismutase

A highly sensitive enzyme immunoassay method for measurement of human Cu/Zn superoxide dismutase (SOD) was established. Antisera were raised in rabbits by injecting SOD purified from human erythrocytes, and antibodies to SOD were purified by the use of Cu/Zn SOD-coupled Sepharose 4B. The assay system consisted of polystyrene balls with immobilized monospecific antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with β- d-galactosidase from Escherichia coli. The assay was highly sensitive, and the minimum detection limit was 3 pg or 0.1 fmol/assay tube. Serum Cu/Zn SOD levels of normal healthy subjects (36.3 ± 15.6, n = 120, 16–64 years old) were not related to age and sex. It was confirmed that Cu/Zn SOD levels in erythrocytes and blood plasma were significantly enhanced in patients with Down syndrome. Immunoreactive Cu/Zn SOD was detectable in all the tissues examined and was present at high concentrations in brain, adrenal gland, liver, heart and kidney.

-Enolase in blood plasma during open heart surgery

The enolase isoenzyme composed of the beta subunit (alpha beta or beta beta enolase) is distributed predominantly in the heart and skeletal muscles. To see whether it may be a useful indicator of myocardial damage, concentrations of beta-enolase (the beta subunit of enolase) were determined in serial blood samples taken from 18 patients who underwent mitral valve replacement, using a highly sensitive enzyme immunoassay method. They were compared with those of creatine kinase MB isoenzyme (CK-MB) in the same samples. The mean arterial beta-enolase concentration was 6.60 (SD 3.84) ng.ml-1 at the beginning of anaesthesia. It increased rapidly after reperfusion and rose to greater than 100 ng.ml-1 at 2 h post-reperfusion. It remained high until 12 h post-reperfusion, and then decreased slowly. The plasma beta-enolase concentrations were significantly higher in coronary sinus samples than in arterial samples in the early phase after reperfusion. Since plasma carbonic anhydrase III, which is known to be localised only in the skeletal muscle, did not increase during the surgery, we suggest that the major portion of the elevated plasma beta-enolase was derived from heart muscle. Plasma concentrations of beta-enolase increased as quickly as those of CK-MB after reperfusion. The CK-MB concentrations had returned to normal at 2 d but the beta-enolase concentrations remained elevated up to 7 d. These results show that the determination of beta-enolase in plasma may be useful for estimating myocardial damage during open heart surgery.

Identification of lung major GTP-binding protein as Gi2 and its distribution in various rat tissues determined by immunoassay

Antisera were raised in rabbits against the 40-kDa alpha subunit of bovine lung GTP-binding protein, which were identified as the alpha subunit of Gi2 (Gi2 alpha) by the analysis of the partial amino acid sequence. Antibodies were purified with a Gi2 alpha-coupled Sepharose column and then were passed through a Gi1 alpha-coupled Sepharose column to remove antibodies reactive also with 41-kDa alpha. Purified antibodies reacted with Gi2 alpha, but not with Gi1 alpha, Gi3 alpha, or Go alpha in an immunoblot assay. A sensitive enzyme immunoassay method for the quantification of Gi2 alpha was developed by using these purified antibodies. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 1 fmol, or 40 pg. Samples from various tissues were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Gi2 alpha were determined. Gi2 alpha was detected in all the tissues examined in the rat. The highest concentration was found in platelets and leukocytes when the data were expressed as picomoles per milligram of protein. The spleen, lung, and cerebral cortex contained relatively high levels of Gi2 alpha. In the bovine brain, Gi2 alpha was distributed almost uniformly among the various regions. The concentrations of Gi2 alpha were constant in the rat brain throughout ontogenic development, in contrast with those of Go alpha which were markedly increased with age.

