Search PubMed⌕ Search

Biomedical subjects

I Julkunen

Publications and source records attributed to I Julkunen.

At least 73 records · Page 4Linked to original sources
PubMed1995

Persistence of anti-mumps virus antibodies after a two-dose MMR vaccination. A nine-year follow-up.

A two-dose vaccination program against measles, mumps, and rubella (MMR) viruses was started in Finland in 1982. In this program the trivalent MMR-II vaccine (MSD, USA) was offered to children at the ages of 14-18 months and 6 years followed by revaccination 4-5 years later. The vaccination coverage has been high (97%) and MMR infections have practically been eliminated in the Finnish population. In a serological follow-up program sequential serum samples were obtained from 254 children (127 14-18-month-old vaccinees and 127 6-year-old vaccinees) during a 9-year follow-up period. Anti-mumps virus antibody titers were determined by enzyme immunoassay using purified whole mumps viruses as the antigen. In seronegative (n = 120) 14-18-month-old vaccinees the seroconversion rate was 86% (geometric mean titer 1/1670 +/- 1/270). The antibody levels fell rapidly (significance p < 0.01) within the first year of follow-up (mean titer 1/1080 +/- 1/190), but remained relatively stable in subsequent years. After revaccination the seropositivity rate was 95% (mean titer 1/2310 +/- 1/260) and declined more slowly thereafter to 86% (mean titer 1/1510 +/- 1/210) at year 9 of follow-up. The mean antibody titer was significantly (p < 0.05) higher 4 years after the second MMR vaccination when compared with the corresponding time point after the first vaccination. In 6-year-old seronegative vaccinees the increase and decay of anti-mumps virus antibodies after the first MMR vaccination was similar to that seen in the group of younger vaccinees. A two-dose MMR vaccination protocol resulted in a high mumps immunity level in the vaccinated population.

Age Factors↗
PubMed1994

Mutational analysis of murine Mx1 protein: GTP binding core domain is essential for anti-influenza A activity.

Interferon-induced resistance to influenza virus in murine cells is mediated by the Mx1 protein, which inhibits viral mRNA synthesis in the nucleus. Murine Mx1 protein is a GTPase specifically targeted into the cell nucleus and it has a C-terminal leucine zipper domain that mediates its oligomerization. In order to determine functionally important elements of the protein we created several substitution, linker insertion, and deletion mutants of murine Mx1 protein. The antiviral activity of mutant Mx1 proteins was analyzed by a transient transfection/influenza A infection assay in COS cells by indirect immunofluorescence. Mx1 proteins carrying mutations in the vicinity or within the consensus GTP binding elements exhibited markedly reduced, but not completely lost, antiviral activity. Baculovirus produced, GTP binding element substitution mutant Mx1 proteins showed very low (< 10%) GTPase activity as compared to wild-type Mx1 protein. Mutations in other portions of the molecule had less effect on antiviral activity, except one mutant, which was situated six amino acids from the C-terminal end. This mutation evidently interrupted the nuclear localization signal rendering the protein cytoplasmic and clearly reduced the anti-influenza activity. Deletions of various sizes and locations further suggested that the N-terminal half of the molecule is more important in the antiviral activity than other regions of the molecule. These results indicate that the GTP binding domain of Mx1 protein is essential for its anti-influenza activity, correlating to the low GTPase activity of the GTP binding element substitution mutants, but other portions of the molecule such as the leucine zipper and the nuclear localization signal are of importance, too.

Animals↗
PubMed1994

Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-gamma.

Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.

Amino Acid Sequence↗
PubMed1994

Enzymatic characterization of interferon-induced antiviral GTPases murine Mx1 and human MxA proteins.

Interferons induce a number of different proteins which mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. Interferon-induced Mx proteins, which confer resistance to influenza, vesicular stomatitis, and measles viruses, contain consensus GTPase sequence elements. Insect cell-produced purified murine Mx1 and human MxA proteins were found to hydrolyze GTP with Km = 65 microM (Vmax, 7.1 min-1) and 62 microM (Vmax, 3.1 min-1), respectively. The GTPase activity of Mx1 and MxA proteins was strictly dependent on Mg2+ ions. Murine Mx1 protein was inactivated at 10 degrees C lower temperatures than MxA protein. As analyzed, by filter binding assay, Mx1 protein (at 1 microM) showed a relatively high affinity for GDP (Kd = 1.0 x 10(-7) M) and approximately 340-fold lower affinity for guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) (Kd = 3.4 x 10(-5) M). The Kd values for MxA protein were 2.0 x 10(-7) M for GDP and 5.9 x 10(-6) M for GTP gamma S, showing approximately a 30-fold affinity difference. ATP, UTP, or CTP did not inhibit the Mx protein-dependent GTPase activity, suggesting that Mx1 and MxA proteins are highly specific for guanosine nucleotides. In conclusion recombinant nuclear murine Mx1 and cytoplasmic human MxA proteins show clear differences in their enzymatic activities and nucleotide binding characteristics. How these differences influence their cellular functions and antiviral potential is presently not known.

