We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.
Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.
Neuraminidase (NA) is a glycoprotein expressed on the surface of influenza virions. Its enzyme activity is essential for release of newly formed virus particles from infected cells 1,2. Antibodies that inhibit NA activity reduce virus titers and disease symptoms in animal models 3 and correlate with resistance against disease in humans 4. An increase in NA inhibition (NI) titers following vaccination may therefore serve an indicator of the vaccine's efficacy, however many past influenza immunogenicity studies did not include this end-point because the traditional assay to measure NI antibody titers is impractical for routine serology.
The traditional method to measure NI titers is based on quantifying the amount of sialic acid that is cleaved from glycoconjugates by NA 1. This method, often referred to as the thiobarbituric acid (TBA) method, uses hazardous chemicals to convert sialic acid to a chromophore that can be quantified by spectrometry. The assay is not suitable for testing large numbers of samples because individual glass tubes are used, making the assay cumbersome. Miniaturization of the assay to a 96 well plates provides a more practical format 5, however this assay still requires the use of toxic chemicals and therefore is not ideal.
An alternate assay to measure NI antibody titers was developed by Lambré et al. 6. This assay quantifies enzyme activity by measuring the amount of the penultimate sugar of glycoproteins, galactose, which becomes exposed when sialic acid is released by NA. Since peanut-agglutinin (PNA) binds specifically to galactose, a PNA-horseradish peroxidase (HRPO) conjugate can be used to obtain a colorimetric read-out. The optical density that is measured is therefore proportional to the NA activity in the sample. NI titers are measured by determining the highest dilution of serum that inhibits at least 50% of the NA activity. This enzyme-linked lectin assay (ELLA) is performed in 96-well plates coated with fetuin, a highly glycosylated serum protein, as the substrate for NA.
An important consideration for assays that measure NI titers, is the source of NA. This is because sera from vaccinated or infected individuals contain antibodies to hemagglutinin (HA) as well as antibodies to NA. Antibodies that bind to HA can interfere with NA activity and therefore, to avoid non-specific inhibition by HA-specific antibodies, purified NA or whole virus containing an antigenically-mismatched HA should be used in the assay. These viruses can be generated by classic reassortment or by reverse genetics; the objective is to rescue a virus that contains HA of a subtype that is not related to the target strain, and NA of the virus that is being studied. The assay described in this article employs influenza A viruses which are generated by reverse genetics to contain HA of subtype H6 and the targeted NA from influenza A viruses, subtypes H1N1 and H3N2 5.
Results generated by ELLA and miniaturized TBA assays show these methods are comparable, with similar subtype specificity and sensitivity 7. ELLA does not require the use of hazardous chemicals and therefore is the preferred method to measure NI titers. It is easy to perform, with only a few steps (Figure 1): samples are diluted and transferred to a fetuin-coated plate to which virus (the source of NA) is added. The plate is incubated O/N and washed before adding PNA-HRPO. After a 2 hr incubation, the plate is washed and peroxidase substrate is added; the color reaction is stopped and finally the optical density is measured and the reciprocal of the sample dilution that resulted in at least 50% inhibition of NA activity is reported as the 50% end-point titer. An intra-laboratory study to assess reproducibility of the ELLA, showed that plate-to-plate variability is minimal and operator-to-operator repeatability resulted in no more than 2-fold differences in titer 7. A subsequent inter-laboratory study of ELLA variability showed that the assay has good reproducibility when performed in different laboratories and that inclusion of a standard can further reduce variability in results 8. The ELLA is suitable for measuring NI antibody titers in serum panels from preclinical and clinical influenza vaccine studies 7 and can also be used to evaluate antigenic differences between the NAs of influenza viruses 9.
All live reassortant viruses with avian components must be handled using biosafety level 2 (BSL2)-enhanced practices in a laboratory approved for use by the United States Department of Agriculture (USDA) and the institutional biosafety committee.
1. Preparation of Reagents and Starting Material
Note: Refer to the Materials Table to obtain the source of all reagents.
2. Determination of the Amount of NA to Use in ELLA
3. Enzyme-linked Lectin Assay
Note: Figure 2 shows the setup of the dilution and assay plates.
4. Data Analysis
The assay presented in this manuscript is shown diagrammatically in Figure 1. Figure 2 shows the 96 well plate layout, and indicates that serial dilutions of the serum samples are prepared in a dilution plate before transferring them to the fetuin-coated plate. A critical parameter of the assay is the amount of neuraminidase that is used in the assay; this is determined through titration of the reassortant virus. Examples of H6N1 and H6N2 virus titrations are shown in Figure 3. At 1:10, 1:20 and 1:40 dilutions of H6N1, the optical density was similar, indicating that the conditions were such that a maximum reading was obtained. At a 1:80 dilution, the read-out was approximately 90% of the maximum and therefore this dilution of virus was used in subsequent assays. Examples of serum titrations are shown in Figure 4. The figure shows a human serum sample that was titrated against H6N1 and H6N2 viruses. Each data point shows the percent inhibition of NA activity that was achieved by serial dilutions of serum; the first serum dilution in the H6N1 assay was 1:80; the first dilution of serum in the H6N2 assay was 5. Since the highest dilution that results in ≥50% inhibition is the NI antibody titer, the titer of the serum shown in this example is 640 against the NA of NC/99 (N1) and 160 against the NA of WI/05 (N2).
