In vitro cell-based assay for activity analysis of staphylococcal enterotoxin A in food

Staphylococcal enterotoxins (SEs) are a leading cause of food poisoning and have two separate biological activities; it causes gastroenteritis and functions as a superantigen that activates large numbers of T cells. In vivo monkey or kitten bioassays were developed for analysis of SEs emetic activity. To overcome the inherent limitations of such bioassays, this study describes an in vitro splenocyte proliferation assay based on SEs superantigen activity as an alternative method for measuring the activity of staphylococcal enterotoxin A (SEA). After incubation of splenocytes with SEA, cell proliferation was measured by labeling the proliferating cells' DNA with bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) and quantifying the incorporated BrdU by immunohistochemistry. BrdU labeling is shown to be highly correlated with SEA concentration ( R ²=0.99) and can detect 20 pg mL⁻¹ of SEA, which is far more sensitive than most enzyme-linked immunosorbent assays. Our assay can also distinguish between active toxin and inactive forms of the toxin in milk. By applying immunomagnetic beads that capture and concentrate the toxin, our assay was able to overcome matrix interference. These results suggest that our in vitro cell-based assay is an advantageous practical alternative to the in vivo monkey or kitten bioassays for measuring SEA and possibly other SEs activity in food.     

Human platelet-derived growth factor: radioimmunoassay and discovery of a specific plasma-binding protein

The platelet-derived growth factor (PDGF) is the principal mitogen in serum for cultured cells of mesenchymal origin. PDGF also is a potent chemotactic protein for inflammatory cells and for cells required for wound repair. Because activity levels of PDGF in biological fluids are difficult to measure, we attempted to develop a radioimmunoassay for PDGF. Rabbits were immunized with purified PDGF; the antiserum obtained was monospecific for PDGF in immunodiffusion analysis against concentrated platelet lysates, serum, and plasma. A radioimmunoassay for PDGF was developed with a sensitivity of congruent to 0.2 ng/ml. Levels of PDGF in plasma/serum were measured and compared with PDGF levels determined by a receptor-competition assay and by a standard biological assay measuring incorporation of [3H]thymidine into 3T3 cells. Radioimmunoassay showed apparent PDGF levels of 50 ng/ml in human plasma and 103 ng/ml in serum. The 50 ng/ml PDGF in plasma was unexpected because the plasma samples contained little or no platelet release products as determined by very low levels of platelet factor 4. We therefore sought an immunologically reactive PDGF molecule in human plasma. No immunologically reactive protein was detected by immunodiffusion analysis or when plasma was treated with an immunoaffinity gel. Subsequently, a 125I-PDGF-binding protein was identified; the 125I-PDGF-plasma-binding protein complex was not reactive with anti-PDGF immunoglobulin. Correction for 125I-PDGF bound by the plasma-binding protein established serum levels of PDGF of congruent to 50 ng/ml; congruent to 50 ng/ml PDGF was found in serum by radioreceptor-competition assays and by mitogenic assays as well. The plasma-binding protein may serve to clear PDGF released in the circulation, thereby limiting PDGF activity to its local interactions at the site of blood-vessel injury.     

Exploration of novel in vitro assays to study drugs against Trichuris spp

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 μg/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.     

A Multiplex Assay for Detection of Staphylococcal and Streptococcal Exotoxins

Staphylococcal and streptococcal exotoxins, also known as superantigens, mediate a range of diseases including toxic shock syndrome, and they exacerbate skin, pulmonary and systemic infections caused by these organisms. When present in food sources they can cause enteric effects commonly known as food poisoning. A rapid, sensitive assay for the toxins would enable testing of clinical samples and improve surveillance of food sources. Here we developed a bead-based, two-color flow cytometry assay using single protein domains of the beta chain of T cell receptors engineered for high-affinity for staphylococcal (SEA, SEB and TSST-1) and streptococcal (SpeA and SpeC) toxins. Site-directed biotinylated forms of these high-affinity agents were used together with commercial, polyclonal, anti-toxin reagents to enable specific and sensitive detection with SD₅₀ values of 400 pg/ml (SEA), 3 pg/ml (SEB), 25 pg/ml (TSST-1), 6 ng/ml (SpeA), and 100 pg/ml (SpeC). These sensitivities were in the range of 4- to 80-fold higher than achieved with standard ELISAs using the same reagents. A multiplex format of the assay showed reduced sensitivity due to higher noise associated with the use of multiple polyclonal agents, but the sensitivities were still well within the range necessary for detection in food sources or for rapid detection of toxins in culture supernatants. For example, the assay specifically detected toxins in supernatants derived from cultures of Staphylococcus aureus. Thus, these reagents can be used for simultaneous detection of the toxins in food sources or culture supernatants of potential pathogenic strains of Staphylococcus aureus and Streptococcus pyogenes.     

