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PubMed1991

An increase of intracellular free Ca2+ is essential for spontaneous meiotic resumption by mouse oocytes.

The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium-free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) at a rate comparable to that shown by oocytes cultured in medium containing 1.7 mM Ca2+. The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca2+ chelator) totally prevents GVBD, while purported antagonists of Ca2+ release from intracellular stores, such as 150 uM 8-(N,N-diethylamino)octyl-3-4-5 trimethoxybenzoate (TMB-8) or 300 uM chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre-incubated for 30 min in CFM supplemented with 100 uM TBM-8 plus 0.2 mM dibutyryl-cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained. Our observations suggest that an increase in intracellular free Ca2+ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca2+, release of the cation from internal stores is sufficient to allow meiotic resumption.

Animals↗
PubMed2005

Regulation of capacitative Ca2+ entry by prothoracicotropic hormone in the prothoracic glands of the silkworm, Bombyx mori.

Measurements of Ca2+ influx in Fura-2/AM loaded steroidogenic cells (prothoracic glands; PGs) of the silkworm, Bombyx mori showed that application of the neuropeptide prothoracicotropic hormone (PTTH) can increase the intracellular [Ca2+]i. This PTTH-mediated Ca2+ influx in PG cells had kinetic patterns and pharmacological characteristics similar to those induced by thapsigargin. Namely, it produced increases in intracellular Ca2+ levels only in the presence of extracellular Ca2+, it was blocked by Gd3+ and 2-Aminoethoxydiphenylborate (2-APB), and it was unaffected by several toxins or compounds that block voltage-activated Ca2+ channels. Moreover, the PTTH-stimulated increase of Ca2+ levels was eliminated in the presence of heparin (an IP3 receptor blocker), and by TMB-8 which also blocked any PTTH-dependent increase of ecdysteroid secretion. The PTTH-mediated increase of Ca2+ levels was not affected by the non-hydrolysable GDP analogue, GDPbetaS, an indication that a G protein is not downstream of the PTTH receptor. These results argue strongly in favor of gating by the PTTH receptor of capacitative Ca2+ entry (CCE) channels (or store-operated Ca2+ channels (SOCs)) by a mechanism that does not involve any G proteins but requires the presence of functional IP3 receptors. Because the ability of PTTH to stimulate the [Ca2+]i levels of PG cells was completely mimicked by thapsigargin and exhibited a pharmacological profile similar to CCE mechanisms, we believe that PTTH directly regulates a CCE pathway in PG cells thereby activating a plethora of downstream regulators responsible for ecdysteroid secretion by the PGs of Bombyx mori.

Animals↗
PubMed1988

Different calcium and oxidative metabolic responses in human blood monocytes during exposure to various agonists.

It has become increasingly apparent that certain agonists of phagocytic cells exert their effects by using calcium as an intracellular messenger. Most studies have employed neutrophils for such analyses. We have investigated the requirement of calcium for respiratory burst activation in human blood monocytes by using three triggering agents, concanavalin A (Con A), N-formyl-methionyl-leucil-phenylalanine (FMLP), and phorbol myristate acetate (PMA). There was no overall correlation between the maximal extent of transient rise in cytosolic-free calcium (FMLP greater than Con A greater than PMA) and superoxide (O2-) release (PMA greater than Con A greater than FMLP). PMA, as the most effective agonist of respiratory burst activity, neither increased ionized cytosolic calcium [Ca2+]i nor plasma membrane 45Ca2+ permeability. FMLP evoked increments in [Ca2+]i occurred at lower concentrations than did respiratory burst activation, whereas with Con A a rise in [Ca2+]i and O2- release were parallel. Intracellular Ca2+ release into cytosol was blocked by 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy benzoate, HCl (TMB-8). This caused a parallel inhibition of Con A or FMLP elicited [Ca2+]i transients and O2- release, while burst activity stimulated by PMA was unaffected. Our data thus suggest that a transient rise in [Ca2+]i is essential for Con A and FMLP activation in human blood monocytes. In addition to the calcium signal, other intracellular messengers are required for FMLP stimulation. Respiratory burst activation by PMA appears to act independently of [Ca2+]i transients.

