Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay

Khan, Muhammad; Gallagher, Marie R.; Bucher, Doris; Cerini, Chiara; Kilbourne, Edwin D.

doi:10.1128/jcm.16.1.115-122.1982

Cited by 20 publications

(9 citation statements)

References 18 publications

(20 reference statements)

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“…NI assays can be susceptible to background interference by HA‐specific antibodies that hinder the substrate’s access to the NA catalytic site 12–14 . Therefore, we generated viral reagents by cloning and reverse genetics to contain the relevant NA gene in combination with H6 HA, a novel subtype for humans which has been used in past NI assays 15,16 . After establishing optimized conditions for the miniaturized assay, its sensitivity and specificity were tested using ferret and human sera against N1 and N2 antigens of recent seasonal influenza vaccine strains, and compared directly with results obtained using the conventional protocol.…”

Section: Introductionmentioning

confidence: 99%

A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

Sandbulte¹,

Gao²,

Straight³

et al. 2009

Influenza Resp Viruses

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Background Antibodies to neuraminidase (NA) contribute to protection during influenza virus infection, but NA inhibition (NI) titers are not routinely analyzed in vaccine trials. One reason is the cumbersome nature of the conventional thiobarbituric acid (TBA) NI assay, which uses chemical methods to quantify free sialic acid following incubation of NA with substrate in the presence of serum. In addition, the assay is complicated by the need to use virus of a hemagglutinin (HA) subtype novel to the host to detect NA‐specific antibodies only. Objectives Our primary objectives were to miniaturize the colorimetric NI assay to a format suitable for quantitative analysis of large numbers of samples, and validate the specificity and sensitivity of the miniaturized format with ferret and human sera. An additional aim was to use reverse genetics to construct HA‐mismatched viral reagents bearing NA of recent influenza A vaccine strains and H6 HA. Results Analysis of ferret antisera by the miniaturized assay demonstrated sensitivity and specificity comparable with the conventional assay. Similar increases in the NI titers in sera from vaccinated human volunteers were measured in miniaturized and conventional assays. Inactivated and live‐attenuated vaccines increased NI titers against a given subtype at approximately the same rate. Conclusions The reagents and miniaturized format of the TBA method described here provide a platform for practical serological monitoring of functional antibodies against NA.

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Section: Introductionmentioning

confidence: 99%

A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

Sandbulte¹,

Gao²,

Straight³

et al. 2009

Influenza Resp Viruses

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“…The H6 NA is immunologically irrelevant to antibody in the human population. Therefore, even when traces of HA persist in these preparations, their usefulness as potential NA-specific vaccines or testing reagents is not significantly diminished (11,13).…”

Section: Resultsmentioning

confidence: 99%

“…d Sole responder to HA antigen; HA inhibition titer of 1:40. tion activity to H6 HA in the microtiter (Dynatech) system (9, 18). A reassortant virus (H6N4; A/duck/France/MB/42/ 76) with heterologous NA was used as the testing antigen (11).…”

Section: Methodsmentioning

confidence: 99%

“…In the present study we found that use of an H6 HA best facilitated the separation of the Ni NAs; reassortants (H6N2) were also generated to provide ready purification of N2, the other antigenic NA subtype found in human influenza viruses. A previous report described the use of Ni purified by this technique as the adsorbent antigen in an enzyme-linked immunosorbent assay for detecting NA antibodies in a clinical population (11).…”

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confidence: 99%

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Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures

Gallagher¹,

Bucher²,

Dourmashkin³

et al. 1984

J Clin Microbiol

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Neuraminidases were purified from reassortant viruses (H6N1 and H6N2) containing the two antigenic subtypes (Nl and N2) found in human influenza viruses. Surface glycoproteins were solubilized with octylglucoside, and the neuraminidase was isolated by chromatography on DEAE-Sephadex. Neuraminidase isolated by this technique coeluted with viral lipids and spontaneously formed liposomes on dialysis. The purified neuraminidase was immunogenic in rabbits, producing a significant antibody response at dose levels as low as 1 ,ug.

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“…Control wells in which there was no antigen or no antiserum and others in which normal allantoic fluid was used as antigen at a protein concentration equivalent to that of the viral antigen were also included in each test. Plates were again incubated overnight (a procedure which we found enhanced the sensitivity of the test [7]) and washed three times with PBS-Tween, and 100 ,ul of an optimal dilution (in PBS-Tween-0.5% bovine serum albumin) of goat antihuman immunoglobulin G Fab serum conjugated with alkaline phosphatase (Dynatech Diagnostics, Inc., South Windham, Maine) was added to each well. Plates were incubated overnight and washed as before, and 100 ,ul of a solution of p-nitrophenylphosphate substrate (1 mg/ml; Sigma Chemical Co., St. Louis, Mo.)…”

Section: Methodsmentioning

confidence: 99%

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay

Khan¹,

Bucher²,

Koul³

et al. 1982

J Clin Microbiol

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An enzyme-linked immunosorbent assay test system was developed in which purified influenza virus M protein was used for the detection of M antibody in human sera. Antibody levels to influenza A virus M protein were monitored in sera from a vaccine study population by using an enzyme-linked immunosorbent assay technique with purified M protein as the adsorbent antigen. A 10-fold variation in titers of preexisting M antibody was observed in this population of young adults. Increases of anti-M titer of 7to 24-fold were observed upon immunization with Formalin-inactivated vaccine or after natural infection. The antibody response to M protein was dissociated from the response to the hemagglutinin or neuraminidase antigens. The M antibody response preceded or was coincident with the antibody response to Hi hemagglutinin upon natural exposure to circulating virus.

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Detection of influenza virus neuraminidase-specific antibodies by an enzyme-linked immunosorbent assay

Cited by 20 publications

References 18 publications

A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

A miniaturized assay for influenza neuraminidase‐inhibiting antibodies utilizing reverse genetics‐derived antigens

Isolation of immunogenic neuraminidases of human influenza viruses by a combination of genetic and biochemical procedures

Detection of antibodies to influenza virus M protein by an enzyme-linked immunosorbent assay

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