Microneutralization Titer Calculate

Influenza Virus Titration, Antigenic Characterization, and Serological Methods for Antibody Detection

Alexander Klimov, Amanda Balish, Vic Veguilla, Hong Sun, Jarad Schiffer, Xiuhua Lu, Jacqueline M. Katz, and Kathy Hancock

Abstract

This chapter describes some commonly used methods of influenza virus titration, antigenic characterization, and serological methods by antibody detection. These methods are essential not only for virus characterization but also for identifying new antigenic variants, vaccine strain selection, and sero-epidemiologic studies of influenza virus transmission and prevalence. Virus titration methods such as the hemagglutination assay, 50% egg or tissue culture infectious dose, and plaque assay are employed to determine the amount of virus particles in a sample. The hemagglutination inhibition assay is a reliable, relatively simple and inexpensive technique to antigenically characterize isolates of influenza viruses. Serological methods such as virus neutralization and hemagglutination inhibition are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. While serological methods rarely yield an early diagnosis of acute influenza virus infection, well-timed, paired acute, and convalescent serum samples may establish the diagnosis of a recent influenza infection even when attempts to detect the virus are negative.

Key words: Virus titration, Hemagglutination assay, Plaque assay, 50% Egg infectious dose, Hemagglutination inhibition assay, 50% Tissue culture infectious dose , Microneutralization, Virus neutralization, Neutralizing antibodies, Influenza

1. Introduction

There are a number of methods of influenza virus titration based on its biological characteristics, such as the virus' ability to agglutinate red blood cells (RBCs) of different species (1). The ability of infectious and noninfectious virus particles to agglutinate RBC is the basis of the hemagglutination assay (HA). The plaque assay, 50% tissue culture infectious dose (TCID50), and 50% egg infectious dose (EID50) are methods used to determine the amount of infectious

Yoshihiro Kawaoka and Gabriele Neumann (eds.), Influenza Virus: Methods and Protocols,

Methods in Molecular Biology, vol. 865, DOI 10.1007/978-1-61779-621-0_3, © Springer Science+Business Media, LLC 2012

virus particles in a sample. Serological methods such as virus neutralization and hemagglutination inhibition (HAI) are the fundamental tools used in sero-epidemiologic studies of influenza virus transmission and prevalence and in the evaluation of vaccine immunogenicity. Both assays are based on the binding of antibodies to hemagglutinin (HA), the major surface glycoprotein of influenza. Neutralizing antibodies directed against HA are the major mediator of protective immunity against influenza.

Proper virus titration is essential for analysis of antigenic properties of major surface proteins of the virus, the hemagglutinin and the neuraminidase. Comparative antigenic analysis of different strains using standard antiserum, particularly from postinfection ferrets, plays an important role in monitoring antigenic evolution of influenza viruses of different types and subtypes. Antigenic characterization is an important tool in the selection of the most updated vaccine strains.

The standard method for influenza virus titration is based on the ability of the virus hemagglutinin (HA) to agglutinate RBCs of different species (see Note 1). Usually avian RBCs, such as from chickens or turkeys, are used in the hemagglutination assay (see Note 2). Avian RBC are small and nucleated, they settle fast and form a compact button on the bottom of the V-well microtiter plate in the absence of the virus, thus providing with clear endpoint dilution determination. Non-nucleated mammalian RBC (human, guinea pig, horse, etc.) appear as a "halo" or circle of settled cells on the bottom of the U-well microtiter plate control wells, making endpoint dilution reading difficult.

The hemagglutination (HA) assay is dependent on the amount of hemagglutinin on the surface of influenza viruses and not the ability of the virus to replicate (2). The HA assay is able to quantify viral particles regardless of infectivity. The HA endpoint is determined by the highest dilution of virus that causes complete hemag-glutination. The HA titer is the reciprocal of the dilution of virus in the last well with complete hemagglutination. An HA unit (HAU) is defined as the amount of virus needed to agglutinate an equal volume of standardized RBCs (3). This "unit" of hemagglu-tination is an operational unit and not a measure of an absolute amount of virus. The influenza virus isolated in cell culture, such as the Madin-Darby Canine Kidney (MDCK) cell line or embryonated chicken eggs, is titrated by performing a twofold serial dilution of the isolated supernatant in a buffer using a 96-well microtiter plate. The first well contains only the isolate, the second and subsequent wells contain 50 ml of buffer and the diluted virus. The HAUs are used in the HAI assay to determine the standardized dilution of antigen needed to perform the assay.

The EID50 is a biological method to determine the amount of infectious virus in a sample by determining the highest dilution of the sample that can infect 50% of the embryonated chicken eggs.

This assay entails performing serial dilutions of the egg sample. To determine the dilution needed to produce a 50% positive result, the Reed-Muench method is used (4). The Reed-Muench method requires the use of three or more eggs per dilution to determine the 50% endpoint by performing a hemagglutination assay for each inoculated egg.

The plaque assay is based on the ability of influenza virus to form plaques in cell monolayer overlaid with agar or agarose (5, 6). The plaques are formed due to cytopathic effect (CPE) caused by the virus and death of the infected cells thus leading to the formation of circular zones of lysed cells on the monolayer. Only infectious virus particles should infect the host cell and be able to produce a plaque. Plaque forming units (PFU) is a quantitative measure of the amount of infectious virus in a sample by determining the number of plaques formed in a cell monolayer (usually MDCK cells are used). At a high dilution of virus stock, each plaque represents the zone of cells infected by a single virus particle. Therefore, the titer of a virus stock can be calculated in PFU per milliliter. The plaque assay is carried out by performing tenfold serial dilutions and then infecting the cell monolayer. An agarose overlay is placed in the wells of the infected cell monolayer. The agarose is removed; the virus inactivated with ethanol and crystal violet is added to the monolayer to visualize the plaques.

Specific antibody attachment to antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on RBCs. This effect inhibits hemagglutination and is the basis for the HAI assay. The HAI test, originally described by Hirst (7) and later modified by Salk (8), is currently performed in 96-well microtiter plates. Briefly, a standardized quantity of HA antigen is mixed with serial dilutions of antiserum, and RBCs are added to determine specific binding of antibody to the HA molecule. The presence of specific anti-HA antibodies will inhibit the agglutination, which would otherwise occur between the virus and the RBCs ( 7). This assay is reliable, relatively simple, and is an inexpensive technique. Limitations of the HAI test include the need to remove nonspecific agglutinins in some serum samples since they may cause false-negative results, the need to standardize the virus concentration each time a test is performed, and the need for specialized expertise in reading the results of the test.

