Patricia Jameson

A sensitive interferon assay for many species of cells: encephalomyocarditis virus hemagglutinin yield reduction.

Summary A new, highly sensitive assay for human interferon was developed in which the reduction in HA yield of a single-cycle infection with EMC virus is measured. The sensitivity and precision of the assay are comparable to or better than the other assays tested, depending most of all upon the cells used in the test. This simple method requires no sophisticated instrumentation, can be used on a microtiter scale, provides objective numerical results, and is readily applicable to testing large numbers of samples. The assay can be used in human lines arid diploid cell strains as well as in monkey, rabbit, cat, mouse, pig, cow, dog, and hamster-mouse hybrid cells. We are grateful to C. K. Schoenherr for her excellent technical assistance. We thank Dr. M. Essex for making it possible for one of the authors (P.J.) to work with the feline cell system in his laboratory. We appreciate the gifts of interferon from Dr. K. Cantell and Dr. J. Valenta, and of cells from Drs. G. G. Jackson, S. Baron, and E. Kilbourne.

[52] Virus yield-reduction assays for interferon: Picornavirus hemagglutination measurements

Publisher Summary This chapter presents the virus yield-reduction assays for interferon by picornavirus hemagglutination measurements. The typical assay consists of four steps, which are (1) preparation of cell cultures, (2) exposure of cells to interferon, (3) infection of washed cell cultures with virus, and (4) measurement of virus yield. The interval between each of these steps is usually 1 day, thereby providing results within 3 days of plating the cell cultures. Considerable reduction in the time required can be achieved by applying techniques used in other rapid methods, such as establishing the cell cultures in interferon-containing growth medium, or adding interferon later the same day, and/or reducing the time of exposure of the cells to the interferon. Some of these modifications may reduce the sensitivity of the assay, and should be evaluated beforehand. The assay done with normally employed volumes (original method) or using an equally sensitive microtiter modification, each of which is described.

Inhibition of feline leukemia virus replication by human leukocyte interferon

The replication of feline leukemia virus (FeLV) is inhibited by treatment of cat cell cultures with crude human leukocyte interferon (HuIFN-alpha) as evidenced by titration of the infectious progeny. The inhibition can be demonstrated in three different cell lines in which the production of hemagglutinin by encephalomyocarditis (EMC) virus, and plaque formation by vesicular stomatitis virus (VSV) are also inhibited by the HuIFN-alpha. The dose dependency of the inhibition of EMC virus by the HuIFN-alpha is similar to that obtained with feline interferon in each of the three cell lines. VSV and EMC virus are less than 10 times more sensitive than FeLV to the inhibitory action of HuIFN-alpha if responses to a single interferon treatment are compared for each of the viruses tested in the most sensitive cell line, FEA. The interferon effect on FeLV is more pronounced when it is added within one day after the inoculation of the cells rather than applied before cell infection. The induction of focus formation by FeLV can also be inhibited by HuIFN-alpha in cat cells (CCC-81) which contain the murine sarcoma virus genome.

Production of interferon by human tumor cell lines.

SummaryFourteen continuous human cell lines, including nine derived from tumors and five from non-neoplastic tissues, produced interferon in response to induction with bluetongue virus (BTV), Newcastle disease virus (NDV), and poly(I) · poly(C) complexed with DEAE-dextran. The seven best interferon-producing cell lines (one from a melanoma, five derived from carcinomas, and one SV40-virus-transformed kidney cell line) responded to at least one of the viral inducers with yields of interferon over 1000 units/ml. Because the HT-1376 bladder carcinoma cell line produced high yields of interferon in this survey, and is easily propagated, the optimal conditions for interferon production were investigated, using BTV as the inducer. Interferon yields in 59 inductions over a period of about two years consistently fell within a 6-fold range, and had a geometric mean titer of about 2700 reference units (RU)/ml, representing the production of about 3 RU/103 cells. This yield is comparable to mean titers of 1 to 10 RU/103 cells obtained by others with human leukocytes, foreskin cell strains, or the Namalva lymphoblastoid cell line. UV-inactivated BTV at a multiplicity corresponding to 10 PFU/cell was as effective an inducer in the HT-1376 cell line as the fully infectious virus at a multiplicity of 1 PFU/cell. The interferon produced by the HT-1376 epithelial cell line has characteristics similar to the interferon induced by poly(I) · poly(C) in human diploid fibroblasts.These studies clearly demonstrate that many different types of tumor-derived cells have the capacity to produce interferon, and that some equal or surpass the efficiency of diploid cells.

Thermal stability of freeze-dried mammalian interferons. Analysis of freeze-drying conditions and accelerated storage tests for murine interferon.

Conditions of Interferon processing were analyzed to select those that promote stability after freeze-drying. The effects of various preparative methods and treatment conditions were assessed by measuring the retention of biological activity by lyophilized interferon samples in two kinds of accelerated storage tests: the linear nonisothermal stability (LNS) test, a rapid method used for direct comparison of two or more preparations of interferon, and the multiple isothermal storage (MIS) test, a slow method requiring weeks to months to obtain data for the prediction of stability of a given preparation stored under various conditions. The most stable preparations of Newcastle-disease-virus-induced mouse L cell interferon were obtained using the following conditions: 1) perchlorate treatment to inactivate residual inducing virus, 2) nonspecific adsorption using zeolite for partial purification, 3) suspending medium of 0.5% bovine serum albumin in 0.1 m sodium phosphate buffer at pH 7, and 4) sublimation of ice in vacuo with a starting temperature of −30 °C to a final residual moisture of about 3%. The final product, reference reagent G002-904-511, was stable throughout the course of the LNS test. From an extensive MIS test, this reference interferon was predicted to lose 1000 units of activity in 110 years at 4 °C and 1000 units in 100 days at 37 °C. After 6 years of storage at 37 °C when the predicted residual activity would be about 20% of the original potency, 35% of initial interferon activity remained, confirming the usefulness of the short-term predictive test.

