Mouse Monoclonal Antibody Screening Set (Flag-tag Antigen)
Mouse Monoclonal Antibody Screening Set (Flag-tag Antigen)
Product Details
Product Details
Product Specification
| Stability & Storage | Store at 2-8°C away from light; product shelf life is 12 months. |
Background
The Homogeneous Immuno Chemiluminescence Assay (HICA) is a homogeneous immunoassay method based on energy transfer between donor beads and acceptor beads at close proximity, resulting in luminescence.
Donor beads recognize Protein 1 (Tag1 label), while acceptor beads recognize Protein 2 (Tag2 label). When Protein 1 binds to Protein 2, the distance between the beads becomes less than 200nm. Upon excitation at 680nm, the donor beads generate singlet oxygen, which diffuses to the acceptor beads. The acceptor beads then undergo a redox reaction, emitting light at 615nm. The signal intensity is directly proportional to the strength of the protein interaction.
This product features a simple operation process, requires no washing, and offers high speed and sensitivity. It is capable of detecting weak interactions.
Components
Name |
Main Components |
3000T |
Reagent A |
Anti-Flag-tag antibody-crosslinked acceptor Beads |
10mL |
Reagent B |
Biotin-labeled anti-mouse IgG antibody |
10mL |
Reagent D |
Streptavidin Donor Beads |
10mL |
Protocol
[Required Reagents]
Name |
Catalog Number |
Universal Buffer 4 |
UA086115 |
[Detection Procedure for Reference]
Detection Process |
Detection Process |
Step 1: |
Add Flag-tag antigen to Reagent A at a concentration of 1.5μg/mL |
Step 2: |
Mix the above Reagent A, Reagent B, and Reagent D uniformly at a volume ratio of 1:1:1,avoid light/green light |
Step 3: |
Add 10μL of test sample + 10μL of mixed reagent per test,avoid light/green light |
Incubation |
37°C with shaking for 60 minutes,avoid light/green light |
Reading |
Instrument reading |
[Sample and Reagent Preparation]
•Pre-dilute mouse anti-IL1B antibody to 1000ng/mL (6.67nM) using DMEM+10%FBS as C8, then perform gradient dilution according to the following scheme:
ID |
Concentration (ng/mL) |
Diluent Volume (μL) |
Antibody Volume (μL) |
C8 |
1000 |
/ |
/ |
C7 |
200 |
160 |
40μL C8 |
C6极> |
40 |
160 |
40μL C7 |
C5 |
8 |
160 |
40μL C6 |
C4 |
1.6 |
160 |
40μL C5 |
C3 |
0.32 |
160 |
40μL C4 |
C2 |
0.064 |
160 |
40μL C3 |
C1 |
0 |
160 |
/ |
[Performance Validation]
•Response curve:
Maximum signal: 1048745
Minimum signal: 280
EC50= 2.106 ng/mL (0.014 nM)
•Limit of blank:
Repeat检测20次C1，计算信号均值及SD，用反应曲线计算 信号均值+2×SD对应的浓度值，即为空白限。
Limit of blank= 0.738 ng/mL (4.922 pM)
•Repeatability:
Repeat testing of high and low concentration samples 10 times, calculate the concentration CV%.
Repeatability |
Low concentration |
High concentration |
CV% |
1.3% |
3.3% |
•Specificity: Dilute the following proteins to 1μg/mL using DMEM+10%FBS and test the cross-reactivity rate.
Test substance |
Cross-reactivity rate |
Human anti-IL1B antibody |
0.00% |
Guidelines
1. This experiment is light-sensitive. Please protect it from light during operation. It is recommended to perform preparation, sample addition, and incubation steps under green light (illuminance below 100 LUX).
2. This product is compatible with multi-functional microplate readers equipped with Alpha detection modules.
3. To ensure comparability of experimental data across different batches, please strictly control incubation temperature and time.
4. Avoid generating bubbles during sample addition.