Evaluation of adenosine deaminase assay for analyzing t-lymphocyte density in vitro

Summary The proliferative capacity of T cells in response to various stimuli is commonly determined by radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly synthesized DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, an alternative method was tested. As an alternative, T-cell proliferation was measured by spectrophotometrically analyzing the presence of an enzyme adenosine deaminase in lymphocytes and also using a standard XTT assay. Jurkat (human) T-cell line (clone E6.1) was used for lymphocyte population. The Jurkat cell concentration was adjusted according to different cell densities and enzyme activity was determined. Cells were also seeded in complete medium up to 72 h and harvested for estimation of enzyme activity. A significant correlation between the standard cell-proliferation assay and adenosine deaminase assay was observed. The present study indicates that the assay of adenosine deaminase is a reliable and accurate method for measuring proliferation of T lymphocytes.     

Development and Evaluation of Simple Dot–Blot Assays for Rapid Detection of Staphylococcal Enterotoxin-A in Food

The present study was aimed to develop and evaluate dot–blot assays for rapid detection of staphylococcal enterotoxin-A (SEA) in food. Dot blots were developed in two formats, indirect and sandwich utilizing mouse monoclonal anti-SEA and rabbit polyclonal anti-SEA antibodies. In indirect dot–blot format, recombinant SEA was directly coated on NCM dot–blot strip and detection was carried out by anti-SEA antibodies. In sandwich dot–blot format, SEA was trapped between anti-SEA capture and detection antibodies. Both the dot–blot assays exhibited a sensitivity of ~48 ng ml^?1 when tested in different food matrices. The developed assays were highly specific as no cross-reactivity was detected with other classical staphylococcal enterotoxins, toxigenic bacteria and foodborne pathogens. Sensitivity and specificity of developed indirect and sandwich dot–blot assays with respect to PCR was found to be 100 and 99%, respectively. The results shows that the developed dot–blot assays can be used as rapid preliminary screening tests for detection of SEA in food or determining the toxigenic potential of staphylococci, especially in resource-limited settings.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Monoclonal antibodies to human recombinant interleukin 1 (IL 1)beta: quantitation of IL 1 beta and inhibition of biological activity

Monoclonal antibodies (McAb) were developed to the Mr 17,500 form of human recombinant interleukin 1, IL 1 beta. Four McAb have been identified that inhibit the biological activity of IL 1 beta. McAb H34 and H67, at 1 microgram/ml (6 X 10(-9) M), completely inhibit the capacity of 1 ng/ml (6 X 10(-11) M) recombinant IL 1 beta to stimulate the proliferation of murine thymocytes or human fibroblasts in vitro. McAb H6 and H21 are approximately 10-fold less potent, and completely inhibit IL 1 beta activity at 10 micrograms/ml (6 X 10(-8) M) in both assays. The McAb do not have a significant effect on the biological activity of human recombinant IL 1 alpha in either assay. These McAb block the binding of recombinant [125I]IL 1 beta to IL 1 receptors on mouse 3T3 fibroblasts and have affinity constants for IL 1 beta in the range of 10(9) to 10(10) liters/mol. Competition studies suggest that two nonoverlapping epitopes on the IL 1 beta molecule are recognized by the McAb. H6 and H34 recognize one epitope, and H21 and H67 another. McAb H6 and H67 have been used together in a two-site ELISA to detect IL 1 beta. The sensitivity of the ELISA, which is 15 pg/ml (0.86 pM), approaches the limit of sensitivity of the thymocyte proliferation assay. The ELISA and thymocyte proliferation assay were used to quantitate IL 1 beta in E. coli LPS-stimulated human monocyte culture supernatants (HMCS). The level of IL 1 beta detected by ELISA in culture supernatants from eight donors ranged from 1.7 to 5.6 ng/ml, with a mean value of approximately 3 ng/ml. By comparison, the thymocyte proliferation assay gave levels of IL 1 in HMCS that were eight fold higher when quantitated by using recombinant IL 1 beta as a standard. This discrepancy with the bioassay used was reflected by the three fold higher maximum stimulation of thymocyte proliferation by HMCS as compared with recombinant IL 1 alpha or IL 1 beta, and only 45% inhibition of HMCS IL 1 activity by McAb. Thus, factors other than IL 1 beta account for the IL 1-like activity in monocyte culture supernatant as measured by the bioassay. The ILB1 McAb and ELISA allow for the first time-sensitive, accurate, and convenient quantitation of IL 1 beta levels in biological fluids or specimens.     