The microneutralization test is a highly sensitive and specific assay for identifying influenza virus-specific, neutralizing antibodies in animal and human sera (9). The 2-day assay is performed in two stages. On day 1 of the assay (1) a virus-antibody reaction step, in which virus is mixed with dilutions of serum and time allowed for antibodies to react and (2) an inoculation step, in which the virus-serum mixture is inoculated into the appropriate host system, MDCK cells in this assay. An ELISA is performed on day 2 of the assay to detect virus-infected cells. The absence of infectivity constitutes a positive neutralization reaction and indicates the presence of virus-specific antibodies in the serum sample. The preferred serum samples in cases of influenza-like illness are paired acute and convalescent serum samples with the acute collected less than 7 days after symptom onset and the convalescent collected at least 14 days after the acute sample and ideally within 2-3 months of illness onset. A fourfold or greater rise in antibody titer demonstrates a seroconversion and is considered to be diagnostic. With single serum samples, care must be taken in interpreting low titers such as 20 and 40. Generally, knowledge of the antibody titers in an age-matched control population is needed to determine a minimum titer that is indicative of a specific antibody response to the virus utilized in the assay.

Conventional neutralization tests for influenza viruses based on the inhibition of CPE formation and/or detection of hemagglutination activity in MDCK cell cultures are laborious and rather slow. Typically, CPE is read at days 3-7. The microneutralization assay described here uses an ELISA on day 2 of the assay to detect virus-infected cells. Studies have shown that neutralization assays using ELISA to detect virus-infected cells are less variable than neutralization evaluated using CPE and/or detection of hemag-glutination activity, possibly because of the extended length of such assays, their use of an endpoint which is more difficult to measure than absorbance, and/or their use of preformed monolayers (10, 11). The steps involved in the influenza virus microneutraliza-tion assay are the following. Serially diluted sera are preincubated with a standardized amount of virus prior to the addition of MDCK cells. Serum neutralizing antibodies to influenza virus hemaggluti-nin inhibit the infection of MDCK cells with virus. After an overnight incubation, the cells are fixed and the presence of influenza A virus nucleoprotein (NP) in infected cells is detected by ELISA. The detection of NP indicates the absence of neutralizing antibodies at that serum dilution.

The microneutralization assay gives the most direct answer to the question of whether an individual has antibodies that can neutralize the infectivity of a given virus strain. The test has several additional advantages for detecting antibody to influenza virus. First, the assay primarily detects antibodies to the virus hemag-glutinin and thus can identify functional, strain-specific antibodies in animal and human sera. Second, since infectious virus is used, the assay can be developed quickly upon recognition of a novel virus and is available before suitable purified viral proteins become available for use in other assays. However, the use of live virus in this assay also predicates the need for adherence to biosafety guidelines as outlined in the Biosafety in Microbiological and Biomedical Laboratories (www.cdc.gov/od/OHS/biosfty/bmbl5/ bmbl5toc.htm).

2. Materials*

2.1. Hemagglutination 1. 96-Well microtiter plates (Nunc, Thermo Fisher Scientific): Titration Assay V-bottom for avian RBCs or U-bottom for mammalian RBCs.

2. 0.01 M Phosphate-buffered saline (PBS), pH 7.2-7.4 (Invitrogen, Corporation, Cat. # 4539).

3. Standardized RBCs (see Notes 1 and 2): 0.5% for avian and 0.75% for mammalian RBCs. Filter RBCs through sterile gauze, centrifuge at 200 xg for 10 min at 4-8°C, aspirate off plasma, alsevers and buffy coat, add PBS, centrifuge and repeat twice. Resuspend RBCs in PBS and adjust to required concentration. Store at 4-8°C (see Note 3).

2.2.50% Egg 1. 9-to 11-day-old embryonated chicken eggs, candled to ensure

Infectious Dose that the embryos are viable (see Note 4).

2. Virus diluent: supplement 10 ml PBS with 1 ml penicillin-streptomycin solution (100 U/ml penicillin G and 100 mg/ml streptomycin) (Invitrogen, Cat. # 15140) and 0.2 ml 50 mg/ ml gentamicin stock (10 mg/ml) (Invitrogen, Cat. # 15750060). Filter with a 0.2-mm membrane filter and can be stored up to 2 months at 4-8°C.

3. Standardized RBCs (0.5% for avian and 0.75% for mammalian), filter through sterile gauze, centrifuge at 200 x g for 10 min at 4-8°C, aspirate off plasma, alsevers and buffy coat, add PBS, centrifuge and repeat twice. Resuspend RBC in PBS to necessary concentration. Store at 4-8°C.

4. 96-Well microtiter plates (Nunc, Thermo Fisher Scientific): V-bottom for avian RBCs or U-bottom for mammalian RBCs.

2.3. Plaque Assay 1. 2x Plaque assay medium: Dulbecco's Modified Eagle's Medium

(DMEM) (Invitrogen) supplemented with penicillin-streptomycin (100 U/ml penicillin G and 100 mg/ml streptomycin), l-glutamine 4 mM and HEPES buffer 50 mM. To sterilize, filter through a 0.2mm membrane filter. Store for not more than 2 months at 4-8°C.

2. Lonza SeaKem Le Agarose (Thermo-Fisher Scientific, Cat. # 5004).

3. MDCK cells (ATCC# CCL-34) confluent in a 6-well tissue culture plates (Nunc, Thermo-Fisher Scientific, Cat. # 150239).

4. Plaque assay wash medium: supplement 490 ml of DMEM with 5 ml of penicillin-streptomycin (100 U/ml penicillin G and 100 mg/ml streptomycin). Filter through a 0.2-mm membrane filter. Can be stored up to 2 months at 4-8°C.

5. l-1-Tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin (2 mg/ml) working solution: supplement 10 ml of identification of particular products is provided as a guide to aid in the selection of equivalent, suitable products

DMEM with 20 mg TPCK-treated type XIII trypsin from bovine pancreas (Sigma-Aldrich, Cat. # T1426-100 mg). Sterilize through filtration. Store in small aliquots at -70 to -80°C until the earliest expiration date of the products.

7. Gram crystal violet primary stain (Becton, Dickinson, and Company, Cat. # 212525).

2.4. Hemagglutination 1. Receptor destroying enzyme (RDE) (II) "Seiken" (Denka Seiken Inhibition Assay Co, Ltd.), dissolved in physiological saline (0.85% NaCl).

3. 96-Well microtiter plates (Nunc, Thermo Fisher Scientific): V-bottom for avian RBCs or U-bottom for mammalian RBCs.

4. Standardized RBCs (0.5% for avian and 0.75% for mammalian): filter RBCs through sterile gauze, centrifuge at 200 xg for 10 min at 4-8°C, aspirate off plasma, alsevers and buffy coat, add PBS, centrifuge and repeat twice. Resuspend RBCs in PBS to required concentration. Store at 4-8°C (see Note 3).