Developmental changes in fiber type-related proteins in soleus, rectus femoris, and heart muscles of normal and dystrophic mice

By using sensitive enzyme immunoassay methods, several isoenzymes or isoproteins related to muscle fiber types were determined in the soleus (SOL), rectus femoris (RFM), and heart muscles of normal and dystrophic ( dy/dy) mice of various ages. In normal adult mice, the S-100 protein α subunit (S-100α) and creatine kinase B subunit (CK-B), which are known to be distributed predominantly in type I muscle fibers as S-100a 0 (αα form of the S-100 protein) and the MB form of CK, respectively, were enhanced several-fold in the “aerobic” SOL muscle as compared with the “anaerobic” RFM muscle. The enolase β subunit (β-enolase) and the M subunit of CK (CK-M) were present in the RFM at levels increased several-fold compared to levels in the SOL of the same mice. In age-matched dystrophic adult mice, however, the compositions of these muscle-related proteins in the RFM muscle shifted to those of the SOL muscle: S-100α and CK-B increased several-fold, β-enolase and CK-M decreased markedly as compared with the normal RFM. On the other hand, the SOL and heart muscles of dystrophic mice showed only a slight increase of CK-B or decrease of CK-M. In the RFM of 3-week-old dystrophic mice, S-100α and β-enolase levels were similar to those in the RFM of control littermates, but a significant increase of CK-B and a decrease of CK-M were already observed in this early stage of dystrophy. These results indicate that changes in muscle-related proteins in the dystrophic muscles are apparently displayed mainly in the anaerobic muscles and feature a decrease in type II fiber-related proteins and a relative increase in type I fiber-related proteins. The mechanism of these changes in dystrophic mice is discussed.

Immunoassay for the beta gamma subunits of GTP-binding proteins and their regional distribution in bovine brain.

Antibodies were raised in rabbits against the beta gamma subunits of bovine brain GTP-binding proteins, and were purified with a beta gamma-coupled Sepharose column. Purified antibodies reacted strongly with 36,000-dalton beta subunit and slightly with 35,000-dalton beta and gamma subunits, but not with other proteins in an immunoblot assay. Using these purified antibodies, a sensitive enzyme immunoassay method for the quantification of brain beta gamma was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimum detection limit of the assay was 3 fmol, or 130 pg. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of beta gamma were determined. The beta gamma were detected in all the regions, and the highest concentrations were observed in the cerebral cortex and nucleus caudatus. The concentrations of beta gamma were higher than those of alpha subunit of GTP-binding protein, Go, in all the regions.

Induction of S-100b (ββ) protein in human teratocarcinoma cells

Human teratocarcinoma NT2/D1 cells undergo differentiation into a variety of cell types, including neurons, treated with retinoic acid. In the present study, the concentrations of α S-100 and β S-100 proteins (α and β subunits of S-100 proteins), and three subunits (α, β and γ) of enolase in NT2/D1 cells were measured using the sensitive enzyme immunoassay method. The concentration of β S-100 was markedly increased in the cells after treatment with retinoic acid, whereas the concentration of α S-100 was undetectably low, indicating that the S-100b (ββ) protein was induced by retinoic acid. On the other hand, the concentrations of the three forms of enolase isozymes did not change in the same culture. The induction of S-100b protein was not observed in the NT2/D1 cells after treatment with forskolin, dibutyryl cyclic AMP or cholera toxin. The indirect double-labeled immunofluorescence, using antibodies specific to β S-100 and monoclonal antibodies specific to neurofilaments, revealed that both the S-100b protein and the neurofilaments were induced in the same subpopulation of cells which underwent neuronal differentiation.

Serum creatine kinase isoenzymes in duchenne muscular dystrophy determined by sensitive enzyme immunoassay methods

Serum levels of creatine kinase (CK) isoenzymes (MM, MB, and BB) were measured by sensitive enzyme immunoassay (EIA) methods in 50 patients with Duchenne muscular dystrophy (DMD) and in 39 controls. MM, MB, and BB levels in DMD patients were higher than in controls, and these three levels decreased with advancing age of DMD patients. Serum MB levels showed a good correlation with serum levels of carbonic anhydrase III (CA-III), but correlated poorly with electrocardiographic findings, suggesting that the main origin of serum MB in DMD is skeletal muscle rather than cardiac muscle.

Highly sensitive immunoassay for the alpha subunit of the GTP-binding protein go and its regional distribution in bovine brain.

