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 Number |
Universal Buffer 4 |
UA086115 |
[Reference Testing Procedure]
Testing Procedure |
Testing Procedure |
Step One: |
Add His-tag antigen to reagent A at a concentration of 2 μg/mL. |
Step Two: |
Mix the above reagent A, along with reagents B and D, in a volume ratio of 2:1:1 until homogeneous.Protect from light/green light. |
Step Three: |
For each test, add 10 μL of the sample to be tested plus 10 μL of the mixed reagent.Protect from light/green light. |
Incubation. |
Incubate at 37°C with shaking for 60 minutes.Protect from light/green light. |
Reading. |
Instrument reading. |
[Sample and Reagent Preparation]
•Use Universal Buffer 4 to pre-dilute the H1 antibody to 1200 ng/mL (8 nM) as C12, then perform gradient dilutions according to the following scheme:
Number |
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 |
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 Verification]
•Reaction Curve:
Maximum Signal: 4,458,991
Minimum signal: 352
EC50 = 45.78 ng/mL (0.31 nM)
•Blank Limit:
Repeat the C1 test 20 times, calculate the average signal and SD, then use the reaction curve to determine the concentration value corresponding to the average signal plus 2×SD—this is the blank limit.
Blank limit = 0.156 ng/mL (1.04 pM)
•Repeatability CV%:
Test high- and low-concentration samples in duplicate ten times each and calculate the concentration CV%.
|
Low concentration |
High concentration |
Repeatability CV% |
2.41% |
2.05% |
•Specificity: Use universal buffer 4 to dilute the following proteins to 1 μg/mL and test the cross-interference rate.
Tested substances |
Cross-reaction 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.
