Product Manual

Influenza A Nucleoprotein ELISA Kit
Catalog Numbers
VPK-5174

96 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
The influenza virus is an enveloped virus that can be divided into three classes, A, B, and C, largely
based upon conserved antigenic differences in the internal nucleoprotein (NP). Only Influenza A and B
are clinically relevant for humans. Influenza A virus, typically encountered more frequently than typeB, is associated with the majority of serious epidemics, and it can be further subdivided into strains or
subtypes based on antigenic differences in the external hemagglutinin proteins (H1-H16) andneuraminidase proteins (N1-N9).
The primary function of NP is to encapsidate the segmented RNA and bind with thethree polymerase subunits, PA, PB1 and PB2, to form ribonucleoprotein particles (RNPs) for RNA
transcription, replication and packaging.
Cell Biolabs’ Influenza A nucleoprotein ELISA Kit is an enzyme immunoassay developed for
detection and quantitation of the Influenza A nucleoprotein. The ELISA antibodies only recognize thenucleoprotein from Influenza A, and will not react with the nucleoprotein from Influenza B nor
Influenza C. The kit has a detection sensitivity limit of 625 pg/mL Influenza A nucleoprotein. Eachkit provides sufficient reagents to perform up to 96 assays including standard curve and Influenza A
lysate samples.

Assay Principle
An anti-Influenza A nucleoprotein monoclonal antibody is adsorbed onto a microtiter plate. Influenza
A nucleoprotein present in the sample or standard binds to the antibody adsorbed on the plate; anFITC-conjugated anti-Influenza A nucleoprotein monoclonal antibody is added and binds to theInfluenza A nucleoprotein captured by the first antibody. Following incubation and wash steps, anHRP-conjugated mouse anti-FITC antibody is added and binds to the FITC conjugated anti-Influenza
A nucleoprotein monoclonal antibody. Unbound HRP-conjugated mouse anti-FITC antibody isremoved during a wash step, and a substrate solution reactive with HRP is added to the wells. Acolored product is formed in proportion to the amount of Influenza A nucleoprotein present in thesample. The reaction is terminated by addition of Stop Solution and absorbance is measured at 450 nm.
A standard curve is prepared from the provided recombinant Influenza A nucleoprotein standard, andthe sample Influenza A nucleoprotein concentration is then determined.

Related Products
1. VPK-108-H: QuickTiter™ Lentivirus Quantitation Kit (HIV-1 p24 ELISA)
2. VPK-150: QuickTiter™ Hepatitis B Core Antigen (HBcAg) ELISA Kit
3. VPK-151: QuickTiter™ Hepatitis C Core Antigen (HCcAg) ELISA Kit
4. VPK-156: QuickTiter™ MuLV Core Antigen (MuLV p30) ELISA Kit
5. VPK-5169: Adenovirus Hexon ELISA Kit
6. VPK-5170: RSV Fusion Protein ELISA Kit
7. VPK-5171: RSV Nucleoprotein ELISA Kit
8. VPK-5175: Influenza B Nucleoprotein ELISA Kit

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Kit Components
Box 1 (shipped at room temperature)
1. Anti-Influenza A Nucleoprotein Antibody Coated Plate (Part No. 51741B): One strip well 96well plate.
2. FITC-Conjugated Anti-Influenza A Nucleoprotein Monoclonal Antibody (Part No. 51742C):
One 20 µL vial.
3. HRP-Conjugated Anti-FITC Monoclonal Antibody (Part No. 310811): One 20 µL vial.
4. Assay Diluent (Part No. 310804): One 50 mL bottle.
5. 10X Viral Lysis Buffer (Part No. 51693B): One 15 mL bottle containing 200 mM Tris, pH 7.5,
1500 mM NaCl, 10% Triton X-100, 1% SDS.
6. 10X Wash Buffer (Part No. 310806): One 100 mL bottle.
7. Substrate Solution (Part No. 310807): One 12 mL amber bottle.
8. Stop Solution (Part. No. 310808): One 12 mL bottle.
Box 2 (shipped on blue ice packs)
1. Recombinant Influenza A Nucleoprotein Standard (Part No. 51743D): One 100 µL vial of 4µg/mL recombinant human Influenza B nucleoprotein (Met1-Tyr560) in PBS containing BSA.

Materials Not Supplied
1. Influenza A Sample: purified virus or unpurified viral supernatant
2.
3.
4.
5.
6.

Microcentrifuge
10 µL to 1000 µL adjustable single channel micropipettes with disposable tips
50 µL to 300 µL adjustable multichannel micropipette with disposable tips
Multichannel micropipette reservoir
Microplate reader capable of reading at 450 nm (620 nm as optional reference wave length)

StorageUpon receiving, aliquot and store the Influenza A Nucleoprotein Standard at -20ºC and avoid
freeze/thaw. Store all other components at 4ºC.

Safety Considerations
Remember that your Influenza A samples contain infectious viruses before inactivation; you must
follow the recommended NIH guidelines for all materials containing infectious organisms.

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Preparation of Reagents•

1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir tohomogeneity.

•FITC-Conjugated Anti-Influenza A Nucleoprotein Monoclonal Antibody and HRP-ConjugatedAnti-FITC Monoclonal Antibody: Immediately before use dilute the FITC-conjugated antibody
1:1000 and HRP-conjugated antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Preparation of Standard Curve
1. Prepare a dilution series of Influenza A Nucleoprotein Standard in the concentration range of 40
ng/mL – 0.625 ng/mL by diluting the stock solution in Assay Diluent (Table 1).

