High Sensitivity Detection of Anti-DFS70 Antibodies by Radioimmunoprecipitation Assay (RIPA).

BACKGROUND: Autoantibodies against the chromosome associated protein DFS70/LEDGF (dense fine speckled 70/ lens epithelial growth factor; anti-DSF70) are increasingly being regarded as biomarkers for the diagnostic exclusion of systemic autoimmune rheumatic diseases (SARD). In routine ANA screening by indirect immunofluores-cence (IIFT) tests the presence of anti-DFS70 may first be presumed because of their characteristic immunofluo-rescence pattern (AC-2 pattern) and then be confirmed by antigen specific assays, a sequential approach, which may underestimate the prevalence of anti-DFS70 because of the inherent shortcomings of the ANA-IIFT. We therefore, for the first time, determined the prevalence of anti-DFS70 in patient sera by means of a sensitive and specific radioimmunoprecipitation assay (RIPA) as compared to a commercial ELISA. METHODS: Blood specimens referred for routine ANA screening (n = 1.100, ANA-Series) or for basic clinical chemistry tests (n = 350, CC-Series) were assayed for the prevalence of anti-DFS70 by RIPA using 35S-methionine labelled full-length DFS70 (FL-DFS70) as well as a C-terminal DFS70 fragment (CT-DFS70) generated by in vitro transcription/translation (ivTT) of the respective cDNAs. ELISA was performed using an anti-DFS70 test-kit (Eu-roimmun) and ANA-IIFT by means of commercial HEp-2 cells (INOVA) and appropriately chessboard titrated conjugates (Dianova). Accessory SARD markers (anti-dsDNA, anti-ENA) were determined in sera positive for anti-DFS70. RESULTS: The detection of anti-DFS70 by RIPA was considerably more sensitive than by ELISA, resulting in an overall detection rate of 9.0% (ANA-Series) and 8.0% (CC-Series) compared to ELISA revealing 4.6% (ANA-Se-ries) and 2.6% (CC-Series) anti-DFS70 positive sera. Of 99 RIPA reactive sera (ANA-Series) 72% were reactive against anti-FL-DFS70, 93% against CT-DFS70, polyspecific antibodies coexisted in 65%, reacting with both antigen specificities, 28% showed monospecific reaction with CT-DFS70 and 7% monospecific with FL-DFS70, indicating also the possible existence of antibodies specific for N-terminal epitopes in DSF70. Similar frequencies were seen in sera of the CC-series. The RIPA measured antibody concentrations (Rratio) obtained with FL-DSF70 antigen and CT-DSF70 antigen showed a correlation. There was also a correlation between the IIFT-ANA titers and Rratio found by RIPA. The consensus of suspected AC-2 pattern in ANA-IIFT and anti-DFS70 measured by RIPA was about 80%. No significant correlation existed between the antibody concentrations measured by RIPA and ELISA. Additional SARD markers were present in 24% of anti-DFS70 positive sera referred for ANA screening. No additional markers were seen in sera of the CC-Series. CONCLUSIONS: RIPA constitutes a highly-sensitive assay for detection of anti-DFS70 in human sera. ANA-IIFT screening performed under consideration of the AC-2 pattern for verification of antibodies to DFS70 under routine conditions may incorrectly estimate a considerable number of not only low but also high titer anti-DSF70 positive sera. The significance of RIPA reactive antibodies, especially of low titer range, in the context of SARD and healthy individuals now has to be scrutinized in further clinical studies.

A fluorescence sedimentation assay for dsDNA antibodies.

The Farr assay is a radioimmunoassay (RIA) for dsDNA antibodies, based on antibody precipitation using ammonium sulphate and quantification using radio-labelled dsDNA. The RIA-Farr assay offers outstanding clinical specificity and sensitivity for systemic lupus erythematosus (SLE) compared to other assays but does also present some disadvantages as it utilizes radioactive-labelled dsDNA and requires high levels of technical expertise for safe handling. Here, a new precipitation assay, 'Fluoro-Farr' assay, is described. This assay maintains a high sensitivity and specificity for SLE but is based on precipitation with polyethylene glycol (PEG) and fluorescence of EvaGreen intercalated in dsDNA as detection principle. As dsDNA antibodies are quantified using fluorescence, the disadvantages of working with radioactivity are eliminated. The Fluoro-Farr assay was developed and validated, and the diagnostic efficiency of the assay was evaluated by testing 57 sera from SLE patients and 60 healthy controls. The Fluoro-Farr assay revealed a diagnostic sensitivity of 68% at a diagnostic specificity of 95% (ROC AUC 0.91). Furthermore, the new Fluoro-Farr assay was compared to the RIA-Farr assay, and showed a correlation of the outcomes from the two assays, but the Fluoro-Farr assay did not outperform the RIA-Farr assay due to its outstanding clinical diagnostic efficiency (ROC AUC 0.99). In conclusion, the Fluoro-Farr assay presents a viable alternative to the traditional RIA-Farr assay; especially in laboratories without facilities to perform assays with radioactivity-based read-out. As the RIA-Farr assay, the Fluoro-Farr assay has the advantage of being a precipitation assay allowing antibody:dsDNA interaction in solution using native dsDNA.

