Bacterial viability and antibiotic susceptibility testing with SYTOX green nucleic acid stain.

A fluorescent nucleic acid stain that does not penetrate living cells was used to assess the integrity of the plasma membranes of bacteria. SYTOX Green nucleic acid stain is an unsymmetrical cyanine dye with three positive charges that is completely excluded from live eukaryotic and prokaryotic cells. Binding of SYTOX Green stain to nucleic acids resulted in a > 500-fold enhancement in fluorescence emission (absorption and emission maxima at 502 and 523 nm, respectively), rendering bacteria with compromised plasma membranes brightly green fluorescent. SYTOX Green stain is readily excited by the 488-nm line of the argon ion laser. The fluorescence signal from membrane-compromised bacteria labeled with SYTOX Green stain was typically > 10-fold brighter than that from intact organisms. Bacterial suspensions labeled with SYTOX Green stain emitted green fluorescence in proportion to the fraction of permeabilized cells in the population, which was quantified by microscopy, fluorometry, or flow cytometry. Flow cytometric and fluorometric approaches were used to quantify the effect of beta-lactam antibiotics on the cell membrane integrity of Escherichia coli. Detection and discrimination of live and permeabilized cells labeled with SYTOX Green stain by flow cytometry were markedly improved over those by propidium iodide-based tests. These studies showed that bacterial labeling with SYTOX Green stain is an effective alternative to conventional methods for measuring bacterial viability and antibiotic susceptibility.     

Development of a short-term assay based on the evaluation of the plasma membrane integrity of the alga Pseudokirchneriella subcapitata

Membrane integrity has been used as a criterion for the definition of cell viability. In the present work, staining conditions (time and dye concentration) for the evaluation of membrane integrity in a fluorescence microplate reader, using the membrane-impermeant nucleic-acid dye SYTOX Green, were optimized. Incubating Pseudokirchneriella subcapitata algal cells with 0.5?μmol/l SYTOX Green for 40?min allowed a clear discrimination between live (intact plasma membrane) and dead cells (with compromised plasma membrane). Algal cell suspensions, labelled with SYTOX Green, exhibited a green fluorescence proportional to the fraction of the cells with a permeabilized plasma membrane. The optimized staining conditions were used to assess the toxicity of 1-pentanol on P. subcapitata in a short-term exposure (6?h) assay. The loss of membrane integrity in the cell population increased with the concentration of 1-pentanol. The 6-h EC(10) and EC(50) values were 7,617?mg/l 1-pentanol (95?% confidence limits 4,670-9,327) and 12,818?mg/l 1-pentanol (95?% confidence limits 10,929-15,183), respectively. The developed microplate-based short-term assay can be useful in the high-throughput screening of toxics or environmental samples using the alga P. subcapitata.     

Paradoxical synergistic effects of tumour necrosis factor and interleukin 1 in murine gut-derived sepsis with Pseudomonas aeruginosa.

The authors evaluated the synergistic effect of tumour necrosis factor (TNF) and interleukin 1 (IL-1) in gut-derived sepsis in mice. After colonization of Pseudomonas aeruginosa strain D4 in the gastrointestinal tract, cyclophosphamide was administered to induce bacterial translocation of the P. aeruginosa and thereby to cause gut-derived sepsis. In this model, treatment either with 8 microg/kg of recombinant human TNF-alpha (rhTNF-alpha) or 2 microg/kg of recombinant human interleukin 1alpha (rhIL-1alpha) solely did not affect the mortality, whereas combined administration of the same doses of rhTNF-alpha and rhIL-1alpha significantly increased the mortality rate in comparison with saline-treated mice. Bacterial counts in liver and blood were significantly higher in rhTNF-alpha and rhIL-1alpha treated mice than in saline-treated mice. Endogenous TNF-alpha and IL-1beta productions were stimulated after combined treatment with rhTNF-alpha and rhIL-1alpha. On the contrary to these adverse effects, combined treatment with 500 microg/kg of rhTNF-alpha and 50 microg/kg of rhIL-1alpha on the day before the administration of cyclophosphamide significantly reduced the mortality from septic infection. We conclude that TNF and IL-1 synergistically affect the mortality of mice after gut-derived sepsis due to P. aeruginosa in mice and the timing of treatment with these cytokines causes both extremes in their effects.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.     

Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide     

Improved flow cytometric analysis of the budding yeast cell cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.     