StandardTubes
12
3
456784 µg/mL Influenza A
NucleoproteinStandard (µL)
10500 of Tube #1
500 of Tube #2
500 of Tube #3
500 of Tube #4
500 of Tube #5500 of Tube #60Assay Diluent
(µL)
990
500
500
500
500
500
500
500

Influenza A
Nucleoprotein(ng/mL)
4020105
2.5
1.25
0.625
0Table 1. Preparation of Influenza B Nucleoprotein Standard
2. Transfer 225 µL of each dilution to a microcentrifuge tube containing 25 µL of 10X Lysis Buffer.
Perform the assay as described in Assay Protocol.

Influenza Virus Sample Inactivation and Lysis
1. (Optional) Dilute Influenza A samples in culture medium. Include culture medium as a negativecontrol.
2. Transfer 225 µL of each sample to a microcentrifuge tube containing 25 µL of 10X Lysis Buffer,vortex well. Inactivate Influenza A sample at 56ºC for 30 min.
3. Centrifuge at 12,000 x g for 5 minutes at 4°C. Collect the supernatant as Influenza A lysate.

Assay Protocol
1. Prepare and mix all reagents thoroughly before use.
2. Each Influenza A lysate sample, Influenza A nucleoprotein standard, blank, and control medium
should be assayed in duplicate.
43. Add 100 µL of Influenza A lysate or Influenza A nucleoprotein standard to Anti-Influenza ANucleoprotein Antibody Coated Plate.
4. Cover with a Plate Cover and incubate at 37ºC for 2 hours.
5. Remove plate cover and empty wells. Wash microwell strips 5 times with 250 µL 1X Wash Buffer
per well with thorough aspiration between each wash. After the last wash, empty wells and tap
microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
6. Add 100 µL of the diluted FITC-Conjugated Anti-Influenza A Nucleoprotein Monoclonal
Antibody to each well.
7. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
8. Remove plate cover and empty wells. Wash the strip wells 5 times according to step 5 above.
9. Add 100 µL of the diluted HRP-Conjugated Anti-FITC Monoclonal Antibody to all wells.
10. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
11. Remove plate cover and empty wells. Wash microwell strips 5 times according to step 5 above.
Proceed immediately to the next step.
12. Warm Substrate Solution to room temperature. Add 100 L of Substrate Solution to each well,including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation
time may vary from 2-30 minutes.
Note: Watch plate carefully; if color changes rapidly, the reaction may need to be stopped sooner
to prevent saturation.
13. Stop the enzyme reaction by adding 100 µL of Stop Solution into each well, including the blankwells. Results should be read immediately (color will fade over time).
14. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave
length.

5Example of Results
The following figures demonstrate typical Influenza A Nucleoprotein ELISA results. One should use
the data below for reference only. This data should not be used to interpret actual results.

Figure 1: Influenza A Nucleoprotein ELISA Standard Curve.

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Figure 2: Influenza A Nucleoprotein in Influenza A Culture Fluid. Influenza A culture fluid (3.16x 106 TCID50/mL) was first diluted 100-fold with culture medium, then heat inactivated and lysed in
Viral Lysis Buffer. Influenza A lysate was subjected to Influenza A Nucleoprotein ELISA Kit
according to Assay Protocol.

References
1. Eisfeld AJ, Neumann G, Kawaoka Y. (2015) At the centre: influenza A virus
ribonucleoproteins. Nat Rev Micro. 13, 28–41.
2. Compans RW, Content J, Duesberg PH. (1972) Structure of the Ribonucleoprotein of Influenza
Virus. J Virol. 10, 795–800.
3. Martin K, Helenius A. (1991) Transport of incoming influenza virus nucleocapsids into thenucleus. J Virol. 65, 232–244.
4. O’Neill RE, Jaskunas R, Blobel G, Palese P, Moroianu J. (1995) Nuclear import of influenza virus
RNA can be mediated by viral nucleoprotein and transport factors required for protein import. JBiol Chem. 270, 22701–22704.
5. Avalos RT, Yu Z, Nayak DP. (1997) Association of influenza virus NP and M1 proteins withcellular cytoskeletal elements in influenza virus-infected cells. J Virol. 71, 2947–2958.
6. Digard P, Elton D, Bishop K, Medcalf E, Weeds A, Pope B. (1999) Modulation of nuclear
localization of the influenza virus nucleoprotein through interaction with actin filaments. JVirol. 73, 2222–2231.
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7. Herz C, Stavnezer E, Krug R, Gurney T. (1981) Influenza virus, an RNA virus, synthesizes itsmessenger RNA in the nucleus of infected cells. Cell 26, 391–400.
8. Jackson DA, Caton AJ, McCready SJ, Cook PR. (1982) Influenza virus RNA is synthesized atfixed sites in the nucleus. Nature 296, 366–368.

Warranty
These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used inaccordance with their instructions. THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSEDWARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR
WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CELL BIOLABS’s sole obligation and purchaser’s
exclusive remedy for breach of this warranty shall be, at the option of CELL BIOLABS, to repair or replace the products.
In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

Contact Information
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San Diego, CA 92111
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E-mail: tech@cellbiolabs.com
www.cellbiolabs.com©2024: Cell Biolabs, Inc. - All rights reserved. No part of these works may be reproduced in any form without permissions
in writing.

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