Animals↗
PubMed1994

Distribution of catechol-O-methyltransferase enzyme in rat tissues.

In the present study we show the distribution of catechol-O-methyltransferase (COMT) in various rat tissues with a highly specific antiserum prepared against recombinant rat COMT. Immunoprecipitation and immunocytochemical controls confirmed the COMT-specificity of the antibodies. The antiserum detected both the 24 KD soluble and the 28 KD membrane-bound forms of the enzyme. By immunohistochemical staining the COMT enzyme was found in most rat tissues. Staining was most intense in the liver and in the kidney, in agreement with previous studies and our immunoblotting results. In the gastrointestinal tract, epithelial cells of the stomach, duodenum, and ileum were immunoreactive for COMT. In pancreas, COMT immunoreactivity was found in insulin-producing beta-cells and somatostatin-producing D-cells but not in glucagon-producing alpha-cells of the islets of Langerhans. In pituitary, COMT immunoreactivity was found in cleft cells, in pituicytes of the posterior lobe, and in the anterior lobe, partly in the same cells containing luteinizing hormone (LH). In other endocrine organs, COMT immunoreactivity was found in epithelial cells of the thyroid gland and in zona glomerulosa of the adrenal cortex. In the brain, brightest immunofluorescence was seen in ependymal cells of the cerebral ventricles and choroid plexus. Weak to moderate immunofluorescence was found in the neuropil of several brain areas, including striatum and cortex. Scattered small neurons in spinal sensory ganglia were also COMT immunoreactive. Previous immunocytochemical studies, enzyme activity determinations, and distribution of the COMT mRNA are in general agreement with the results presented here. The wide distribution of COMT in different tissues suggests an important role for this protein in inactivation of catechol compounds.

Animals↗
PubMed1993

Control of IFN-inducible MxA gene expression in human cells.

MxA is an IFN-induced human protein which is located in the cytoplasm of induced cells. MxA makes the cells resistant to infection by influenza and vesicular stomatitis viruses. In the present work we used baculovirus expression system to produce MxA protein. The protein was purified to homogeneity and highly specific polyclonal anti-MxA antibodies were prepared. In human mononuclear cells, and A549 lung carcinoma cells expression of MxA protein is induced by very low (< 1 IU/ml) doses of leukocyte IFN-alpha (nIFN-alpha), whereas IFN-gamma does not seem to induce it or potentiate the induction by IFN-alpha. In mononuclear cells stimulated with high doses of leukocyte IFN-alpha concentrations, the amount of MxA mRNA was induced 10-fold at 4 h after IFN induction and up to 10-fold higher MxA protein levels were observed at 24-48 h postinduction. The gene can be reinduced by IFN-alpha 24 h after the initial induction suggesting for a lack of negative feedback after this time point. The protein is very stable, the half-life being approximately 2.3 days. Flow cytometric analysis revealed that monocytes have higher basal and induced MxA protein levels than lymphocytes but the dose-dependency of MxA expression is very similar in both cell types. Granulocytes are producing very low amounts of MxA protein.

Animals↗
PubMed1993

Lysosomal aspartylglucosaminidase is processed to the active subunit complex in the endoplasmic reticulum.

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency of which leads to a human storage disease, aspartylglucosaminuria (AGU). Although numerous mutations have been identified in AGU patients, elucidation of the molecular pathogenesis of the disease has been hampered by the missing information on the cellular events resulting in the maturation and activation of the enzyme. Here we used the expression of in vitro mutagenized constructs of the AGA cDNA to define three specific proteolytic trimming steps resulting in mature AGA. Removal of the signal peptide is immediately followed by proteolytic cleavage of the precursor into two subunits and results in biologically active enzyme already in the endoplasmic reticulum. This early activation has not previously been described for lysosomal enzymes. The subsequent lysosomal trimming does not influence the enzymatic activity of AGA. It consists only of a single proteolytic cleavage which removes 10 amino acids from the C-terminal end of the larger subunit, in contrast to the multistep lysosomal processing observed in several other hydrolases.

