bioaffinity Year
Date 2025-06-14
Type
Feel
Area
Details of results
Results
The experiment was conducted to evaluate the sensitivity of labeling antibody with Alkaline phosphatase (AP) as the label reagent. Totol 4 types of AP labelled antibodies were prepared: C1R scFv-HAP fusion, C1R-T3AP, C1R-AP conjugate and whole antibody-AP conjugate proteins.
Method:
500 μg/ml whole anti-CRP-mouse IgG in PBS was dispensed onto the NC membrane and dried at room temperature. A total volume of 200 μl of running buffer containing 0.2 M arginine and 0.2% BSA-TBST was prepared by mixing 100 μl of CRP antigen (at concentrations ranging from 1- 1×10-6 μg/ml) with 100 μl of 100 ng/ml labeled scFv-AP. The test strip was immersed in 2 ml Eppendorf tube containing 200 μl running solution. After 20 minutes, the nitrocellulose membrane was isolated and washed 3 times with TBST. 100 μl of BCIP-NBT was added to the membranes and incubated for 10 minutes to develop the color. The color development was stopped by washing 3 times with TBST. The membranes were then dried at room temperature and scanned.
Results:
C1R-T3AP possessed the best signal development, followed by C1R-AP conjugate, C1R-HAP and finally whole Ab-AP, respectively. C1R-T3AP could detect CRP at 10 pg/ml while other labelled antibodies could not.
No background signal was found.
keywords: LFIA, scFv-AP, NC membrane