Description
PRINCIPLE OF THE ASSAY This assay employs the quantitative competitive enzyme immunoassay technique. Recombinant Monkeypox virus E8L antigen has been pre-coated onto a microplate. Standards or samples are premixed with biotin-labeled antibody and then pipetted into the wells. Human Anti-Monkeypox virus IgG (E8L) in the sample competitively binds to the pre-coated protein with biotin-labeled Human Anti-Monkeypox virus IgG (E8L). After washing away any unbound substances, Streptavidin-HRP is added to the wells. Following a wash to remove any unbound enzyme reagent, a substrate solution is added to the wells and color develops in inversely proportion to the amount of Human Anti-Monkeypox virus IgG (E8L) bound in the initial step. The color development is stopped and the intensity of the color is measured.
Catalog No.
KAV13201
Tested applications
Used for the quantitative determination of Human Anti-Monkeypox virus IgG (E8L) concentration in serum and plasma.
Target
E8L
Stability and Storage
When the kit was stored at the recommended temperature for 6 months, the signal intensity decreased by less than 20%. For unopened kits, if you want to prolong storage time, please store the Standard, Detection A, Detection B and Microplate at - 20 ℃, the rest reagents should be store at 4℃.
Detection method
Colorimetric
Precision
MATERIALS PROVIDED & STORAGE CONDITIONS
|
PART |
Format |
Description |
STORAGE CONDITIONS |
|
Pre-coated Microplate |
1 plate |
96 well polystyrene microplate (12 strips of 8 wells) precoated with recombinant Monkeypox virus E8L antigen. |
Store in sealed at -20℃. |
|
Human Anti-Monkeypox virus IgG (E8L) Standard |
2 bottles |
1,000 ng/bottle of lyophilized Human Anti-Monkeypox virus IgG (E8L). Reconstitute in 1mL Standard Diluent before used. |
Store at -20℃. |
|
Detection A |
1 vial |
60 μL/vial of Biotin labeled Human Anti-Monkeypox virus IgG (E8L) antibody (including preservative), 1:100 diluted by Assay Diluent before used. |
Store at -20℃. |
|
Detection B |
1 vial |
120 μL/vial of Streptavidin-HRP (including preservative), 1:100 diluted by Assay Diluent before used. |
Store at -20℃. |
|
Standard Diluent |
1 bottle |
25 mL/bottle diluent (including preservative) was used to dilute the Standard and Samples. |
Store at 4℃. |
|
Assay Diluent |
1 bottle |
25 mL/bottle diluent (including preservative) was used to dilute the Detection A and Detection B. |
Store at 4℃. |
|
20 × Wash Buffer |
1 bottle |
25 mL/bottle of a 20-fold concentrated solution of buffered surfactant with preservative, 1:20 diluted by deionized water before used. |
Store at 4℃. |
|
Color Reagent |
1 bottle |
12 mL/ bottle of TMB (Tetramethylbenzidine). |
Store at 4℃. |
|
Stop Solution |
1 bottle |
6 mL/ bottle. |
Store at 4℃. |
|
Plate Sealers |
4 strips |
Adhesive strips. |
Store at RT. |
* Provided this is within the expiration date of the kit.
OTHER SUPPLIES REQUIRED
• Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 630 nm or 620 nm.
• Pipettes and pipette tips.
• Deionized or distilled water.
• 500 mL graduated cylinder.
• Squirt bottle, manifold dispenser, or automated microplate washer.
• Test tubes for dilution of standards.
SAMPLE COLLECTION & STORAGE
The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated.
Handle all blood and serum as if capable of transmitting infectious agents. The NCCLS provides recommendations for handling and storing serum and plasma specimens (Approved Standard-Procedures for the Handling and Processing of Blood Specimens, H18-A. 1990).
Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20℃. Avoid repeated freeze-thaw cycles.
Plasma - Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20℃. Avoid repeated freeze-thaw cycles.
REAGENT PREPARATION
Bring all reagents to room temperature before use.
20-fold Wash Buffer Concentrate - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Add 25 mL of Wash Buffer Concentrate to 475 mL of deionized or distilled water to prepare 500 mL of Wash Buffer.