Calcium↗
PubMed2005

Trans- and cis-resveratrol increase cytoplasmic calcium levels in A7r5 vascular smooth muscle cells.

The effects of trans- and cis-resveratrol on cytosolic Ca2+ concentration ([Ca2+]i) were studied using fura-2 in vascular smooth muscle cells (A7r5). Both isomers of resveratrol caused a sustained elevation in [Ca2+]i, cis-resveratrol being significantly more effective than the trans-isomer. The resveratrol-induced increase in [Ca2+]i was significantly potentiated by the previous application of low concentrations of thapsigargin, partially inhibited by nifedipine or Ni2+, and not affected by SKF 96365. In the absence of extracellular Ca2+, both isomers of resveratrol induced a transient, slow increase in [Ca2+]i, which was inhibited by the previous depletion of intracellular stores with thapsigargin and completely blocked by preincubation with TMB-8, an inhibitor of intracellular calcium release. Reintroduction of Ca2+ in the external solution after the resveratrol-induced release of Ca2+ activated the Ca2+ influx through store-operated calcium channels. The resveratrol-induced increase in [Ca2+]i in the absence of extracelullar Ca2+ partially reduced the increase in [Ca2+]i evoked by the subsequent application of thapsigargin. Our results suggest that trans- and cis-resveratrol induce a depletion of Ca2+ from the same intracellular stores released by thapsigargin and subsequent capacitative influx of Ca2+. Additionally, a direct activation of transmembrane Ca2+ influx through another type of channel may be also implicated.

Animals↗
PubMed2002

Overproduction of pentose phosphate pathway enzymes using a new CRE-loxP expression vector for repeated genomic integration in Saccharomyces cerevisiae.

Two new vectors are described, the expression vector pB3 PGK and the CRE recombinase vector pCRE3. The pB3 PGK has a zeocin-selectable marker flanked by loxP sequences and an expression cassette consisting of the strong PGK1 promoter and the GCY1 terminator. The S. cerevisiae genes RKI1, RPE1, TAL1 and TKL1 were cloned in pB3 PGK and integrated in the locus of the respective gene, resulting in overexpression of the genes. S. cerevisiae TMB 3026, simultaneously overexpressing the RKI1, RPE1, TAL1 and TKL1 genes, was created by successive integrations and removal of the loxP-zeocin-loxP cassette using pCRE3. The 2mu-based pCRE3 carries the aureobasidin A, zeocin and URA3 markers. pCRE3 proved to be easily cured without active counter-selection. The zeocin marker is present on both the pB3 PGK and on pCRE3, so that screening for zeocin sensitivity indicates both chromosomal marker loss and loss of the pCRE3 vector. This feature saves time, since only one screening step is needed between successive chromosomal integrations. Marker recycling did not lead to increased zeocin resistance, indicating that the zeocin marker could be used for more than four rounds of transformation. The use of the CRE/loxP system proved to be a practical strategy to overexpress multiple genes without exhausting available markers.

Gene Expression Regulation, Fungal↗
PubMed1997

Thrombin regulates interleukin-6 synthesis through phosphatidylcholine hydrolysis by phospholipase D in osteoblasts.

We previously reported that thrombin stimulates Ca2+ influx and activates phosphatidylcholine-hydrolyzing phospholipase D in osteoblast-like MC3T3-E1 cells. In this study, we investigated the effect of thrombin on interleukin-6 (IL-6) synthesis in these cells. Thrombin stimulated IL-6 synthesis dose-dependently in the range between 0.01 and 1 U/ml. The depletion of extracellular Ca2+ by EGTA suppressed the thrombin-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, also inhibited the IL-6 synthesis by thrombin. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, enhanced the IL-6 synthesis by thrombin. Calphostin C, a highly potent and specific inhibitor for protein kinase C, significantly amplified the IL-6 synthesis by thrombin. The thrombin-induced IL-6 synthesis was enhanced in PKC down-regulated MC3T3-E1 cells. These results strongly suggest that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilization mainly from extracellular space in osteoblasts, and that the IL-6 synthesis by thrombin is regulated due to thrombin-activated protein kinase C through phosphatidylcholine-hydrolyzing phospholipase D.