Amino Acid Sequence↗
PubMed1993

Effect of carbohydrates on the pharmacokinetics of human interferon-gamma.

Human interferon-gamma (IFN-gamma) has two N-linked glycosylation sites at positions 25 and 97 of the 143-amino-acid-long secretory form. To study the role of glycan residues in the pharmacokinetics of IFN-gamma, we produced recombinant IFN-gamma molecules lacking either one or both of the glycosylation sites (Asn mutated to Gln) by baculovirus expression in insect cells. In addition, we produced the fully glycosylated forms both in insect cells and in human leukocyte cultures. Two million units of each IFN were injected intravenously or intramuscularly into rabbits. The glycosylated IFN-gamma molecules from the insect cells were rapidly eliminated from the blood. This is probably due to the fact that their oligosaccharides are of a high mannose type that are rapidly taken up by the liver. The unglycosylated IFN-gamma persisted longer in the blood than the glycosylated recombinant forms. However, the natural IFN-gamma exhibited the longest survival in the blood. The results emphasize the importance of the carbohydrate groups in human IFN-gamma to its pharmacokinetic properties.

Animals↗
PubMed1993

Establishment and characterization of a rat glomerular endothelial cell line.

BACKGROUND: Primary cell cultures have been widely used for research purposes, but kidney glomerular and vascular endothelial cells and, particularly, stable endothelial cell lines from these have been difficult to obtain. EXPERIMENTAL DESIGN: We used electroporation and lipofectin transfection to introduce various transforming constructs, and direct infection with an oncogenic adeno 31 (Ad31) virus to immortalize cells from freshly isolated rat kidney glomeruli and characterized the cells obtained at their 20th, 40th, 60th, and 80th passage for markers of various glomerular cell types. RESULTS: Direct infection with an oncogenic adenovirus type 31 (Ad31) resulted in a stable cell line (over 120 passages) morphologically resembling epithelial/endothelial cells line (over 120 passages) morphologically resembling epithelial/endothelial cells. These cells were constantly positive for factor VIII-related antigen, podocalyxin and for OX-43 anti-rat endothelial cell antibodies, Bandeiraea simplicifolia (BSI-B4) lectin, and expressed receptors for acetylated low density lipoprotein. Anti-Thy 1.1 and anti-desmin antibodies recognizing glomerular mesangial cells and antibodies against O-acetyl GD3 and gp330 antigens of podocytes, as well as anti-thrombospondin and anti-cytokeratin antibodies identifying parietal epithelial cells failed to bind to our cells. These rat glomerular endothelial cells (RGE cells) showed decreased serum requirements for growth, changes in morphology, loss of contact inhibition, and loose adherence to growth support. The transforming E1A gene could not be found by Southern blotting or polymerase chain reaction amplification analysis. Neither did the RGE cells produce any adenoviral proteins tested by immunoprecipitation analysis. CONCLUSIONS: The RGE cells represent a stable cell line with various characteristics of endothelial cells. Furthermore, they remain responsive to basic FGF and grow on type IV collagen and fibronectin matrices forming capillary-like structures. Thus, we consider RGE cells a cell line of rat glomerular endothelial origin.

Adenoviridae Infections↗
PubMed1992

Interferon-induced Mx proteins form oligomers and contain a putative leucine zipper.

Interferons induce a number of different proteins that mediate the antiproliferative, antiviral, and immunomodulatory functions of interferons. At least three different proteins mediate the antiviral response, and one of them, Mx protein, specifically inhibits the replication of influenza virus and (vesicular stomatitis virus). Mouse and rat Mx1 proteins are nuclear, whereas other presently known Mx proteins are cytoplasmic. The cellular functions of Mx proteins are unknown, but all of them contain a consensus GTP binding site. Very little information is available on the structure and characteristics of the mouse Mx1 protein itself. For biochemical characterization, we expressed mouse Mx1 protein in a baculovirus system and purified it to homogeneity. The purified protein as well as the authentic murine cellular Mx1 protein exists in dimers and trimers in the presence of dissociating solvents, whereas in physiological buffers they form aggregates. Cross-linking experiments done on Mx-expressing cells from various species revealed that mouse, rat, and human Mx proteins exist predominantly in trimers. Amino acid sequence analysis shows that all known Mx proteins have conserved leucine repeats typical for a leucine zipper at their COOH-terminal end. In vitro translation of chimeric catechol O-methyltransferase-Mx1 gene constructs revealed that the leucine zipper domain of Mx1 protein is responsible for the oligomerization. The COOH terminus also functions as a nuclear localization signal. Microinjection of purified oligomers into the cell cytoplasm resulted in a fast accumulation of the protein in the resulted in a fast accumulation of the protein in the nucleus. Immunoelectron microscopy revealed that nuclear murine Mx1 protein exists in distinct, electron-dense structures separate from nuclear membrane, and chromatin, or nucleolus. These observations reveal that a COOH-terminal leucine zipper domain is an important structural element of all Mx proteins. Its relevance to the biology and functions of Mx proteins is presently not known.

