Human Monoclonal Antibody Screening Set (His-tag Antigen)
Human Monoclonal Antibody Screening Set (His-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
Homogeneous Immuno Chemiluminescence Assay (HICA) is a homogeneous immunoassay method based on energy transfer between donor beads and acceptor beads for 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, no washing steps, fast speed, and high sensitivity, enabling the detection of weak interactions.
Components
Name |
Main Component |
2000T |
Reagent A |
Anti-His-tag antibody-crosslinked acceptor Beads |
10mL |
Reagent B |
Biotin-labeled anti-human IgG antibody |
5mL |
Reagent D |
Streptavidin Donor Beads |
5mL |
Protocol
[Required Reagents]
Name |
Catalog No. |
Universal Buffer 4 |
UA086115 |
[Detection Procedure for Reference]
Detection Step |
Procedure |
Step 1: |
Add His-tag antigen to Reagent A at a concentration of 2μg/mL |
Step 2: |
Mix Reagent A, Reagent B, and Reagent D uniformly at a volume ratio of 2:1:1,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 H1 antibody to 1200ng/mL (8nM) using Universal Buffer 4 as C12, then perform serial dilutions as follows:
ID |
Concentration (ng/mL) |
Universal Buffer 4 Volume (μL) |
High Concentration Addition Volume (μL) |
C12 |
1.20E+03 |
/ |
/ |
C11 |
3.60E+02 |
210 |
90μL C12 |
C10 |
1.20E+02 |
180 |
90μL C11 |
C9 |
3.60E+01 |
210 |
90μL C10极 |
C8 |
1.20E+01 |
180 |
90μL C9 |
C7 |
3.60E+00 |
210 |
90μL C8 |
C6 |
1.20E+00 |
180 |
90μL C7 |
C5 |
3.60E-01 |
210 |
90μL C6 |
C4 |
1.20E-01 |
180 |
90μL C5 |
C3 |
3.60E-02 |
210 |
90μL C4 |
C2 |
1.20E-02 |
180 |
90μL C3 |
C1 |
0 |
180 |
/ |
[Performance Validation]
•Response curve:
Maximum signal: 4458991
Minimum signal: 352
EC50= 45.78 ng/mL (0.31nM)
•Limit of blank (LoB):
Repeat C1 testing 20 times, calculate mean signal and SD. Use the response curve to determine the concentration corresponding to mean signal + 2×SD, which is the LoB.
LoB= 0.156 ng/mL (1.04pM)
•Repeatability CV%:
Repeat testing high and low concentration samples 10 times, calculate concentration CV%.
|
Low concentration |
High concentration |
Repeatability CV% |
2.41% |
2.05% |
•Specificity: Dilute the following proteins to 1μg/mL using Universal Buffer 4 and test cross-reactivity rates.
Test substance |
Cross-reactivity rate |
Mouse IgG |
0.00% |
Rabbit IgG |
0.00% |
Guidelines
1. This experiment is light-sensitive. Ensure light protection 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 multifunctional microplate readers equipped with Alpha detection modules.
3. To ensure comparability of experimental data across different batches, strictly control incubation temperature and time.
4. Avoid generating bubbles during sample addition.
