Product Manual

RSV Nucleoprotein ELISA Kit
Catalog Numbers
VPK-517196 assays

FOR RESEARCH USE ONLY
Not for use in diagnostic procedures

Introduction
Respiratory syncytial virus (RSV) infection is a significant cause of hospitalization of children and oneof the leading causes of death of infants less than 1 year of age worldwide. RSV is a member of
the Pneumoviridae genus and contains a single-stranded non-segmented negative-sense RNA genome
approximately 15,200 nt in length. There are three surface proteins that coat the virion: smallhydrophobic (SH), attachment (G), and fusion (F). G and F proteins are the major antigenic proteins.
RSV is categorized into subgroups A and B based on the sequence of the G protein.
RSV nucleoprotein (N) is essential for virus assembly and replication. It is essential for virus
encapsidation by coating the entire viral RNA genome to form the ribonucleoprotein. The RSV
nucleoprotein is highly conserved among RSV isolates from both A and B subgroups, with over 95%
amino acid sequence identities.
Cell Biolabs’ RSV Nucleoprotein ELISA Kit is an enzyme immunoassay developed for detection andquantitation of the RSV nucleoprotein. The ELISA antibodies recognize the nucleoprotein of both
RSV type A and type B. The kit has a detection sensitivity limit of 313 pg/mL RSV nucleoprotein.
Each kit provides sufficient reagents to perform up to 96 assays including standard curve and RSV
lysate samples.

Assay Principle
An anti-RSV nucleoprotein monoclonal coating antibody is adsorbed onto a microtiter plate. RSV
nucleoprotein present in the sample or standard binds to the antibodies adsorbed on the plate; anFITC-conjugated anti-RSV nucleoprotein polyclonal antibody is added and binds to the RSV
nucleoprotein captured by the first antibody. Following incubation and wash steps, a HRP-conjugatedmouse anti-FITC antibody is added and binds to the FITC conjugated anti-RSV nucleoprotein
polyclonal antibody. Unbound HRP-conjugated mouse anti-FITC antibody is removed during a washstep, and a substrate solution reactive with HRP is added to the wells. A colored product is formed inproportion to the amount of RSV nucleoprotein present in the sample. The reaction is terminated byaddition of Stop Solution and absorbance is measured at 450 nm. A standard curve is prepared fromrecombinant RSV nucleoprotein standard and sample RSV nucleoprotein concentration is thendetermined.

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-5174: Influenza A 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-RSV Nucleoprotein Antibody Coated Plate (Part No. 51711B): One strip well 96-wellplate.
2. FITC-Conjugated Anti-RSV Nucleoprotein Polyclonal Antibody (Part No. 51712C): 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 RSV Nucleoprotein Standard (Part No. 51713D): One 100 µL vial of 2 µg/mL
recombinant human RSV nucleoprotein (Met1-Leu391) in PBS containing BSA.

Materials Not Supplied
1. RSV 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 RSV Nucleoprotein Standard at -20ºC and avoid freeze/thaw.
Store all other components at 4ºC.

Safety Considerations
Remember that your RSV samples contain infectious viruses before inactivation; you must follow therecommended NIH guidelines for all materials containing infectious organisms.

Preparation of Reagents•1X Wash Buffer: Dilute the 10X Wash Buffer Concentrate to 1X with deionized water. Stir tohomogeneity.
3•FITC-Conjugated Anti-RSV Nucleoprotein Polyclonal Antibody and HRP-Conjugated Anti-FITC
Monoclonal Antibody: Immediately before use dilute the FITC-conjugated antibody 1:1000 andHRP-conjugated antibody 1:1000 with Assay Diluent. Do not store diluted solutions.

Preparation of Standard Curve
1. Prepare a dilution series of RSV Nucleoprotein Standard in the concentration range of 20 ng/mL –0.313 ng/mL by diluting the stock solution in Assay Diluent (Table 1).

StandardTubes
12345
6782 µg/mL RSV NucleoproteinStandard (µL)
10500 of Tube #1
500 of Tube #2
500 of Tube #3
500 of Tube #4500 of Tube #5500 of Tube #60

Assay Diluent(µL)
990500
500
500
500
500
500
500

RSVNucleoprotein(ng/mL)
201052.51.25
0.625
0.313
0Table 1. Preparation of RSV 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.

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

Assay Protocol
1. Prepare and mix all reagents thoroughly before use.
2. Each RSV lysate sample, RSV nucleoprotein standard, blank, and control medium should beassayed in duplicate.
3. Add 100 µL of RSV lysate or RSV nucleoprotein standard to Anti-RSV Nucleoprotein Antibody
Coated Plate.
4. Cover with a Plate Cover and incubate at 37ºC for 2 hours.
4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-RSV Nucleoprotein polyclonal Antibody to eachwell.
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.

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

Figure 1: RSV Nucleoprotein ELISA Standard Curve

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Figure 2: RSV Nucleoprotein in RSV Culture Fluid. RSV A culture fluid (1.05 x 106 TCID50/mL)
was first diluted 1000-fold with culture medium, then heat inactivated and lysed in Viral Lysis Buffer.
RSV lysate was tested in the RSV Nucleoprotein ELISA Kit according to the Assay Protocol.

References
1. Afonso, C. L. et al. (2016) Taxonomy of the order Mononegavirales. Arch. Virol. 161, 2351–2360.
2. Shi, T. et al. (2017) Global, regional, and national disease burden estimates of acute lower
respiratory infections due to respiratory syncytial virus in young children in 2015: a systematicreview and modelling study. Lancet 390, 946–958.
3. Bhella D., Ralph A., Murphy L. B., Yeo R. P. (2002) Significant differences in nucleocapsid
morphology within the Paramyxoviridae. J Gen Virol 83, 1831–1839.
4. Tawar R. G., Duquerroy S., Vonrhein C., Varela P. F., Damier-Piolle L., Castagné N., MacLellanK., Bedouelle H., Bricogne G.. (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNAcomplex of respiratory syncytial virus. Science 326, 1279–1283.

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.

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In no event shall CELL BIOLABS be liable for any proximate, incidental or consequential damages in connection with theproducts.

Contact Information
Cell Biolabs, Inc.
5628 Copley Drive
San Diego, CA 92111
Worldwide: +1 858-271-6500
USA Toll-Free: 1-888-CBL-0505
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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