Detection of anti-ESA antibodies in human samples from PRCA and non-PRCA patients: an immunoassay platform comparison.

BACKGROUND: The immunological methods for detecting antibodies to erythropoiesis-stimulating agents (ESAs) differ in assay sensitivity. However, this parameter, routinely determined in clinical assays using a high-affinity non-human polyclonal antibody, gives a one-dimensional assessment of antibody detection. We compare three widely used immunological methods and evaluate the ability of each to detect mature human antibodies and human antibodies characteristic of an early immune response. METHODS: The detection of anti-ESA antibodies was compared between a radioimmunoprecipitation (RIP) assay, an electrochemiluminescence (ECL) bridging enzyme-linked immunosorbent assay and a surface plasmon resonance (SPR)-based immunoassay. All three methods were validated for sensitivity, specificity and precision. Specimens from clinical studies or post market testing were categorized as pure red cell aplasia (PRCA) or non-PRCA and then analyzed in each method. RESULTS: Among the antibody-mediated PRCA samples, which contain high affinity neutralizing antibodies, there was strong correlation between all methods. The results from non-PRCA sample analysis show high correlation between RIP and ECL methods; however, differences between the SPR immunoassay and the ECL and RIP were demonstrated. The samples that scored positive in the SPR immunoassay and negative by RIP and ECL were characterized to be of low antibody concentration, contained a high percentage of rapidly dissociating antibodies, or were antibodies of the IgM isotype. CONCLUSIONS: All three immunological methods are appropriate for detection of antibodies associated with antibody-mediated PRCA. However, the SPR immunoassay platform detected an early, low affinity IgG and IgM antibody response as well as detected and characterized a pathogenic antibody response associated with antibody-mediated PRCA.

A clinico-radiological phenotype of voltage-gated potassium channel complex antibody-mediated disorder presenting with seizures and basal ganglia changes.

In childhood, central nervous system (CNS) presentations associated with antibodies to voltage-gated potassium channel (VGKC) complex include limbic encephalitis, status epilepticus, epileptic encephalopathy, and autistic regression. We report the cases of two individuals (a 6-year-old male and an 11-year-old female) who presented with an acute-onset explosive seizure disorder with positive VGKC complex antibodies and bilateral basal ganglia changes on magnetic resonance imaging (MRI). Both patients made a complete clinical recovery, without immunotherapy, with resolution of the MRI changes and normalization of the antibody levels. Extended antibody testing, including testing for leucine-rich glioma-inactivated 1 (LGI1), contactin-associated protein 2, and contactin-2 was negative. This could suggest that the clinico-radiological phenotype in our patients may in fact be associated with a novel autoreactive target(s) within the VGKC complex, as may be the case in other children with VGKC complex-mediated CNS disorders.

Specific anti-nuclear antibodies in systemic sclerosis patients with and without skin involvement: an extended methodological approach.

OBJECTIVES: To determine how sensitively SSc-associated ANAs are detected by a standard identification algorithm compared with an extensive panel of ANA identification assays, and to assess the distribution of SSc-associated ANAs and SSc organ system involvement in patients without skin involvement (limited SSc). METHODS: Serum samples from 145 consecutive monocentric SSc patients fulfilling LeRoy and Medgser's criteria for early SSc were studied. ANAs were detected by IIF on HEp-2000 cells and identified by western blotting, protein radio-immunoprecipitation, RNA immunoprecipitation and line immunoassay (LIA). SSc organ involvement was assessed according to a modification of Medsger's disease severity scale. RESULTS: At least one specific ANA reactivity was present in 88% of the patients. The standard algorithm (IIF and LIA) found at least one specific ANA in 74% of the patients. The main reactivities missed by this algorithm were anti-RNA polymerase III, anti-PM/Scl and anti-Th/To. Eighty-three percent of the patients with limited SSc had at least one ANA. ACAs and anti-Th/To antibodies were significantly associated with limited SSc, whereas anti-topoisomerase I and anti-RNA polymerase III were observed less frequently. SSc organ system involvement was present in 63% of the patients with limited SSc, most of whom had lung involvement. CONCLUSIONS: Standard algorithms for ANA identification lack sensitivity for the detection of SSc-associated ANA and should be supplemented with additional assays, especially in a clinical environment that has particular interest in SSc. The spectrum of SSc-associated ANA differs according to the presence or absence of skin involvement.