2.5. MDCKCell Culture 1. MDCK medium prepared from DMEM (Invitrogen, Cat. #

11965-092), supplemented with 10% fetal bovine serum (FBS) (Hyclone Cat. # SH30070.03), 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Cat. # 15140-122), and 2 mM l-glutamine (Invitrogen Cat. # 25030-081). Sterilize by filtration.

2. Solution of trypsin (0.05%) and ethylenediamine tetraacetic acid (EDTA) (Invitrogen, Cat. # 25300-054) (see Note 5).

2.6. TCID Assay The materials needed for the TCID are the same as those listed for to Determine Working the microneutralization assay.

Dilution of Virus for Microneutralization

Assay

2.7. Microneutra 1. Virus diluent prepared using DMEM, supplemented with 1% lization Assay bovine serum albumin (BSA) (fraction V, protease free, Roche,

Cat. # 03117332001, prepared as a 10% w/v solution in dH2O, filter sterilized, and stored at 4-8°C) 100 U/ml penicillin, 100 mg/ml streptomycin, and 20 mM HEPES (Invitrogen, Cat. # 15630-080). Prepare fresh for each assay. Sterilize by filtration.

2. PBS (0.01 M PBS, pH 7.2). Sterilize by autoclaving.

3. Fixative: Cold 80% acetone in PBS. Prepare and use 80% acetone in a BSC that is externally vented or in a chemical hood. Wear standard PPE which includes latex or nitrile gloves, lab coat, and safety glasses. Prepare the day before use and store at -20°C. It is critical that the fixative is cold when used. After use, discard appropriately.

4. Wash Buffer: PBS + 0.3% Tween 20 (Sigma-Aldrich, Cat. P1379). Prepare fresh daily.

5. Antibody diluent: PBS + 0.3% Tween 20 + 5% w/v milk (nonfat dry milk). Prepare fresh daily. About 1 L is needed for 40 microtiter assay plates.

6. 1° antibody: Anti-influenza A NP mouse monoclonal antibody (Millipore, Cat. # MAB8257 and MAB8258, both purified Ig; mix equal quantities). Dilute in antibody diluent at optimum concentration as determined by titration (see Subheading 3.7.2, steps 5-11).

7. 2° Antibody: Goat anti-mouse IgG conjugated to horseradish peroxidase (hRP) (KPL, Cat. # 074-1802). Dilute in antibody diluent at optimum concentration as determined by titration (see Subheading 3.7.2, steps 1-4).

8. Substrate: a-phenylenediamine dihydrochloride (OPD) in citrate buffer. Prepare citrate buffer by mixing one buffer capsule (Sigma-Aldrich Cat. # P4922) with 100 ml dH2O. The contents of one capsule dissolved in 100 ml of dH2O yields 0.05 M phosphate-citrate buffer, containing 0.03% sodium perborate as a substitute for H2O2, pH 5.0 at 25°C. Handle OPD tablets in a BSC that is externally vented or in a chemical hood. Wear standard PPE which includes latex or nitrile gloves, lab coat, and safety glasses. After the tablets are dissolved, the substrate may be used on the BSL-2 laboratory bench top. Prepare OPD immediately before use by adding OPD tablets (Sigma-Aldrich, Cat. # P8287) to citrate buffer, one tablet for each 20 ml of citrate buffer. After use, discard appropriately.

9. OPD stop solution (0.5 M sulfuric acid). Add 28 ml of concentrated sulfuric acid (18 M) to 972 ml dH2O to obtain 0.5 M sulfuric acid. Prepare this solution in a BSC that is externally vented or in a chemical hood. After the addition of dH2O, the stop solution may be used on the BSL-2 laboratory bench top. Wear standard PPE which includes latex or nitrile gloves, lab coat, and safety glasses. After preparation, store 0.5 M H2SO4 on bench top. After use, discard appropriately.

10. Influenza virus (see Note 6). The TCID (tissue culture infective dose) must be determined for each lot number of virus before beginning an MN assay. Virus propagated in MDCK cells or in the allantoic fluid of eggs must be stored at -70°C or colder in single use aliquots. Thaw quickly shortly before use and put on ice. Once the virus dilution for the assay is made, any remaining virus in the thawed aliquot must be appropriately discarded. Virus that has been refrozen should not be used.

11. Serum samples for testing. All serum samples must be stored frozen at -20 to -80°C. Thaw serum samples rapidly in a 37°C

water bath. As soon as thawed, place on ice. Keep on ice or at 4-8°C until returned to freezer. If testing from the same aliquot the next day, it is acceptable to keep serum samples at 4-8°C overnight and return to freezer the next day. This method of rapid thawing and handling at 4-8°C minimizes antibody denaturation. Before use in the microneutralization assay, human serum samples must be heat-inactivated at 56°C for 30 min. Animal serum samples, for example positive and negative control sera, must be treated with RDE before use (see Subheading 3.4.1). RDE treatment includes a heat-inactivation step.

12. Purified mouse IgG (Thermo-Fisher Cat. # 31202) is needed for the determination of the working dilution of the 2° antibody (conjugate).

3. Methods

3.1. Hemagglutination In the hemagglutination assay, serial virus dilutions are mixed with Assay a constant amount of RBCs. The RBCs contain numerous recep tors for virus hemagglutinin and agglutination occurs, if the virus concentration is high enough. If serum is added, the agglutination of RBCs by the influenza virus will be blocked; this is the bases of the HAI assay. Control antiserum and antigens should be included in every HAI assay to verify the specificity of the test.

1. An aliquot (100 ml) of each influenza virus isolated in tissue culture or embryonated chicken eggs (see Note 7) is placed in a well at the first row (row 1) of the 96-well microtiter plate (Fig. 1). Add 50 ml of PBS to rows 2 through 12. Perform a serial twofold dilution series down the 96-well plate transferring 50 ml from row 1 to row 2, etc., disposing of the final 50 ml from the last well. Add 50 ml of the standardized RBCs to all wells. Tap the plate gently to mix or use a mechanical plate shaker. Incubate the plates at room temperature (20-25°C) for the incubation time required for the RBCs used (Table 1).

2. After the incubation period, the HAU are observed for their endpoint of agglutination (Fig. 2). The RBCs will settle to the bottom of the V-well microtiter plate in negative samples, and in positive samples they will agglutinate. The wells in column 8 contain no virus. RBCs that agglutinate will evenly distribute within the well, whereas RBCs that do not agglutinate will form a button in the bottom of the V-well. The HA titer is the last dilution that shows complete hemagglutination activity (see Note 8).

ABCDEF GH

lOOul virus 50ul

OOOOOOOCS serial

OOOOOOOO^dilution

OOOOOOOO' 00000000' 00000000' 00000000' oooooooo; 00000000 00000000 00000000 00000000 + oooooooo.

Fig. 1. The 96-well microtiter plate layout for the hemagglutination titration assay.