Animals↗
PubMed1992

Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain.

The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.

Animals↗
PubMed1992

Secretion and biological activities of heparin-binding growth-associated molecule. Neurite outgrowth-promoting and mitogenic actions of the recombinant and tissue-derived protein.

The cDNA for the developmentally regulated, neurite outgrowth-promoting protein HB-GAM (heparin-binding growth-associated molecule) was recently cloned and shown to encode a novel lysine-rich sequence that is homologous with retinoic acid-induced sequences suggested to function in cell differentiation (Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724). The same sequence was found for the mitogenic and neurite outgrowth-promoting protein pleiotrophin (Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). In this study, we have constructed a recombinant baculovirus using the cDNA that encodes the putative preprotein of HB-GAM. The putative secretion signal of HB-GAM is cleaved off in the baculovirus expression system, and the recombinant protein is rapidly secreted to the culture medium. Recombinant HB-GAM purified from the culture medium retains the biochemical characteristics and the neurite outgrowth-promoting activity found for the tissue-derived protein. Studies on the neurite outgrowth-promoting activity suggest that HB-GAM functions as an extracellular matrix-associated protein that enhances axonal growth in perinatal cerebral neurons of the rat. Since the same predicted amino acid sequence has been ascribed to a mitogenic protein, mitogenic activities of the recombinant HB-GAM and of tissue-derived HB-GAM fractions were also studied. Recombinant HB-GAM did not display any significant mitogenic activity, suggesting that tissue-derived HB-GAM preparations may contain other heparin-binding mitogenic factors. We identified in brain-derived HB-GAM fractions a 17-kDa protein (p17) that is detached from heparin by a slightly higher salt concentration as compared to HB-GAM. We suggest that p17 is structurally distinct from HB-GAM and responsible for the mitogenic actions of tissue-derived HB-GAM fractions.

3T3 Cells↗
PubMed1992

Differential inactivation of interferons by a protease from human granulocytes.

Human leukocyte suspensions produced interferon-alpha (IFN-alpha) without induction during incubation at 37 degrees C. The highest titers were obtained at about 30 million cells/ml. The best yields, approximately 2 IU per 10(6) cells, were achieved in medium with or without albumin; serum inhibited the production. The uninduced IFN-alpha peaked at 24 h. The titers dropped on further incubation due to release of a protease from polymorphonuclear cells. The protease inactivated all tested human class I IFNs, but recombinant IFN-alpha 1 was clearly more resistant to the enzyme than rIFN-alpha 2. Human IFNs-gamma from different sources exhibited striking differences in their sensitivity to the protease. Glycosylated natural IFN-gamma from human leukocytes and glycosylated rIFN-gamma from CHO cells were relatively resistant, whereas unglycosylated rIFN-gamma from Escherichia coli was rapidly degraded by the protease. The protease was inhibited by PMSF and by greater than or equal to 1% human or fetal bovine serum but not by EDTA or less than or equal to 1% human albumin. Its optimum pH was between 7 and 8. It was resistant to treatment for 30 min at 56 degrees C.

Cells, Cultured↗
PubMed1991

Gm allotypes influence the production of IgG3 but the effect is age-dependent.

Serum concentrations of IgG3 were found to be higher in Gm-f-positive (= b-positive) than in f-negative individuals except in young children. Young children aged 3-4 months had a mean concentration of 0.24 g/l of IgG3 regardless of allotype. The concentration gradually rose with age in f-positive individuals to a geometric mean of 0.56 g/l in adults but it remained essentially unchanged in f-negative people. A corresponding allotype effect was seen in influenza-specific antibody responses. While the total IgG response (mainly IgG1) was equally strong in f-positive and in f-negative patients, f-positive (= b-positive) patients produced more IgG3 antibodies than f-negative patients. The difference between geometric mean values of opposite homozygotes (f/f versus f-negative) was 2.3-fold (p = 0.0113). This finding indicates that the b-positive gamma-3 allele is more productive than the g-positive allele.