Antisera were raised in rabbits against the alpha subunit of a GTP-binding protein, Go. Because the antisera cross-reacted weakly with the alpha subunit of inhibitory GTP-binding protein of adenylate cyclase (Gi), they were purified with a Go alpha-coupled Sepharose column. Purified antibodies reacted only with Go alpha and did not cross-react with the Gi alpha subunit or beta gamma subunits in an immunoblot assay. Using these purified antibodies, a highly sensitive enzyme immunoassay method for the quantification of bovine brain Go alpha was developed. The assay system consisted of polystyrene balls with immobilized antibody F(ab')2 fragments and the same antibody Fab' fragments labeled with beta-D-galactosidase from Escherichia coli. The minimal detection limit of the assay was 0.1 fmol, or 4 pg. The assay was specific for Go alpha, and it did not cross-react with Gi alpha or beta gamma. Samples from various regions of bovine brain were solubilized with 2% sodium cholate and 1 M NaCl, and the concentrations of Go alpha were determined. Go alpha was detected in all the regions, and the highest concentration was observed in the cerebral cortex. The immunohistochemical study showed that the neuropil was rich in Go alpha.

Extrahypophyseal distribution of α-melanocyte stimulating hormone (α-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer-type dementia patients

Using a sensitive double-antibody solid-phase enzyme immunoassay method α-melanocyte stimulating hormone-like immunoreactivity (α-MSH-LI) was measured in 21 regions of postmortem brains from 8 normal subjects and 5 patients with Alzheimer-type dementia (ATD). In the brains from the normal subjects, the highest concentration of α-MSH-LI was found in the hypothalamus. Relatively high concentration were also measured in the locus coeruleus, substantia innominata, substantia nigra, amygdala and medial nucleus of thalamus. α-MSH-LI in other regions was approximately1/100 of the hypothalamic content. This data is consistent with the existence of α-MSH in extrahypophyseal regions and indicates its regional distribution in the human brain. In the Alzheimer brains, although the temporal cortex and hippocampus had normal concentrations of α-MSH-LI, the cingulate cortex, caudate and substantia nigra showed significantly lower concentrations of α-MSH-LI than those of the control brains. This data suggests that further studies of α-MSH content in a larger number of ATD brains would be useful.

Highly sensitive enzyme immunoassay for human creatine kinase BB isozyme

A sensitive sandwich-type enzyme immunoassay method for measurement of brain-type isozyme of human creatine kinase (CK-BB) was developed using purified antibodies specific to the B subunit. The assay system consisted of polystyrene balls with immobilized antibody F(ab') 2 fragments and the same antibody Fab' fragments labelled with β-D-galactosidase from Escherichia coli. The assay was highly sensitive and 1 pg of CK-BB was measurable. The assay was specific to the B subunit of creatine kinase (CK-B), and it cross-reacted about 25% with CK-MB, the heart-type isozyme. However, the assay showed no cross-reactivity with CK-MM, the muscle type-isozyme or with neuron-specific αα enolase. Coefficients of variation in withinrun and between-run precision studies for serum CK-B were < 8%. Serum CK-B levels in healthy adults of various ages (16–59 yr old) ranged from 0.25–1.44 ng/ml, whereas the CK-B concentrations in children (< 10 yr old) were relatively high, ranging from 1.3–7.4 ng/ml. The CK-B levels in the cerebrospinal fluids (CSF) could be determined by the present method, and they ranged from 0.10–0.76 ng/ml in the samples from patients with non-neuronal disorders. Determination of immunoreactive CK-B in the extracts of various human tissues confirmed previous reports that CK-B was distributed at high concentrations in the central nervous tissue, prostate, uterus, bladder, gastrointestinal tract and heart muscle.

Sensitive enzyme immunoassay for the quantification of aclacinomycin A using beta-D-galactosidase as a label.