Animals↗
PubMed1995

Pentoxifylline augments but does not antagonize TNF alpha-mediated neuroblastoma cell differentiation: modulation of calcium mobilization but not cAMP.

We described here that pentoxifylline (PTX), which is well known to counteract tumor necrosis factor alpha (TNF alpha)-mediated inflammatory responses, augmented TNF alpha-induced neuroblastoma cell differentiation in conjunction with growth inhibition and cell-cycle arrest in G1 phase. PTX also enhanced TNF alpha-induced down-regulation of acetylcholine-mediated [Ca2+]i mobilization in neuroblastoma cells. Furthermore, we found that addition of cAMP failed to induce neuroblastoma cell differentiation, whereas blockade of [Ca2+]i mobilization by 8-(N,N-diethyl-amino)octyl-3,4,5-trimethoxybenzoate HCl (TMB-8, 10 microM) did induce neuroblastoma cell differentiation. Taken together, these results indicated that PTX possessed a novel signal transduction, down-regulation of [Ca2+]i mobilization, to augment but not counteract TNF alpha-mediated functions.

Acetylcholine↗
PubMed1995

Extracellular calcium causes the release of calcium from intracellular stores in chick osteocytes.

We have recently demonstrated that a rise in the extracellular divalent cation concentration induces a rapid elevation of cytosolic calcium in chick osteocytes. Here, we demonstrate that cytosolic calcium elevation that occurs in osteocytes on exposure to elevated extracellular calcium is independent of membrane voltage and is insensitive to modulation by organic calcium channel modulators, namely, BAY K 8644, nicardipine, and nifedipine. However, the calcium elevation was sensitive to modulation by an intracellular calcium antagonist, TMB-8, suggesting that the cytosolic calcium elevation was due to mobilization of this cation from an intracellular store.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1997

Role of intracellular calcium in the angiotensin II-mediated tyrosine phosphorylation and dephosphorylation of PLC-gamma 1.

Angiotensin II induces the rapid temporal tyrosine phosphorylation and activation of phospholipase C-gamma 1 (PLC-gamma 1) and the elevation of intracellular calcium levels. To investigate the relationship of these intracellular signaling events, rat aortic smooth muscle cells were treated with the calcium chelator BAPTA-AM, the calcium channel blocker verapamil, the intracellular calcium antagonist TMB-8, and the calcium ionophore ionomycin. The effects of these agents on PLC-gamma 1 tyrosine phosphorylation were then measured. We found that treatment of these cells with the calcium inhibitors augmented the basal level of PLC-gamma 1 tyrosine phosphorylation, without changing the peak level of tyrosine phosphorylation induced by angiotensin II. The rapid dephosphorylation of PLC-gamma 1 that follows angiotensin II stimulation was prevented by these calcium antagonists. In contrast, angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 was inhibited by ionomycin. These results suggest that the angiotensin II-induced tyrosine phosphorylation of PLC-gamma 1 is calcium-independent, while the dephosphorylation is calcium-dependent.

Angiotensin II↗
PubMed1998

Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin5 water channel via cytosolic Ca2+ elevation in rat parotid glands.

To evaluate the role of aquaporin5 (AQP5) in salivary secretion induced by cholinergic stimulation, the alteration of the distribution of AQP5 in rat parotid tissues induced by acetylcholine (ACh) was studied by immunobolt analysis. The treatment of the tissues with ACh within 1 min induced the translocation of AQP5 from intracellular membranes (ICM) to apical membranes (APM), but that for more than 5 min resulted in the converse translocation from APM to ICM. The ACh-induced increase in the amount of AQP5 in APM was inhibited by atropine, p-F-HHSiD and TMB-8, but not by methoctramine, staurosporine or H-7. The calcium ionophore A-23187 alone stimulated the translocation of AQP5 between APM and ICM. These results indicated that ACh acted on M3 muscarinic receptors and induced the translocation of AQP5 between ICM and APM, and that the cytosolic Ca2+ elevation by ACh may play a key role in this translocation in rat parotid glands.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Glibenclamide induces apoptosis through inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and intracellular Ca(2+) release in HepG2 human hepatoblastoma cells.

