A bioassay employing polyethylene glycol for measuring neutralization of interferon by specific antibodies.

A bioassay employing polyethylene glycol (PEG) for measuring the level of interferon-neutralizing antibodies is described. The method is based on incubating a concentrated preparation of interferon with an appropriately diluted anti-interferon serum or globulin for 1 h at 37 degrees C, precipitating the antigen-antibody complexes as well as some free serum protein with sterile PEG at a final concentration of 10% for 18 h at 4 degrees C, separating the sediment by centrifugation, and titrating the residual interferon in the supernate. It is not necessary to remove PEG from the samples since it is not toxic for tissue culture at a final concentration of less than 1%.

Biological activity of sera obtained by hyperimmunizing rabbits with interferon from mouse L cells.

Rabbits immunized with interferon from mouse L cells for long periods of time produced interferon-neutralizing antibodies with titers from 1:80 to 1,2000. The anti-interferon sera also contained antibodies against antigens contaminating the interferon preparations such as albumin, bovine gamma-globulin, chicken albumin, extract from L cells, and Sindbis virus antigens. Some sera also displayed cytotoxic activity against cells of transplantable murine leukemia. These antibodies could be removed by specific absorption. Titers of antibodies neutralizing interferon were not correlated with the titers of antibodies against concomitant antigens. In hyperimmunized rabbits interferon-neutralizing antibodies persisted for long periods of time in spite of interrupting immunization.

Dextran T fractions substituted with cibacron blue F3G-A as carriers for mouse interferon.

Commercial dextran T fractions covering the molecular weight (Mt) range 10,000-2,000,000 were substituted with a covalently linked chromophore: Cibacron blue F3G-A. Mouse interferon (IF) was found to bind firmly to the blue dextran (BD) fractions. Fractions of high Mt (BD 2000 and BD 500) associated with IF are precipitated from the solution by polyethylene glycol (PEG). Fractions of lower Mt (BD 70, BD 40 and BD 10) associated with IF are only partially, if at all, precipitated by PEG. However, when BD-IF complexes of various Mt were analyzed by chromatography on Sephadex G-150, IF activity migrates together with BD and not at the distance characteristic of native IF. These data suggested that IF is bound to various BD fractions. BD-IF complexes of various Mt have almost the same antiviral and anticellular activities as native IF. BD-IF complexes are neutralized to a greater extent by antibody than native IF. Clearance of BD 70-IF complex from the mouse peritoneal cavity is slightly slower than native IF. However, the i.v. clearance of both forms of IF is almost the same.

Use of polyethylene glycol-treated calf serum for cell cultures in virus and interferon studies.

Immunoglobulins and lipoproteins can be efficiently removed from calf serum by precipitation with polyethylene glycol (PEG) 6000 under sterile conditions. The PEG-treated serum is suitable for cell cultures used for virus growth and assays. Moreover, PEG was found to slow down the growth of L cells and to enhance the production and activity of mouse interferon.

Enchancement of leukemogenesis in mice after prolonged administration of anti-interferon or normal rabbit globulin.

The prolonged adminstration of rabbit anti-mouse L cell interferon globulin had a marked potentiating effect on Rauscher Murine Leukemia Virus (MuLV-R) infection in BALB/c mice, as shown by spleen size. Normal rabbit globulin had a lesser, but still significant, augmenting effect on splenic enlargement. It was possible to discriminate quantitatively between the non-specific enhancement of splenomegaly in MuLV-R infected mice due to antigenic stimulation with normal rabbit globulin and the effects due to elimination of endogenous interferon by specific antibodies. The difference in the spleen-enlarging activity between the anti-interferon IgG and normal rabbit IgG was found to be maximal 3-4 weeks after infection when potent, diluted anti-interferon IgG (58 microgram protein per dose) was used. It would appear that the endogenous interferon, even prodcued in undetectable amounts, plays an essential role in controlling infection with an oncogenic virus.

Platelet-derived growth factor (PDGF) inhibits priming in synchronous mouse or human fibroblasts producing interferon.

Monolayer cultures of Lpa and FS-4, mouse and human fibroblasts that are good producers of interferon were synchronized by contact inhibition and by incubation in 5% platelet poor plasma serum (PPPS) medium. When the quiescent cultures were stimulated for growth by PDGF, pre-treatment with homologous interferon before induction with virus or poly (rI) . poly (rC) did not increase interferon yields. Under similar experimental conditions priming (increased interferon synthesis) was demonstrated in the asynchronous or quiescent Lpa or FS-4 cells pre-treated with homologous interferon. The unprimed cells, whether stimulated for growth with PDGF or not, produced normal amounts of interferon after appropriate induction. We suggest that growth factor (PDGF), a hormone with opposing action to interferon, can inhibit priming but it has little, if any, effect on the basic process of induction of interferon synthesis.