A sensitive enzyme immunoassay method (EIA) for an anticancer drug, aclacinomycin A (ACM), has been developed. With a double-antibody technique, ACM at a concentration as low as 100 pg/tube can be detected. An antibody to ACM was obtained by immunizing rabbits with an antigen prepared by coupling ACM with mercaptosuccinylated bovine serum albumin via N-maleoyl aminobutyric acid (MABA) as a coupling agent. Enzyme labeling of ACM was performed with beta-D-galactosidase (beta-Gal; EC 3.2.1.23) via m-maleoyl benzoic acid (MBA). The standard curve of the assay was linear on a logit-log plot over a concentration range of 30 pg to 10 ng. The antibody detected ACM and its metabolites, MA144 M1 (M1), MA144 N1 (N1), MA144 S1 (S1), and aklavin (T1) equally well, but was only minimally reactive with aklavinone (D1) and 7-deoxyaklavinone (C1), thus suggesting that this EIA can detect the total amounts of ACM and its biologically active glycosides among metabolites of ACM. This EIA is practically free from interference by any other anticancer drugs. Using this assay, serum levels of ACM equivalents can be determined accurately after administration of the drug to rats at a single dose of 10 mg/kg. Since ACM is now undergoing clinical trial, the EIA of the drug will be a valuable tool in clinical pharmacological studies.

ラットα‐フェトプロテインのエンザイムイムノアッセイ ペルオキシダーゼ標識抗体を用いた高感度測定法

A sensitive sandwich enzyme immunoassay method for measurement of rat α-fetoprotein(AFP) was established by use of affinity purified antibodies to rat AFP. The assay system consisted of filter paper discs with immobilized antibodies and the same antibodies labeled with horseradish peroxidase(POD). The optimal conditions for the assay and for the preparation of POD labeled antibodies were determined. The assay was as sensitive as radioimmunoassay(RIA) and rat AFP at concentrations of 5 to 1280ng/ml was measurable. AFP values obtained by the present method were in good agreement with those by RIA and the correlation coefficient was 0.99.

Different Tissue Distributions of Two Types of Thiol Proteinase Inhibitors from Rat Liver and Epidermis1

The concentrations of two types of endogenous inhibitors of thiol proteinases were determined in soluble extracts of various rat tissues by means of a sensitive enzyme immunoassay method, which consisted of solid-phase immobilized anti-rat liver inhibitor or anti-rat epidermal inhibitor and antibodies labeled with horseradish peroxidase. The minimum detectable amounts of inhibitors from liver and epidermis were 30 pg and 3 pg/assay, respectively. The tissue distributions of the epidermal and liver-type inhibitors were found to differ. The liver-type inhibitor was found to be widely distributed in various tissues at levels of 76-420 ng/mg protein, whereas the epidermal-type inhibitor was found at high levels in the skin, tongue, esophagus, stomach, intestine, and vagina, but at quite low levels in other tissues tested.

Distribution of immunoreactive carbonic anhydrase III in various human tissues determined by a sensitive enzyme immunoassay method

A sensitive sandwich enzyme immunoassay method for measurement of carbonic anhydrase III (CA-III) was established by use of purified antibodies to CA-III. The assay system consisted of polystyrene balls with immobilized antibody F(ab') 2 fragments and the same antibody Fab' fragments labeled with β- d-galactosidase from E. coli. The assay was highly sensitive and pg levels of CA-III were measurable. Coefficients of variation in within-run and between-run precision studies for serum CA-III were less than 10%. Serum CA-III levels in healthy subjects of various ages ranged from 0.8 to 24 ng/ml. Concentrations of immunoreactive CA-III in the extracts of various human tissues were also determined. Tissues composed of striated muscle contained more than 10 μg/mg protein of CA-III, whereas other tissues, including heart muscle, contained less than 0.5 μg/mg protein. These results were consistent with other data showing that serum CA-III levels were raised in patients with progressive muscular dystrophy but not in those with acute myocardial infarction.

Regulation of nervous system-specific S-100 protein and enolase levels in adipose tissue by catecholamines.

