Introduction
The RayBio® Monkeypox Virus (MPXV) E8L ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of MPXV E8L in serum, plasma (collect plasma using EDTA as an anticoagulant. Heparin or citrate are not recommended in this assay), and cell culture supernatants. This assay employs an antibody specific for MPXV E8L coated on a 96-well plate. Standards and samples are pipetted into the wells and MPXV E8L present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-MPXV E8L antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of MPXV E8L bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Product Features
- Strip plates and additional reagents allow for use in multiple experiments
- Quantitative protein detection
- Establishes normal range
- The best products for confirmation of antibody array data
Standard Curves
Spiking & Recovery Results
| Sample Type | Average % Recovery | Range (%) |
|---|
| Serum | 106.0 | 96.9-118.3 |
| Plasma | 103.4 | 99.6-107.2 |
| Cell culture media | 108.1 | 102.5-120.3 |
Linearity Results
| Sample Type | Serum | Plasma | Cell Culture Media |
|---|
| 1:2 | Average % of Expected | 96.6 | 104.1 | 99.9 |
| Range (%) | 80.1-114.9 | 99.1-109.1 | 93.9-105.8 |
| 1:4 | Average % of Expected | 98.4 | 98.7 | 86.3 |
| Range (%) | 83.7-123.2 | 85.4-112.0 | 80.4-92.2 |
Application Notes
Kit Components
- Pre-Coated 96-well Strip Microplate
- Wash Buffer
- Stop Solution
- Assay Diluent(s)
- Lyophilized Standard
- Biotinylated Detection Antibody
- Streptavidin-Conjugated HRP
- TMB One-Step Substrate
Other Materials Required
- Microplate reader capable of measuring absorbance at 450 nm
- Protease and Phosphatase inhibitors
- Precision pipettes to deliver 2 µL to 1 µL volumes
- Adjustable 1-25 µL pipettes for reagent preparation
- 100 µL and 1 liter graduated cylinders
- Log-log graph paper or computer and software for ELISA data analysis
- Tubes to prepare standard and sample dilutions
- Distilled or deionized water
- Absorbent paper
Protocol Outline
- Prepare all reagents, samples and standards as instructed.
- Add 100 µL of each standard and sample to each well.
- Incubate 2.5 hours at room temperature or overnight at 4°C with gentle shaking.
- Add 100 µL prepared detection antibody to each well.
- Incubate 1 hour at room temperature with gentle shaking.
- Add 100 µL prepared HRP-Streptavidin solution.
- Incubate 1 hour at room temperature with gentle shaking.
- Add 100 µL TMB One-Step Substrate Reagent to each well.
- Incubate 30 minutes at room temperature.
- Add 50 µL Stop Solution to each well.
- Read at 450 nm immediately.
Storage/Stability
The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.