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PubMed1994

Epidermal growth factor enhances proliferation, migration, and invasion of follicular and papillary thyroid cancer in vitro and in vivo.

The prognosis of patients with follicular (FTC) and papillary (PTC) thyroid cancer depends on age and the size and extent of the tumor. Differentiated thyroid cancers bind more epidermal growth factor (EGF) than normal thyroid tissue, but the role of EGF in the proliferation and invasion of thyroid cancer is unknown. We investigated the effects of EGF on growth, migration, and invasion in a follicular thyroid cancer that metastasized to cervical lymph nodes and the lung (FTC 133, primary; FTC 236, lymph node; and FTC 238, lung metastasis) and in a papillary thyroid cancer (PTC-UC3). As measured by the formazan method (dimethylthiazol-diphenyltetrazolium bromide), EGF caused a dose- and time-dependent increase in the growth of FTC 133 and PTC-UC3 by 25%, but its stimulatory effect on growth of the metastatic FTC subclones was smaller (FTC 236, 14%; FTC 238, 8%; P < 0.001). EGF also enhanced the ability of all cell lines to migrate (through 8-microns pore membranes without Matrigel) or invade (membranes with Matrigel). Migration of FTC 133 was enhanced from 86% migrated tumor cells to 95% after 72 h (P < 0.02). Again, stimulation by EGF was lower in FTC 236 and FTC 238. EGF increased migration in PTC-UC3 from 49% to 58%. EGF stimulated invasion of FTC 133 from 17.5% to 24.9%. In the absence of EGF, FTC 238 was the most invasive tumor, but, again, the EGF stimulatory effect was less pronounced than in the primary tumor. EGF stimulated the invasion of PTC-UC3 from 10.9% to 14.3% (P < 0.03). EGF also stimulated the growth of thyroid cancer xenografts in nude mice. Although all FTC cell lines were 100% tumorigenic in nude mice, PTC-UC3 was less tumorigenic. However, after sc inoculation of EGF-pretreated tumor cells, 7 of 10 animals developed tumors (mean size, 2.3 cm3) compared to 2 of 10 animals (mean size, 1.4 cm3) in the control group (P < 0.02). In summary, EGF stimulates the growth and invasion of differentiated thyroid cancer cells in culture and in nude mice. Escape from growth factor control, such as in FTC 236 and FTC 238, may be an important step in the development of metastatic thyroid cancer.

Adenocarcinoma, Follicular↗

PubMed1995

The measurement of growth hormone bioactivity in patient serum using an eluted stain assay.

We have adapted the MTT-ESTA bioassay for human GH (hGH) to measure the lactogenic bioactivity of the hormone in human serum. This highly quantitative in vitro colorimetric bioassay is based upon the reduction of a tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT), to its formazan by lactogen-activated Nb2 cells. Relatively high concentrations of human serum (1-10%) modified responses to the hormone in a complex manner. As the serum effects varied between samples, it proved impossible to adapt the bioassay by the conventional approach of using a lactogen-depleted serum as a representative matrix. However, as the Nb2 cells were exceptionally sensitive to hGH, the serum effects could be diluted out. We adopted a dilution strategy by which all samples of human serum were included in the bioassay at a concentration of 0.625% or less. A valid assay was obtained, as judged by the criteria of parallelism between diluted samples and hGH standards, and recoveries of spiked samples that were close to 100%. Hormonal specificity was achieved with the use of a highly specific anti-PRL antiserum. A within-assay precision of between 2-5% over the dose range of 0.03-0.96 microgram hGH/L was attained. As only highly diluted samples could be used, the sensitivity of the clinical bioassay was 1.2-2.4 micrograms hGH/L. The between-assay precision was estimated to be 11% and 9% at initial hGH concentrations in serum of 4.8 and 19.2 micrograms hGH/L, respectively. By exploiting the high sample capacity of the eluted stain bioassay system, we followed the changes in bioactivity and immunoactivity of hGH in multiple timed samples after stimulation of hGH secretion in an adult by GHRH. Systematic and progressive changes were observed in the bioactive/immunoactive ratios. Analogous changes were observed after insulin-induced hypoglycemia in a child with short stature. We speculate that the changes in the bioactive/immunoactive ratios reflect alterations in the proportions of the isoforms of hGH in the circulation after acute stimulation.

Animals↗

PubMed1996

Metabolic activation of islet cells improves resistance against oxygen radicals or streptozocin, but not nitric oxide.