Table 1

Recommended time of incubation at 20-25°C for the hemagglutination assay with the use of different species of RBCs

Avian

Mammalian

Chicken Turkey

Guinea pig

Human type O

Concentration

O.50/0

0.5%

0.75% v. 75%

0.75%

Microtiter plate

V

V

U

U

Incubation time

3O min

30 min

l h

l h

Appearance of control cells

Buttona

Buttona

Halo

Halo

a When tilted at a 45° angle the RBCs in the V-well plate will flow a When tilted at a 45° angle the RBCs in the V-well plate will flow

Add 450 ml of the virus diluent to 10 vials labeled 1 through 10. Aliquot 50 ml of the influenza virus stock to be tested into the first vial, then perform a tenfold dilution by transferring 50 ml from vial 1 to subsequent vials, changing tips between each vial.

2. Inoculate three (or more) embryonated eggs with 100 ml of each virus dilution (see Note 9). Incubate eggs at 35-37°C for 48 h for influenza A and at 33-35°C for 72 h for influenza B. After incubation, chill eggs overnight to halt embryonic growth. Harvest eggs and perform a hemagglutination assay using 100 ml of allantoic fluid from each egg. Observe the endpoint of agglutination and record the results as positive or negative for agglutination.

3.2. Egg Infectious 1.

Dose Assay

Wells

Wells

Hemagglutination Test Picture Story
8 0

Fig. 2. Interpreting the hemagglutination titration assay.

Table 2

Example of EID50 calculation using the Reed-Muench method

Log of virus

Infected

Cumulative

Cumulative

Percent

dilution

samples

positive (A)

negative (B) Ratio of A/(A+B)

infected (%)

-5

3/3

6

0 6/6

100

-6

2/3

3

1 3/4

75

-7

1/3

1

3 1/4

25

-8

0/3

0

6 0/6

0

Proportional distance formula:

[(»/»positive value > 50% - 50%)/(%positive value > 50%) - (»/»positive value < 50%)]

Infectious dose50 titer: 1065 ID50/0.1 ml or 1075 ID50/ml

3. Determine the EIDS0 titer per 100 ml by the Reed-Muench method (Table 2).

3.3. Plaque Assay 1. Pre-warm the 2x plaque assay medium in a 37°C water bath and prepare 1.6% (w/v) solution of agarose in a 56°C water bath.

2. Remove the growth medium from the 6-well MDCK tissue culture plates, wash cell monolayer with room temperature plaque assay wash medium (see Note 10). Do not pipette medium directly onto the monolayer as this may disrupt the cells.

3. Thaw virus in cool water. Perform tenfold dilution series starting at 10-1 and diluting virus samples down to 10-10 (or less) dilution in plaque assay wash medium. Changing tips between dilutions is required. Inoculate 6-well tissue culture wells in duplicate with 100 ml diluted virus samples and gently rock tissue culture plate to cover monolayer with inoculums. Incubate plates at 33-37°C for 30-60 min (see Note 11).

4. After incubation, wash wells twice with room temperature (20-25°C) plaque assay wash medium, taking care not to add medium directly onto the monolayer as this may disrupt the cells.

5. Add 1 ml of 2 mg/ml TPCK-trypsin working stock to 2x plaque assay medium. Mix (1:1) 2x plaque assay medium with Lonza SeaKem Le Agarose and immediately add 2 ml of this mixture to each inoculated well. Allow to solidify at room temperature (20-25°C). Incubate 6-well tissue culture plates at 33-37°C. Observe MDCK plates daily with an inverted microscope for plaque formation.

6. After 72 h, remove the agar plug from each well using sterile forceps. Pipette 2 ml of 70% ethanol into each well and incubate 20 min at room temperature (20-25°C) to fix the MDCK cell monolayer. Remove ethanol and add 1 ml crystal violet solution to each well. Incubate at room temperature (20-25°C) for 10 min to stain MDCK cell monolayer. Remove crystal violet solution and wash wells with water to rinse away excess stain solution. Allow plates to dry overnight at room temperature (20-25°C) prior to counting plaques.

7. Count plaques (Fig. 3) in each well and determine the PFU per milliliter using the following formula: PFU/ml = number of plaques x dilution factor x 10 (see Note 12). The PFU/ml calculation should be based on the dilution of virus sample that gives >10 plaques per well but is still countable. The number of plaques are counted, and then multiplied by the reciprocal of the amount

Fig. 3. Well of a 6-well plate indicating plaques visualized. The plaques will appear as clear circular areas on the background of the crystal violet-stained monolayer.

of samples added to the plate, then multiply by the reciprocal of the dilution factor. For example, if 50 plaques were counted in the 10-4 well that had 0.1 ml ofsample added, 50 plaques x 10-1 ml sample x 10-4 dilution factor = 5.0 x 106 PFU/ml.

3.4. Hemagglutination Inhibition Assay

3.4.1. Treatment of Sera

1. The serum samples should be treated with RDE (see Note 13) to remove nonspecific inhibitors of hemagglutination. Add 3 volumes of RDE to the tube with 1 volume of serum. Incubate in 37°C water bath for 18-20 h. Remove tubes from the 37°C bath and place them in a 56°C water bath for 30 min to inactivate the RDE (see Note 14).

2. Remove tubes from 56°C water bath and allow to equilibrate to room temperature (20-25°C). Add 6 volumes of physiological saline (0.85% NaCl).

3. To remove nonspecific agglutinins from the serum samples, add 1 volume of packed RBCs to 20 volumes of RDE-treated serum. Mix thoroughly and incubate at 4-8°C for 1 h shaking tubes periodically to resuspend cells. Centrifuge at 400 x g for 10 min at 4-8°C.

3.4.2. Standardization 1. Based on HAUs determined in the HA assay, standardize the of Antigens antigens to 8 HAU/50 ml. For the HAI assay, the antigens will be 4 HAU/25 ml, since only 25 ml of standardized antigen is added to each well (1 ml of standardized antigen will test 5 sera). Homologous control antigens corresponding to the control antisera used in the assay should be included into the HAI assay. Calculate the antigen dilution dividing the HA titer by 8. Example: 64 HAU/8 = 8 HAU, thus add one part of antigen with titer 8 HAU to seven parts of PBS. To confirm that the antigen has 8 HAU/50 ml, make twofold dilutions of the standardized antigen with PBS and perform a back titration (see Note 15).

2. A back titration is performed similar to the hemagglutination assay. The standardized antigens should all equal to 8 HAU/50 ml. The titer is equal to 8 HAU when the first three dilutions of virus will show complete hemagglutination and the fourth and further dilutions will have partial or no hemagglutination. If the standardized antigens do not equal to 8 HAUs/50 ml, add antigen or PBS according to Table 3. After adjustment, perform another back titration to ensure all of the antigens have a titer of 8. Store the standardized antigens at 4-8°C.