Figure 1. A schematic of the ELLA protocol. (A) Determination of the dilution of antigen (virus) to use in the assay (step 2 of the protocol): Virus dilutions are added to a fetuin coated plate and the NA activity measured by quantifying terminal galactose residues through binding of PNA-HRPO as described in step 2.3 of the protocol; (B) Determination of serum NI antibody titers (step 3 of the protocol). Samples are diluted and then transferred to a fetuin-coated plate where virus is added. The plate is incubated O/N before adding PNA-HRPO and completing the steps needed to generate a color reaction. Finally, the color reaction is stopped and the optical density is measured. The reciprocal of the sample dilution that results in at least 50% inhibition of NA activity is reported as the 50% end-point titer. Please click here to view a larger version of this figure.
Figure 2. Diagram to show the ELLA plate set up. Serial dilutions of serum samples are made in a dilution plate and then transferred to a fetuin-coated plate. Please click here to view a larger version of this figure.
Figure 3. Examples of H6N1 and H6N2 virus titrations. Serial dilutions of H6N1BR/07 (NA of A/Brisbane/59/2007 (H1N1) shown in blue symbols) and H6N2UR/07 (NA of A/Uruguay/716/2007 (H3N2) shown in red symbols) were incubated for 18 hr in fetuin-coated plates and the reactivity with PNA-HRPO determined as described. Average OD490nm of 2 wells is plotted against the reciprocal of the virus dilution. The dilution of H6N1 selected for use in the ELLA was 1:80 and the dilution of H6N2 selected was 1:40 because these dilutions resulted in approximately 90% of the maximum optical density and were within the linear range. Please click here to view a larger version of this figure.
Figure 4. Titration of serum against NA of N1 (red symbols) and N2 (blue symbols) subtypes. NA inhibition titers were measured against reassortant viruses H6N1NC/99 (contains the NA of A/New Caledonia/20/1999 (H1N1)) and H6N2WI/05 (contains the NA of A/Wisconsin/67/2005 (H3N2)). Percent enzyme inhibition for serial dilutions of serum is shown with the dashed horizontal line indicating 50% inhibition. The NA inhibition titers against the NAs of A/New Caledonia/20/1999 (H1N1) (NC/99) and A/Wisconsin/67/2005 (H3N2) (WI/05) were 29.3 (640) and 27.3(160), respectively. Please click here to view a larger version of this figure.
Problem | Possible cause(s) | Solution |
Weak signal or no plateau reached in virus titration | i) low NA enzyme activity ii) virus stock not stored under optimal conditions |
i) confirm that the diluent has pH that is optimal for NA activity; if pH is optimal, prepare a new virus stock or concentrate virus ii) Regrow and aliquot stock; snap-freeze vials on dry ice before storing at -80 °C |
Weak or no color in positive cell control wells | i) Virus dilution incorrectly assigned ii) Vial-to-vial variability in frozen virus aliquots iii) PNA- HRPO denatured or diluted too much iv) OPD incorrectly prepared |
i) Repeat virus titration ii) Titrate several vials from the same batch to ensure there is no variability. If significant variability, prepare fresh aliquots iii) Use optimum virus dilution to retitrate PNA- HRPO iv) Repeat with fresh preparation of OPD |
Weak or no inhibition by positive control sera | i) Too much virus used in assay ii) Serum deteriorated |
i) Repeat virus titration ii) Obtain new antisera Check storage conditions |
Inhibition by negative control sera | i) Inadequate heat-treatment of serum ii) Too little virus used in assay |
i) Repeat heat-inactivation of serum ii) Repeat virus titration |
High background | i) Possible contamination of plates ii) PNA- HRPO concentration too high/too low |
i) Repeat using freshly coated plates ii) Titrate PNA-HRPO to identify correct dilution to use |
Virus titration shows apparent inhibition of NA activity at low dilutions | i) Allantoic fluid may contain substrate for NA | ii) Use virus that has been pelleted through a sucrose cushion |
Table 1. Root cause analysis of assays that do not meet performance criteria. This table provides possible causes and solutions for problems that may occur when performing the ELLA.
The enzyme-linked lectin assay is a practical method to measure NI antibody titers in sera. Although ELLA was described by Lambre et al., in 1990, its acceptance as a standard serologic assay has been more recent, with numerous laboratories performing the assay to measure NI antibody titers of clinical samples 12-16. Some modifications can be made to protocol steps without a large impact to the NI titers that are measured. For example, PBS can be used as diluent, however, it is very helpful to compare the virus titration curves in the recommended buffer (MES, pH 6.5) and PBS before a decision is made, as some NA subtypes have significantly reduced enzyme activity at pH >7.0. When the optimal pH is not used, the maximum signal of virus may be reduced, resulting in the use of an excessive amount of antigen (i.e., virus) in the assay; under these conditions, the assay may have reduced sensitivity.