Rapid screening test for the diagnosis of rotavirus infection.

The early diagnosis of human rotavirus infection is essential for effective patient management and infection control. We report here a rapid, easy-to-perform, and inexpensive test for rotavirus detection. The viral RNA is extracted directly from the stools and electrophoresed on 1% agarose gels. Currently available immunoassays for routine diagnostic purposes are directed at the common group A-specific antigen. As reports become available on human gastroenteritis caused by the atypical or novel rotaviruses, this technique presents an added advantage in that it can detect both group A and non-group A rotaviruses.

Repeated 'superinduction' of interferon in human diploid fibroblast cultures.

Human diploid fibroblast cultures induced to make interferon by the combination of polyriboinosinic acid-polyribocytidylic adic, cycloheximide and actinomycin D degenerate thereafter, owing to the irreversible nature of the inhibition induced by actinomycin D. However, cultures superinduced with the DNA-dependent RNA synthesis inhibitor 5,6-dichloro-I-beta-D-ribofuranosylbenzimadazole (DRB) survive, owing to the reversible nature of the inhibition induced by DRB, and can again be superinduced on several occasions.

Interferoids: in vitro and in vivo conversion of native interferons to lower molecular weight forms.

Mouse interferons appear as two distinct molecular forms, one migrating at 38,000 daltons in sodium dodecyl sulfate/polyacrylamide gels and one migrating at 22,000 daltons; these interferons comprise about 80% and 20% of total activities, respectively. When such interferon preparations are briefly exposed to acidic periodate buffer, the larger interferon species is apparently converted to the smaller form since the activity at 38,000 daltons is completely eliminated while the activity at 22,000 daltons increases significantly; upon further oxidative cleavage, antiviral activity becomes detectable migrating at 15,000 daltons. Because no native mouse interferon has been reported as such small molecules, this antiviral activity is designated mouse "interferoid" to distinguish it from the native, naturally occurring interferon forms. Prolonged acidperiodate treatment fails to quantitatively convert the 22,000-dalton interferon to the 15,000-dalton interferoid since both are inactivated. When L cells are induced to make interferon in the presence of glycosylation inhibitors, either D-glucosamine or 2-deoxy-D-glucose, they produce approximately normal levels of antiviral activity. However, when such preparations are analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, little activity (<10%) migrates as either the 38,000-dalton or 22,000-dalton native interferons. The interferons and interferoid are antigenically and hydrophobically indistinguishable. These data suggest that induced mouse cells normally synthesize the interferoid as a precursor polypeptide that is either partially or extensively modified by carbohydrate additions to produce, respectively, the 22,000- and 38,000-dalton mouse interferons. Because interferoid is apparently fully biologically active without these moieties, chemical synthesis of such unmodified polypeptides or active fragments from them appears feasible.

Unusual human interferons produced by virus-infected amniotic membranes.

Interferon (IFN) induced in the human amniotic membrane contains at least five different molecular species, as shown by analysis in NaDodSO4/polyacrylamide gels after heating and under reducing conditions. Three of the IFN components reported here--migrating at 26, 43, and 80 kilodaltons--are of unusual antigenic structure because they are neutralized to about the same extent by anti-IFN-alpha and anti-IFN-beta antibodies. The 15- to 17-kilodalton species belongs to the IFN-alpha group, while the 21- to 22-kilodalton species, the most frequently detected major peak, is IFN-beta. In addition to their unusual size and antigenic structure, these IFNs could play a role during embryonic development and in the immune tolerance of the mother with regard to the fetus.

Molecular alterations of interferons.

Interferons can be chemically and enzymatically altered to obtain molecular forms which differ in size, charge, and/or host-range from native interferons. These data suggest that small active cores of interferons can be obtained which may possess more desirable characteristics than native interferons, such as lack of "species-specificity"" and increased stabilities. Studies for assessing alterations of interferons due to chemical or enzymatic treatments must monitor the treated interferons for changes in ratios of each of the various activities attributed to native interferons and must characterize the altered interferons in terms of each of the physical and biological markers.