In the present study we investigated the effect of metabolic activation on the susceptibility of isolated rat pancreatic islet cells to the alkylating beta-cell toxin streptozocin (SZ), reactive oxygen intermediates (ROI), and nitric oxide (NO). The latter two represent physiologically occurring mediators involved in the autoimmune destruction of islet cells. ROI were generated by the enzyme xanthine oxidase, and NO was released from sodium nitroprusside. During 18 h of culture at a physiological glucose concentration (5 mmol/L), 75% of the islet cells were lysed by SZ, 81% by ROI, and 74% by NO, as determined by the trypan blue exclusion assay. Increasing concentrations of glucose or the nonnutrient stimulators theophylline and glibenclamide dose-dependently reduced SZ- and ROI-mediated islet cell lysis. In the presence of 29 mmol/L glucose, 5.5 mmol/L theophylline, or 10 micrograms/mL glibenclamide, SZ-induced lysis was reduced to 15%, 22%, or 15%, and ROI-induced lysis was reduced to 20%, 34%, or 15%, respectively. In contrast, stimulation by glucose, theophylline, or glibenclamide did not improve resistance against NO. The protection against SZ and ROI was associated with preserved mitochondrial activity, as determined by the ability of the islet cells to convert the tetrazolium salt 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide into its formazan. Elevation of the glucose concentration from 5.5 to 29 mmol/L increased the residual mitochondrial activity from 45% to 80% in SZ-exposed islet cells and from 21% to 78% in ROI-exposed cells. Conversely, the lack of protection against NO correlated with no preservation of mitochondrial activity in the presence of high concentrations of glucose, theophylline, or glibenclamide. In conclusion, our results show that metabolic stress does not render islet cells more susceptible to inflammatory insults in vitro. Rather, an increased mitochondrial energy supply improves the resistance against SZ and ROI, whereas the toxicity of NO was independent of islet cell activity.

Animals↗

PubMed2005

Cytotoxic, antiviral (in-vitro and in-vivo), immunomodulatory activity and influence on mitotic divisions of three taxol derivatives: 10-deacetyl-baccatin III, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate) and N-benzoyl-(2'R,3'S)-3'-phenylisoserine.

The aim of this study was to evaluate cytotoxic, antiviral (in-vitro and in-vivo) and immunomodulatory activity, as well as the influence on mitotic division, of three taxol derivatives representing modified parts of its molecule: 10-deacetyl-baccatin III, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate) and N-benzoyl(2'R,3'S)-3'-phenylisoserine. The cytotoxicity of the compounds, assessed by the formazane method, was relatively low, with a 50% cytotoxic concentration (CC50)>500 microg mL-1. Moreover, all tested compounds inhibited Herpes simplex type 1 virus (HSV-1) replication in non-cytotoxic concentrations in-vitro. Selectivity indices were in the range 9.5-46.7. Anti-HSV-1 activity of the compounds may be associated with their influence on mitotic division. All of the compounds decreased the number of cell divisions. Mitotic indices ranged from 40/1000 (4.0%) to 62/1000 (6.2%). One compound, 10-deacetyl-baccatin III, influenced the growth of tumours induced in mice by infection with Moloney murine sarcoma virus. The effect of the tested compounds on T lymphocyte proliferation was evaluated by measurement of the activity of tritiated thymidine incorporated into DNA of dividing cells. One compound, methyl (N-benzoyl-(2'R,3'S)-3'-phenylisoserinate), inhibited T lymphocyte proliferation. This paper demonstrates that modified parts of the taxol molecule possess various types of biological activity in-vitro and in-vivo. Further experiments, focused on revealing their mechanisms of action, are necessary.

Adjuvants, Immunologic↗

PubMed1992

Enhancement of radiosensitivity by tamoxifen in C6 glioma cells.