Some non-specific inhibition is observed when untreated sera are tested in the ELLA. This is removed by heat-treatment (56 °C for 45 min), indicating the presence of thermolabile β-inhibitors. Other non-specific inhibitors (α and γ-class) of influenza HA have been described, particularly in relation to H3N2 virus hemagglutination and infectivity. Indeed, non-specific inhibition is also observed in ELLA when H3N2 viruses are used as the source of NA; this inhibition is removed by treatment of the serum samples with a small amount of sialidase 9.
As for other serologic assays, negative and positive controls should be included in each assay to provide a means to evaluate assay performance. When the assay has not met acceptance criteria, the reason should be identified. Table 1 provides a list of potential steps in the assay that can be addressed to troubleshoot problems.
The ELLA protocol provided in this report uses reassortant viruses that contain the HA originating from an avian virus, as the source of NA. This presents a limitation to performing the assay because a permit is required to work with low pathogenic avian viruses. H6Nx reassortant viruses can be shared between laboratories after they have been inactivated. Therefore the assay can be conducted through collaborations once the laboratory generating the reassortant virus has demonstrated that the inactivation is complete and the preparation has retained its NA activity. The constraint of using H6Nx reassortant viruses can be overcome by using non-infectious sources of NA. For example, some laboratories have used purified recombinant NA 17, while others have used virus-like particles (VLPs) 15. However, neither recombinant NA nor VLPs are readily available and therefore we continue to use influenza viruses with an antigenically-mismatched avian HA.
The traditional method to measure NI antibody titers is not practical for several reasons; of most concern are the harmful chemicals that are needed to produce a color reaction. In addition, large volumes of sample are needed and the assay is cumbersome to perform. In contrast, the ELLA is easy to perform and is in a format that allows titration of a reasonable number of samples. Data from studies that examined ELLA variability show that the assay yields reproducible results 7,8. The ELLA has consequently made routine measurement of NI antibody titers possible, and it is anticipated that it will be used more frequently by laboratories conducting serologic studies.
There are several critical steps that need to be considered when performing the ELLA. First, non-specific inhibitors of NA activity must be removed. These inhibitors are generally thermolabile and are destroyed by heat treatment at 56 °C for 45 min. Heat treatment is sufficient to remove non-specific inhibitors from samples when H6 reassortant viruses are used as the source of NA, however, when H3N2 viruses are used in the ELLA, serum factors that bind to hemagglutinin also interfere with the ELLA and should be removed by treatment with a small amount of sialidase prior to heat treatment 9. Second, an excessive amount of antigen, i.e., reassortant H6Nx virus, should not be used as this reduces the sensitivity of the assay. Careful titration of virus is therefore an essential step; the amount of antigen used in each assay must provide a signal that is well above the background, and must be within the linear range of the titration curve, i.e., the signal (optical density) must be proportional to the virus dilution.
In addition to routine serology, the ELLA can be used to evaluate antigenic differences between NAs of seasonal influenza viruses. This information may be very helpful when selecting virus strains for inclusion as vaccine candidates and will facilitate a greater understanding of immune pressures that result in antigenic drift of NA. In addition, antigenic analysis of NAs from swine or avian influenza viruses may provide critical information in determining the pandemic potential of emerging strains.
The authors have nothing to disclose.
This project was funded by intramural PanFlu funds from the Center for Biologics Evaluation and Research (CBER). Plasmids used to prepare reassortant viruses were kindly provided by Robert Webster, St Jude Children’s Research Hosptial, Memphis, TN. We are indebted to Ewan Plant and Haruhiko Murata for critical review of the manuscript.
coating buffer | KPL | 50-84-01 |
fetuin | Sigma | F3385 |
5X MES, pH 6.5 | KD-Medical | PBS-0134 |
CaCl2 | Sigma | C7902 |
30% BSA | Sigma | A8327 |
Tween 20 | Sigma | P1379 |
Lectin PNA-HRPO | Sigma | L7759 |
PBS-T | Sigma | P3563-10PAK |
o-Phenylenediamine dihydrochloride | Sigma | P8287 |
phosphate-citrate buffer | Sigma | P4922 |
Sulfuric acid | Sigma | 258105 |
96-well Maxisorp plates | NUNC | 439454 |
96-well round bottom well plates | NUNC | 267245 |
Plate sealers | Thermo Scientific | 14-245-192B |
chicken eggs | Charles River Laboratories | 9 day old embryonated specific pathogen free (SPF) chicken eggs |
multichannel pipette | Variety of suppliers e.g., Rainin, Eppendorf | 8 or 12 channel manual pipettem 50-250 µl volume |
Pipette tips | Depends on pipettor brand | Depends on pipettor brand |
Plate reader, with 490 nm filter | Perkin Elmer | Victor V |
water bath set to 37 °C | Variety of suppliers | Variety of models |
water bath set to 56 °C | Variety of suppliers | Variety of models |
refrigerator set to 4 °C | Variety of suppliers | Variety of models |
incubator set to 37 °C | Variety of suppliers | Variety of models |