3.4.3. The Hemagglutination Inhibition Assay Setup

1. Add 50 ml of the RDE treated serum to row 1 of the microtiter plate. Add 25 ml of PBS to rows 2 through 10 (see Note 16). Perform a serial twofold dilution mixing 3-5 times starting at row 1 and discarding the 25 ml of diluted serum into row 11.

Table 3

To adjust antigens based on the results of the back titration. If the back-titration result is less that 8 HA units, add more antigen and if greater than 8 HA units add buffer

Interpretation of HA units ADD undiluted antigen ADD volume of PBS

Table 3

To adjust antigens based on the results of the back titration. If the back-titration result is less that 8 HA units, add more antigen and if greater than 8 HA units add buffer

0

7 volumes

1

5 volumes

2

3 volumes

2%

2 volumes

4

Equal volume of antigen

4%

% volume

8

No adjustment

8%

% volume

16

E qual volume

16%

2 volumes

32

3 volumes

32%

5 volumes

2. Add 25 ml of standardized antigen into all wells with diluted serum samples. Shake plate gently or use a mechanical plate shaker. Incubate at room temperature for 15-30 min.

3. Add 50 ml of standardized RBCs to all wells with serum-antigen mixtures. Shake plate gently or use a mechanical shaker. Incubate at room temperature for 30-60 min, depending on the RBCs used in the assay.

4. The interpretation: the HAI titer is the reciprocal of the last dilution of antiserum that completely inhibits hemagglutination (Fig. 4). The row 1 is read as 1:10, row 2 is read as 1:20, row 3 as 1:40, etc.

3.5. MDCK Cell Culture for TCID and Microneutralization Assays

MDCK cells were originally derived from a kidney of an apparently normal adult female cocker spaniel in September 1958 by S.H. Madin and N.B. Darby (12). The line of MDCK cells used here was obtained from Dr. John Wood at the National Institute for Biological Standards and Control (NIBSC) in the UK. This cell line originated from the Common Cold Laboratory in Salisbury UK at some point prior to 1985. This "London line" of MDCK cells exhibited greater sensitivity for isolation of virus from clinical

10 20 40 80 160 320 640 1280 2560

Fig. 4. I nterpretation of an HAI assay. Columns 1, 2, 3, and 7 have HAI titers of 1:10. Column number 4 has an HAI titer of 80. Column 5 has an HAI titer of 20 and column 6 has an HAI titer of 40. Column 8 was the negative control.

specimens in studies during the mid-1980s than other MDCK cell lines. Other MDCK cell sublines may be used but should first be tested for their abilities to adhere to microtiter plates after being added as a suspension and to support infection of influenza viruses such that virus control wells achieve optimal ELISA O.D. values in the microneutralization assay.

Prior to the passage of MDCK cells, the cell monolayer should be 90-95% confluent. It is critical that the cells do not overgrow and enter stationary phase. Cells must be in log phase growth for maximum virus infectivity. Cells for use in an assay may be at 75-95% confluence. Split the confluent monolayer 1:10 2 days before use in an assay for optimum yield and growth. One T-162-cm2 flask at 95% confluence should yield enough cells to seed about 4-5 microtiter plates. If the flasks are at 75% confluence, there will be enough cells to seed about four microtiter plates. A microneutralization assay testing 100 serum samples in duplicate against two viruses requires 40 microtiter assay plates and between 8 and 12 flasks of MDCK cells depending upon cell confluency.

1. To passage the MDCK cells, remove cell culture medium and gently rinse monolayer with 5 ml trypsin-EDTA. After removing, add another 5 ml trypsin-EDTA to cover the cell monolayer. Incubate at 37°C in 5% CO2 until monolayer detaches, usually 3-10 min. As soon as most of the monolayer has been dislodged from the bottom of the flask, the remaining cells can be dislodged by gently tapping the side of the flask. Add 15 ml of MDCK medium to each flask containing trypsinized cells, bringing the total volume to 20 ml for each flask. Pipette the cells up and down about five times to separate from one another. Add cells to new 162-cm2 tissue culture flasks, each

Hemagglutination Test Chicken Rbcs

Fig. 4. I nterpretation of an HAI assay. Columns 1, 2, 3, and 7 have HAI titers of 1:10. Column number 4 has an HAI titer of 80. Column 5 has an HAI titer of 20 and column 6 has an HAI titer of 40. Column 8 was the negative control.

Table 4

Dilutions of cell suspensions for passage in T-162-cm2 flasks

Volume of cells to add Number of cells to add Dilution to new flask (ml) to new flask When confluent

Table 4

Dilutions of cell suspensions for passage in T-162-cm2 flasks

1:5 (4 ml of the 20 ml)

4.0

5-6 x 106

~24 h (1 day)

1:10 (2 ml of the 20 ml)

2.0

1.75-2 x 106

~48 h (2 days)

1:20 (1 ml of the 20 ml)

1.0

5-7 x 105

~72 h (3 days)

containing 30-50 ml MDCK medium, using the volumes of suspended cells described in Table 4. The dilutions shown refer to the proportion of suspended cells that are added to the new flask. These dilutions are based on optimal growth conditions and some variations may exist. Alternatively, the cells may be passed based on cells counts also shown in Table 4.

2. To use the MDCK cells in a TCID or MN assay (see Note 17), remove cell culture medium and gently rinse monolayer with 20 ml PBS. After removing, add 7 ml of trypsin-EDTA to cover the cell monolayer. Incubate at 37°C in 5% CO2 until monolayer detaches. Add 7 ml of virus diluent to each flask. Pipette the cells up and down to separate the cells from one another. Remove cells and transfer to 50-ml conical tube. Combine cells from two or three flasks in one, 50-ml conical tube. Fill conical tube with virus diluent, centrifuge at 485 x g for 5 min. This is the first wash.

3. Remove the supernatant and resuspend the cells in 10 ml of virus diluent. Pipette the cells up and down to separate the cells from one another. Combine all cells in one, 50-ml conical tube. Fill the conical tube with virus diluent and centrifuge again at 485 x g for 5 min. This is the second wash.

4. Remove the supernatant and resuspend cells in 10 ml virus diluent. Pipette the cells up and down to separate the cells from one another. Determine the total volume of cells needed, 10 ml per microtiter plate. Divide the total volume needed by 3 to obtain the volume of virus diluent needed to resuspend the cells in. If the cells were less than 90% confluent, then decrease the amount of virus diluent added. Gently mix.

5. Count cells using a hemacytometer or a cell counter. Adjust the cell number to 1.5 x 105 cells/ml using virus diluent.