Nonspecific reactions of a commercial enzyme-linked immunosorbent assay kit (TECRA) for detection of staphylococcal enterotoxins in foods.

A staphylococcal enterotoxin visual immunoassay kit (TECRA) has recently become commercially available. Since the kit is an enzyme-linked immunosorbent assay system equipped with polyvalent antisera against staphylococcal enterotoxin types A to E (SEA to SEE) and the test is simple and rapid to perform (4 h), it has been widely used for screening purposes. In this study, the sensitivity of the kit for detection of SEA, SEB, and SEC in ham, cheese, and mushrooms was similar to those of kits based on an enzyme immunoassay and reversed passive latex agglutination: 0.75 to 1.0 ng of SEA per ml, 0.5 to 0.75 ng of SEB per ml, and 1.0 to 1.25 ng of SEC per ml. However, the TECRA kit showed nonspecific reactions with food samples contaminated by microorganisms other than Staphylococcus aureus, such as Enterobacter agglomerans, Enterobacter cloacae, Proteus mirabilis, Pseudomonas aeruginosa, and Serratia marcescens. The substance contributing to the false-positive results differed from true staphylococcal enterotoxins in that it was (i) heat labile (completely inactivated by heating for 2 min at 100 degrees C, whereas true staphylococcal enterotoxins were inactivated by about 10% with this treatment), (ii) lower in molecular weight than staphylococcal enterotoxins, and (iii) not bound to a copper chelate Sepharose gel (all of the substance remained in the unbound wash fraction, whereas staphylococcal enterotoxins were quantitatively bound to the gel). The problem of false-positive results with the TECRA kit could be resolved by heat treatment (2 min at 100 degrees C) or by cleanup procedures involving metal chelate affinity chromatography with copper chelate Sepharose for 4 h before use of the TECRA kit.     

Development of a cell culture assay for Bordetella parapertussis heat-labile toxin

A cell culture assay for heat-labile toxin isolated from Bordetella parapertussis has been developed. In this assay, the ability of heat-labile toxin to induce contraction of vascular smooth muscle cells is measured. The method allows for detection of as little as 0·6 ng/ml of the toxin. The results obtained from this in vitro assay correlated well with those obtained with in vivo assays indicating that the cell culture assay may be a useful alternative to animal assays.     

A gas chromatographic-mass spectral assay for the quantitative determination of oleamide in biological fluids.

Oleamide is a putative endogenous sleep-inducing lipid which potently enhances currents mediated by GABAA and serotonin receptors. While a quantitative assay would aid in determining the role of oleamide in physiological processes, most of the available assays are lacking in sensitivity. We now describe a quantitative assay for measuring low nanogram amounts of oleamide in biological fluids using GC/MS in the selective ion-monitoring mode. The internal standard (13C18 oleamide) was added to known concentrations of oleamide, which were converted to the N-trimethylsilyl or N-tert-butyldimethylsilyl derivatives before analysis by GC/MS, yielding linear calibration curves over the range of 1-25 ng of oleamide when monitoring the m/z 338/356 fragments. Using this technique, oleamide levels were determined following solvent extraction of normal rat cerebrospinal fluid and plasma to be 44 and 9.9 ng/ml, respectively. This technique constitutes a sensitive and reliable method for determining low nanogram quantities of oleamide in biological fluids.     

Statistical analysis and quality control in radioimmunoassays for staphylococcal enterotoxins A, B, and C.

The objective of these studies was to set up a reliable radioimmunoassay (RIA) for staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC) in a food system. Significant differences (95% confidence limits) were obtained between the 0- and 1-ng/ml enterotoxin standards, so the sensitivity of the RIAs was 1 ng/ml. Polystyrene tubes coated with anti-SEB and stored at 4 degrees C were unstable. The percentage of iodinated SEB bound to these tubes decreased at a rate of 0.33%/day, in contrast to the rate of 0.07%/day obtained with tubes prepared the day before the analyses. Satisfactory precision and maximum sensitivity were obtained by using six replicates for each sample and freshly coated tubes. The antisera used for coating the tubes were reused four times and were frozen between coatings. The process of drum drying mashed potatoes containing 1 mug of SEB per g of mashed potatoes inactivated 83% (wt/wt) of the SEB. Statistical quality control parameters were used to insure that RIAs were performing reliably with a sensitivity of 1 ng/ml. Over 450 samples of potato flakes and granules, which represented different production lots from 12 different manufacturers, were examined for SEA, SEB, and SEC. No enterotoxins were detected.     

