Product Manual

RSV Fusion Protein ELISA Kit
Catalog Numbers
VPK-5170

96 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 ofthe 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-F protein is a trimeric transmembrane glycoprotein that mediates binding of RSV to cellular
receptors and induces pH-independent fusion between the viral envelope and the cellular plasma
membrane. The F glycoprotein is highly conserved among RSV isolates from both A and B
subgroups, with over 90% amino acid sequence identities.
Cell Biolabs’ RSV Fusion Protein ELISA Kit is an enzyme immunoassay developed for detection andquantitation of the RSV fusion protein. The ELISA antibodies recognize the fusion protein from both
RSV type A and type B. The kit has a detection sensitivity limit of 156 pg /mL RSV-F. Each kit
provides sufficient reagents to perform up to 96 assays including standard curve and RSV lysate
samples.

Assay Principle
An anti-RSV fusion protein monoclonal coating antibody is adsorbed onto a microtiter plate. RSV
fusion protein present in the sample or standard binds to the antibodies adsorbed on the plate; anFITC-conjugated anti-RSV fusion protein monoclonal antibody is added and binds to the RSV fusion
protein captured by the first antibody. Following incubation and wash steps, a HRP-conjugated mouseanti-FITC antibody is added and binds to the FITC conjugated anti- RSV fusion protein monoclonal
antibody. Unbound HRP-conjugated mouse anti-FITC antibody is removed during a wash step, andsubstrate solution reactive with HRP is added to the wells. A colored product is formed in proportion
to the amount of RSV fusion protein present in the sample. The reaction is terminated by addition ofStop Solution and absorbance is measured at 450 nm. A standard curve is prepared from RSV fusion
protein standard and sample RSV fusion protein 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

Kit Components
Box 1 (shipped at room temperature)
1. Anti-RSV-F Antibody Coated Plate (Part No. 51701B): One strip well 96-well plate.
22. FITC-Conjugated Anti-RSV-F Monoclonal Antibody (Part No. 51702C): 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. RSV Fusion Protein Standard (Part No. 51703D): One 100 µL vial of 1 µg/mL recombinanthuman RSV fusion protein 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 Fusion Protein 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.

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

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Preparation of Standard Curve
1. Prepare a dilution series of RSV Fusion Protein Standard in the concentration range of 10 ng/mL –0.156 ng/mL by diluting the stock solution in Assay Diluent (Table 1).
StandardTubes
123456781 µg/mL RSV Fusion ProteinStandard (µL)
10500 of Tube #1500 of Tube #2500 of Tube #3500 of Tube #4500 of Tube #5500 of Tube #60Assay Diluent(µL)
990
500
500
500
500
500
500
500

RSV F(ng/mL)
105
2.5
1.25
0.625
0.313
0.156
0Table 1. Preparation of RSV Fusion Protein 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 fusion protein standard, blank, and control medium should beassayed in duplicate.
3. Add 100 µL of RSV lysate or RSV fusion protein standard to Anti-RSV-F 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-RSV-F Monoclonal Antibody to each well.
7. Cover with a plate cover and incubate at room temperature for 1 hour on an orbital shaker.
4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 RSV Fusion Protein ELISA results. One should use the data
below for reference only. This data should not be used to interpret actual results.

Figure 1: RSV Fusion Protein ELISA Standard Curve

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Figure 2: RSV fusion protein 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 subjected to RSV Fusion Protein ELISA Kit according to 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 systematic
review and modelling study. Lancet 390, 946–958.
3. Teng, M. N., Whitehead, S. S. & Collins, P. L. (2001) Contribution of the respiratory syncytial
virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro andin vivo. Virology 289, 283–296.
4. McLellan, J. S. et al. (2013) Structure of RSV fusion glycoprotein trimer bound to a prefusionspecific neutralizing antibody. Science 340, 1113–1117.
5. Swanson, K. A. et al. (2011) Structural basis for immunization with postfusion respiratorysyncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl.
Acad. Sci. USA 108, 9619–9624.
6. Gonzalez-Reyes, L. et al. (2001) Cleavage of the human respiratory syncytial virus fusion protein
at two distinct sites is required for activation of membrane fusion. Proc. Natl. Acad. Sci. USA 98,9859–9864.
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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’sexclusive 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
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
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in writing.

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