3.6. TCID Assay to Determine Working Dilution of Virus for Microneutralization Assay

The microneutralization assay requires that a standardized amount of virus is added to each serum-containing well of the microtiter plate to test for neutralizing antibodies. To quantify the amount of virus, a TCID for the microneutralization assay is determined. Generally, the virus working dilution is 200 or 400 times the log10

virus dilution at the cut-off point. We typically use 200 times the virus dilution at the cut-off point for avian influenza viruses such as H5N1 to obtain 100 TCID in 50 ml and 400 times the virus dilution for seasonal H3N2 and seasonal or pandemic H1N1 viruses to obtain 200 TCID in 50 ml (see Note 18). The minimum dilution of the virus stock for the microneutralization assay is 1:100. The virus titration is done on day 1 and the ELISA to detect viral-infected cells is done on day 2.

1. Rapidly thaw a vial of virus at 37°C and immediately place on ice. Virus should be thawed just before use for optimal infectivity. Test each virus at two or more different starting dilutions. Dilutions of 10-2, 10-3, and 10-4 in virus diluent are suggested depending upon how well the virus replicates in eggs or cells. Test each dilution on separate microtiter plates.

2. Add 100 ml of virus diluent to all wells, except column 1, of a 96-well microtiter plate. Add 146 ml of the virus starting dilution to all wells in column 1. Transfer 46 ml serially from column 1 through column 11. Change pipette tips between wells. After mixing, discard 46 ml from column 11. This dilution scheme results in Yh log10 dilutions. Dilutions will be 10 2, 10

10 3, ... 10 7 if the starting dilution was 10 2. Column 12 contains virus diluent only (no virus) and is the cell control (CC).

3. Stack plates and cover with an empty plate. Place in 37°C, 5% CO2 incubator for 1 h to mimic the virus + serum incubation step of the microneutralization assay.

4. Prepare MDCK cells for use in assay as described. Add 100 ml of diluted cells to each well of the microtiter plate. Each well will contain 1.5 x 104 cells. Incubate at 37°C in 5% CO2 for 18-20 h.

5. The procedures for day 2 of the TCID determination, plate fixation, 1° antibody, 2° antibody, and substrate, are the same as these procedures for the microneutralization assay.

6. To analyze the data, calculate the median absorbance (O.D.) of the cell controls (column 12) and multiply by two to obtain the cut-off value. Prepare a table like Table 5. Once all test wells have been scored positive or negative for virus growth based on the cut-off value, the TCID of the virus suspension can be calculated by the method of Reed and Muench ( 4). Record the number of positive and negative values at each dilution. Select the region of the data where the transition from all wells positive at one dilution to all wells negative occurs. This region is shown in the box in Table 5. Calculate the cumulative numbers of positive wells at each dilution. The cumulative number positive is obtained by adding the positives at each dilution starting at the bottom. Calculate the cumulative numbers of negative wells at each dilution. The cumulative number negative is obtained by adding the

Table 5

Calculation of TCID by the Reed-Muench method

Observed value (O.D.) Cumulative value Dilution # Positive # Negative ST Positive Si Negative Ratio % positive

10-2

8

0

10-25

8

0

10-3

8

0

10-35

8

0

10-4

8

0

16

0

1 6/1 6

100

10-45

7

1

8

1

8/9

89

10-5

1

7

1

8

1/9

11

10-55

0

8

0

16

0/16

0

10-6

0

8

10-65

0

8

10-7

0

8

negatives at each dilution starting at the top. Determine the ratio at each dilution as follows:

„ . sum of cumulative positive

sum of cumulative positive + sum of cumulative negative negatives at each dilution starting at the top. Determine the ratio at each dilution as follows:

„ . sum of cumulative positive

sum of cumulative positive + sum of cumulative negative

Convert the ratio into percent positive. If the dilution with exactly 50% positive wells is identified, then the proportional distance does not need to be determined.

If not, then calculate the proportional distance between the dilution showing >50% positive and the dilution showing <50% positive as follows:

Proportional distance =

% positive value above 50% - 50 % positive value above 50% - % positive valuebelow 50% x Correction factor for 12 log10 dilutions 89 - 50

Calculate the MN TCID by adding the proportional distance to the dilution showing >50% positive. For the example in Table 2, add 0.25 to 4.5 to obtain 10-475. The virus working dilution that is 200 times the cut-off dilution is 10-475 x 200 = 10-475 + 10230 = 10-2 45 = 1/102 45 = 1:2 8 2. This dilution will give 100 TCID per 50 ml. The virus dilution that is 400 times the cut-off is 1:141. This dilution will give 200 TCID per 50 ml.

3.7. Microneutra-lization Assay (see Note 19)

A virus titration, to determine the MN TCID and therefore the working dilution of the virus stock, must be done prior to running a microneutralization assay. The minimal acceptable working dilution of virus in the microneutralization assay is 1:100. For the microneutralization assay, the serum titration is combined with a standard amount of virus day 1 and the ELISA to detect viral-infected cells is done on day 2. The ELISA portion of the assay uses a mouse monoclonal antibody to influenza A virus nucleopro-tein to detect virus replication in the MDCK cells. All serum samples are tested in duplicate against each virus. Each assay includes positive and negative control serum samples and a virus back titration. If animal serum samples are used as controls, then they must be RDE treated before use. Each microtiter plate includes four virus control (VC) wells and four cell control (CC) wells. See Fig. 5 for an overview of the microneutralization assay.

Hemagglutination Test Picture Story

Fig. 5. Overview of microneutralization assay. The main steps of the microneutralization assay are shown. Steps 1 and 2, the neutralization portion of the assay and step 3, the infection of MDCK cells with non-neutralized virus, are done on day 1. Steps 4 and 5, the detection of virus infected cells using an ELISA, are done on day 2.

Fig. 5. Overview of microneutralization assay. The main steps of the microneutralization assay are shown. Steps 1 and 2, the neutralization portion of the assay and step 3, the infection of MDCK cells with non-neutralized virus, are done on day 1. Steps 4 and 5, the detection of virus infected cells using an ELISA, are done on day 2.

3.7.1. Microneutraiization 1. Quickly thaw frozen sera in 37°C water bath. Immediately after Assay Procedure thawing, place sera in ice or proceed with heat inactivation for

30 min in a 56°C water bath. The heat-inactivated sera may be stored at 4°C overnight or at -20°C for a longer period of time.

2. Prepare initial serum dilutions of 1:5 using virus diluent. We recommend preparing the serial twofold dilutions of serum samples in Titertubes (Biorad, Cat. # 223-9390). To prepare serial dilutions for testing in duplicate against two viruses, add 50 ml of heat-inactivated sera to 200 ml of virus diluent. Add 125 ml of virus diluent to the other Titertubes and serially dilute transferring 125 ml. Discard 125 ml from the last Titertube after mixing. Add 125 ml of virus diluent to all Titertubes to make the serum dilutions 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. The total volume in each tube is 250 ml.