The antiestrogen drug tamoxifen, which is used extensively in the treatment of breast cancer, has also been reported to inhibit the proliferation of some estrogen receptor-negative cell lines, including malignant glioma in vitro. To explore the possible role of tamoxifen in the treatment of malignant glioma, we have investigated its effects on cell growth and radiosensitivity in C6 glioma cells using a colony-forming assay and a tetrazolium-formazan growth rate assay. Pretreatment of C6 cells with tamoxifen resulted in dose-dependent inhibition of cell growth and enhancement of the antitumor effects of ACNU and irradiation. The radiosensitivity of the treated cells was enhanced by the administration of 5 mumol/L of tamoxifen either before and during irradiation or continuously before, during, and after irradiation [37% survival dose (Do) = 2.68 +/- 0.19 and 2.64 +/- 0.04 Gy, respectively, P < 0.01)], as compared with controls (Do = 3.79 +/- 0.25 Gy). In addition, protein kinase C activity was also inhibited by tamoxifen at the concentration in which the radiosensitivity was augmented in C6 cells. Taken together, our results demonstrate a synergistic effect of tamoxifen with radiation on intracellular damage in C6 glioma cells, which may in part be due to the inhibition of protein kinase C, suggesting that tamoxifen serves as a useful agent in combination therapy of glioma.

Brain Neoplasms↗

PubMed1993

Diagnosis of drug allergy by the lymphocyte stimulation test with the MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay.

The lymphocyte stimulation test (LST) is one of the most useful laboratory tests for the identification of allergy to a specific drug. The present study was conducted to examine utility of the LST using the [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) assay as a method for diagnosing drug allergy. The basic experimental conditions for the MTT assay were determined by using CTLL cells from human T-lymphoma, dependent on IL-2, and human peripheral blood lymphocytes. The amount of MTT needed was 0.5 mg per well and the peak maximum of MTT formazan was close to 565 nm. The blastogenesis of the lymphocytes was expressed as a Stimulation Index (SI). The value of the SI was almost constant at cell numbers ranging from 5 x 10(5) to 1 x 10(6) cells/ml. The SI of some drugs using normal peripheral blood lymphocytes was less than 1.3. We used the MTT assay in the LST to examine the allergenicity of 83 drugs which were used in 43 cases of suspected drug-induced hepatitis. The range of SI was 0.92-2.02. An SI of 1.2 or more was seen in 62.8% of cases, 1.3 or more in 51.2% of cases, and 1.4 or more in 41.9% of cases. There were 26 drugs with SI greater than 1.3. Seven of these were antibiotics, while the rest included antihypertensives, analgesics, psychotropics, antiallergics and antiepileptics. This method is extremely well suited for the diagnosis of drug allergy in a clinical situation. The greatest benefit of the MTT assay is that the complete test procedures can be carried out in a general laboratory.

Adult↗

PubMed1996

A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet.

Cell viability and in vitro cytotoxicity assay methods were developed using a combination of dyes, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), neutral red (NR) and crystal violet (CV), with HeLa cells as a bioindicatior. As WST-1 produces a highly water soluble and non-cytotoxic formazan dye, ti allows each assay to be carried out in one culture dish. The combined cell viability assay using WST-1, NR and CV gave an absorbance that correlated linearly with the number of cells over the range 1000 to 50,000 cells/well. The combined assay was applied to the evaluation of IC50 values for sodium dodecyl sulfate as a model toxicant, which yielded similar values to those obtained with each assay independently.

Cell Survival↗

PubMed2006

Effects of poly-L-arginine on the permeation of hydrophilic compounds through surface ocular tissues.

We investigated whether poly-L-arginine (PLA) could improve permeation of the hydrophilic compounds, FITC-labeled dextran (MW 3800, FD-4) and pyridoxamine, through ocular surface tissues. Samples of cornea, conjunctiva, or conjunctiva/sclera composite from Japanese white rabbits were mounted in Ussing chambers to measure FD-4 and pyridoxamine permeation and transepithelial electric resistance (TEER). The integrity and viability of the conjunctiva were assessed by chronological TEER monitoring and MTT assay, respectively. The permeability coefficient (Papp) of FD-4 in the cornea, conjunctiva, and conjunctiva/sclera composite was increased by the addition of PLA (MW 38 kDa, PLA (50) at 0.1 mg/ml by 6.81-, 9.78-, and 7.91-fold, respectively. The Papp of pyridoxamine was also increased in the presence of PLA (50) by 7.98-, 4.67-, 8.31-fold, respectively. A corresponding reduction in TEER was observed in all tissues. However, the reduced TEER in the case of the conjunctiva had recovered to ca. 70% 120 min after replacing the mucosal fluid with fresh bicarbonated Ringer's solution. MTT assay results indicated that treatment of the conjunctiva with 0.1 mg/ml PLA (50) did not change the production of formazan compared to that without PLA (50), indicating that the conjunctival viability is not significantly affected by PLA (50). Our findings suggest that PLA may be useful in promoting the ocular delivery of hydrophilic drugs without producing significant epithelial damage.