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients' and blood donors' sera.

AIM: To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. METHODS: Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunofluorescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. RESULTS: FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. CONCLUSION: CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity.

Eliminating false-positive results in serum tests for neuromuscular autoimmunity.

In this study we describe the false-positive frequency in radioimmunoprecipitation assays for muscle acetylcholine receptor (AChR) and neuronal voltage-gated potassium channel (VGKC) autoantibodies, attributable to 125I-ligand immunoprecipitation. Sera were evaluated for AChR autoantibody (n = 34,095) and VGKC autoantibody (n = 11,028). We retested sera that yielded apparently positive results with 125I-ligand with and without detergent-solubilized cation-channel protein, indentified clinically validated fals-positive rates of 0.05% and 1.7% for AchR and VGKC autoantibodies, respectively. Specificity assurance in radioimmunoprecipitation assays requires subtraction of values for 125I-ligand binding.

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Comparison of insulin autoantibody: polyethylene glycol and micro-IAA 1-day and 7-day assays.

BACKGROUND: Older studies of diabetes development typically utilized a 7-day incubation polyethylene glycol competitive insulin autoantibody assay (CIAA). Our standard micro-IAA assay (mIAA) utilizes precipitation with proteins A/G and 1-day incubation (1-day mIAA), but is less sensitive compared to the CIAA assay. METHODS: We performed CIAA and mIAA assays in various conditions. We analyzed serum samples from 446 type 1 diabetes patients, from another set of 247 type 1 diabetes patients within 2 weeks of initiation of insulin treatment, from 150 healthy control donors, from 22 healthy participants in the diabetes autoimmunity study in the young (DAISY), and also coded sera from 50 patients with newly diagnosed type 1 diabetes and 50 blood donor control samples. RESULTS: In the process of our study, we found that the key condition was the incubation time. Therefore, we extended the incubation time to 7 days (7-day mIAA assay). No CIAA-negative control was positive with either 1-day or 7-day mIAA. In a new onset type 1 diabetes and at risk cohorts (DAISY study), the 7-day mIAA identified an additional 18% as being positive along with 16% of those who were initially 1-day mIAA negative and CIAA positive. Most subjects detectable only with the 7-day mIAA assay had intermediate levels of CIAA (80-300 nU/mL) (p = 0.01). CONCLUSIONS: The 7-day mIAA assay identifies a small but significant additional subset of individuals positive on the CIAA assay, while preserving specificity.

Autoantibodies against dsDNA measured with nonradioactive Farr assay-an alternative for routine laboratories.

Autoantibodies against dsDNA are utilized for the diagnosis and prognosis of SLE as they are highly specific and correlate with disease activity/renal involvement. However, different detection methods are used in routine diagnostic laboratories. Farr radioimmunoassay (Farr-RIA) has been designated as the preferred method, since it provides very specific and at the same time quantitative results, enabling follow-up of level variations over time. Using intercalating fluorescent dsDNA dye would enable all the benefits of Farr-RIA without the radioactive material and organic solvents. To develop a modified fluorescent Farr method (Farr-FIA) and compare it to the classical Farr-RIA in regard to laboratory parameters, as well as clinical utility. Assays were tested on sera of 70 SLE patients, 78 other autoimmune patients, and 145 healthy blood donors. DNA for Farr-FIA was isolated from healthy donor, for Farr-RIA, 14C-labeled dsDNA from E. coli was used and mixed with sera in borate-buffered saline, followed by precipitation with saturated ammonium sulfate solution and centrifugation. The supernatant (S) was separated from the precipitate (P), and content of dsDNA was measured with PicoGreen (Invitrogen) in Farr-FIA or radioactive isotope in scintillation solution in Farr-RIA. The results were calculated as a ratio (P-S)/(P+S). Farr-FIA has a diagnostic sensitivity of 53% and diagnostic specificity of 100% (ROC AUC 0.781). Good correlation and agreement were shown between Farr-RIA and Farr-FIA. Also, there is good correlation between Farr-FIA and SLEDAI, comparable to that of Farr-RIA. Farr-FIA differs from Farr-RIA in the changed detection system yielding comparable results and thus could represent a nonradioactive replacement for Farr-RIA.

Detection and characterization of antibodies against recombinant human erythropoietin by RIPA, ELISA and neutralization assay in patients with renal anemia.