3. Transfer 50 ml of diluted sera from the Titertubes into four microtiter plates (two plates per virus) starting with row H and going to row A. Change the pipette tips after transferring the sera in row A to the microtiter plate. It is critical that the tips are changed before beginning to pipette the next set of serum samples. Cover and hold in 37°C, 5% CO2 incubator while the diluted virus is being prepared. It is critical that the proper pH is maintained so that there will be no deleterious pH effects on the virus when it is added.

4. Dilute the virus in virus diluent to the correct working dilution as determined by the TCID assay. Five milliliters of diluted virus are needed per plate. Hold on ice. Add 50 ml of diluted virus to wells containing sera and the VC wells (A12, B12, C12, and D12). Do not add virus to the CC wells (E12, F12, G12, and H12) and do not add virus to the column of wells reserved for the virus back titration. Tap the plate gently to mix. Add 50 ml of virus diluent to the CC wells.

5. In each assay, include a back titration of the working dilution of virus. Add 50 ml of virus diluent to all wells in one column. Add 50 ml of the working dilution of virus to the first well and make serial twofold dilutions by transferring 50 ml from the first well to each successive well through well 12. Discard 50 ml from well 12. To avoid virus carryover, change pipette tips between wells. Add an additional 50 ml of virus diluent to all wells in the column to bring the total volume to 100 ml. Cover and incubate at 37°C, 5% CO2 for 1 h. During this incubation, prepare the MDCK cells as described in Subheading 3.5.

6. Add 100 ml of diluted cells to each well of the microtiter plates. Each well contains 1.5 x 104 cells. Stack all plates in four plate stacks and cover each stack with an empty plate. Incubate at 37°C in 5% CO2 for 18-20 h.

7. On the second day, after incubation, remove the medium from microtiter plates by carefully decanting into an autoclavable container. Add 200 ml PBS to each well to wash and remove PBS wash by carefully decanting. Tap inverted plate on laboratory paper towels.

8. Add 100 ml cold fixative to each well. Do not allow wells to dry out before the fixative is added. Stack and cover each stack with an empty plate. Incubate at room temperature (RT) for 10-12 min.

9. Remove the fixative by carefully decanting. Spray a paper towel with 70% EtOH and wipe exterior of plates. Let the plates air-dry inside the BSC for 10 min or until dry. After the microtiter plates have been wiped with EtOH and have air dried, they may be removed from the BSC.

10. Visually check the cell monolayer in each well and note any wells with reduced cell confluence on the process sheet. Compare the cell confluence to that observed in the CC. Cell toxicity due to the serum sample is most likely to be noticed in the wells with the lowest dilution of serum (see Note 20). Plates may be held at 4°C for up to 2 days before proceeding with the ELISA portion of the assay. Cover with an empty plate, place in plastic bags, and seal.

11. Dilute the 1° antibody (mouse anti-influenza A NP) in antibody diluent to a predetermined optimum working dilution. Mix well. Wash plates 3x with 300 ml wash buffer per well. Add 100 ml of diluted 1° antibody to each well. Stack plates and cover with an empty plate. Incubate for 1 h at RT.

12. Dilute 2° antibody (goat anti-mouse IgG HRP labeled) in antibody diluent to a predetermined optimum working dilution. Mix well. Wash plates 3x with 300 ml wash buffer per well. Add 100 ml diluted 2° antibody to each well. Stack plates and cover with an empty plate. Incubate for 1 h at RT.

13. Prepare citrate buffer. Wash plates 5x with 300 ml wash buffer per well. Prepare OPD substrate in citrate buffer immediately before use. Tap washed plates on a laboratory paper towel. Add 100 ml freshly prepared OPD substrate to each well. Incubate at RT until the color change in the VC wells (column 12, rows A-D) is intense and the corresponding color change in the CC wells is minimal. Add 100 ml stop solution to each well. The O.D. in the VC wells should be at least 0.8 and is typically in the range of 1.0-1.5 though higher is acceptable. The O.D. in the CC wells must be <0.2. The incubation time required to obtain the targeted O.D. may vary between viruses. Read the absorbance (O.D.) of wells at 490 nm using a microtiter plate spectrophotometer. See Fig. 6 for the image of a developed and stopped MN plate.

Fig. 6. I mage of developed microneutralization assay plate. Ten serum samples were tested on this plate in columns 1 though 10 as noted at the bottom of the plate. The serum dilution is shown on the right-hand side of the microtiter plate. Column 11 shows the virus back titration (BT). The virus control (VC) is shown in the first four wells of column 12; the cell control (CC) in the second four wells of column 12. The titers obtained for these serum samples after data analysis are shown across the top of the plate.

14. The data are analyzed as follows: The VC and CC medians are determined for each plate. In order for the ODs on each plate to be considered valid, the CC must have a median OD of <0.2. The VC must have a mean OD of >0.8. The virus back titration is evaluated to determine if the working dilution of virus used in the assay contained the desired amount of virus. The cut-off value for the virus back titration is the mean of the VC median and the CC median. This is the same cut-off that is used to evaluate neutralizing antibody titers. The dilution of the first well below the cut-off value is the back titration titer. The dilutions in the back titration wells are beginning at well A: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. In general, back titration titers of 16, 32, and 64 are acceptable (see Note 21).

15. Neutralizing antibody titers are determined by calculating the cut-off value to determine a 50% neutralizing antibody titer for each plate based on the equation: (median O.D. ofVC + median O.D. of CC)/2 = X, where X= the 50% cut-off value. All values below or equal to X are positive for neutralization. Read each column which contained diluted serum from the bottom, beginning at well H. Note the first well with an OD of less than the 50% cut-off. The reciprocal serum dilution corresponding to that well is the 50% neutralization antibody titer for that serum sample. Serum dilutions are well A 1:10, well B 1:20, well C 1:40, well D 1:80, well E 1:160, well F 1:320, well G 1:640, and well H 1:1,280. Serum samples with a titer of >1,280 may be repeated beginning at a 1:80 starting dilution to obtain an

Fig. 6. I mage of developed microneutralization assay plate. Ten serum samples were tested on this plate in columns 1 though 10 as noted at the bottom of the plate. The serum dilution is shown on the right-hand side of the microtiter plate. Column 11 shows the virus back titration (BT). The virus control (VC) is shown in the first four wells of column 12; the cell control (CC) in the second four wells of column 12. The titers obtained for these serum samples after data analysis are shown across the top of the plate.

endpoint titer. For an assay to be valid, the control serum samples must meet the following criteria: (1) the GMT of the duplicate titers of all negative control serum samples must be <10 and (2) the GMT of the duplicate titers of all positive serum samples must be >80 and must be within twofold of a previously determined GMT based on 10 or more assays done in duplicate. See Table 6 to troubleshoot potential problems associated with the interpretation of the microneutralization assay.