Serum and Plasma - Serum and plasma samples require a 10-fold dilution. A suggested 10-fold dilution is 10 μL of sample + 90 μL of Standard Diluent (diluted 1:9). If the sample value is outside the range of the standard curve, the dilution can be adjusted appropriately and the assay can be redetermined. If the antibody concentration in the sample can be estimated and the assay can be performed simultaneously by diluting several gradients prior to the experiment.
Standard - Reconstitute with 1mL Standard Diluent, this reconstitution produces a stock solution of 1,000 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. 1,000 ng/mL is the first standard point, and the concentration of the 7 standard sample were 1,000 ng/mL, 500 ng/mL, 250 ng/mL, 125 ng/mL, 62.5 ng/mL, 31.25 ng/mL, 15.6 ng/mL respectively. The appropriate Standard Diluent serves as the zero standard (0 ng/mL).
Detection A (working solution) - Shake and mix before used. Centrifuge instantaneously with palm centrifuge to make the liquid at the bottom of the tube. Dilute the Detection A 1: 100 times to the working concentration with Assay Diluent.
Detection B (working solution) - Shake and mix before used. Centrifuge instantaneously with palm centrifuge to make the liquid at the bottom of the tube. Dilute the Detection B 1: 100 times to the working concentration with Assay Diluent.
ASSAY PROCEDURE
Bring all reagents and samples to room temperature before use. It is recommended that all standards and samples be assayed in duplicate.
1. Prepare all reagents and working standards as directed in the previous sections.
2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
3. Add 100 μL premixed solution (50 μL diluted standard/sample + 50 μL Detection A working solution) to each well. Cover with the adhesive strip provided. Incubate for 2 hours at 37℃.
4. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 μL of Detection B (working solution) to each well. Cover with a new adhesive strip. Incubate for 30 minutes at 37℃.
6. Aspirate each well and wash, repeating the process five times. Wash by filling each well with Wash Buffer (300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
7. Add 100 μL of Color Reagent to each well. Incubate for 15 minutes at 37℃. Protect from light.
8. Add 50 μL of Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well within 10 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 630 nm or 620 nm. If wavelength correction is not available, subtract readings at 630 nm or 620 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
PRECISION
Intra-Assay Precision (Precision within an assay): <20%
Three samples of known concentration were tested sixteen times on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays): <20%
Three samples of known concentration were tested in twenty four separate assays to assess inter-assay precision.
TROUBLESHOOTING GUIDE
|
Problem |
Probable Cause |
Solution |
|
Poor Precision |
Wells are not washed or aspirated properly |
Make sure the wash apparatus works properly and wells are dry after aspiration |
|
Bubbles in the wells |
Tap plate gently to disperse bubbles |
|
|
Wells are scratched with pipette tip or washing needles |
Dispense and aspirate solution into and out of wells with caution |
|
|
Particulates are found in the samples |
Remove any particulates by centrifugation prior to the assay |
|
|
High background |
Plate is not washed properly |
Make sure the wash apparatus works properly |
|
Incorrect incubation times and/or temperatures |
The OD value increased gradually along with the time. Reduce the color developing time properly |
|
|
Weak/No Signal |
Pipetting errors |
Make sure the pipette is calibrated |
|
The working solution not be prepared immediately before use |
The working solution should be prepared immediately before use and should not be stored |
|
|
Volumes errors |
Repeat assay with the required volumes in manual |
|
|
The plate is not incubated for proper time or temperature |
Follow the manual to repeat assay |
|
|
Detection A working solution is not completely mixed with the samples |
After adding the Detection A into the wells, make sure the detection A and the samples are mixed thoroughly |
Sample type
Plasma, Serum
Assay type
Quantitative
Range
15.6 ng/mL - 1,000 ng/mL
Recovery
80-110%
Alternative Names
E8L, Cell surface-binding protein, Carbonic anhydrase homolog, Monkeypox virus/MPXV
Shipping
2-8 ℃
Note
For Research Use Only.
For research use only. Not for human or drug use.