Statistical analysis and quality control in radioimmunoassays for staphylococcal enterotoxins A, B, and C.

The objective of these studies was to set up a reliable radioimmunoassay (RIA) for staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC) in a food system. Significant differences (95% confidence limits) were obtained between the 0- and 1-ng/ml enterotoxin standards, so the sensitivity of the RIAs was 1 ng/ml. Polystyrene tubes coated with anti-SEB and stored at 4 degrees C were unstable. The percentage of iodinated SEB bound to these tubes decreased at a rate of 0.33%/day, in contrast to the rate of 0.07%/day obtained with tubes prepared the day before the analyses. Satisfactory precision and maximum sensitivity were obtained by using six replicates for each sample and freshly coated tubes. The antisera used for coating the tubes were reused four times and were frozen between coatings. The process of drum drying mashed potatoes containing 1 mug of SEB per g of mashed potatoes inactivated 83% (wt/wt) of the SEB. Statistical quality control parameters were used to insure that RIAs were performing reliably with a sensitivity of 1 ng/ml. Over 450 samples of potato flakes and granules, which represented different production lots from 12 different manufacturers, were examined for SEA, SEB, and SEC. No enterotoxins were detected.     

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner’s fiber

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.     

Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods.     

A Novel ELISA Format for the Rapid and Sensitive Detection of Staphylococcal Enterotoxin A

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.     

A spectrophotometric assay for nanogram quantities of biotin and avidin

Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 min incubation at 21 degrees C and 5 min to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphate dehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 min with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method.     

COMPARISON of ANALYTICAL SENSITIVITIES of THREE ENZYME IMMUNOASSAY PROCEDURES FOR the DETECTION of SALMONELLA LIPOPOLYSACCHARIDE

The analytical sensitivities of three different enzyme linked immunoassays (ELISA), two competitive and a capture format were assessed. the assay systems employed monoclonal antibodies to Salmonella lipopolysaccharide (LPS) outer core epitopes to detect crude LPS antigens from Salmonella typhimurium. the most sensitive ELISA was the capture procedure, being capable of detection 1.3 ng/ml of LPS. This technique, however also gave the greatest between-test variation and as a result, the lowest amount that could be detected with a 95% confidence limit was actually 12.8 ng/ml and it took the longest time to perform (3 h, 30 min). A competitive ELISA using limiting monoclonal antibody to compete between solid phase antigen and soluble antigen in the sample, ranked second in sensitivity, and can detect 2.8 and 3.8 ng/ml of LPS when tested with two different monoclonal antibodies. However, because of the slight between test variation, the actual sensitivities that could be detected with a 95% confidence limit were 3.1 and 4.6 ng/ml, respectively. This test takes approximately 1 h and 30 min to perform.
The classical type of competitive assay, employing a labelled antigen, was the least sensitive being capable of detecting 5.8 ng/ml if the LPS was conjugated with horseradish peroxidase and 16.0 ng/ml if alkaline phosphatase was used as a label. to account for the between-test variation, the sensitivities with a 95% confidence limit were 8.6 and 18.7 ng/ml for the respective assays, which take 2 h and 15 min to perform.
These sensitivities compare favorably with those published for similar assays, but all of the procedures were judged insufficiently sensitive for direct use on food samples to be tested for the presence of Salmonella species. However, the assays would be quite suitable for demonstration of Salmonella sp. after an enrichment procedure.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Development of an Immunofluorometric, High-Capacity, Cell-Based Assay for the Measurement of Human Type I and Type II Interferons

We have developed a cell-based 96-well microtiter plate, high throughput assay for measuring both type I and type II interferon (IFN) activity on human cells. This assay makes use of a previously described IFN-specific reporter stably expressed in human HT 1080 cells. The induction of the reporter by IFN is determined by measuring the IFN-dependent expression of CD2 on the cell surface. The cytokine-induced expression of CD2 occurs within 48 h and is measured using a time-resolved fluorometric immunoassay. The limit of detection for type I IFN is.0.4 IU/ml. Interassay and intraassay coefficients of variation were 1.1% and 1.3% for the medium control (31 IU IFNb1b/ml), respectively. The limit of detection for type II IFN is.8 IU/ml, and the assay coefficients of variation are similar to those determined for type I IFNs. The level of sensitivity for this assay is comparable to other assays commonly used to measure IFN activity on cells. The current assay has an advantage over antiviral and antiproliferative assays, in that there is no requirement for the use of pathogenic virus or for determining viable cell numbers. The current assay is ideally suited for increasing sample screening and high-capacity automation, making it an excellent tool for drug discovery.