Animals↗

PubMed1993

Swine interleukin 2 activity produced by mesenteric lymph node cells.

Swine interleukin 2 (IL-2) activity was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay using murine IL-2 dependent cell line (CTLL-2). The culture supernatant of mesenteric lymph node (MLN) cells stimulated with phytohemagglutinin-P (PHA) induced a generation of MTT formazan in a dose-dependent manner, suggesting dose-dependent proliferation of CTLL-2. The maximal swine IL-2 activity was observed in the culture of MLN cells at 1 to 2 x 10(7) cells/ml when stimulated with 20 to 40 micrograms/ml PHA for 48 hr. Based on these findings, a large culture of MLN cells to prepare swine IL-2 were performed under the following condition; cell concentration of 1 x 10(7) cells/ml, PHA concentration of 20 micrograms/ml, a culture scale of 200 to 400 ml, and a stirring speed of 30 rpm. Swine IL-2 activity was detected from 4 hr after PHA stimulation, and rapidly increased until 16 hr. Almost maximal IL-2 activity in stirring culture was observed at the incubation of 20 hr. Swine IL-2 was partially purified by Sephacryl S-200 gel filtration and the estimated molecular weight was about 32 kD based on the peak of IL-2 activity. The pI value of swine IL-2 was estimated to be approximately pH 5.3. Swine IL-2 was sensitive to acid (pH 3.2) or alkaline (pH 10.5), 4 or 8 M urea, trypsin, and the heating at 70 degrees C. These physico-chemical properties of swine IL-2 was similar to those of human, murine or feline IL-2.

Animals↗

PubMed1990

Morphologically and functionally distinct subpopulations of steroidogenic cells in corpora lutea during pregnancy in rats.

On days 5, 10, 15 and 20 of pregnancy, rat corpora lutea (CL) were dissected and dissociated into single cell suspensions by enzyme treatments. The suspended luteal cells were allowed to sediment in a BSA gradient at 4 degrees C for 3.5 hours. Five fractions were collected from the top (Fraction (Fr.) 1) to the bottom (Fr. 5) of the gradient. Cells were incubated in serum-free DME-F12 for 20 hours with or without hCG (100 ng/ml) to test them functionally, and the accumulation of progesterone and testosterone was determined by radioimmunoassay. To assess 3,3-hydroxysteroid dehydrogenase (HSD) activity, a histochemical suspension-staining procedure was used. Cells were examined by light microscopy, and the percentage of cells containing dark blue formazan deposits and their diameters were determined in at least 40 microscopic fields. The number of cells staining for 3 beta-HSD did not vary by day 15 but decreased from 141.6 +/- 16.5 X 10(3) cells/CL on day 15 to 113.8 +/- 13.2 X 10(3) cells/CL on day 20 of pregnancy. However, 3 beta-HSD-positive cells maintained the same levels of progesterone secretion until the advent of luteolysis, then they increased in size progressively throughout pregnancy. In BSA gradients, the relatively larger 3 beta-HSD-positive cells migrated faster than the smaller 3 beta-HSD-positive cells on each day of pregnancy. The diameters of 3 beta-HSD-positive cells differed significantly in Frs. 2, 3, 4 and 5 on days 15 and 20 of pregnancy. On day 15 of pregnancy, less progesterone accumulated in wells containing 3 beta-HSD-positive cells from Fr. 2 (mean diameter; 24.96 microns) than from Fr. 3, Fr. 4 and Fr. 5 (mean diameters; 27.20, 30.79 and 31.28 microns, respectively) but the Fr. 2 cells responded more to hCG stimulation. Fr. 2 also showed a higher ratio of testosterone accumulation to progesterone accumulation than the other fractions. The response to hCG stimulation of cells in Fr. 2 tended to be higher than that in Fr. 3 on day 20 of pregnancy. These data suggest that the steroidogenic rat luteal cells are comprised of morphologically and functionally different cell types after day 15 of pregnancy. No stimulating nor inhibiting effects were observed in co-incubation of cells from Fr. 2 with cells from Fr. 3 or Fr. 4 on days 15 and 20 of pregnancy.

3-Hydroxysteroid Dehydrogenases↗

PubMed1975

Superoxide dismutase micro assay in biological material.