Glibenclamide, an inhibitor of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels, induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoblastoma cells. Glibenclamide increased intracellular Ca(2+) concentration, which was significantly inhibited by Ca(2+) release blockers dantrolene and TMB-8. BAPTA/AM, an intracellular Ca(2+) chelator, and the Ca(2+) release blockers significantly inhibited glibenclamide-induced apoptosis. Glibanclamide also increased intracellular Cl(-) concentration, which was significantly blocked by CFTR Cl(-) channel activators levamisole and bromotetramisole. These activators also significantly inhibited both intracellular Ca(2+) release and apoptosis induced by glibenclamide. The expression of CFTR protein in the cells was confirmed by Western blot analysis. These results suggest that glibenclamide induced apoptosis through inhibition of CFTR Cl(-) channels and intracellular Ca(2+) release and that this protein may be a good target for treatment of human hepatomas.

Apoptosis↗
PubMed1999

The putative morphogen, DIF-1, of Dictyostelium discoideum activates Akt/PKB in human leukemia K562 cells.

The differentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces stalk-cell formation in the lower eukaryote Dictyostelium discoideum. This molecule has been shown to inhibit cell growth and induce erythroid differentiation in human leukemia K562 cells. In the present study, to clarify the mechanism of the actions of DIF-1, we examined the effect of DIF-1 on Akt/protein kinase B (PKB) in K562 cells. Akt/PKB is a serine/threonine kinase that plays a pivotal role in the regulation of cell survival and differentiation in a variety of cells. A nonphosphorylated (inactive) form of Akt/PKB was ordinarily expressed in K562 cells. However, Akt/PKB was phosphorylated and potently activated within several hours of incubation with 5-30 microM DIF-1, and this activation was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). Calcium-increasing agents thapsigargin and A23187 also activated Akt/PKB slightly, which was inhibited by wortmannin. By contrast, calcium-reducing agents TMB-8 and EGTA together with A23187 inhibited the DIF-1-induced activation of Akt/PKB. PMA (PKC activator) also activated Akt/PKB but this activation was not inhibited by wortmannin. DIF-1 exhibited no marked effect on the activation of PKCalpha, beta, and gamma, which were activated by PMA. These results indicate that DIF-1 activates Akt/PKB possibly via cytosolic calcium and subsequent activation of PI3-kinase and also that PMA activates Akt/PKB in a PI3-kinase-independent manner.

Androstadienes↗
PubMed1999

alpha(1)-adrenoceptor-induced trafficking of aquaporin-5 to the apical plasma membrane of rat parotid cells.

Incubation of rat parotid tissue with 10 microM epinephrine resulted in a transient and marked trafficking of aquaporin-5 (AQP5) from intracellular membranes to the apical plasma membrane (APM) that was maximal at 1 min. This effect of epinephrine was mimicked by phenylephrine, but not by clonidine, dobutamine, or salbutamol, and it was inhibited by phentolamine, but not by propranolol. Furthermore, the epinephrine-induced trafficking of AQP5 was inhibited by phospholipase C inhibitor U73122 as well as dantrolene and TMB-8, both of which inhibit the release of Ca(2+) from intracellular stores. Cytochalasin D and tubulozole-C also inhibited this action of epinephrine. These results indicate that epinephrine, acting at alpha(1)-adrenoceptors, induces the trafficking of AQP5 to the APM by triggering the release of Ca(2+) from intracellular stores through inositol 1,4,5-trisphosphate and ryanodine receptors. In addition, the potent involvement of the cytoskeleton was shown in the epinephrine-induced trafficking of AQP5.

Adrenergic alpha-Agonists↗
PubMed1999

Vasopressin stimulates the induction of heat shock protein 27 and alphaB-crystallin via protein kinase C activation in vascular smooth muscle cells.