The aim of the present study was to analyze the relations between the serum anti-erythropoietin antibody (AEAb) levels and the antibodies' neutralizing activity in 20 patients with renal anemia and rhEPO-induced antibodies. AEAb levels were determined by the enzyme-linked immunosorbent assay (ELISA, double antigen-bridging) and by radioimmunoprecipitation assay (RIPA). The bone marrow neutralization test was used to determine the neutralizing activity of the antibodies. RIPA and ELISA data resulted in closely correlated measurements. The relations between AEAb levels and the neutralizing activity of the antibodies are variable as shown by follow-up and cross-sectional evaluations of the data. Serum samples with a high antibody level (>1000 ng/ml) are associated with 100% neutralizing activity, whereas serum samples with lower AEAb levels show partial neutralizing activities or have no effect. Determining the neutralizing activity might be helpful when it comes to deciding of whether or not rhEPO therapy should be continued, specifically in patients who have low antibody levels. The apparent affinity of the AEAb as defined by inhibition of the binding of rhEPO (IC(50)) did not change in the course of the disease, nor did it correlate to the AEAb levels or the neutralizing activities.

Column-based method to simultaneously extract DNA, RNA, and proteins from the same sample.

We describe a procedure for the simultaneous extraction of proteins and nucleic acids from the same experimental sample allowing for direct correlations between genetic, genomic, and proteomic data. This approach, using commercially available column-based nucleic acid extraction kits, requires no hazardous chemicals and is a quick, reliable, and consistent method for concomitant protein extraction. Buffer choice is critical to completely solubilize all proteins in the sample. Proteins solubilized in radioimmunoprecipitation assay (RIPA) buffer did not represent the entire profile when compared with conventionally extracted proteins using the same buffer at the one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) level, however proteins extracted from the columns and solubilized in a two-dimensional (2-D) electrophoresis lysis buffer showed a similar profile to conventionally extracted proteins when analyzed at both the 1-D and the 2-D level. We further showed that proteins extracted using these methods were compatible with Western blot analysis. This technique provides a simple and effective way to analyze protein and nucleic acids simultaneously from the same sample without affecting yield and quality.

Quantification of photoproducts in mammalian cell DNA using radioimmunoassay.

Over the past 20 yr, the use of polyclonal and monoclonal antibodies to quantify damage in DNA has burgeoned. Immunoassays offer distinct advantages over other anaytical procedures currently used to measure DNA damage including adaptability, sensitivity and selectivity. This combination of attributes allows for the development of powerful analytical techniques to visualize and quantify specific types of DNA damage in cells and organisms exposed to subtoxic levels of xenobiotics with distinct advantages over the other procedures in the analysis of DNA damage in human and environmental samples. Radioimmunoassay (RIA) is readily applied to a variety of biological materials and has typically been used to measure DNA damage in cell and organ cultures, tissue sections and biopsies, buccal cells, bone marrow aspirates, peripheral blood lymphocytes, and urine. Here we describe the use of a very sensitive RIA for the specific quantitation of cyclobutane dimers and (6-4) photoproducts in DNA extracted from mammalian cells and tissues.

Transforming growth factor-beta enhances connective tissue growth factor expression in L6 rat skeletal myotubes.

Transforming growth factor (TGF)-beta plays an important role in fibrosis of various organs and tissues. TGF-beta1 stimulates fibroblastic cells to form extracellular matrix (ECM), and regulates all critical events in wound healing. Connective tissue growth factor (CTGF), a TGF-beta-inducible molecule, has recently been reported to promote fibroblast proliferation, migration, adhesion and extracellular matrix formation, both in vivo and in vitro. In this study, we demonstrated that TGF-beta1 enhances CTGF mRNA and protein levels in L6 rat skeletal muscle myotubes. TGF-beta might, therefore, play a role in fibrosis of skeletal muscle by stimulating CTGF expression in the muscle tissue itself.

Molecular characterization of proteolytic processing of the Gag proteins of human spumaretrovirus.

Molecular characterization of proteolytic processing of the human spumaretrovirus (HSRV) Gag proteins and the precise determination of cleavage sites was performed. For in vitro processing of recombinant HSRV Gag proteins, a recombinant enzymatically active HSRV protease was employed. Recombinant Gag proteins and protease were cloned and expressed as hexa-histidine-tagged proteins in pET-32b and pET-22b vectors, respectively, in the E. coli BL21 expression strain. The recombinant proteins were purified by affinity chromatography on an immobilized metal ion matrix. To determine the precise processing sites, recombinant Gag proteins or synthetic peptides derived from Gag sequences were cleaved in vitro by the recombinant protease. Proteolytic processing reactions were carried out under optimal reaction conditions of HSRV protease in sodium phosphate buffer, pH 6.0, supplied with 2 M NaCl at 37 degrees C. The cleavage sites were determined by amino-terminal amino acid sequencing as well as by matrix-assisted laser desorption/ionization mass spectrometry analysis of the reaction products. Fluorescence spectrophotometry was used to determine cleavage kinetics of peptides mimicking different cleavage sites within the HSRV Gag proteins.

