Human Monoclonal Antibody Screening Set (Flag-tag Antigen)

Homogeneous Immuno Chemiluminescence Assay (HICA) is a homogeneous immunoassay method based on energy transfer between donor beads and acceptor beads at close proximity to generate 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 proportional to the strength of 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.

Name	Main Component	3000T
Reagent A	Anti-Flag-tag antibody-crosslinked acceptor Beads	10mL
Reagent B	Biotin-labeled anti-human IgG antibody	10mL
Reagent D	Streptavidin Donor Beads	10mL

[Required Reagents]

Name	Catalog No.
Universal Buffer 4	UA086115

[Reference Detection Protocol]

Assay Procedure	Assay Procedure
Step 1:	Add Flag-tag antigen to Reagent A at a concentration of 1.5 μg/mL
Step 2:	Mix Reagents A, B, and D uniformly at a volume ratio of 1:1:1,Protect from light / Green light
Step 3:	Add 10 μL of test sample + 10 μL of mixed reagent to each test,Protect from light / Green light
Incubation	Shake incubate at 37°C for 60 minutes,Protect from light / Green light
Reading	Instrument reading

[Sample & Reagent Preparation]

•Pre-dilute human anti-IL1B antibody to 1000 ng/mL (6.67 nM) using DMEM + 10% FBS as C8, then perform serial dilutions according to the following scheme:

ID	Concentration (ng/mL)	Diluent Volume (μL)	Antibody Volume (μL)
C8	1000	/	/
C7	200	160	40μL C8
C6	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]

•Standard Curve:

Maximum Signal: 935617

Minimum Signal: 447

EC50= 2.045 ng/mL (0.014 nM)

•Limit of Blank (LOB):

Test C1 repeatedly 20 times, calculate the mean signal and SD. Use the standard curve to calculate the concentration corresponding to mean signal + 2×SD, which is defined as the LOB.

LOB = 0.393 ng/mL (2.618 pM)

•Repeatability:

Test high and low concentration samples repeatedly 10 times each, and calculate the concentration CV%.

Repeatability	Low Concentration	High Concentration
CV%	5.0%	3.7%

•Specificity: Dilute the following proteins to 1 μg/mL using DMEM + 10% FBS, and test for cross-reactivity interference.

Analyte	Cross-reactivity (%)
Mouse anti-IL1B antibody	0.00%

1. This experiment is light-sensitive; ensure light protection during operation. It is recommended to perform preparation, sample loading, and incubation 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 loading.