In the present study, we examined the effect of vasopressin on the induction of the low-molecular-weight heat shock proteins heat shock protein 27 (HSP27) and alphaB-crystallin in an aortic smooth muscle cell line, A10 cells. Vasopressin induced a time-dependent accumulation of HSP27 and alphaB-crystallin. The stimulatory effects of vasopressin were dose-dependent over the range 0.1 nmol/L to 0.1 micromol/L. The EC50 values for vasopressin were 2 (HSP27) and 4 nmol/L (alphaB-crystallin). Vasopressin induced increases in the levels of the mRNAs for HSP27 and alphaB-crystallin. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, induced an accumulation of HSP27 (EC50, 20 nmol/L) and alphaB-crystallin (EC50, 2 nmol/L). In contrast, 4alpha-phorbol 12,13-didecanoate, a non-PKC-activating phorbol ester, had no such effect. Staurosporine and calphostin C, inhibitors of PKC, significantly reduced the vasopressin-induced accumulation of HSP27 and alphaB-crystallin as well as that induced by TPA. BAPTA/AM and TMB-8, inhibitors of intracellular Ca2+ mobilization, significantly reduced the vasopressin-induced accumulation of HSP27 and alphaB-crystallin. These results strongly suggest that vasopressin stimulates the induction of HSP27 and alphaB-crystallin via PKC activation in vascular smooth muscle cells and that this effect of vasopressin is dependent on intracellular Ca2+ mobilization.

Animals↗
PubMed1998

Transmembrane Ca2+ fluxes associated with zoospore encystment and cyst germination by the phytopathogen phytophthora parasitica

Phytophthora spp. can infect plants within 30 min of a zoospore arriving at a host surface. The infection sequence from zoospore encystment to cyst germination and host penetration seems to be largely preprogrammed and dependent on calcium signaling. Here we present evidence that the encystment process leading to autonomous germination in Phytophthora parasitica may be coordinated by sequential net Ca2+ fluxes across the cell membrane, which we assessed by fluorimetry of the probe fura-2 in zoospore bathing medium. Encystment was associated with a large initial net Ca2+ influx, which was abolished by the channel blockers La3+ and verapamil. The net influx was followed by a larger, progressive net Ca2+ efflux over 20-30 min, which was associated with cyst germination but was inhibited by TMB-8, implicating Ca2+ release from intracellular stores. The effects of inhibitors were overcome by high (1 mM) exogenous Ca2+, enabling encystment and germination. Copyright 1998 Academic Press.

Journal Article↗
PubMed2000

Ca2+ regulation of Phyllosticta ampelicida pycnidiospore germination and appressorium Formation.

Phyllosticta ampelicida conidia germinate only after making contact with and attaching to a substratum. Previous studies suggested a role for Ca2+ in this process. A Ca2+ buffering system was used to control the external free Ca2+ concentration. Both germination and appressorium formation were reduced or abolished with low Ca2+ (less than or equal to nanomolar levels) but were nearly 100% at millimolar levels of Ca2+. Germination initiation required Ca2+ within 10-25 min after the spore made contact with the substratum. Appressorium initiation required Ca2+ 90-120 min following initial contact. Ca2+ channel blockers nicardipine and lanthanum abated spore development. TMB-8, a blocker of internal Ca2+ channels, reduced both developmental events. Gadolinium, a putative stretch-activated Ca2+ channel blocker, abolished both developmental events at nanomolar levels. Calmodulin antagonists, compounds R-24751 and 48/80, abated spore development at micromolar levels. Together, these results suggest that Ca2+ signaling is involved in both germination and appressorium formation in P. ampelicida pycnidiospores.

Ascomycota↗
PubMed2000

Pancreatitis-associated ascitic fluid increases intracellular Ca(2+) concentration on hepatocytes.