Characterization of metabolically labelled antigen following immunoprecipitation with antibody bound on ELISA plates.

Immunoprecipitation is a powerful technique for the immunochemical characterization of antigens. In combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) a number of features, e.g., presence of antigen, rate of synthesis, relative molecular weight of the polypeptide chain or post-translational modifications can be determined. Four different steps are basically involved in the immunoprecipitation procedure: (1) metabolic labelling of the antigen by incubation of viable cells with a radioactive precursor, (2) harvesting of the labelled antigen from the cells by lysis or in the case of secretory proteins from the supernatant, (3) formation and (4) purification of antibody-antigen complexes. The last step relies on secondary agents which bind to the antibody.

Metabolic radiolabelling of Mycoplasma gallisepticum on Vero cells and radioimmunoprecipitation assay.

A monoclonal antibody (MAb) A2 was produced against a major polypeptide of Mycoplasma gallisepticum with a molecular mass of 64 kDa. MAb A2 reacted in immunoblot at a titre of 10(4.33) and had a titre of 10(4.5) in an enzyme-linked immunosorbent assay. In a radioimmunoprecipitation assay (RIPA) using metabolic [35S]methionine radiolabelling of M. gallisepticum suspension in Vero cell culture, MAb A2 was able to precipitate the 64 kDa protein and another protein of 47 kDa. The present study involving [35S]methionine labelling of M. gallisepticum in Vero cells represents a novel approach for labelling and characterizing the conformation-dependent mycoplasmal antigens in a RIPA system.

Clinical evaluation of a radioimmunoprecipitation assay for IA-2 antibody and comparison of GAD antibody in type 1 diabetes mellitus.

We evaluated a insulinoma-associated protein (IA-2) antibody assay kit using 125I-labelled recombinant IA-2. IA-2 antibodies were present in patients with early-onset type 1 diabetes mellitus (DM) at frequencies of 74%, 67%, 57%, and 50% for respective periods <1 year, 1 < or =years<2, 2< or =years<3, and 3< or =years<4 after onset. IA-2 antibody frequency was low throughout the DM course as compared with glutamic acid decarboxylase (GAD) antibody frequency. No one had IA-2 antibody, but 29% still had positive GAD antibody titers after 11 years. Of the patients with 0

Serial immunoprecipitation assays for interferon--(IFN)-beta antibodies in multiple sclerosis patients.

We devised a sensitive, radioimmunoprecipitation assay (RIPA) for anti-interferon (IFN)-beta-binding antibody (BAB) detection. Our RIPA showed good agreement with a reference RIPA (mean difference, -3.2 +/- 10.6 AU), and detected BAB to both IFN-beta-1a and IFN-beta-1b. Neutralizing antibodies to IFN-b (NAB) were also determined with a standard method. BAB and NAB were measured in 393 serum samples from 77 multiple sclerosis (MS) patients treated with IFN-beta-1a or -1b, who were studied over two years, and subsequently classified as responders and non-responders. BAB were found at higher concentrations, and more frequently detected, in IFN-beta-1b- than in IFN-beta-1a-treated patients, and, at highest titres, preferentially in patients who were positive for NAB. However, in our series of MS patients, both titres and frequency of detection of BAB or NAB did not differ between IFN-b responders and non-responders.

Loss of glycosylation at Asn144 alters the substrate preference of the N8 influenza A virus neuraminidase.

Role of asparagine-linked (N-linked) oligosaccharide side chains in the maturation and the function of influenza virus neuraminidase (NA) subtype N8 was examined by site-directed mutagenesis and vaccinia virus expression system. Mutations in the consensus sequence for N-linked glycosylation at Asn 84 or 398 prevent the proper maturation of mutant NAs. On the contrary, mutation at Asn 144, that is conserved in all except two strains of influenza virus NA ever sequenced, did not affect the proper maturation and the transport of the mutant NA to the cell surface. Furthermore, this mutation led the alternation of substrate preference of this enzyme. These observations indicate that N-glycosylation at Asn 144 of N8 NA may be conserved from the functional requirement, but not from the structural necessity.