3.7.2. Determination of Working Dilution for Primary and Secondary Antibodies in the Microneutralization Assay

To detect all influenza nucleoprotein expressed in the MDCK cells, an excess of primary and secondary antibodies must be used. The working dilution, to ensure excess reagent, is determined as described below.

1. The working dilution of the secondary antibody, goat anti-mouse IgG HRP labeled, is determined first. Purified mouse IgG is used to coat microtiter plates at a concentration of 2 mg/ml in PBS, 100 ml/well. Incubate for 2 h or longer at room temperature with shaking using a mini-orbital shaker.

2. Wash plates 3x with 300 ml wash buffer per well. Prepare serial twofold dilutions of secondary antibody in antibody diluent buffer, typically beginning at 1:250 and continuing through 1:32,000. Add 100 ml of each dilution, in triplicate, to the coated wells. As a control, include a no conjugate control that contains antibody diluent only. Incubate for 30 min at room temperature with shaking.

3. Wash plates 3x with 300 ml wash buffer per well. Add 100 ml of OPD substrate in citrate buffer and develop until the color is intense in the wells with the highest concentrations of secondary antibody. Add 100 ml of stop solution to each well. Read the absorbance (OD) of wells at 490 nm using a microtiter plate spectrophotometer.

4. Analyze the data by graphing the mean OD of each dilution versus the secondary antibody dilution. It is expected that the curve will plateau at the lower dilutions. Choose the highest dilution in the plateau region. When a second lot of secondary antibody is evaluated, titrate both lots in the same assay to aid in the selection of a comparable working dilution.

5. The working dilution of the primary antibody, anti-influenza A NP mouse monoclonal antibody, is determined second. Test using two viruses, preferably of different subtypes. Add 50 ml of virus diluent to all wells in columns 1 through 8. Add 100 ml of virus diluent to all wells in columns 9 through 12.

6. Prepare the working dilution of each virus by diluting in virus diluent. Add 50 ml of the first diluted virus to all wells in columns 1 through 4. Add 50 ml of the second diluted virus to all wells in columns 5 through 8. Cover and hold in 37°C, 5% CO2 incubator while the MDCK cells are being prepared.

Table 6

Potential problems associated with interpretation of the microneutralization assay

Problem Possible cause(s) Solution

Table 6

Potential problems associated with interpretation of the microneutralization assay

Weak or no color in virus control (VC) wells

Problem with ELISA: using wrong antibodies or substrate Buffer solutions incorrect Too little virus used in assay or forgot to add virus to VC wells Virus inactivated during incubation step MDCK cells not optimal: too old (>30 passages), not in log phase growth, or contaminated

Check the antibodies and substrate Prepare fresh buffers

Redetermine virus TCID or adjust the dilution of virus used or repeat and do not forget to add virus to VC wells Check incubator temperature and CO, level

Thaw a new vial of cells, do not allow cells to enter stationary phase

Weak or no neutralization by positive control sera

Too much virus used in assay Serum deteriorated

MDCK cells not optimal: too old (>30 passages), not in log phase growth, or contaminated

Redetermine virus TCID or adjust the dilution of virus used Obtain new antisera, check storage conditions

Thaw a new vial of cells, do not allow cells to enter stationary phase

Neutralization by negative control sera

Nonspecific reaction or cross-reactivity Too little virus used in assay

Check the RDE treatment

Check samples for cross-reactive antibodies

Redetermine virus TCID or adjust the dilution of virus used

Nonspecific virus inactivation

Serum not heat inactivated or contains nonspecific viral inhibitors

Heat-inactivated serum (56°C, 30 min). Treat animal serum with RDE

MDCK cell monolayer toxicity

Serum not heat inactivated or is toxic to cells

Heat-inactivated serum (56°C, 30 min). Run serum toxicity check on normal MDCK cells and check microscopically for toxicity

7. Prepare the MDCK cells as described in Subheading 3.5. Add 100 ml of diluted cells to each well of the microtiter plates. Each well contains 1.5 x 104 cells. Stack all plates in four plate stacks and cover each stack with an empty plate. Incubate at 37°C in 5% CO2 for 18-20 h.

8. Fix the plates as described in Subheading 3.7.1. As the plates are air drying, prepare serial twofold dilutions of anti-NP antibody in antibody diluent buffer, typically beginning at 1:250 and continuing through 1:32,000. Wash plates 3x with 300 ml wash buffer per well. Add 100 ml of 1:250 dilution to all wells in row A. Add the next dilution to row B and continue through row H. Following this design, each primary antibody dilution is tested against each virus in quadruplicate. There are cell controls, in quadruplicate, for each antibody dilution. Testing in triplicates is also acceptable. Incubate at room temperature for 1 h.

9. Dilute the 2° antibody (goat anti-mouse IgG HRP labeled) in antibody diluent to the optimum working dilution as determined above. Mix well. Wash plates 3x with 300 ml wash buffer per well. Add 100 ml diluted 2° antibody to each well. Stack plates and cover with an empty plate. Incubate for 1 h at RT.

10. Prepare citrate buffer. Wash plates 5x with 300 ml wash buffer per well. Prepare OPD substrate in citrate buffer immediately before use. Tap washed plates on a laboratory paper towel. Add 100 ml freshly prepared OPD substrate to each well. Incubate at RT until the color is intense in the wells with the highest concentrations of secondary antibody. Add 100 ml of stop solution to each well. Read the absorbance (O.D.) ofwells at 490 nm using a microtiter plate spectrophotometer.

11. Analyze the data by graphing the mean O.D. of each dilution versus the primary antibody dilution for the wells with virus and the wells without (the cell controls). It is expected that the curve will plateau at the lower dilutions. Choose the highest dilution in the plateau region and also take into account background noise in the cell control. When a second lot of primary antibody is evaluated, titrate both lots in the same assay to aid in the selection of a comparable working dilution.

4. Notes

1. Blood from a farm should be prescreened, it is recommended to find a commercial source. The RBCs used for all testing should be as fresh as possible. The blood should be preserved in Alsever's or sodium citrate at a 1:1 dilution.

2. In the 1990s, human influenza A (H3N2) lost its ability to agglutinate chicken RBCs (13) RBCs such as turkey or guinea pig RBCs are preferred when testing for currently circulating influenza types and subtypes.

3. Standardized RBCs may be used up to 1 week or until hemo-lyzed for the HA assay and the EID50. RBCs used for the HAI assay should be made the day of the test for optimal sensitivity.

4. Use of dead, broken or nonembryonate

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  • JOHANNA
    What is a negative titer for microneutralization?
    6 months ago
  • jörg
    How to prepare dilution samples of influenza virus?
    4 months ago

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