Characterization of SYBR Gold nucleic acid gel stain: a dye optimized for use with 300-nm ultraviolet transilluminators

The highest sensitivity nucleic acid gel stains developed to date are optimally excited using short-wavelength ultraviolet or visible light. This is a disadvantage for laboratories equipped only with 306- or 312-nm UV transilluminators. We have developed a new unsymmetrical cyanine dye that overcomes this problem. This new dye, SYBR Gold nucleic acid gel stain, has two fluorescence excitation maxima when bound to DNA, one centered at approximately 300 nm and one at approximately 495 nm. We found that when used with 300-nm transillumination and Polaroid black-and-white photography, SYBR Gold stain is more sensitive than ethidium bromide, SYBR Green I stain, and SYBR Green II stain for detecting double-stranded DNA, single-stranded DNA, and RNA. SYBR Gold stain's superior sensitivity is due to the high fluorescence quantum yield of the dye-nucleic acid complexes ( approximately 0.7), the dye's large fluorescence enhancement upon binding to nucleic acids ( approximately 1000-fold), and its capacity to more fully penetrate gels than do the SYBR Green gel stains. We found that SYBR Gold stain is as sensitive as silver staining for detecting DNA-with a single-step staining procedure. Finally, we found that staining nucleic acids with SYBR Gold stain does not interfere with subsequent molecular biology protocols.     

Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-alpha spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-alpha value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-alpha concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-alpha levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.     

Optimal collection of blood samples for the measurement of tumor necrosis factor alpha

We have examined how delayed separation of plasma from cells affects the recovery of recombinant human tumor necrosis factor alpha (rhTNF alpha) from whole blood. Storage of heparinized whole blood samples at room temperature for 1 hr results in a significant (p = 0.036) fall in recovery of plasma TNF alpha from 788 +/- 119 pg/mL to 472 +/- 77 pg/mL, measured by specific enzyme-linked immunosorbent assay (ELISA). Storage of whole blood samples at 4 degrees C for 1 hr reduces but does not prevent the fall in recovery of plasma TNF alpha: 725 +/- 82 pg/mL at time 0, 472 +/- 81 pg/mL after 1 hr, p = 0.038. Recovery of bioactive TNF alpha (cytotoxocity for L929 cells) after 1 hr at room temperature is also significantly reduced from 576 +/- 139 pg/mL to 450 +/- 154 pg/mL, p = 0.036. Studies with 125I-rhTNF alpha confirmed the fall in plasma activity and revealed a rapid commensurate increase in 125I-rhTNF alpha activity in the cell fractions. We recommend that clinical samples for the measurement of cytokines should be kept at 4 degrees C and separated rapidly (within half an hour) before storing the plasma at -70 degrees C.     

Recombinant tumor necrosis factor: species specificity for a variety of human and murine transformed cell lines

Tumor necrosis factor (TNF) exhibits cytotoxic or cytostatic activity on a wide range of animal and human transformed cell lines. Using pure, recombinant human and mouse TNF, we examined the degree of species specificity of the in vitro TNF activity on a variety of human and murine transformed cell lines. This species specificity was studied for the TNF activity alone or in synergism with IFN-gamma. Recombinant human and mouse TNF behave remarkably similarly regarding the in vitro cytolytic/cytostatic activity. However, a certain degree of species-specific preference could be revealed as human cell lines needed a higher concentration of recombinant mouse TNF than of recombinant human TNF to attain a similar effect, while on mouse cells the reverse was true. Also, synergism with IFN-gamma seemed more effective when the target cell was treated with homologous TNF.     

Use of SYTOX green dye in the flow cytometric analysis of bacterial phagocytosis

BACKGROUND: Fluorescein isothiocyanate (FITC) is used widely to label the targets used in flow cytometric phagocytosis assays. Unfortunately, the fluorescence intensity of phagocytosed FITC-labeled targets is influenced by changes in intracellular pH level, making quantitative measurements with this fluorophore problematic. We describe the use of SYTOX green nucleic acid stain to measure phagocytosis by flow cytometry. METHODS: Suspensions of isopropyl alcohol-permeabilized Escherichia coli DH5alpha were stained with the SYTOX green dye and then incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytosis was determined by gating the cells. RESULTS: Results are expressed as percentage of phagocyte-associated green fluorescent cells. The validity of the method was shown by the effects of a phagocytosis inhibitor (incubation at 4 degrees C) or enhancer (gamma interferon [IFN- gamma] treatment) being accurately assessed with this assay. CONCLUSIONS: The method described was reproducible and provides an advantageous alternative to the use of FITC to label bacteria for the flow cytometric measurement of target uptake by phagocytic cells.     