A micro assay for the rapid and convenient determination of superoxide dismutase activity in limited amounts of biological material was devised and successfully employed. The combination of the formazan derivative colour formation induced by reaction of O2theta with nitroblue tetrazolium and a suitable analytical polyacrylamide gel electrophoresis system was used. It was possible to show that the reactivity of soluble superoxide dismutases in polyacrylamide gels was proportional to the enzyme concentrations employed. Bovine erythrocyte Cu, Zn-superoxide dismutase (EC 1.15.1.1) (erythrocuprein) served as a standard throughout. To measure the degree of superoxide dismutase activity, a gel-scanning apparatus was usedThe integrated scanning curve of the unstained portions of the gel proved linearly proportional to the logarithm of the superoxide dismutase activity in the range between 10(-12) and 7 X 10(-11) mol of the bovine enzyme. Although the absolute integral is dependent on the different staining conditions, the slope of the superoxide dismutase calibration curve is highly reproducible. Superoxide dismutase added to crude liver and brain homogenates could be fully detected using this assay. Thus, biological material including nucleic acids, enzymes, lipids etc. do not inhibit this reaction.

Animals↗

PubMed1996

Determination of choline dehydrogenase activity along the rat nephron.

A radioenzymatic microassay was developed to quantitate choline dehydrogenase activity in single microdissected nephron segments. This enzyme is the rate limiting step in the biosynthesis of betaine, which serves as an intracellular osmoregulatory organic solute in mammalian kidney. The enzyme localized in renal mitochondrial inner membrane forms betaine aldehyde, which in the assay is converted to betaine by oxidative treatment. A histochemical procedure based on the formazan detection of tetranitroblue tetrazolium chloride was applied in parallel. The results show that activities in proximal convoluted and straight tubules are more than 5 times higher (21 to 25 pmol h-1 mm tubule-1) compared to distal nephron segments with no significant differences along the proximal tubule. Along the osmotic gradient from the outer medullary towards the papillary structures enzyme activities increased in ascending limbs of Henle's loop and collecting tubules. Collecting ducts showed two times higher activities than ascending loop segments when corrected for tubular cell volumes. The quantitative data were confirmed by the histochemical procedure. The results allow for the conclusion that betaine synthesis is sufficient to build up renal betaine, but cannot explain the distribution pattern of betaine along the corticopapillary axis. Additional mechanisms like intrarenal and tubular transport have to be postulated.

Alcohol Oxidoreductases↗

PubMedDate not supplied

The antiproliferative agents trans-bis(resorcylaldoximato)copper(II) and trans-bis(2,3,4-trihydroxybenzaldoximato)copper(II) and cytopathic effects of HIV.

trans-Bis(resorcylaldoximato)copper(II) and trans-bis-(2,3,4-trihydroxybenzaldoximato)copper(II) (CuRES2 and CuTRI2, respectively) have been tested for antiviral properties against HIV, using an in vitro assay that measures the ability of the test compounds to prevent the killing of susceptible human cells by HIV. In the case of CuTRI2, T4 lymphocytes (CEM-V and CEM-Z cell lines) were exposed to HIV at a virus to cell ratio approx. 0.05 in microtiter plates. In the case of CuRES2, a human leukemia cell line (MT-2) was used instead. The tetrazolium salt XTT was added to all wells, and the cultures were incubated and analyzed spectrophotometrically to quantitate formazan production and viewed microscopically for detection of viable cells. In spite of their antiproliferative properties, neither agent had any detectable ability to prevent the cytopathic effects of HIV in cultures of the target cells used. Because the test system employed was constructed in such a way as to detect antiviral agents acting at any stage of the virus reproductive cycle, the results obtained strongly suggest that neither studied agent has any value as the direct prevention of the cell destruction caused by HIV is concerned.

Anti-HIV Agents↗

PubMedDate not supplied

Inhibition of functionalized 1,3-dienes against Trypanosoma cruzi.

Six functionalized 1,3-dienes were synthesized using cross-coupling reactions, catalyzed by palladium complexes, between alkenylboronic acids and alpha-bromo-alpha,beta-unsaturated carbonylic compounds. Their cytotoxicity against epimastigotes of Trypanosoma cruzi and fibroblastic Vero cells was evaluated, using concentrations ranging from 100 microM to 2.5 mM in experiments with three incubation times (4, 8 and 16 h). These tests were performed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric bioassays and its further reduction to formazan, according to the viability of the parasites and cells. With the exception of (5E,6E)-5-benzylidene-2-methylundec-6-en-4-one, all compounds were cytotoxic to both Trypanosoma cruzi and Vero cells, however differential values of IC50 were observed for two of these compounds. A possible structure-activity relationship is discussed.