The effect of catecholamines on the levels of S-100 protein and nervous system-specific enolase (NSE) in epididymal adipose tissue of Wistar rats in vivo was examined by sensitive enzyme immunoassay methods. Soluble S-100 protein levels in the adipose tissue of 9-12-week-old rats (1.46 +/- 0.19 microgram/mg protein) were decreased to less than 50% of those of controls by serial injection (for 4-7 days) of epinephrine (0.1 mg/day) or norepinephrine (0.15 mg) with, however, little effect on the levels of membrane-bound (pentanol-extractable) S-100 protein. A significant decrease in the soluble S-100 protein levels was observed at 2 h after a single injection of epinephrine (1.04 +/- 0.13 microgram/mg protein). On the other hand, levels of NSE subunit (gamma subunit or 14-3-2 protein) in adipose tissue (0.51 +/- 0.03 gamma gamma-equivalent pmol/mg protein) were increased to 170% of control by serial injection (for 7 days) of epinephrine or norepinephrine with little change of the level of enolase alpha subunit on a mg protein basis. Isoproterenol had no apparent effect on the levels of soluble S-100 protein and NSE subunit. These results suggest that the levels of S-100 protein and NSE in adipose tissue are regulated by catecholamines.

Gamma-Glutamyl transpeptidase of rat liver and hepatoma tissues: an enzyme immunoassay and immunostaining studies.

A sensitive enzyme immunoassay method for rat gamma-glutamyl transpeptidase was developed that was able to quantitate between 5 to 500 ng of the enzyme. The enzyme contents of fetal liver, adult liver, hyperplastic nodules, and various tissues including hepatoma were determined. Normal rat liver contained very little enzyme, while fetal liver and hepatoma tissue contained high levels. The gamma-glutamyl transpeptidase content of the liver was assayed during the process of 3'-methyl 4-dimethyl-aminoazobenzene-induced hepatocarcinogenesis. The content was increased at an early stage, then decreased, and again increased at the stage when hepatoma developed. The change of the enzyme content determined by an enzyme immunoassay was highly consistent with that determined by enzymatic assay. This indicates that the increased activity of the enzyme as previously reported was accompanied by an increased immunoreactive enzyme protein. Immunohistochemical studies on the hyperplastic nodule revealed the presence of gamma-glutamyl transpeptidase in the hepatocyte membrane.

Production of antibodies to calmodulin in rabbits and enzyme immunoassays for calmodulin and anti-calmodulin.

The production of anti-calmodulin antibodies in rabbit was examined with the use of performic acid-oxidized calmodulin from bovine brain or an emulsion of native calmodulin and methylated bovine serum albumin as the antigen. The antibodies in rabbit sera were determined serially by means of a sensitive enzyme immunoassay method that uses a mini-column of goat (anti-rabbit IgG) IgG-coupled Sepharose 4B for separation. The antibodies could be produced in rabbits with either antigen. Although the native calmodulin with methylated bovine serum albumin seemed to be a better antigen, the titer of antiserum was not raised to levels detectable in the double immunodiffusion test. Monospecific antibodies were purified from the antiserum, and a competitive enzyme immunoassay method for the assay of calmodulin was developed with the use of the column-separation technique employed in the assay of anti-calmodulin. The method could determine from 10 ng to 10 micrograms (0.6 pmol to 0.6 nmol) of rat calmodulin and gave no cross-reaction with S-100 protein. Calmodulin contents in rat brain determined by the present method were consistent with those measured by the bioassay using calmodulin-deficient phosphodiesterase.

24] Assay of S-100 protein by an enzyme immunoassay method

Publisher Summary This chapter discusses the rapid purification procedure for S-100 protein and a sensitive enzyme immunoassay method for this same protein. S-100 protein can be readily purified from the bovine brain by the combined use of diethylaminoethyl (DEAE)-Sephadex A-50 column chromatography, followed by N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7)-coupled Sepharose. A sandwich-type enzyme immunoassay system that involved a silicone rubber solid-phase with immobilized S-100 antibody F(ab') 2 fragments and the Fab' fragments labeled with β-D-galactosidase was used for the assay for S-100 protein. The labeling of the antibody Fab' fragments with β-D-galactosidase was carried out by using a bifunctional coupling reagent, N , N '-o-phenylenedimaleimide. The method was sufficiently sensitive for a precise determination of S-I00 protein in the crude extracts of tissue. The chapter shows the amounts of S-100 protein in various rat tissues determined by this sensitive enzyme immunoassay system. The results are expressed as nanograms of bovine brain S-100 protein equivalents per milligram of protein.