New fluorochromes, compatible with high wavelength excitation, for flow cytometric analysis of cellular nucleic acids

The spectroscopic properties of three new dyes, EK4, VL772, and LL585, free and bound to nucleic acids, are presented, with particular emphasis on their potential use in flow cytometry. Two of these dyes, EK4 and LL585, exhibit red fluorescence, while dye VL772 exhibits yellow fluorescence. Dye LL585 exhibits specificity for DNA, relative to RNA, and a marked enhancement of fluorescence efficiency upon binding to DNA, needed for a red fluorescent DNA-specific stain for flow cytometry. The dye penetrates live cells, although uniformity of nuclear fluorescence, as evidenced by DNA flow histograms, is better if the cells are first permeabilized with Triton X-100. Dye VL772 exhibits yellow fluorescence and little DNA-RNA discrimination, but may prove useful in conjunction with dye LL585 when simultaneous assay of cellular RNA and DNA is desired. Dye EK4 shares properties of the other two dyes but fluoresces with much less efficiency. Dyes LL585 and VL772, used singly, as a pair, or in combination with blue-fluorescing DNA specific dyes, such as bisbenzimidazole derivatives, should permit new, convenient analyses of the content and organization of cellular nucleic acids.     

Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas

Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.     

Microplate fluorescence assay for the quantification of double stranded DNA using SYBR Green I dye

A high-throughput, 96-well microplate fluorescence assay (MFA) was developed for DNA quantification using the double-stranded DNA-binding dye SYBR Green I. Samples mixed with SYBR Green I in the wells of a microtiter plate produced fluorescence in proportion with DNA concentration which was measured using a fluorescence plate reader. The performance characteristics of the assay were compared with spectrophotometric quantification based on ultraviolet absorption and the Hoefer DyNA Quant assay utilizing the fluorescent dye, Hoechst 33258. The MFA accurately quantified different types of DNA over a broad linear dynamic range of concentrations (0.25–2,500 pg/μl), and was not affected by a variety of contaminants in the assay mixture.     

Development of specific adsorbents for human tumor necrosis factor-alpha: influence of antibody immobilization on performance and biocompatibility

To develop adsorbents for the specific removal of tumor necrosis factor-alpha (TNF) in extracorporeal blood purification, cellulose microparticles were functionalized either with a monoclonal anti-TNF antibody (mAb) or with recombinant human antibody fragments (Fab). The TNF binding capacity of the adsorbents was determined with in vitro batch experiments using spiked human plasma (spike: 1200 pg TNF/mL; 1 mg particles in 250 muL plasma). Random immobilization of the full-sized monoclonal antibody to periodate-activated cellulose yielded particles with excellent adsorption capacity (258.1 +/- 48.6 pg TNF per mg adsorbent wet weight). No leaching of antibody was detectable, and the adsorbents retained their activity for at least 12 months at 4 degrees C. We found that the conditions used during immobilization of the antibody (pH, nature of the reducing agent) profoundly influenced the biocompatibility of the resulting adsorbents, especially with respect to activation of the complement system. Particles obtained by random immobilization of the monovalent Fab fragments on periodate-activated cellulose using the same conditions as for immobilization of the mAb exhibited only low adsorption capacity (44 +/- 7 pg/mg adsorbent wet weight). Oriented coupling of the Fab fragments on chelate-epoxy cellulose via a C-terminal histidine tag, however, increased the adsorption capacity to 178.3 +/- 8.6 pg TNF/mg adsorbent wet weight. Thus, in the case of small, monovalent ligands, the orientation on the carrier is critical to retain full binding activity.     

Production of recombinant porcine tumor necrosis factor alpha in a novel E. coli expression system.