Alkenes↗

PubMed1979

Activity of ovarian delta 5-3 beta-hydroxysteroid dehydrogenase in the domestic fowl (Gallus domesticus) with respect to age.

The distribution of delta 5-3 beta-hydroxysteroid dehydrogenase was examined in the ovaries of hens aged 8--30 weeks. At all ages the interstitial cells (both thecal and stromal) gave a positive formazan reaction, but a positive reaction was not obtained in the granulosa cells of follicles less than 5 mm diameter in mature or immature birds. Since activity was restricted to follicles of larger size than this, it is concluded that steroidogenesis occurs in the granulosa cells for only the last 7--8 days of their life in the preovulatory follicle.

3-Hydroxysteroid Dehydrogenases↗

PubMed2002

In vitro and in vivo assays of 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives against Trypanosoma cruzi.

Cytotoxicity assays of 24 new 3,5-disubstituted-tetrahydro-2H-1,3,5-thiadiazin-2-thione derivatives were performed. The 17 compounds with higher anti-epimastigote activity and lower cytotoxicity were, thereafter, screened against amastigote of Trypanosoma cruzi. Out of these 17 derivatives S-2d was selected to be assayed in vivo, because of its remarkable trypanocidal properties. To determine toxicity against J774 macrophages, a method based on quantification of cell damage, after 24 h, was used. Cell respiration, an indicator of cell viability, was assessed by the reduction of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to formazan. Anti-amastigote activity was estimated after 48 h by microscopic counts of May Grünwald-Giemsa-stained monolayers. Nifurtimox and benznidazole were used as reference drugs. For the in vivo experiences, mice were infected with 10(4) blood trypomastigotes and then treated during 15 days with S-2d or nifurtimox by oral route. All of the compounds were highly toxic at 100 micro g/ml for macrophages and a few of them maintained this cytotoxicity even at 10 microg/ml. Of the derivatives assayed against amastigotes 3k and S-2d showed an interesting activity, that was held even at 1microg/ml. It is demonstrated that the high anti-epimastigote activity previously reported is mainly due to the non-specific toxicity of these compounds. In vivo assays assessed a reduction of parasitemia after administration of S-2d to infected mice.

Animals↗

PubMed1988

ESTA: a bioassay system for the determination of the potencies of hormones and antibodies which mimic their action.

A bioassay system named ESTA (eluted stain assay) has been developed to measure hormones and antibodies which mimic their action. It is derived from approaches used for cytochemical bioassays. Unlike the latter which use tissue segments or sections, ESTA is based upon uniform microcultures of target cells maintained in microtitre plates. Direct elution of the cytochemical stain from these microcultures into the wells of the microtitre plates permits rapid quantification with a microtitre plate reader. We describe ESTA systems for GH, prolactin, thyroid stimulators and human chorionic gonadotrophin which utilize the reduction of a tetrazolium salt to a formazan by intracellular dehydrogenase as the cytochemical system. These provide examples of ESTA systems in which the assay signal depends solely upon an increase in cell number in response to the hormone, or in which there is additional enzymic amplification.

Animals↗

PubMed1992

A simple bioassay for epidermal growth factor using pig thyrocytes.

A bioassay for epidermal growth factor (EGF) is described using an eluted stain assay (ESTA) of dehydrogenase activity in pig thyrocytes. Optimal responsiveness to EGF was obtained in confluent cultures of primary pig thyrocytes cultured with EGF or biological samples containing EGF for either 24 or 48 h. Dehydrogenase activity was determined by measuring the production and then the release of a coloured formazan product produced by reduction of a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide substrate added to the cells. The assay responded equally to mouse, human and recombinant EGF and was suitable for measuring EGF activity in some but not all biological fluids. Specificity of detection of EGF activity was confirmed using antibody to EGF. The ESTA assay compared favourably with the radioreceptor assay for EGF in terms of sensitivity to EGF with half-maximal activation at 0.24 +/- 0.06 nmol/l (mean +/- S.E.M., n = 22 experiments) for the ESTA assay and 0.60 +/- 0.13 nmol/l (n = 7 experiments) for the radioreceptor assay.

Animals↗