| Product Description |
Lentivirus
vector is derived from human immunodeficiency virus-1 (HIV-1), which has been
widely used to transfer gene(s) of interest into dividing and non-dividing
cells. The lentiviral titer,
as assessed by the p24 protein content, is an
essential characterization procedure for routine quality control and lot
release, as well as for purification process optimization. However, HEK-293T
cells will release free p24 protein into the supernatant during transfection,
and this part of p24 would account for a large percentage of the total p24. The
traditional p24 ELISA kit detects both lentivirus associated p24 and free p24,
which will overestimate the titer of the lentivirus.
GenScript’s
Lentivirus Titer p24 ELISA Kit Pro(Cat. No. L00974) is a Sandwich ELISA
designed for quantitatively measuring the physical titer of any HIV-1 based
lentivirus in cell culture supernatant. After removing free p24 (patented
technology), the amount of lentivirus-associated p24 can be measured by the p24
ELISA kit. The kit has the same linear range, detection capability, and precision
with Lentivirus Titer p24 ELISA Kit (Cat. No. L00938) in the detection of p24
concentration. This kit makes lentivirus titration easy, accurate and
reproducible. |
| Linearity Range |
31.25 to 2000 pg/mL
|
| Sensitivity |
LoB: 14.13 pg/mL
LoD: 29.42 pg/mL
LoQ: 39.03 pg/mL
|
| Precision |
Intra-assay: CV ≤ 10%;
Inter-assay: CV ≤ 15%
|
| Kit Contents |
| Component |
Quantity/Size |
Part No. |
| Capture Plate |
1 plate |
E1-80 |
| Standard Stock |
1 vial (100 μL) |
E1-10 |
| Lysis Solution |
1 vial (1.6 mL) |
E1-A0 |
| Biotin Anti-p24 Antibody |
1 bottle (12 mL) |
E1-20 |
| Streptavidin-HRP |
1 bottle (12 mL) |
E1-30 |
| Lentivirus Free p24 Removal Reagent |
1 bottle (2 mL) |
E1-40 |
| 10× Beads Wash Solution |
1 bottle (40 mL) |
E1-50 |
| TMB Solution |
1 bottle (12 mL) |
A1-40 |
| 20× Wash Solution |
1 bottle (40 mL) |
A1-70 |
| Stop Solution |
1 bottle (6 mL) |
A1-50 |
| Plate Sealer |
2 pieces |
N/A |
|
| Storage |
The
unopened kit is stable for at least 12 months from the date of manufacture at
2°C to 8°C, and the opened kit is stable for up to 1 month from the date of
opening at 2°C to 8°C. |
| Assay Principle |
The Lentivirus Titer p24 ELISA Kit Pro consists of two parts, including Lentivirus Free p24 Removal Reagent (patented technology) and Lentivirus Titer p24 ELISA Kit. Before detecting the concentration of p24 protein, free p24 in lentiviral supernatant can be specifically removed by Lentivirus Free p24 Removal Reagent.
The Lentivirus Titer p24 ELISA Kit utilizes two HIV-1 p24 monoclonal antibodies which bind to different epitopes of the p24 protein. When standards or samples are added to the capture plate, the p24 protein in the sample can be captured on the plate coated with the HIV-1 p24 capture antibody. Then the HIV-1 p24 detection antibody conjugated with biotin is added to interact with the p24 protein bound on the plate. Streptavidin-Horseradish Peroxidase conjugate (Streptavidin-HRP) is added to interact with the biotin-conjugated anti-p24 antibody (Biotin Anti-p24 Antibody). After washing steps, 3,3',5,5'-Tetramethylbenzidine solution (TMB Solution) is added resulting in the formation of blue color. The reaction is stopped by adding Stop Solution. Application of the Stop Solution results in the color changing from blue to yellow. The intensity of the color can be read at 450 nm by a microplate reader.
The quantity of p24 protein in the sample is precisely quantified against a p24 standard curve. p24 values are then correlated to the lentivirus titer of packaging cell culture supernatants.
Figure 1: Schematic diagram of Lentivirus Titer p24 ELISA kit Pro |
| Reference |
1. CLSI document EP06-A (Evaluation of the Linearity of
Quantitative Measurement Procedures: A Statistical Approach; Approved
Guideline, 2nd Edition).
2. CLSI document EP17-A2 (Evaluation of Detection
Capability for Clinical Laboratory Measurement Procedures, 2nd Edition).
3. Kahl C A,
Marsh J, Fyffe J, et al. Human immunodeficiency virus type 1-derived lentivirus
vectors pseudotyped with envelope glycoproteins derived from Ross River virus
and Semliki Forest virus[J]. Journal of virology, 2004, 78(3): 1421-1430.
4. White S M, Renda M, Nam N Y, et al. Lentivirus
vectors using human and simian immunodeficiency virus elements[J]. Journal of
virology, 1999, 73(4): 2832-2840.
5. Kafri T, van
Praag H, Ouyang L, et al. A packaging cell line for lentivirus vectors[J].
Journal of virology, 1999, 73(1): 576-584. |
Figure 3: Lentivirus Titer p24 ELISA kit standard curve.
Schematic diagram of Lentivirus Titer p24 ELISA kit Pro
Figure 4 Different concentrations of p24 protein in cell culture media were detected by traditional p24 ELISA kit or Lentivirus Titer p24 ELISA Kit Pro
Figure 5 Samples containing different ratios of free p24 and purified lentivirus were detected by traditional p24 ELISA kit or Lentivirus Titer p24 ELISA Kit Pro
Figure 6 Different lentivirus samples at the same production stage were analyzed by traditional p24 ELISA kit or Lentivirus Titer p24 ELISA Kit Pro
For research use only.
Not intended for human and animal therapeutic or diagnostic use.