DNA sequences encoding porcine tumor necrosis factor alpha (TNF alpha) were reconstructed from a genomic-derived PCR product for expression in Escherichia coli. A synthetic DNA primer containing most of exon III was fused to exon IV sequences by means of PCR. The fused product was then inserted into the novel FLAG vector by restriction and ligation. This initial recombinant construct was propagated in single-strand form through use of a helper phage and subjected to oligonucleotide-directed mutagenesis for the purpose of introducing additional coding sequences from exons II and III. The final construct encoded a fusion protein consisting of the Omp-A signal peptide, a seven-amino acid FLAG peptide and the soluble form of porcine TNF alpha. Bacteria containing this construct produced a protein which was recognized by anti-FLAG monoclonal antibody in Western blots and which was purified by anti-FLAG immunoaffinity chromatography. The purified material was cleaved with enterokinase to remove the FLAG peptide. Both the enterokinase-cleaved form and the uncleaved form were shown to have TNF activity in a WEHI cell cytotoxicity assay.     

Cell surface tumor necrosis factor (TNF) accounts for monocyte- and lymphocyte-mediated killing of TNF-resistant target cells

WEHI164 cells are susceptible to cytotoxicity by soluble recombinant or monocyte-derived TNF alpha, as well as to cell-mediated cytotoxicity by monocytes or lymphocytes. In contrast, K562 cells are resistant to lysis by soluble recombinant or natural TNF alpha, but are killed by monocyte or lymphocyte effector cells. Cell-mediated cytotoxicity against both target cell lines is enhanced by treatment of monocyte effector cells with recombinant interferon gamma or lymphocyte effector cells with interleukin-2. However, treatment of monocytes with LPS, or of lymphocytes with PHA, although inducing secretion of soluble TNF alpha in the medium, does not increase cell-mediated cytotoxicity. Anti-TNF alpha neutralizing antibodies partially inhibit monocyte- as well as lymphocyte-mediated cytotoxicity against WEHI164 and K562 cells. Formaldehyde-fixed effector cells are cytotoxic to both target cell lines. Cytotoxicity by fixed effector cells can be inhibited by anti-TNF alpha antibodies. The extent of cell-mediated cytotoxicity induced by treatment of effector cells with stimulators prior to fixation corresponds to the expression of TNF on monocyte membranes, but not to the titers of secreted TNF. The data suggest that membrane-associated TNF alpha may be a mechanism of human monocyte- as well as lymphocyte-mediated cytotoxicity, regardless of whether the target cells are sensitive or insensitive to soluble TNF.     

CELLULAR DNA CONTENT OF MARINE PHYTOPLANKTON USING TWO NEW FLUOROCHROMES: TAXONOMIC AND ECOLOGICAL IMPLICATIONS1

Two new fluorochromes, PicoGreen® and SYTOX Green? stain (Molecular Probes, Inc.), are useful with flow cytometry for quantitative detection of cellular DNA in a variety of marina phytoplankton. The basic instrument configuration of modern low-power flow cytometers (15 mW, 488 nm excitation) is sensitive enough to detect the DNA signal in nearly all of the 121 strains (from 12 taxonomic classes)examined. The major advantages of these dyes over others are 1)suitability for direct use in seawater, 2)green fluorescence emission of the DNA-dye complex (wavelength 525 ± 15 nm) showing no overlap with the autofluorescence of the plankton pigments in the red band, 3) high fluorescence yield of the DNA-dye complex with an increase in fluorescence > 100-fold compared to the unstained cell, and 4)dyes can be used to quantify double-stranded DNA. The high sensitivity allowed the quantification of the DNA of the smallest known phyto-plankter (Prochlorococcus) as well as bacteria found in some of the algal cultures. Of the 12 taxonomic classes tested, only the 3 Nannochloropsis spp. (Eustagmatophyceae) stained poorly, and a few members of the Chlorophyceae and Pelagophyceae showed poor staining occasionally. In general, maximal fluorescence was achieved within 15 min after addition of the dye. Although the PicoGreen dye stained some living phytoplankton species, preservation is recommended for quantitation. SYTOX Green did not stain live cells. The combination of the dyes, therefore, allows the discrimination between live and dead cells in some algal groups (Prochlorococcus, diatoms, prasinophytes, and pelagophytes). Paraformaldehyde was preferred over glutaraldehyde for fixation to avoid (induced) green autofluorescence. Total DNA values measured in 90 algal species (ca. 121 strains) varied by a factor of 20,000. The lowest values were found in Prochlorococcus and the highest in a large dinoflagellate (Prorocentrum micans). DNA content appears to be a scaleable cell component covarying with the carbon and nitrogen contents of the phytoplankton cells. This covariation allows the total DNA content to be used as an accurate, independent estimate of total cell carbon biomass in unicellular pelagic phytoplankton.     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.