HPV (59) Fluc Pseudovirus

应用说明（Application）

Pseudovirus Neutralization Assay
a. Mix 89% DMEM medium, 10% Fetal bovine serum and 1% Penicillin-Streptomycin to prepare complete DMEM medium.
b. Thaw the pseudovirus at room temperature. Dilute the pseudovirus with complete DMEM medium according to your pre-test results. In general, we recommend a 25-fold to 250-fold dilution, among which 250-fold dilution is optimal at ACROBiosystem's experimental platform.
c. Dilute your samples with complete DMEM medium in a 96-well white flat bottom plate to reach a volume of 75 μL per well, then add 25 μL pseudovirus dilution per well to reach a final volume of 100 μL per well. Gently flap to mix well. Incubate the plate at suitable temperature for 60 min. We suggest that 4℃ is an optimal incubation temperature, while 37℃ is an alternative.
d. Digest and resuspend SV40 Large T antigen overexpressing HEK 293T cells with complete DMEM medium. Adjust the cell density to 5 × 10⁵ cells per milliliter with complete DMEM medium. Seed 100 μL the cell suspension per well into the 96-well plate. Gently flap to mix well. Incubate the plate in a 5% (vol/vol) CO₂, 37°C incubator for 48 h.
e. Prepare firefy luciferase detection reagent (britelite plus Reporter Gene Assay System (PerkinElmer, Cat. No. 6066761)) and balance it to room temperature.
f. Take out the 96-well plate and discard 100 μL medium per well, which makes there is approximately 100 μL medium left in each well. Balance the plate to room temperature for 10 min. Add 100 μL detection reagent and mix well. Incubate for 2~5 min at room temperature.
g. Read the luminescence values (RLU) of the wells with a luminescence meter (PerkinElmer, Cat. No. HH34000000).
h. Calculate the inhibition rate with the following formula:

产品背景（Background）

Human papillomavirus (HPV) is a common sexually transmitted infection (STI), which is related to several diseases, such as genital warts and tumours. The virus has no envelope and uses major capsid protein (L1) and Minor capsid protein (L2) as its capsid with a double-stranded circular DNA inside.
HPV (59) Fluc Pseudovirus is packaged with L1 and L2 proteins of HPV type 59 expressed in SV40 Large T antigen overexpressing HEK 293T cells, as well as a double-stranded DNA containing firefly luciferase gene. The pseudovirus can mimic the structure of HPV, while does not contain oncogenes from HPV, such as HPV E6 / E7, and replication deficient. It can effectively infect SV40 Large T antigen overexpressing HEK 293T cells and can be used in determining neutralizing antibody titer, screening for inhibitors, studying virus invasion and HPV vaccine development.
The reference sequences are: Uniport # Q81971 (L1) and Uniport # B9UPE5 (L2).

注意事项（Notice）

Basic advice
a. Though pseudovirus particles has no pathogenicity and cannot replicate, the assays should be carried out carefully in a Biosafety Level 2 or higher-level laboratory with a biosafety cabinet.
b. Serum samples from animals or humans should be inactivated in a water bath at 56°C for 30 min before being tested.
c. Please avoid freezing and thawing, which would influence the titer of the pseudovirus.
d. The product is for Research Use Only.

For more information and advice, please check the Data Sheet of this product.