Adult↗
PubMed1990

Parental influenza virion nucleocapsids are efficiently transported into the nuclei of murine cells expressing the nuclear interferon-induced Mx protein.

The interferon-induced murine Mx1 protein, which is localized in the nucleus, most likely specifically blocks influenza virus replication by inhibiting nuclear viral mRNA synthesis, including the mRNA synthesis catalyzed by inoculum (parental) virion nucleocapsids (R. M. Krug, M. Shaw, B. Broni, G. Shapiro, and O. Haller, J. Virol. 56:201-206, 1985). We tested two possible mechanisms for this inhibition. First, we determined whether the transport of parental nucleocapsids into the nucleus was inhibited in murine cells expressing the nuclear Mx1 protein. To detect the Mx1 protein, we prepared rabbit antibodies against the Mx1 protein with a CheY-Mx fusion protein expressed in bacteria. The fate of parental nucleocapsids was monitored by immunofluorescence with an appropriate dilution of monoclonal antibody to the nucleocapsid protein. The protein synthesis inhibitor anisomycin was added to the cells 30 min prior to infection, so that the only nucleocapsids protein molecules in the cells were those associated with nucleocapsids of the parental virus. These nucleocapsids were efficiently transported into the nuclei of murine cells expressing the Mx1 protein, indicating that this protein most likely acts after the parental nucleocapsids enter the nucleus. The second possibility was that the murine Mx1 protein might act in the nucleus to inhibit viral mRNA synthesis indirectly via new cap-binding activities that sequestered cellular capped RNAs away from the viral RNA transcriptase. We show that the same array of nuclear cap-binding proteins was present in Mx-positive and Mx-negative cells treated with interferon. Interestingly, a large amount of a 43-kDa cap-binding activity appeared after interferon treatment of both Mx-positive and Mx-negative cells. Hence, the appearance of new cap-binding activities was unlikely to account for the Mx-specific inhibition of viral mRNA synthesis. These results are most consistent with the possibility that the Mx1 protein acts directly to inhibit the viral transcriptase in the nucleus.

Animals↗
PubMed1987

Proportions of immunoglobulin isotypes in paralytic poliomyelitis and after vaccination.

Immunoglobulin isotype composition of poliovirus antibodies was studied by isotype-specific solid-phase radio-immunoassay (RIA) in four patients with paralytic poliomyelitis, five adults receiving live poliovirus vaccine as a booster immunization, and seven children receiving first doses of inactivated poliovirus vaccine. In paralytic poliomyelitis serum and cerebrospinal fluid (CSF) poliovirus antibodies were mainly of IgG1, IgG3, and IgA isotypes. IgM antibodies were found in sera but not in CSF. Either IgG2 and IgG4 antibodies were undetectable or the titers were low. In adults who had received live trivalent poliovirus vaccine, antibodies against poliovirus type 3 were detected in IgG1 (53% of total antibodies), IgG3 (25%), IgM (9%), IgA (8%), IgG2 (3%), and IgG4 (2%) isotypes. In prevaccination and late postvaccination sera the share of IgG3 antibodies was exceptionally high (35%). In children who received inactivated poliovirus vaccine, antibodies developed in IgG1 (53-61% of total antibodies for poliovirus types 1, 2, and 3), IgG3 (12-21%), and IgM (23-33%) isotypes. Antibody levels in IgG2, IgG4, and IgA isotypes were low and observed only in a few cases. Like other viral antibodies IgG1 and IgG3 isotypes were the major IgG subclasses in poliovirus antibodies.

Adult↗
PubMed1987

Preparation of nasopharyngeal secretions for immunofluorescence by one-step centrifugation through Percoll.

A simple method was developed for separation of cells from nasopharyngeal secretion for the diagnosis of respiratory virus infections by immunofluorescence microscopy. The diluted specimen, containing dithiothreitol to break up the mucus, was centrifuged once through a cushion of 20% Percoll (colloidal silica, a density gradient medium), which permitted sedimentation of cells through the cushion, but retained mucus on top of it. The pelleted cells were resuspended, and microscope slides were then prepared by standard techniques. The Percoll centrifugation method was also applicable for sputum and bronchoaveolar lavation specimens. Immunofluorescent antibody staining of nasopharyngeal secretions prepared by the described method was more sensitive than enzyme immunoassay for the detection of respiratory syncytial virus.

Antigens, Viral↗