Production of interferon in human cell cultures by a new, potent viral inducer.

A new discovered double-stranded RNA inducer of interferon, bluetongue virus (BTV), stimulates the production of large amounts of interferon in animals as well as in many types of mammalian cell cultures, including human leukocytes, and continuous cell lines. The exceptional pH lability of BTV and its lack of pathogenicity for man further recommend its use as an interferon inducer. Among several human cell lines tested, the most efficient producer of interferon was a continuous cell line designated HT-1376, derived from a bladder carcinoma. With infectious BTV as the inducer, the HT-1376 line produced more interferon per cell than did leukocytes; interferon yields ranged from 10,000 to 60,000 units per ml of crude, unconcentrated supernatant fluid. Noninfectious BTV, inactivated by ultraviolet irradiation, was as effective as infectious virions. The interferon produced in HT-1376 cells has physicochemical and antigenic properties resembling those of fibroblast interferon produced in diploid cells.

The Neuraminidase Yield-Reduction Bioassay of Human and Other Interferons

Summary The production of neuraminidase by the recombinant influenza virus X7(F1) in human, monkey, rabbit, hamster, mouse, and chicken cell cultures is inhibited by interferon. Described is a new enzyme assay for neuraminidase that can be applied to the bioassay of interferons. The advantages of this interferon bioassay are its sensitivity, reproducibility, rapidity, and convenience. We thank Mary Dixon, Joyce F. Otto, and Christine Schoenherr for their excellent technical assistance. Note added in proof: Experiments subsequent to this manuscript have revealed that the production of neuraminidase in the RK-13 cell line is variable and at times too low for use in an interferon assay. Note added in proof: Low yields of neuraminidase have recently been obtained in the RK-13 rabbit cell line, an observation which makes this line unsuitable for the neuraminidase bioassay. However, high neuraminidase yields are obtained in another interferon-sensitive line of rabbit skin (RS) cells.

[83] Procedures for stabilization of interferons

Publisher Summary This chapter discusses the procedures for stabilization of interferons. The stability of interferons, especially as they are processed and purified, has proved to be a greater problem than is appreciated from early observations on the stability of crude materials. It is now realized that interferons in solution can be inactivated by a variety of physical and chemical treatments, but a degree of stability can be provided by means of certain additives. For long-term storage of interferon preparations, particularly those intended for clinical use, freeze-drying is preferred. One of the most effective methods of preserving interferon activity for extended periods, such as storage of reference reagents or preparations for clinical use, is freeze-drying in the presence of low concentrations of added protein and a sodium phosphate buffer. Two accelerated storage tests are used to predict the stability of freeze-dried interferons. The accelerated multiple isothermal stability test is used to provide a conservative estimate of the expected stability of these standards under long-term storage conditions.

Thermal and vortical stability of purified human fibroblast interferon.

The loss of biological activity upon heating or agitation of human interferons is markedly altered by changing their aqueous environment. Low pH significantly stabilizes liquid fibroblast interferon at 68 degrees C and 37 degrees C whereas chaotropic salts stabilize at 68 degrees C but not at 37 degrees C; this anomalous result may be due to reactivation of biological activity at the higher temperature. The concentration of extraneous proteins influences the apparent thermal stability at any temperature and pH; thus, interferon was not stable even at low pH at protein concentrations less than 5 microgram/ml. Solutions of partially purified fibroblast interferon can be inactivated by mechanical stress; the addition of proteins or nonionic detergents prevents such inactivation. Freeze-dried preparations show the greatest thermal stability. The use of high-temperature, accelerated storage tests makes it possible to predict the shelf-life of freeze-dried interferon.

[36] Induction of interferon with bluetongue virus in various cells

Publisher Summary Bluetongue virus (BTV) is an exceptionally potent interferon inducer; it has properties that make it suitable for laboratory use, for example, for evaluating the ability of cells of various species to produce interferon or for the production of high-titered interferon preparations. The methods and results described may pertain only to the attenuated BT-8 vaccine strain of serotype 10 that have tested; others working with another strain of BTV of the same serotype obtained only low levels of interferon. The induction procedure for human leukocytes, the HT-1376 human cell line, and primary rabbit kidney cell cultures, are detailed. Cultures of HT-1376 cells (derived from a human bladder carcinoma) in 32-oz bottles are prepared by subculturing a confluent monolayer into 3-6 new bottles, and waiting for 4-7 days for confluent monolayers to develop; the cell density of a confluent monolayer is 15 to 20 x 10 6 cells per 120 cm 2 surface area. HT-1376 cell interferon is characterized by serological neutralization and by physicochemical tests. It has been found to satisfy the criteria of an interferon resembling the fibroblast type induced by exposure of foreskin fibroblast cells to poly(I) . poly(C).