BACKGROUND: We have reported that pancreatitis-associated ascitic fluid (PAAF) contains a cytotoxic factor(s) inducing apoptosis on renal tubular cells and hepatocytes. It has been suggested that elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is associated with the development of cell damage and apoptosis. METHODS: To clarify the mechanism of hepatocellular injury in acute pancreatitis, the effect of PAAF on hepatocyte [Ca(2+)](i) was investigated. Primary cultures of rat hepatocytes were loaded with Fura-2/acetoxymethyl, and the changes of [Ca(2+)](i) were measured using spectrofluorometer. RESULTS: The baseline of hepatocyte [Ca(2+)](i) was 172 +/- 17 nM. [Ca(2+)](i) increased from 1 min after the addition of PAAF in a dose-dependent manner. Fractionation of PAAF revealed only one fraction (molecular weight >/= 5 x 10(4)) possessed both [Ca(2+)](i) elevation activity and cytotoxic activity. Neither 8-(N,N-diethyl-amino) octyl-3,4,5-trimethoxybenzoate (TMB-8) nor thapsigargin inhibited the PAAF-evoked [Ca(2+)](i) elevation. Chelation of extracellular Ca(2+) by ethylene glycol bis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) prevented the elevation of [Ca(2+)](i), but verapamil did not prevent it. Platelet-activating factor antagonist (TCV-309) blocked the PAAF-elicited [Ca(2+)](i) elevation. Pancreatitis-associated serum also increased hepatocyte [Ca(2+)](i). Moreover, PAAF increased [Ca(2+)](i) on Madin-Darby canine kidney cells in a dose-dependent manner. CONCLUSIONS: These results suggest that the dramatic elevation of hepatocyte [Ca(2+)](i) due to PAAF may be closely related to the hepatocellular injury in severe acute pancreatitis and that platelet-activating factor may play a pivotal role in increasing hepatocyte [Ca(2+)](i). Elevation of [Ca(2+)](i) in various cells may be involved in the mechanism of multiple organ failure in this disease.

Acute Disease↗
PubMed2000

Antibodies and immunoassays.

As a glycoprotein hormone, human chorionic gonadotropin (hCG) is not a single molecular entity. This term comprises not only the bioactive heterodimer hCG but also an array of molecular protein backbone and glycosylation variants, such as its free beta (hCGbeta) and alpha (hCGalpha) subunits and clipped, cleaved, terminally differently sialylated, and overglycosylated forms. This heterogeneity places great demands on selective detection systems for hCG-derived molecules. Measurement of hCG and/or its derivatives is highly dependent on the selection of target molecules and the natural variability of hCG in the specimens analyzed. Monoclonal antibody (mAb)-based immunoassays are still the state-of-the-art technique for both clinical and research applications but a major problem is the different extents of recognition of hCG variants by mAbs used in different immunoassays. On the whole, construction of sandwich-type assays obviously must take into consideration mAb characteristics, such as main and fine specificities, cross-reactivities, epitope locations and compatibilities, overlap and overhang in specificities (pairs of mAbs), and, finally, overspecificity. Consequences of overhang and overlap in antigen recognition of coating and detection mAb specificities are nondesirable assay cross-reactions and competitive interference by antigenic variants. The general agreement on the most favorable assay design is contrasted by the variety of isotopic and nonisotopic detection systems in current use. The immunoenzymometric assay (IEMA) technique is hampered by a relatively small measuring range and limited sensitivity. By measuring substrate absorption values off the absorption maximum, the measuring range of any IEMA can be extended significantly, as shown for 3,3',5, 5'-tetramethylbenzidine (TMB), without jeopardizing assay characteristics. Sensitivity of the IEMA can be enhanced by modifying the horseradish peroxidase (HRPO) labeling technique by using highly purified mAb preparations and higher-input HRPO/mAb ratios. We have also compared the assay characteristics of time-resolved fluoroimmunoassay (IFMA), IEMA, immunoradiometric assay (IRMA), and competitive radioimmunoassay (RIA) based on identical mAbs. Reasons for the observed superiority of the IFMA lie in its concept of signal detection and the high specific labeling of the detection mAb which on a molar basis can be up to 7-fold and 15-fold higher compared with (125)I and HRPO, respectively.

Antibodies, Monoclonal↗